Preface

Contents

iii

I. The Need for Sterility I

2. Sterility and Steril,ity Assurance 17

3. Sl'crilization by Gamma Radiation 53

4. Slerilization by S:uumted Steam 81

5. Dry Heat Sterilization and De~enation 109

6. Sterilization by Ethylene Oxide 123

7. Sterilization by Fihration 153

8. Aseptic ~'1anUr.3CIUre 179

9. Maintenance of Stcrilily 241

10. Paramelric Release and Oilier Regulatory Issues 259

1nJjl't 275

v

Copyrighted leria

AChievil SterilitY•

I

•

I

Copyrighted leria

1The Need for Sterility

I. Slerile Pharmaceutical Products 3II. Slerile Medical Devices 6

III. Consequences o( Nonsterility 9A. The 1971n2 Devonport lncidenl in the U.K. 9B. The 1970171 Rocky Mounllncident in the U.S.A. 10C. The 197m3 Cu'ter Laboralories Incident in the U.S.A. 12D. The 1964 Imported Eye Ointmenllncident in Sweden 13E. The 1981 Imported Indian Dressings Incident in the U.K. 13F. Incidents Originaling (rom Other Sources 14

Why are some medical products required 10 he slerile? What distinguishes lhe..products from olber medic31 products that are nol required to be sterile? Whatare the consequences of nonsterility?

Sterility is defined academically as the total absence of viable life forms.Some partS of the human body are always exposed to and contaminated by otherforms of life. For insrance.1.he external surfaces afme body, skin, hair. airways.etc.• are unavoidably in conlaCt with the general (microbiologically contami­nated) environment The bucca! cavity and intestinal trael are regulady broughtinto conlaCl wilh food~ and water-borne microorgani ms ingested with the dietIn many cases, some of these microorganisms colooize the surfaces of lIle human

1

2 Chapler 1

body and exist in hannaD)' with the human host. sometimes even beneficially.Internal tissues are, however. expected 10 be totally free from microbial con­tamination.

The external surfaces of the "nannal," fit. healthy human being haveevolved to be effective barriers against penetration by opponunistic microor­ganisms to inlema! tissues that might provide them with nourishment at theexpense of the host Sometimes lhe external physical barriers fail. and then otherantimicrobial defense mechanisms come into play. the immune system forinstance. These internal mechanisms are combating infection. The varioussymptoms of infectious disease are the result of the interaction between theanempls by the infecting agent to colonize the internal tissues of lhe body andthe auempts by the body's defense mechanisms to overcome this invasion.

From the sterility standpoint, no distinction can be made between themicroorganisms that are known to be specific causative agents of disease and(hose that are nol. It would of course be a major disaster if a specific pathogensuch as Haeillu$ anthracis (the causative agent of anlhrax) were to be introducedinto the human (or animal) body through the administration of a supposedlytherapeutic agenl. On the other hand, microorganisms that are frequently foundon man or in man's immediate environment are often assumed to be hannlessbec-duse they are nol associated with any specific disease. However. this is awholly invalid assumption once the body's antimicrobial defensive baniers arebroken down. as they usually are in the administration of parenteral preparations.These microorganisms may often be opponunistic pathogens. This is panicu­1arly applicable in the case of weak and debilitated patients who are ill-equippedto resist infection, even from microorganisms that have not evolved to be spe­cially invasive. Complete freedom from all microorganisms is the only criterionfor sterility.

Many therapeutic procedures quite deliberately break down the body'sexternal physical barriers. From the application of an ointment or cream to bro­ken skin, to simple or complex surgery. to injection, 10 implantation of. say. acardiac pacemaker, all of these procedures risk infecting the patient by breakingthrough the body's ex.temal physic,aI barriers. Infection will only occur. how­ever. if these procedures carry viable microorganisms to internal tissues. On theother hand. if the devices and substances that are brought into contacl with inter­nal tissues are free from viable microorganisms-in other words, "slerile"­there ought to be no infection. This is the first and most fundamental reason whysome medical products are required 10 be sterile.

There are other reasons.Devices that are intermediales in the delivery of therapeutic substances 10

internal tissues. say infusion sets or catheters. ought to be sterile. It is quiteobviously inappropriate to convey a sterile nuid from ils sterile reservoir toimemal tissues by a nonsterile route.

The Need for Sterility 3

GarmenLS. gloves. drapes and other operating theater paraphernalia oughtto be sterile to prevent tnlnsfer of microorganisms to exposed internal tissueduring surgical procedures.

Ophthalmic prepamlions. eye drops and eye ointments. ought to be sterile.There are three: basic reasons for Ihis. First. the cornea and other tr.Insparentpans of the eye have a particul:>.rly poor supply of blood and Ihe",fore a lessresponsive immune reaction than other pans of the body. Second, the trans·parency of the·se pans of the eye may be irreversibly damaged as a result ofinfection. wilh resultant permanent loss of vision. Third. infectious damage tothe optic nerve is irreparable.

Numerous items of laboratory equipment. for instance piptues. petridishes. tissue culture plates. etc., have to be sterile. 11 is not within the scope ofthis text to address these. except 10 indicate striking similarities or differences inpassing. In medical laboratory sciences particularly. conlwners for collection oftissues and body fluids for diagnostic analysis oughl 10 be !t1erile. This is 10

ensure true resuhs. Microbial contamination may perven biochemical leslresults. Microbial COnlaminalion in containers may prevent accurate diagnosisof infectious conditions.

Numerous olher medical producis are not required to be sterile. Medicinesto be taken by moulh. enemas, inhalations, most topical products. etc., need nO(be sterile. In some cases Ihere may be a need to ensure Ihat these products aremicrobiologically "clean:' or free from specific pathogens or from microbiologi­cal conlamination indicators. but there is no obligal'ion 10 sterility.

Sterility is. however. required in some unusual circumstances for medicaland nonmedical prodUCIS that would nOI nonnaJly be associated wilh this type ofneed. For insmnce. you may consider sterile diets for hospital patients who arebeing Ircated with immunosuppressive lherapeutic agents.

1be scale of manufacturelpreparat'ion of sterile medical products and thecomplex.ity of sl'c.rile producLS is extremely wide ranging. Nothing is truly typi­cal. nor can any lext claim to be genuinely comprehensive. In this lexl we shallbe addressing industrial manufacture of sterile products because governmentalregulalory agencies and olhcr elhical purchasing organil:ltions have led industryto a certain consistency of approach thal allows sensible generalizations lO bemade.

I. STERILE PHARMACEUTICAL PRODUCTS

Although il is conceivable thal there are occasions when almoil any phannaceu­ricaJ produci may be required 10 be sterile. there are only two broad groups ofslerile p/larmaceulical prodUCIS. parenteral prodUCIS and ophthalmic prodUCIS.

The European Pharmacopoeio is panicularly succinct in ils definition ofpreparations for parenleral use. It states IhaI Ihey are sterile preparations

4 Chapter 1

intended for administration by injection. infusion. or implantation into thehuman or animal body. Funher. parenteral preparations are supplied. according10 the European Pharmacopoeia. in glass ampoules. hollies, or vials. or in othercontainers such as plastic bottles or bags or as prefilled syringes.

This colorless but clear definition of parenteral products has pretty welluniversal acceptance and is likely almost timeless as well: current United StatesFDA thinking is thai no new forms of presentation of sterile parenteral productsare likely to be approved without strong justification of their being of benefit 10

the patient. Commercial reasons are nOI accepuble.Nonetheless, there is a huge variety and wide range of parenteral products.

Some parenteral dosage fonns may be filled into their presenLation Conns orsystems of containment under controlled but nonsterile conditions and thenexposed to a sterilization process~ these are referred 10 as tenninally sterilizedproducls. Terminal sterilization must be the method of first choice for all sterilepharmaceutical products. This is good sense and reflects current FDA thinking.There are a variety of terminal sterilization processes. thermal. chemical, or byionizing radiation. bUI quite frequently dosage forms cannot withstand any ofthese treatments without loss of efficacy. In I.hese cases. recourse is made toaseptic manufacture. With aseptiC manufacture. product conlact componentsmaking up the system of cOnlainmem are sterilized before filling: the dosageform is sterilized before filling, preferably by filtration but possibly by somechemical treatment that mayor may not be pan of its initial synlhesis, .md lhewhole final presentation is filled and sealed in a sterile or as near sterile as possi­ble environment.

The first broad division among parenteral products is between those usedfor infusions and those used for other forms of administration. Infusions areprincipally intended for administration in large volumes and are frequentlyreferred (0 as large volume parenterals (LVPs). With me exception of slerileWater jor Jnj~clion. LVPs are usually made to be isolonic Wilh blood. for exam­ple saline. dextrose. etc.

The widest range of parenteral products are however. the small volumeparenterals (SVPs). These may be sierile solulions for injecting directly into lhepatient. They may be concentrated solutions or suspensions or emulsions oreven solids (solid dosage forms may be anhydrous. crystalline. or freeze dried[lyophilizedD for dilution or reconstitution in LVPs for direct injection or infu~

sian into the patient.Table 1 lists some ex.amples of sterile parenteral products classified as

LVPs or SVPs. as aseptically filled or tenninaUy sterilized. and as solutions.suspensions. or solid dosage forms.

The therJpeutic applic31ion of sterile parenteral products is almost bound­less. Some products can only be adminislered via the parenteral route; othersmay be administered orally. as suppositories. intranasally. CIC. This begs the

The Need fo' Ste,ility

Table 1 Some Examples of Sterile P:lrenteraJ Products

S

ProdUCt

LVPs

WatftrIor Injection USPDtxtrose Injection USP

SVPs

Ranitidin~ Injection USPSlLUJ11U!thonium ChlorideInjection BP

ProgesleroM Injection SPEpiMphriM OilSwpe.nsion USP

Sruilt! Cejlotidime USPDiamorphine Injection BP

Condition

"Solution"Solution

SolutionSolution

SolutionSuspension

SolidSolidOyophilized)

Achievement ofSterility

Terminal (steam)Terminal (steam)

Aseptic fiUTerminal (steam)

Tenninal (dry heat)Aseptic fill

Aseptic fillAseptic fill

question of why they are being manufactured for parenteral adminiSlJ'ation al all.The answer may tie with the prodUCl'S efficacy. with the acuteness of the condi­tion it is being used to treat, or with the speed at which relief of symptoms isrequired.

Taking the products listed in Table 1. Ceftazidime has only two COllies inthe USP. Ceftazidime for Injecrion USP and Slerile Ceftalidime USP. Bo'" arerestricted to parenteral adminisLration because of loss of efficacy when deli\'eredby other routes.

Ranltidine.. on the other hand. has entries as Ranitidine Injection USP andas Ranilidine Tablets USP. Epinephrine bas entries as an inhalation aerosol. aninjection, an inhalation solution. a nasal solution. an ophthalmic olution, and anoil suspension.

The question of whic:h Ranitidine preparation to use for ulcer treatment isbased primarily on the acuteness of the condition and with regard 10 conveniencefor maintenance therapy after the condition has been brought under control Par­enteral administration is in the main restricted to acute symptoms under hospitalsupenlisioo; oral administration is used for maintenance of the condition oncestabilized.

Epinephrine is ralher more complicated. because it may be used in con·nection with a varicl'Y of symptoms. Subcuraneous or intramuscular injecti.onmay be life~savlng for anaphylactic shock or acute allergic reactions. or il may

6 Chapt.r 1

be used to connol bronchial spasm in acute attacks of asthma. The other prepa­rations are used for local applic.ation in less extreme circumstances. Forinstance. the ophthalmic solution may be used for pupillary dilation in connec­tion with ophthalmic treatment or glaucoma.

Sterile Epinephrine Ophlhalmic Solution USP lakes us out of the realm ofsterile parenteral produclS into ophthalmics. The manner of presenullion ofophthalmics (i.•.• as drops or ointments) is likely 10 be quile familiar. For themost pan (bul not exclusively) lhey are in multidosc: presentations. As such,most (onnutations include some fonn of preservative to control proliferation ofany microorganisms Ihal may by chance contaminate the product on one or ornerof the occasions when il is open, or during the time when it is left standing on thebathroom shelf. The inclusion of preservatives in a muhido e formulation of anophlhalmic (or parenteral) is not a primary part of the process of achievingsterility. Il has quite a separate purpose.

Even when preservatives are included in single-dose presenlations (as Lheyoften arc). their efficacy against particular types of microorganisms can never belegitimately used as an excuse for tolerating in-process contaminatlon by preser­vative-sensitive types. or can the inclusion of preservatives in products beused to shorten or reduce the intensity of sterilization processes applied to prod­ucts or Lheir containers to lower than normaJ levels of sterility assur.mce. ~r­vatives are supplementary. 001 intrinsic to industrial-scale processes ofachie\'ingsterility.

An important distinction to draw between sterile parenteral producls andsterile ophthalmic produ lS concerns pyrogens. We will discuss pyrogens insome detail at a later stage in this text. They are substances that induce feverwhen injected into mammals. As such. aU sterile products for parenteral admin­istration are expected to be pyrogen free. and if dilution is required they must bediluted in a sterile pyrogen-free diluent The tie-up be'ween sterility. absence ofpyrogens. and administration by injection is reflected in the USP distinctionbetween the two types of water recouunended for ingredient purposes. PurifiedWatu and Water for Jnj~clion. The fonner is not required to be pyrogen fn:e.and only the laner is to be recommended for use in preparations intended forparenteral administration.

Sterile ophthalmic products have no requirement to be pyrogen free.

II. STERILE MEDICAL DEVICES

1be term med;cal d~vice includes iosuuments. apparatus. implementi. con~

uivances. implants. or other similar or related articles used in meclicallI'eaunent.A medical device does nOl achieve its principal intended purpose through chenu­calor phannacological action within or on the body. Some medical devices needto be sterile.

lVrl(,> led

The Need for S'erility 7

For lhe most pan (measured as numbers of devices used per annum). ster­ile medical devices are for single use only ("use once and then discard''), Hypo­demUc products and infusion sets are probably the most familiar types of single·use medical device. They are a comparatively modem concept thai had it's ori­gins in econonUcs and in an increasing concern over hospital-acquired infectionsin the "antibiotic era." Bdore the 19505. most medical devices were washed,resteriJized. and reused repeatedly. As antibiotics became widely available inmal decade. "background" infections diminished in proponion to lhosc lhat wereassociated with the reuse of equipment. At the same time the cost of labor forreprocessing was increasing while the cost of plastics was decreasing. Single.use industrially sterilized plastic medical devices grew from a practical alterna­tive to be the current nonn.

There are a huge range of different types of medical device. Approval tomarket is. as with pharmaceuticals. subject to regulalory control. Most sieriledevices in the U.S.A. would require premarket approval and fall into Class lIJ ofPan 860 of the Code of Federal Regula/ions. This classification places greatemphasis on devices that are life-supporting or life-sustaining. or those that areof substantial imponance in preventing impairment of human healLh. or thosemat present potential unreasonable risks of illness or injury.

Less formally, sterile devices may be classified in terms of lhc severity ofthe consequences of their nonsterility.

(a) Devices making no direct conl3et with patients. Mainly we are think­ing here of diagnostic devices. bearing in mind that contamination couldaffect the patient through adversely influencing the outcome of the diag­nostic process.

(b) Devices that contact intael eXlemaJ surfaces, such as !irerile dressings,or heavily contaminated internal surf:iilces such as the gut. for instanceexamination gloves. Patients arc not really likely to die as a resuh of non­sterility of these products unless a chance contaminant bas unusually inva­sive properties competitive with the innate micronora. 'Their sterility is ofgrealer significance with susceptible patients, an example being those withsevere bums, where infeclion is a major and possibly life-threatening issue.The range of products in this category is impossible to ex.emplify. but itmay be of value to consider sterile ceUulosic dressings. Almost inevitably,cellulosics are microbiologically contaminated, often with bacterial endo­spores. and lherefore pose a severe challenge to whatever sterilizationprocess is being applied,

(e) Devices that cont.aet directly or indirectly with the intnlvascular sys­tem. say "giving" sets. Here we are tall..ing about a vastly imponant routeof administr.ltion, often for severely ill patients. Tne consequences ofmicroorganisms being delivered directly to the blood, with the risk of 'hem

8 Chple.r 1

being carried throughout the body and inducing generalized infection. isself-evidenl. The principal ponion of a "giving" set is tubing. possiblyrubber but nowadays more likely extruded plastic tubing. The tempera­lures reached in plastic extrusion processes are quite high enough to bringabout significant reductions in numbers of vegetative microorganisms.However. cooling in waler and subsequent assembly and packing may leadto recontamination.

(d) Invasive devices. This category probably contains Lhe largest numberof items marketed, because it embraces hypodermic needles and syringes.scalpel blades. catheters. etc. These l1re lhe mechanisms that break downthe body barriers. If we take hypodermic syringes as an example of thistype of device. we can consider a variety of different types of manuractur~

ing technology versus their effeclS on microorganisms. The charJclerislicsingle-use disposable hypodennic syringe is made up of Ihree pieces; thebarrel, Ih< plunger, and Ih< plunger tip ("stopper"), Plastic plungers andbarrels are aimosl always injection-molded; rubber "SIOPpers" are com­pression-molded. The temperatures achieved wiLh these technologies killmost microorganisms. Like "giving" sets, contaminalion may occur dur­ing assembly and packaging; the numbers and types of microbial con·taOlinants on pack:tged hypodennic syringes prior 10 sterilization are verylargely relaled to the number of manual steps involved in these processes.In modem automated high-VOlume manufacture the final biological chal­lenge (bioburdrn) on these prodUClS tends (0 be quite low (I).

(e) Implantable devices. Some of lhese may have a purely mechanicalfunction, like (he very widely used anificial hip- and knee-joinls; Olhershave more complex and life-sustaining functions. such as cardiac pace­makers. In both cases there is a critical necessity for sterility. Again thetechnologies of manufacture and the complexity of lhe devices are diverse.The technology of manufacture of cardiac pacemakers is that of Ihe elec·tronies industry, where cleanliness is of the highest imponance to functiona.~ well as LO the control of bioburdeo. The lechnology of manufacture ofanificiaJ hip-joints is the lechnology of the machine shop, casting, millingelc. Cleanliness is an additional conSlraint to the traditional praclice ofthese crafts.

As with sterile phannllceulicals. pyrogens are of significant importance 10 medi­cal devices. Any device intended for adminiSlration of a sterile parenteral phar·maceutical must (like the pharmaceutical preparation) be pyrogen free. So muslall invasive and implantable devices.

The- Ne-HI for Sterility

III. CONSEQUENCES OF NONSTERILITY

9

Hospilal·acquired (nosocomial) infections are not uncommon. However. thosethat have been conclusively attributed to supposedly sterile but actually Ronster­ile pharmaceutical products or medical devices are quite rare. The consequencesof these incidenlS have not confined themselves to the companies responsible forthe failure to achieve sterility but have reverberated throughout the whole"steri1es" industry. No company wishes to face the litigation. loss of sales. lossof goodwill. and generally bad publicity that accompanies nonsterility. Most ofaU. ethic,at companies are in the business of preserving life. not in the business ofkilling people and death is often the consequence of nonsterility.

Although aU Lhe incidents described below occurred quite a long time agoand technology has changed and improved, and regulatory control has becomemore demanding and explicit, we believe that because sterility can only beachieved consistenlly by constant vigilance there are important lessons to belearned from reviewing them again.

A. The 1971/72 Devonporllncident in the U.K.

The Devonpon incident occurred in Lhe U.K. Some pDSloperative patients whohad been given supposedly sterile but actually contaminated infusion fluids died;others made unnecessarily long recoveries. The incident summarized below isdescribed fully in a U.K. government enquiry, the Clothier ReporrI2).

A series of untoward reactions were seen among postoperative patients inthe Devonpon Section of Plymouth General Hospital in March 1972. Sevenpatients were involved; five died. A commonality among the patients wa thataU had received intravenous administration of Dextrose Injection BP (5% dex­trose infusion fluid). AU intravenous infusion fluids containing dextrose werepromptly withdrawn from use, and samples were examined in the laboratory.

A batch of OOhles of "sterile" Dextrose Injection BP manufactured byEvans Medical Ud. (at that time a major U.K. producer of lbese types of prod­UClS) was found to be contaminated by Klebsiella aerogenes and other gram­negative colifonn bacteria. Approximately one-third of all of the OOllles fromthe incriminated batch were found to be nonsterile. The concentralion of bacte­ria in lbe bottles of fluid was sufficiently high to be visually perceptible to lbenaked eye; this would typically mean more than I()6 bacteria per mL.

An urgenl investigation was initiated. The possibility of olber batchesbeing contaminated could nOI be ruled out, and all Evans Medical infusion fluidswere placed under U.K. government embargo.

The contaminated product was traced to incorrect operation of EvansMedical's sterilizing autoclaves. The Committee of Enquiry [2) concluded thattoo many people believed that sterilization of fluids was easily achievable with

10 CNpt~r 1

simple equipment operated by men of littJe skill under 3 minimum of supervi­Ion.

What had happened was lhi . The sterilization process for houles of Dex­lros~ Injufion SP was by exposure to saturated steam at a temperature of 116-C(specified as 240·F) for 30 min. Evans Medical"s autoclaves were equipped withtwo temperature measuring devices. The first and most important of these was arecording thermometer located in the chamber drain. This is normally thecoolest pan of any autoclave. and it is from the sensor located at this point thaithe decision should be made thaI the autoclave has reached its specified operat­ing temperature and exposure timing begun. The second temperature measuringdevice on the autoclaves was a dial thermometer located near the steam inletpipe at the lOp of the chamber. This location usually reaches high temperaturesmore rapidly than any other location in the chamber. The recording thennome­lcrs in the chamber drains of Evans Medical's aUloclave were subject to faultyoperation... and it had become "custom and practice" for the steriliz.er operators togive more credence to (he dial thermometers. It had been quite common forb3tches of autoclaved infusion fluids to be released as sterile despite the temper·ature rec<>rt.kr chan showing an inadequate cycle.

The batch implicated in the Devonpon IncideOl had been sterilized in April1971. The recording thc:nnomeler did DOl. indicate the expecled rise in temper3­lure. On past experience, the manager of the area ignored this device and can·(ioued lhe process through reliance on the dial thennometer. With hindsight il ispossible (0 conclude that all of the air had not been vented from lhe oonom ofthe chamber at the beginning afthe cycle and consequently the correct oper.llingtemperature was not being achieved throughout the load; panicularly it was notbeing achieved for bottles in the lower pan of the chamber nor in the chamberdrain. In other words, the recording Lbennometer had been operating correctly.If the correct procedure had been followed Ihe process would nOI have beenapproved nor allowed t'O continue.

II is not peninenl to go into the detail of the likely technical problems thatmay have led to stratification of st.eam over air in the bouom of this aUloclave.but delails are given in the Clorhier Report [2).

The contaminated bottles were not delected by end·product sterilil)' test­ing. The batch was released 10 a wholesaler and distributed to the DevonportSection of Plymouth General Hospital in March 1972. The high concentrationsof microorganisms found in me infusion fluid can be attributed to the period oftime belween sterilization. distribution. and [mal administralion (0 the patients.

B. The 1970/71 Rocky Mount Incident in the U.S.A.

The Rocky Moun' Incidenl. which began in July 1970. affected al leasl 378patients in at least 25 US hospitals 13.4]. Forty patients died.

Th. Need 10< Sterility 11

As with the Devonport Incident. the Rocky Mount Incident was caused bycontaminated bouJes of infusion fluids. The fluids were all made by AbbottLaboratories in their Rocky Mount, Nonh Carolina. plant The company's infu­sion products were recalled in March 1971.

The clinical features seen with patients who received these contaminatedOuids included extreme fever, shaking chills, systemic toxicity, abdominalcramps. nausea, vomiting, diarrhea. delirium, and seizures. With hindsight lbeseare the symptoms of gram.negative septicemia. but with sudden onscllhey weresometimes misdiagnosed [3J. Confirmed cases were mainly drawn from largehospilals, often university teaching hospila1s. using significantly large volumesof infusion fluids. It is possible that many more patients in small hospitals wereimplicated, but the cases were nol diagnosed or reponed.

The microorganisms associated wilh the epidemic were identified withEnterobacler cloacae. Enrerobacler agglomuans. and olher Enterobacur spe·cies. 1be precise cause of the incidenl was traced 10 a program of gradualreplacemenl of Gilsonile cap liners for the infusion fluid bottles wilh anelastomer cap liner (Fig. I). The replacement program was operaling only inAbbon's Rocky Mount planl and nol on any oilier Abball operating sile.

FellS el at (4] examined 93 bottles containing a variety of different infu­sion fluids. They looked for microbiological conlamination of the closures.

CAP SHELL

"ETAl SLIP DISCRUBBER DISC ~- i:::r-GILSONrTE LINER

THREADED NECKOF BOmE

CAP SHELL

"ETAl SLIP DISC

PLASTIC DISC WITHElASTOMER FACE

THREADED NECKOF BOmE

Fig. 1 Simplified drawings of boule cap differences in Rocky Mount Incident (not 10scale).

t j

12 Chapter 1

Twenty-five bottles were contaminated; all were made at Abbott's Rocky Mountplant. and all had elastomer cap liners.

None of the twenty bottles from Abbott's Nonh Chicago plant were con­taminated; all had Gilsonite cap liners. Neither of two houles from batchesmade at the Rocky Mount plant with Gilsonite cap liners was contaminated.

The study also showed lhal dye could penetrate from closures with elas­tomer cap liners into infusion fluids in the course of normal usage. No dye pen­etration was seen in bottles with Gilsonite cap liners. It was also shown inexperimental conditions thai the types of microorganisms implicated in the epi­demic and found on contaminated closures could increase to concentrations ofup to 1()6 per mL in AbOOn's 5% De.trose, Normal Saline, and Waler for Injec.tion products.

What seems to have happened was that the elastomer-lined caps becamecontaminaled after sterilization. Invesligations showed thai tap water used incooling could readily penetrate the interslices of the screw thread and gel into thecap assemblies. These microorganisms most likely got into lhe fluids when lhebottles were being set up for infusion, through removal and replacement of thecap, through shaking the batLie to distribute additives throughout the fluid. orthrough seepage during lhe lime the bottles were hanging invened for infusion.

The principle that the Rocky Mount Incident exemplifies is lhe criticalityto achieving sterility of even the seemingly most trivial change in components ormethods or what have you. The concept is validaLion-<lemonstidting thai achanged process is capable of doing what it is supposed to be doing. In this casethe change of cap liner had not been demonstrated to be capable of maintainingsterility, and lives were lost.

C. The 1972/73 Culler Laboratories Incident in the U.S,A.

In March 1973. Culler Laboratories withdrew from the market all I,OOO-mL bol·ties of 5% Dextrose in Lactated Ringers InjecLion produced at its Chattanooga.Tennessee, plant since September 1972. Five cases of clinical septicemia hadbeen associated with administration of these fluids; three of the five patients died[5].

The microorganisms implicaled in the clinical cases were £merooocteragglomerans. Entuobaclu cloacae, and Citrobacter freundii. Unopened bottlesfrom the same plant were found to be contaminalcd with the same and similarmicroorganisms. in some instances to the point of visible turbidity.

This incident is cited here only as an indication that there have been otherincidents similar to the (wo main examples described above. Nonsterile par­enteral products have a real potential 10 kill. Death is 3 quite probable conse·quence of administration of nonsterile parenteral prcxJuclS. so that achievingsterility for these products concerns the sanctity of human life.

The Need for Sierilily

D. The 1964 Imported Eye Ointment Incident in Sweden

13

Posloperative iofec,tions after ophthalmic ~urgery may arise from anyone of sev­eral sources. In 1964. eigh[ patients in [wo Swedish hospi131s developed Ps~u·

domonns aeruginosa iofeelions from conlaminaled hydroconisone eye ointment[6].

The consequences were 10lal blindness in the infected eye of one palienl.considerable loss of vision in two patients, and reduced visual acuily in Iheremaining five patienls.

The levels of Pseudomonas contamination in unopened tubes of ointmentfrom lhe balCh that had been implicated in the infection, and in other batchesfrom the same manufacturer, were higher than 2.()(x) colony-forming unils pergram. 100 isolate was resist:lnt 10 neomycin and amphomycin, the Iwo antibi­otics included in the formulation.

The oinlmenl had nol been terminally slerilized. It had been manufacturedaseptically. no preservalives were included in the formulation. and the manufac­turers had laboratory data to show Ihat microbial growth in lhe oinunenl wasvery unlikely due to Ihe low water content and the presence of two antibiotics inhigh concentrations.

in lhe case of this ointment, lhe petrolalum base was dry heat sterilized inbulk prior to lhe addition of solubilized active ingredients. Evidently a film ofwater had condensed on the surface of the cooling petrolatum and this hadbecome conlaminated by Pseudomonas aerugitlOsa shown 10 be present on tow·cis. shoes. gloves. and hands of personnel engaged in manufac,lure of lhe oinl­ment. The conlaminanl was shown by simulation to be capabl.e of muhiplying inthe film of moisture over periods of storage similar to those lhal might havearisen routinely in praclice.

Antibiotic-resistance may have developed over time in the premises.

E. The 1981 Imported Indian Dressings Incident in the U.K.

This incident of contaminated first aid dressings was detected by regulatory vig­ilance [7]. No patienl infection arose. The U.K. regulatory aulhorities werealerted to the po sibilily of supposedly sterile flf'St aid dressings of Indian manu­faelure being contaminated from reponed concern in Australia.

Thiny·three of 38 batches of Indian dressings were found 10 be eonlami­nated by aerobic spore.Corming bacilli; levels of contamination by these micro­organisms were Iypically less than 10 per dressing. Low levels of contaminationby Clostridium sp and by fungi were also seen. The spore formers and clostridiawere attributed to inadequate steam sterilization (8). Because the fungal can·lamination was heaviest in the outcr pan of the dressings. and because there wasvisual evidence of Ihe paper wrappings having been wet at one time. it wasassumed mal this had ariscn from poststerilization contamination.

14 Chapter 1

The U.K. official repon on this incident 171 concluded thai the contami­nating microorganisms were of low palhogenicity. They also commented thatthe types of first aid dressings involved were most commonly applied to woundsthat were already contaminated. They evaluated the risk. to heallh arising fromuse of these conlaminated dressings to be small. Nonetheless they severely crili­ci7..ed the manufacturers for labelling these products as "sterile" and judged therisk to be unacceptable.

F. Incidents Originating from Other Sources

h should nol be assumed that all serious consequences arising from the use ofnonsterile products are of industrial origin. Many are from products preparedand sterilized in hospil.a.1 pharmacies. 1bose which have been confirmed to be ofindustrial origin art: usually more widespread because of the scale of manufac­ture and distribution. A few examples are included below to indicate the rangeof problems encountered over many years.

Michaels and Ruebner (9) reponed two cases of patients whose tempera·tures rose dramatically while receiving intravenous therapy and then settlednonnaJly when the infusion apparatus was taken down. These authors anributedthese reaclions 10 in-use contamination of giving sets left in place over severaldays and over several changes ofboules of infusion fluid.

In 1969. crncks in bottles led to inadvertent infusion of fungal-contami­nated glucose·saJjoe solution to two patients [10). Fungal m.i rocolonies werevisible in the Ouids. Both patients recovered satisfactorily after prompt treat­ment with ampicillin and amphotericin B.

In the latter half of 1971. 40 patients in a U.K. hospital acquired bac­teremia. urinary tract infections, or respiratory infections from in-house manu·factured sterile parenteral solutions. The infective agent. Pseudomonm Ihomll.fii.was traced to water used for cooling sterili1.ed parenteral solutions in a rapid­cooling autoclave. The microorganism had penetrated beneath the cap seah andentered the boules of fluid either in the autoclave or when lhe caps were dis­turbed on selUp [II].

Postoperotive eye infections from conwninated instruments. Ouids. andeyedroppen have been periodically reported over many years [12.13.14]. P"u­domonas auug;nosa and StrrDl;o marUSCtns. among other microorganisms. havebeen implicated. Some permanent visual impairment was common.

REFERENCES

l. Halls, N. A.. Joyce. T. M.. Doolan, P. T.. and Tlllltntire. A. (1983). The occurrenceor 3lypical1y high presterili1..ntion microbial counts ("spikes") on hypodennic prod­ucts. Radiation Physics and Ch~mistf)' 22 (3-5): 663-666.

The N.... 10< Sterility IS

2. R~por1 of the CommillU Appointed to Inquire into the Circumstance. Including theProduction, Which Led to the US~ ojConltlm;naud InfusiOlI Fluids;n 'he De\'onponSection of Plymouth General Hospital (C. M. Clothier. Chainnan). London: HerMajesty's SIaHODery OffICe. 1972.

3. Fells. S. K.. Schaffner. W.. Melly. A.. and Koenig. M. G. (1972). Scpsis caused byconlaminated intravenous fluids-Epidemiological. clinical and laboratory lovesli·galion of an outbreak in Doe hospital. Anno's of Internal Medicine 77 (6): 881-890.

4. Milki. D. G.. Rhame. F. S.. Mackel. D. C.. and Bennel. J. V. (1976). Nationwideepidemic of septicemia caused by contaminated intravenous products. AmericanJoumolojMedicine60: 471-485.

5. Ceoler for Disease Control (1973). Follow-up on scpticemias associated with con·taminated intravenous fluids. Moroidiry and MOrloJiIy Weekly Report 22 (13): 115­116.

6. KaJlings. L 0 .• Ringenz. 0 .• Silversto!pe. L.. and Emcrfeldl. F. (1966). Microbialcontamination of medical preparations. Acta PJuJnn. Suedea 3: 219-228.

7. Marples. R. R. (1983). Conlaminated first-aid dressings: Repon of a working partyof the PHLS. Journal ofHygiene (Cambridge) 90: 241-252.

8. Thomas. S.• Dawes, C. E.. and Hay, N. P. (1981). Microbiological contamination ofimported wound dressings. PhamuJceutical Joumo/l981: 783.

9. Michaels. L. and Roebner. B. (l953). Growth of bacleria in intravenous infusionfluids. !Ancet 1953: 772-774.

10. Robenson. M. H. (1970). Fungi in fluids-A hazard of intravenous t.herapy. JoumtJlofMedical MicrobiologyJ: 99-102.

II. Phillips. I.. Eykyn. S.. and Laker. M. (1972). Ou.b=!< of hospital infection causedby contaminaled autoclaved fluids. !Anat 1m: 1258-1260.

12. I...epard. C. W. (1941). 8. P)'OC)'OMUS ulcer. Report of three cases: Resulls of sul­fapyridine lherapy in one case. Transactions of the American Academy of Ophthol·mology and Otolaryngology 46: 55-60.

13. Ayliffe. G. A. I.. Bany. D. R.. Lowbury. E. J. L.• Roper-Hall. M. J.. and MartinWalker. W. (1966). PosIoperative infection with PseudomOlla.J aeruginosa in an eyehospiral. U1ncetl966: 1113-1117.

14. Templeton. W. C.• Eiferman. R. A., Snyder. J. W., Melo. J. C. and Raff. M. J.(l982). Suratia keratitis transmitted by contaminaled CyL-droppers. American Jour­tldl ofOphthalmology 93: 723-726.

t j

Copynghled aena

2Sterility and Sterility Assurance

I. Detection of Nonsterili.y 18A. Compendial Melhods of Detecting Nonsterility 19B. Newer Technologies 22

IJ. Conlinning Sterility of Balches by End-Produci Testing 25III. Conlinning Sterility by Compliance wilb Process Specifications 28IV. Slerility Assurance 29

A. Ex:ponentiallnactivaLion 30V. Determinants of Sterility Assurance 34

A. Bioburden 34B. Survival Curves 38

Sterility is defined as the total absence of viable life-Conns (Table I). The con­cept of sterility is absolute and acknowledges no boundaries. However. life-­forms are ubiquitous on this eanh. and sterile conditions can only exist wiLh.insome form of boundary. Once these boundaries are broken down the sterile con­dition is inevitably lost

St'crile conditions exist in nature-within solid rock for instance. Butshould Ibe rock be broken open it will become nonslerile unless Ibis is donewilhin some other set of barriers that protect the rock's internal sterility. Sterileconditions will exist in the hean of a volcano, protected by temperatures

17

t j

•

J

18 Chapler 2

Table 1 Defmition ofTenns Used in Chapler 2: Sterility and Slerility Assurance

Tenn

Sterility

Viable (of microorganisms)Discemable level (of viablemicroorganisms)

SAL (Sterility Assurance Level)

Validate (of a SleriHzation process)

Definition

Condition in whi.ch an item is totally freeof alllife·fomisCapable of reproducing to discemahle levelsEilh~r as a colony. film, or slime visible tothe naked eye or under the microscope onsolid nutrienl media. or as a physical orchemical change (usually turbidity) in thecomposition of fluid nutrient mediaThe probability of a supposedly sterile itembeing contaminated by one or more micro­organismsDemonS1r;lIe thai the process is capable ofachieving what il is supposed to achieve

deslrUctive (0 the essential molecules of life. In this case lhe barriers protectingsterililY an: barriers of heat; nonsterile conditions will exist outside these barri­ers.

In real \enns. sterilil)' is therefore a descriptive and limited concept. Asterile item is one thai does not contain. carry, or harbor any viable life-fonns.The sterile item must be prolected from conlamination from the general envi­ronmenl; otherwise it becomes nonslerile.

Thus far me concept of sterility is still academic in that we have nOIaddressed the question of how sterility or (conversely) nonslerilit)' can be identi·fied or demonSlrated for 3n item. The situation (hen becomes immediatelymurkier. No melhod exists whereby an ileffi can be examined and be shownconclusively to be sterile. All methods approach the problem from the viewpointof demonslrating nonsterility; if nonslerility cannol be demonstrJ.ted, the item istherefore assumed to be sierile, This is not necessarily true,

I. DmCTION OF NONSTERILITY

Demonstration of nonsterilily has its origins in the very beginnings of microbiol·ogy as a science in the eighteenth and nineleeoth ceolUries. In Ihose times therewas considerable debate over the origins of microorganisms ("animaJcules'')­by spontaneous formation (spontaneous generation) from non-living materials.on the one hand. versus formation from living "seeds" or "genns," which weresupposed to be always present in the atmosphere. on the other.

Sterilily and Sterility Assurance 19

Pasteur produced some pretty conclusive evidence in favor of the germtheory through his famous swan-necked flask experimenls. This experimenta­tion was based upon detection of microorganisms by visible changes in organicinfusions. The infusions were boiled up in the Oasks. and as long as l1leasureswere in place to prevent microorganisms from entering the nasks from the .1lmo­sphere. the infusions remained clear to the naked eye. and microbial growth wasundetectable on microscopic examination. If the necks were bro.ken off, theinfusions became lurbid 10 the naked eye. and microorganisms became delect­able under the microscope. Pasteur was using growth or absence of growth inorganic infusions as a memod of distinguishing between Lhe slerile and lite non­sterile slates.

Considering Pasteur's twbid organic infusions in more detail. we canenlarge upon the concept of viabiJily. which features 3S a qualification within lhedefinition of sierilily. Viability has various meanings for differentlire-fonns; inlhis text we are concerned with viability among microorganisms, a concept quitedifferent from viability when used in connection with mammals, for instance.For a microorganism 10 be viable, it musl have the capability of reproducingitself, in practice not just once but through sufficie.nl successive divisions 10 forma colony on solidified nutrient media or visible grov.'lh in fluid nutrient media.In these terms, Pasleur's lurbid organic infusions were quite clearly nonslcrile.

But can we reasonably infer that Pasteur's clear organic infusions werenecessarily sterile? The answer 10 this question is not stmighlforward. The clearinfusions were clearly not conLQminated by any microorganisms that had thecapability of repnxlucing in these media under the conditions within which theywere incubated. The queslion remains whelher lhere might have been microor­ganisms presenllhal if transferred 10 another medium, or incubated under differ­enl conditions, would have multiplied to discemable levels. Naturally this ques·tion is now unanswerable.

The general point to be made is that detectable microbial growth in anytest system is positive confumation of nonsterility, but absence of growth canonly give an assurance of sterility limited by the conditions of the leSt. 11 is fun·damenw] thlll srcrility for an ilem can never be conftnned with 100% cenaintyby any te I melhod.

A. Compendial Methods of Detecting Nonsterilily

In essence, the pharmacopoeias are compendia of end-product specifICations fortherapeutic substances and descriptions of methods :.pproved for testing sub­stances againsl these specifications. Methods for detecting nonsterility wereintroduced inlo the British Plwrmacopoeio in 1932 and into the United StalesPharmacQp<Jeio in 1936. In the years since, the melhods have changed in derailand in application. and have differed between the two major phannacopoeias.

20

Table 2 Evolution of the Test for Sterility in the British Pharmacopoeia

Ch41pter 2

1932 Media and melhods specified, direct inoculation, incubation al 37·C for 5days

1948 Thioglycollate medium included as an alternative recovery medium foranaerobes

1953 No change from 19481958 Accommodation made for solid dosage forms. "neulraliZ(rs" allowed. and

specified for cenain antibiotics1963 Incubalion conditions changed to 30-32'C for 7 day. membrane filtration

allowed for certain antibiotics1968 Sample sizes included to allow cenification of batches as sterile1973 Inclusion of European Pharmacopoeia 1971. recomme.nded thai the mem­

brane filtration technique be usai wherever possible. inclusion of mediaconltOl tests; incubation conditions changed to 3O-35"C for bacteria and20-25·C for fungi over 7 days

1980 General cll:pansion of the detail1988 No significant change from 1980

Therefore, ahhough the concept of sterility is absolute and unchanging. the stan­dard required to confirm its achievement has never been consistent.

Consider the development of !.he Ttst for Sterility in the British Plumna·copoeia since 1932 (Table 2). The test in 1932 was an appendix of only twoparagraphs. It described the media (0 be used in the test and a method of testing.

From this first compendial method it was recognized that the selection ofappropriate media was imperative to the detection of as wide a range ofmicroorganisms as possible. Replicate samples were required to be tested ineach of two fluid media. The first of these. intended for recovery of aerobicmicroorganisms. was defined as meat extract containing I % peptone. and the pHafter sterilization was required to be in the range 7.2to 7.8. Recovery of Ilnaero­bic microorganisms was recommended in the same medium but wilh the additionof heat-coagulated muscle to a deplh of I cm at the bottom of the tube.

The recommended method was to inoculate Ihe preparation directly intoeach of the two media. taking care that the final concentration of any phenolicantiseptic in the preparation under test was diluted to less than 0.01 %. Mediawere to be incubated at 37·C for 5 days.

The 1932 OP method recommended that the complete contents of a con­tainer should be tested only when the volume was less than 2 mL (two equalpans. one part for each medium). For volumes of 2 mL and greater, I mL was tobe tested in each medium.

If there was no growth under these conditions. the preparation was con·finned as sterile (passed the Test for Sterility). [f growth was delected, retests

Sterility and Sterility Assurance 21

and second retests were allowed on fresh samples. The preparation could onlybe failed if growth was seen in all three test~, or if the same microorganismswere found in twO of lhe te ts.

TIle presentation of the Test for Sterility in BP 1932 was in the same styleas any of the o!.her chemical or physical test methods in the phannacopoeia, i.e.,withoul any guidance on lhe number of items from a batch of items required 10make up a valid sample.

Three themes contained in the BP 1932 Test for Sterility merit some em·phasis.

(a) The leSt presumed sterility. Even with the limitations of the steriliza·tion technology of the 19305. abe phannacopoeia was presuming sterilityunless nonsterilily could be convincingly and conclusively demonstrated.Tbis is rather unusual because it goes against the grain of scientific criti·cality to assume abat a hypolhesis is valid unJess it can be proven other­wise. The test was far less a critical test for sterility, as one might supposeit was intended to be. than a test for nonsterility-Le., false nonsterileresults were !.hought to be more likely than false sterile results (the phar.macopoeia had more faith in the potential of the recommended media torecover microorganisms than it had in the ability of laboratories to performsuccessful aseptic manipulations).

(b) The test did not address total freedom from microorganisms for prepa­rations in 2 mL volumes or greater. For these larger volumes it was reallya microbial limit lest with a lower sensitivity of detection of one microor·ganism per mL.

(c) The tesl gave no guidance on interpretation of dala from replicaterecovery conditions.

These three themes, which are common to the BP, USP. and olher pharma·copoeias, remain fundamentally unchanged up to and incJuding the currenleditions of Ihe compendia. despile numerous oaber modifications and alterationsto broaden recovery condil'ions and improve methods.

After 1932 very little changed in lhe next edition. BP 1948, except for theintroduction of an alternative medium for recovery of anaerobes, a medium verysimilar in composition to present-day Thiog/ycollate Medium USP. The BP Testfor Sterility remained at about two paragraphs in this edition and in its 1953edition.

By 1958 the test increased in complexity by accommodating solid dosageforms and antibiotics. The weight separating most solid dosage presentationsfor which the total contents were to be tested and those for which only a ponioowas to be tested was 100 mg. For preparations of 100 mg or more. only 50 mgwas to be tested in each medium. Less than 100 mg. equal parts were to bedivided belween the two media. Substances 10 "ocutralize" microbial growth

22 Chapter 2

inhibitors were allowed to be included in test media. Details of neutralizers wecegiven for various antibiotics.

In SP 1963 there were two imponant innovations: incubation changedfrom 37-C for 5 days to 3O-32"C for 7 days, and a membrane filtration techniquewas introduced for certain antibiotics.

The membrane filtration technique. now almost universally used for phar­maceutical preparations but not for devices, was an advance of major signifi­cance. SP 1963 advised dissolution of the antibiotic in saline. followed by pas­sage through a sterile membrane filler. The principle is that soluble substancespass through the membrane in solution and any microbial contaminants areretained. The membrane was to be CUi in two and onc half tested for aerobicmicroorganisms and the olher for anaerobes. This technique is more amenableto all of the contents of a single presentation being rested. and also. if necessary.for the contents of several presentations to be bulked and tested together in oneset of media.

Only in t968 does the BP provide any guidance on the number of items(2% of the (otal containers or 20 conminers. whichever is the fewer) that shouldcomprise a sample whereby the result of the Tesr for Sleriliry can be extended toapply to batches of product needing to be certified as sterile. These samplingrequirements only renected those given in the UK Therapelllic Sl4bsranc:es Reg­ulations [t} lhat had existed since 1952. Importantly it was a move away fromthe straightforward specification of a tesl rncthod that had predated it

The Tests for Steriliry in the 1973. 1980, and 1988 revisions of the BririJihPharmacopoeia are greatly expanded from earlier editions. with up 10 five pagesto include control systems ensuring thai leSI media are themselves sterile andcapable of supporting microbial growth. and thai subslances under lest do nOIinhibit the growth of selected microorganisms. The conditjons became 3G-35-Cfor bacteria and 2G-2S"C for fungi over 7 days incubation since 1973 onwards.

Through aJlthese changes the three nawed themes described above remainunchanged: as a means of confinning steriljty the test is flawed because it pre·supposes sterility rather than nonsterilily~ in many cases it o(X:rales only 3S amicrobial limit test; and it avoids addressing the interpretation of data obtainedfrom replicate samples in different media. Nonetheless. the (wo techniques ofthe compendia! sterility lests (direct inoculation and membrane filtration). theconsideration given over many decades to broad·spectrum microbial recoveryconditions. and the recommendalions concerning media control logether providea very powerful basis for a method of delcc[ing nonsteriliry if that is what isrequired.

B. Newer Technologies

The speed of response from compendial methods of testing for nonslerilily islimited by the incubation period. Of course. it often happens that a nonslerile

Sterility .1M Sterility Assurance 23

item will produce discel1\3ble growth within 24 or 48 h under compendiaJ condi­tions: t'O demonstrate the converse. i.e., that an item is sterile. the tesl mUSI runthrough its allolted incubation period.

The food indusuy has invesled a considerable amount of effon and exper­tise into Ihe development of quicker methods of delecting microorganisms. Sofar none of ahese have found application 10 sterility/nonsteril,iry as it is under­stood in the phnnnaceutical and medical products industries. This is because themain drive from the food industry has been toward microbial limit rests based onthe presupposition that SOIT'le microorganisms will always be present in mostproducts. Where microorganisms are required to be totally absent, e.g., Clostri­dium botulinum from canned foods, the food industry has recognized the inade­quacy of end-product testing and relies on verification of a rigorous processspecification. The presupposition of the pharmaceutical and medical prodUClSindustries is that viable microorganisms are in fact absent from sterile productsand the purpose of the lest for nonsterility is only to confirm this. This presup­position is much hanler to reconcile, both philosophically and technically_

Data from newer technologies is challenging even the basic assumptions ofthe traditional definition of viability and the compendia! approach to determiningnonsterility. Many types of common microorganisms. including E. coli. Salmo­neilD, and Stnptococcus fa~calis [2,3] have been shown to be capable of devel­oping viable but nonculturable forms in stressed environments.

The first source of evidence for the existence of viable but noncuhurablemicroorganisms is from the newer technologies of detection. Typically this iswhere counts of microorganisms by newer technologies fail to correlate withcounts by culture methods, or exceed counts obtained by cuhure methods. Theissues of viability and cuhurability are of course always chalJengable on thebasis of lhere being always another set of culture conditions not studied and ofthe application of the newer technology being overzealous.

However. the definition of viable bUI nonculturable microorganisms hasbeen in the main based on pathogens thaI have been shown 10 be capable ofgrowth in warm-blooded hosts while defying recovery by microbiological cui·ture techniques. There is a panicularly striking epidemiological example of this[4] in which treatment against CampyloMeler of the water supply 10 a chickenshed resulted in the disappearance of a panicular serotype from the chicken pop­ulation even though il had not at any time been recovered by culture techniquesfrom the waler supply.

Summaries of the main applicalions of newer technologies lO del'a'tion andenumeration of microoganisms merit consideration.

J. Determination of Microbial ATP: ATP is presenl in all living cells. Theconcentration of ATP decreases through .phosphorylal'ion reactions that occurwhen microorganisms lose their viability. Methods for delecting microorgan­isms based on ATP determination presume thai the presence of detee-table levels

24 Chapter 2

of ATP confirms the presence of viable microrganisms, and conversely theabsence of ATP contions the absence of viable microorganisms.

ATP can be detected through 3 luminescent reaction catalyzed by an ATP­specific enzyme:

E + ATP + LH2 E-LH-AMP + PP

E-LH-AMP +~ E-L-AMP + H20 2+ light

where LH2 is luciferin. an oxidizable substance obtained from fireflies rnat afterreaction with ATP becomes the subslrate for an oxidative reaction c3lalyzed byan enzyme, lucifemse (E). The reaction results in production of light.

1be intensity of light produced may be detected by a phOiomultiplier andrelated to the concentration of ATP present.

The amOUD' of AT? in vegetative bacterial cells can be expecled 15) to bearound 10.13 [0 10-1'2 mg. less in spores. more in yeasts. The lower limit of thesensitivity of the technique is usually quoted 10 be on the order of 10- 10 mg.This value may be even higher if oockground ATP has 10 be taken into ac.count.The technique has two difficulties. therefore. for the detection of nonsterility­low sensitivity and the possibility of false alanu~ (false positives) due to extra­neous ATP.

2. Detectiml of Changes jn Electrical R~sisul1Jce: Viable microorganismsmetabolize. End-products of metabolism. when released into the surroundingmedia. cause detectable changes in electrical resistance. Detection of thesechanges is the basis of several wcll-developed conductance. impedance. andcapacitance melhods of detecting microorganisms.

In the main. these types of instruments have been used to deteffiline num­bers of microorganisms in nonsterile milieux. A sample of the preparation underexamination would typically be incubated in an appropriate growth mediumcontained within an electrode-equipped well. Changes in electrical resislance(conductance. impedance. andlor capacitance) can be monitored and recordedduring incubation. or alternatively inslruments can be sci to a threshold valuelinked to a lower sensitivity of detection. With commercially available instru­ments the threshold of detection has usually been quoted at around 101 microor­ganisms per mL. For enumeration of microorganisms. the incubation timerequired 10 reach the threshold "lrigger" is inversely proportional to the initialconcentration of microorganisms in the sample.

The ab olute lower limit of sensitivity of methods based on electricalresistance and existing tectmologies is somewhere around 102 microorganismsper sample versus background effects. This is really too insensitive for delcctionof nonsterility. Furthermore. there are significant media-te-media, batch-lO­batch, and microorganism-to·microorganism differences in senSitivity. Mediarecipes that have stood the test of time for growth promotion may contain e1ec-

Sterility ,and Sterility Assurance 25

trically active substances thai make them totally unsuitable for deleCLion ofmicroorganisms via resistance changes. Some microorganisms may even in lhebest of circumstances give only minimal changes thai are not detectable againslthe background. Optimal conditions for one microorganism may not be optimalfor another. So overall these methods lack the robustness of traditionalapproaches for detecting broad-spectrum nonsterilily, and they leave some doubtabout the absence of a response confirming sterility.

3. Near-In/mud Spectrometry: Near-infrared spectrometry is an exciting newtechnology for detecting microorganisms in fluids. The principle is based onscanering of near infrared light by solid objects in Ouids; suspended microor­ganisms by absorption at wavelengths between 1100 and 1360 nm: microorgan­isms adhering to container walls by absorption in the region of 1600 to 2200 nm.

Research wilh deliberately contaminated intravenous infusion bags (6] hasdemonstrated lower limits of detection of around 102 colony·forming units permL of fluid. Some distinction between different types of microorganisms wasalso indicated. Whereas lhese recenl1y published limits of sensitivity would bewholly unS3lisfactory 10 lhe detection of nonslerility (and conversely (he confir­mation of sterility). the near infrared approach has the advantage oyer otherexisting melhods of being noninvasive and nondestructive. WiLh development.this lechnology could add an extra dimension (0 existing microbial-deteetionpractices.

II. CONFIRMING STERILITY OF BATCHES BY END-PRODUCTTESTING

SterililY is an attribute. An item can exist in only one of two conditions, sterileor nonsterile. Methods are available (sec above) for tesling ilems to determine inwhich of lbese states the item exists. All technologies involve sacrificing theitem or al the very least compromizing its slerilily. After tesling. the ilem is nolonger in 3 sterile useable stale. Therefore the value of testing an item must liewilh Ihe item being a sample or pan of a sample from a greater population oruniverse about which we are trying to gain infonnalion.

This was Ibe principle Iha. led '0 Ibe pharmacopoeias adding samplingschemes to the basic method of the Test/or Sterility. As mentioned previously.the original pharmacopoeial enlries comprised a simple tesl method applicable tofinding oul whelher items tested were nonsterile. Sampling schemes appeared inthe U.K. TMrapeunc Substances Regulations in 1952, in the USP in 1955. and inthe SP in 1968. The actual sample size has varied. usually related to a percenl­age of lIle number of containers in the batch but limited 10 a maximum samplesize (Fig. I).

26 Chop'•• 2

{JJ§!P

NO

1970

1975

1955

UNITSU.

NITSU

PI.

AnON

1973

1980

1968

Fig. 1 Sampling recommendations in the phannacopoeial tests for sterility.

With Lhe inlroducl'ion of sampling schemes. the compendiaJ Tests forSterility became approved methods for confirming Ihe sterility of batches ofsupposedly sterile products by means of end-product testing.

As a means of confirming sterility. all phannacopoeial sampling schemesfor lhe Test for Sterility are totally inadequate. This has been well known at leastsince Iile 1940. [7].

Sterility ..nd Sterility Assurance 27

The usual sample size is 20 items. Imagine a batch of 100 ilems of whichonly 99 were sterile. This would be tOlally unacceplable for release if the singlenonsterile irem were known (0 be present But the odds are in favor of that batchpassing a phannacopoeial sterility test Divide lhe batch into five sets of twentyand consider how many of those samples would pass the Tesl for Sterility. Fourwould pass and only one would fail. If the sample tested contained the contami­nated item. lhen the retest sample wouJd not contain a contaminalCd item-so!he balCh would be passed as slerile.

Tbe ronnal slatistics sunounding this are nal complicated [8.9.10]. Theprobability of drawing n consecutive sterile items from a population of items isgiven by Ihe expression

(I - p)" (2.\)

where p = the proponion of Donsterile items in the population and n = the num·ber of items in the sampJe.

Conversely, the probabiJity of selecting one or more nonsterile items in asample of n items is given by !.he expreSSion

I-(l-p)" (2.2)

Annex I describes the use of these expressions to calculate the effectiveness ofthe phannacopoeial Test for Sterility as a means of confirming sterility. QuitesimpJy, the tesl does nOI confirm slerility. Batches of producl released on Iheb:asis of a Test for Sterility alone could 100 easily conlain signific,ant proponionsof nonslerile items.

1be inadequacies of the Trsl for Sterility are acknowledged in the pharma­copoeias themselves. The European PhamlOcoPMia does nol have a long his­tory of publication; lhree of its four fasdcuJes carry a ··disclaimer" to the Te~·t /or$"ri/;/y. Only in 197\ did !he EP publish an unembellished method descriplionand sampling plan. Each of its neXl revisions. EP \978. EP 1980. and EP \986start their sections beaded Test /or Sterility with an ilalicized paragraph statingthe following:

(a) ... 3 salisfaclory result (from the Test for Sterility) onJy indicales thatno contaminating microorganism has been found in the sample examinedin the conditions of the leSI.

(b) ... extension ... to lhe whole of a batch ... requires tbe assurance thaievery unit in the b~uch has been prepared in such a manner lhal il wouldalso have passed !.he lest. Clearly this depends on the precautions takenduring manufacture.

(c) ... (for) products sierilised in their final sealed containers physica.lproofs, biologkaJly based and automatically documented. showing correcl

28 Chapter 2

treatment throughout the batch during sterilisation are of greater assuranceth.:m the sterility test

(d) (The Test/or Slerilily) ... is the only analytical method available to !.hevarious authorities who have to examine a product for sterility.

The same messages appear, but initially less explicitly staled. in all revisions ofthe USP since USP XVIII (J 970) under the heading of Slal/i,alioll. USP XX(1980), Section <1211> introduces the very direct Slalemenl, 'The sterility test isintended as the official referee Ie t in the event thai a dispute arises concerningthe sterility status of the lot."

In summary, end-product tesling is not a defendable option. either scien­tifically or officiaJly, for confirming lile sterility of batches of supposedly sterileproducts.

III. CONFIRMING STERILITY BY COMPLIANCE WITH PROCESSSPECIFICATIONS

If sterility cannot be confirmed by end-product testing, one alternative is that itmay be confinned by some positive affinnation that every item in a batch ofproduct has been Ireated by some officially recognized process. An appropriateanalogy might be the hard·boiled egg. Imagine that any method of lesting boiledeggs for hardness must be destructive, So rather than boiling eggs for arbitrarylengths of time and periodically sampling we could decide to boil for 20 min.and so the definition of a hard·boiled egg by its process speCification is any eggthat has been held in boiling water at IlXtC for 20 min.

Table 3 lists the sterilization cycle specifications currently recognized bythe three major pharmacopoeias ("compendial cycles"). A lxxly of knowledgeexist's to suppon the view that products that are properly exposed to anyone ofthese compendial cycles will be free from all viable microorganisms. Theorigins of I.he compendial cycles are lost in history except to say thai they wereprobably based on available technology (l21-C is achieved by S31unued steamheld at a pressure of 15 Ibs per inch2, which is equivalent to I bar or one atmo­sphere).

There are two points to be considered in relation to compendial cycles.The first is common to all approache.'Ii to sterility control: there must be reliableassurance of every treated item in the batch having been exposed to the specifiedparameters that deliver leLhality. The second point is lhal compendia) cycles donot take account of any differences in the numbers and types of microorganismscontaminating the product before treatmenl. Returning to the analogy of a hard­boiled egg, definition by process specification would embrace everything from aquail's egg to an ostrich's egg being hard-boiled by holding in water at 100°C for20 min.

Sterility and Sterility Assurance 29

Table 3 Currenl Compendia} Slerilizalion Cycles

Slerilization Cycleprocess specification Reference Commenl.s

Saturaled steam 15 min al121"C EP(198J).BP(1988). For aqueous

p~panttions

andUSP xxn (1990) When "auto-

claved" is statedin the monograph

3 min at 134-c BP (1988) For dressingsDry heat 30 min .1180'C}

1 h.1 170'C EP (l983).nd

2h at 160'C BP (1988)

Gamma radialion 2.5 Mrads (25 kGy) BP(1988) andUSP xxn (1990)

Filb'ation Passage lllrough • EP (1983).0.22 Ilm membrane BP (1988), and

USP xxn (1990)

There have been huge technological advances in contamination conlrolduring manufacture and in precise control of sterilization processes since most ofthe compendial cycles nrst came to be recognized. Under these changing cir­cumstances it has DOl made sense to many sterilization scientists and practition­ers to standardize a definition of sterility on something as rigid as a processspecification. This has led 10 the concept of sterility assurance that now appearsalongside the Test for Sterility and the compendial cycJes in all the major phar-

•macOpoela5.

IV. STERILITY ASSURANCE

The concepl of sl'eritity assurance invok.es the idea of confidence. How confi­denl should we be that an item is Slerile? All three of the major phannacopoeiasnow require assurance of less man I chance in 1.000.000 lhal viable microor­ganisms are presenr in a sterilized anicle or dosage form (JO-6 probability ofnonsterilily). To obIain this assurance we must have good knowledge of theeffects of sterilization processes on microbial populations.

Microbial inactivation has been well researched. This is panicularly lruefor populalions of bacleria. Although each Iype of microorganism responds dif­ferenlly to the various available sierilization processes. Ihe form of inactivation

30 Chapter 2

of microbial populations is broadly similar enough to depict a general case basedon exponential death.

A. Exponential Inadivalion

The death of a single microbial cell is a biochemical process (or series of pro·cesses); the entrapment of individual microbial cells in or on fillers is due tophysical forces. These effects on individual cells are peculiar 10 individualsterilization processes. On the other hand. the effeelS of inactivating processesand filtering processes on populations of microbial cells are sufficiently similarto be described by one general fonn-cxponential death. Exponenlial kineticsare typical of first-order chemical reactions. For inactivation this can be attri­buted 10 cell death arising from some reaction thai causes irreparable damage toa molecule or molecules essential for continuing viability.

For fillrd.tion it can be assumed lhat binding of me microorganism to thefilter is due to interactive effects between molecules on the surface of Ihe fillerand molecules on me surface of the microorganisms, again first-order kinetics.

Chemical kinetics cannOl be assumed for aseptic manufacture; nonetheless,protection of ilems (rom microbiological contamination in clean rooms cannever be perfect and may merefore be assumed to be subject to the laws ofchance. The sterility assurance concept is ccnainly applicable 10 aseptic manu­facture. bUI nol in a manner directly comparable 10 temlinal sterilization pro·cesses.

For simplicity. the kinetics of exponential inactivation of populations ofmicroorganisms will be illustrated by reference to the application of some formof inimicallreatment to a pure culture of bacteria. There are plenty of data in thescientific literature to suppon the general case.

Let us suppose that the inimical process can be applied 10 a population ofbacleria in an incremenlal manner. Initially and after various periods of expo.sure we can withdraw samples from the population and count the number ofviable bacteria preient. When we plot these data as a survival curve on arith·metic axes we get me fonn of Fig. 23. The relationship of survivors to lime ofexposure shows an initial rapid decline and then levels oul and becomes asymp­totic to the time axis. IJ we were to cooven Ihe urvivor data to logarithms andthen plot Ihese logarithmic data against time of exposure we would oblain astraight-line relationship (Fig. 2b). This is the general form for exponential orlogarithmic survival curves.

There are two imponant related conclusions to be drawn from this generalform of exponential or logarithmic inaClivalion

(a) The logarithmic axis has no zero point. Succes.... ive poinls on the axisbecome proponionately smaller. bu( zero can never be reached. Whal thismeans to sterilization processes is that you can never define a lime of

Sterility ~nd Sterility Assurance 31

'.000.000

W

~ ._- W 100.000

~~ § 10,000

I =.= z0

I'.=

Ii =.=

I.~

"=- 0

ffi "~ m

~ffi~.-

,m~

~ ~ •••• • , • , • • • • • ,

(a) ,... (b) TWE

Fig. 2 Kinetics of microbial im,ctivation (generalized). (a) Arithmeticlarilhmetic; (b)logarithmic/arithmetic.

exposure that will guarantee sterility in the sense of total absence of viable• •nucroorganlsms.

(b) For each tixed increment of exposure time, a constant proportion ofthe surviving population is inactivated. If a particular exposure timedecreases the number of viable microorganisms in a population by 90%,and that period of exposure is then applied 10 the survivors, it will againdecrease the population by a runner 90%, Le., the number of survivors willbe decreased to 10% by the first treatment. I% by the second treatment.etc. The time of exposure required from a sterilization process to reduce amicrobial population by 90% is called the decimal reduction value (D­value). D-values are specific for panicular microorganisms but may differfor the same microorganism according to its condition during treatment.

But what does this mean to sterilization? Fig. 2b shows an initial popula-tion of 1()6 bacteria. After six equal increments of time (applied D-values) thereis only one survivor (Fig. 2b). After seven D-values. the graph suggests thatthere is only one tenth of a bacterium left surviving, and common sense wouldsuggest that single tenths of hacleria have no survival capability. In fact, whatthe graph tells us is Chal after application of seven D-values there is only a one­in-ten chance of a single inlact bacterium surviving, after eight D·values a one­in-one-hundred chance of a single intact bacterium surviving. and so on.

32 Chapter 2

In summary. there is always a finite probability of a survivor occurring, nomatter lhe strength or duration of a sterilization process. In other words, abso­lute sterility, in !.he sense oftOlal freedom from all viable life fonns, can never beachieved in practice. The acceptance of exponential inaCllvation kinetics has ledto two different approaches to the establishment of standards of satisfactory sler­ilization trealment. l.he inactivation factor approach and me sterility assurancelevel (SAL) approoch.

J. I"activation FaclOrs ("Overkill"); When the canning indu try addressed lhequestion of how much heat lreatmenl was needed to protect the public frombotulism through ingestion of viable spores of Clostridium botulinum. theydecided that twelve D-values was a safe level. In other words. they reckoned ona treatment thai would reduce a population of spores of C/o botulillum by a factorof one thousand billion.

The USP allows a very similar approach (termed ··overkill") to the deter­mination of valid sterilization processes for compendial preparations ("'a lethalityinput of 120 may be used in a typical "overkill approach'·).

To adopt an overkill approach it is necessary to fix on a reference microor­ganism. In food canning. Ihis was easily chosen-the toxin of C/. bOlu/illum isthe single major hazard from canned products. and the spore of C/o botulinum isconsiderably heal-resistant For medical sterilization the situation is less clear:any microorganism could lead to a fatality. Microorganisms (biological indica­tors) used for this purpose are usually chosen to be among the most resistanttypes known against the chosen sterilization lreatmenl. Spores of Bacillussrearorhermophilus are generally recommended for sterilization by saturatedsteam. Other microorganisms are more appropriate for other sterilization pro­cesses.

There are both practical and academic problems associated with settingstandards for sterility on the basis of inactivation fac,tors.

In the first instance. the intensity or duration of the sterilization treatmentmay be very high, possibly 100 high to be tolerated by some products. This begsthe practical question of why such an intense process must be used. It might beargued that the actual contaminants on the product are far less resistant to thesterilization treatment than Ihe reference microorganisms.

Secondly. as with defining sterility in relation to process specific.ations,there is something incongruous about using the same treatment in this case 12D-values versus some reference microorganism. to all products regardless of theinitial number and responsiveness of the acluaJ contaminanlS.

2. SterWry Assurance Levels: The concept of the sterility assurance level(SAL) not only considers the kinetics of inactivation of microbial populatjonsbut also addresses the numbers of contaminants on product items prior to steril-

Sterility and Sterility Assurance 33

izadon treatment. In some applications it may also take account of the resistanceof actual conlaminants to particular stcriHzation treatments and any effects thatthe condition of the product and its immediate environment may have on resis­tance.

An SAL is a target to be achieved for the treated product items. It is aprobability. h is defined as the probability of a treated item remaining contami­nated by one or more viable microorganisms.

The major pharmacopoeias. and most regulatory documents governingsterile medical devices. require an SAL of 10-6 or betler (less than one chance inone million of an item being nonsterile after treatment) to be achieved for termi­nally sterilized products. Why 10.6 rather than to-5 or 1O-1? This is nol known.

The Canadian Standard for Industrial Sterilization of Medical Devices bythe Steam Process. 1979 (II) differentiates between prodUClS intended to comeinto contact with compromized tissue. which are required to have an SAL of 10-6(quoted as a probability of being sterile of 99.9999%) and products not intendedto come inlO contact with compromised tissue. which are required only to havean SAL of (().) (a probability of being sterile of 99.9%). No other formallyacknowledged exceptions to the general rule of sterility being defined as a 10--6SAL are known.

SALs are not dircclly measureable. A supposedly sterile item can only besterile or nonsterile. as indicated by testing or usage. Estimates of probabililiesof nonsterility can be obtained by testing samples of item populations oruniverses of items lhat have been treated identically. For instance, if each itemin a population has a probability of nonsterility of 10-1, we will find thatapproximately one item in ten will be nonsterile. etc. The sampling dimensionsrequired to suppon SALs of (()-6 by end-product testing are impossibly large forboth routine and nonroutine purposes.

SALs for terminally slerilized products are subSlanliatcd (validated) byextrapolation of measurable responses of microbial populations at sub-processlrealment levels 10 process trealments that ought 10 be providing the specifiednonmeasureable SALs. Extrapolation can only be justified when a responsetakes a regular form and can be supponed by Iheery. This is clearly the case forthe lcineues of inactivation of mkrobial populations.

Simulation trials (media fills) do not validate SALs for aseplically filledproducts. The frequenlly encountered regulatory requirement for aseptic fillingprocesses to be valjdated by simulation versus a standard of no rnore than Icontaminated item in 1.000 items is nol intended to imply that an SAL of 10-3 issatisfactory for these products. The SAL is a complex. function of contaminationrate and probability of survival; the simulation trial measures only the first ofthese factors. SALs for aseptically filled products are ill all likelihood muchbetter than 10"3. only they are nonmeasureable. and there is no basis Of generallyaccepted theory to support extrapolation.

34 Chapter 2

(2.4)

The concept of the SAL specifies a single target for all sterilization pro­cesses. This takes account of the different initial levels of contamination thatmay be encountered for different products manufactured in different places andunder different circumslances. This is its strength. Versus the other standardsfor sterility. it places rar greater demands on sound knowledge of the product'sbioburden (numbers and types of microorganisms conUlminating the proouctprior to sterilization), its response to the sterilization treatment, and the unifor­mity of the pankuw sterilization processes. Ultimalely it relies on the confi­dence in Ihe extrapolation required to demonstrate that lhe process is capable ofachieving what it is supposed 1'O achieve.

V. DETERMINANTS OF STERILITY ASSURANCE

When validation depends on eXlrapohuion, it is of utmost importance 10 haveconfidence in the base data from which the extrapolation is to be made. Thedeterminants of steriLity assurance are defined by an equation describing expo·nential inacI'ivation. When time is the variable. as in Fig. 2b, the expression iswritten as

N, = NO . e·I , (2.3)

where Nt = Ihe number of microorganisms surviving after time t, No :::: thenumber of microorgani ms prior to treatment, t = the time of treatment, and k = aconstant expressing the resistance of the microorganisms to the particularsterilization treatment, i.e., a measure of the slope of the survival curve.

To determine an appropriate time I of trealment to achieve an SAL of 10-6;:: Nt it is necessary to have a fix on the bioburden NO on lhe items prior to ster·ilization and the slope k of the dose/response curve. The teoo k is most usuallyexpressed via the D·value. when the expression becomes

loSIONo + loslOSAL1=

D

The deten:ninanlS of sterility assurance that must be considered in determiningappropriate treatment levels to achieve particular SALs (in this text it will beassumed that the target SAL is 10-6) or in validating existing treatment levelsare. therefore. bioburden (microorganisms contaminating the item prior to treat­ment) and the shape and slope of the survival curve.

A. Bioburden

Bioburden is a jargon word that can assume at least two different meanings. Asa delenninant of sterility (N~ it means each and every viable Iife-fonn contami·nating the product item. As a practical concept it usually means an estimate ofthe numbers of microorganisms contaminating a product item, subject 10 all (he

Sterility and Sterility Assurance 3S

usual limitations of microbiological technique. Any bioburden claimed to be agood estimate of No is required to have been obtained from a fully validatedprocedure.

All practical bioburdens are estimates. Estimates of what? Usually lheyare measures of average. expressed as the mean number of colony-fanning unilsper product item or per unil volume or per unit weighl of the preparation. Agood. estimate of mean numbers of mi4;roorganisms per ilcm will describe a typi­cal item drawn randomly from the population. However. wjlh every popultltjonof nonslerile items, there will be some degree of variation in numbers of con­taminanls from one hem to another. Some ilems will possess fewer contami­nanlS than would be deduced from the mean: others will possess more items.

Is it therefore proper to use the mean number of microorganisms conlami­nating an item prior to sterilizaljon as a valid measure of bioburden in lhe senseof No? Alternatively. should the highest recorded number of colony-formingunits per item be used as the estimate of No. or should the estimate of No be themean plus one or two or lhree standard deviations?

This issue was addressed by Doolan and coworken; [12) in 1985.Allhough their model was derived from experience with radiation sterilization. itis a general case for all forms of exponential inactivation. These aUlhors consid­ered the case of a sterilization treatment being applied 10 a population of itemscontaminated with a pure culture of microorganisms where the mean number oforganisms per item was initially greater than one. At some level of treatment themean number of survivors would drop to one per item, so that the 101411 numberof contaminating microorganisms would be equal 10 the number of items. Atthis level of treatment. the most unfavorable way, from a sterilization smndpoint.in which the survivors could be distributed over items would be if each individ­ual ilem were to have one contaminant. In such circumslances every item in thepopulation would be nonslerile. If Ihis number of microorganisms were to bedistributed over the population of items in any other manner it would only leadto Donconlaminated (sterile) items appearing in the population. They lenned thismost unfavorable disLribution "the limiting case."

In the limiting case lhere are Iwo phases associated wilh application of asterilization treatment to a populat.ion of items with an initial mean number ofcontaminants grealer than one. In Lhe ft.rst phase the effeci of increasing lreat­ment is to decrease the to('aI number of microorganisms wilhoul effecting anychange in the proportion (Pu::) of nonsterile items in the population:

PLC = I

In the second phase. the mean number of contaminants begins to dropbelow one, and the proportion of nonslmle items also begins to drop below one.The effecl of increasing dose beyond this point is to decrease this proponionsuch that always for lilt limiting case

3& Chapter 2

(2.5)

where m = mean number of survivors per item. For microorganisms that respondto sterilization trealmenls according to the simple function Nt = No . e-kl (Eq.2.3), the behavior of PLe with increasing lrealmenl is therefore completely inde­pendent of the initial distribution of microorganisms on items and is delemlinedsolely by nJo. the average number of microorganisms per item prior to lrealmenl(Fig. 3).

The limiting case treatment for an SAL of 10.6 can be calculated from

'I\.c= D(Jog lO mo -logIOPlC)

or when loglo PLe = -6 for an SAL of 10-6

'I\.c = D(loglOmo + 6)

(2.6)

where D ::; the D-value, !.he time required (0 reduce the number of survivors in apopulation by 90%, and "'0 = NO' !.he average number of microorganisms con­taminating the product prior to treatment.

These equations illustrate the imponance of the mean number of microor·ganisms conlaminating prodUCl"i prior to sterilization and show that SALs calcu-

Umiting Case lines for twovalues of 1110 according toincreasing time of treatment

, , ,,

- ., " UMmNG CASE...l ",('"0 = 1(0)-<"l. ·2

,, ,- ,.. ,0 ,- ,

-3 ,11

, ,- ,UMIT1NG CASE ,,

~-4 (m, = I )

~.. ·sQ-

-7

D = 3 mins (approx)

2 4 6 8 10 12 14TIME (min)

Fig. 3 Limiting case model.

Sterility and Sterility Assurance 37

lated using the mean are not influenced by the occurrence of occasional highnumbers of microorganisms contaminating the odd item in the population.

Finally, on theoretical considerations relating ('0 bioburden, mo I experi­menial work on sterilizalion kinetics has addressed lIle inaclivalion of purecullures of microorganisms. Mosl "real life" bioburdens are mixed cullures. Animportanl assumption surrounding lhe application of experimental pure culruresituations 10 praclical mixed cuhure situations is that each component of a mixedcuhure should behave independently of the others. As far as is known there is noevidence to doubl this assumption.

J. Delenninalioll of Bioburden: Estimates of bioburden are no bener than Ihemicrobiological techniques used for their detennination. There is no single stan·dard approach for dosage forms for medical devices. Therefore atl techniquestend to be particular. and generalizations are difficult to make.

With medical devices, the first stage in bioburden detennination is 10remove microorganisms from the device and suspend them in a fluid diluent forsubsequenl manipulation.

Removal of microorganisms from devices usually requires some means ofcounteracting the forces that retain them on the device 113]. In the simplesttechnologies, devices may be nushed through with diluenl. They may beshaken. either manually or mechanically after immersion in diluent. They maybe sonicated. These fechniques should not be inimical, and it may be convenientto complete a composite validation technique for physical removal and for theeffects of the diluent

Removal of microorganisms from me product is less complicated for liquidand soluble pharmaceutical dosage forms. Removal and recovery methods arcthose of the microbial limit tesls contained in the compendia. Liquid dosageforms are passed through 0.45 IJm membrane filters and nushed with a diluent ora diluenl plus neutraJiz.er if the dosage fonn is antimicrobial. The membmnesare plated on suitable media and incubated to recover viable contaminants.Weighed amounts of soluble solid dosage forms are dissolved at aboul 1:10 in adiluent, fihered, flushed, and plated. When products are heavily contaminated,microbial recovery may be by direct plating methods rather than membr.lf1efiltration. Many laboratories still prefer to use membrane filtration techniques inthese cases even if secondary dilution is required.

The first issue in bioburden detenninalion thai merits validation is thechoice of fluid used in preparatory stages of removal of microorganisms fromdevices and for suspending, dissolving, and diluting dosage forms. Phosphatebuffer pH 7.2. buffered sodium chloride-peptone solution pH 7.0. and lactosebrolh are recommended in the various compendia. Saline, Ringer's solution, and0.1 % peptone waler are also quite commonly used. These fluids should neitherpromote me growth of microorganisms nor inhibit their growth. The compendiasuggest StaphylococcllS aureus, Pseudomonas aemginosa, E. coli, Salmonella,

38 Chapter 2

Bacillus .subtiUs. alld Candida albicans for valid31ing microbial limit (ests andby inference for validating diluents. elc. The addition of local isolmcs should beconsidered.

The second stage in hioburden determination is the recovery of microor­ganisms in cuhure. For both medical devices and phannaceuticals (as slaledabove), the rncthods of choice are membrane counts and plate counts. Soybeancasein digest agar is recommended for pharmace.utical dosage forms. In a surveyof bioburden recovery methods used with medical devices [141. most laboralo­ries appear to use either variations on soybean casein digest agar or variationsthe American Public Health Associalion's standard methods media recom­mended for plale CQunlS of microorganisms in milk and dairy producis. Incuba­tion times range from 2 to 21 days~ temperatures are usually in the range 30 to37'C.

Recovery conditions should also be validated 3gainst a range of microor­ganisms as described above. Variation in recovery characteristics of laboratorymedia should be guarded against lhrough application of rigorous batch-I,o-batchquality conllol techniques.

B. Survival Curves

Beside bioburden. the olher determinant of sterility assurance is the survivalcurve and ilS shape and its slope. It is not correct to assume that all survivalcurves are of the simple linear type when dala is plotted on semilogarithmicgraph paper. Three general Iypes of survival curve have been reported. theexponential curve. the "shouldered" curve, and the "tailed" curve (Fig. 4).

J. Technical Considerations: Survival curves cannot be derived theoreticallyfor panicuJar microorganisms versus particular sterilization treatments; they canonly be determined through laboratory studies.

If it is assumed that the technology of the sterilization treatment in ques­tion can be controlled sufficiently well to deliver reasonably precise incrementaltreatment levels. then the procedures and precautions required to derive repro­ducible survival curves are quite similar.

Most survival curves have been construcled for pure cultures of microor·ganisms. Reproducible curves. however, will nol be obtained unless cuJwres aregenetically homogeneous, physiologically homogeneous. and free from clumps.Assurance of genetic homogeneily resides with sound microbiological technique.Physiological homogeneity demands thai all cells within the population are allhesame stage in their growth cycles and have been grown in me same media underthe same conditions of incubalion. Microorganisms respond quite differenlly tosterilization trealments according to their physiological stale; in the grossest caseabe bacterial endospore responds quile markedly differently to sterilizationtrealmenlS lhan Ihe vegetative cell of the same microorganism. Microorgan-

, I

Sterility and Sterility Assurance 39

'SHOULDERED'

,•

"•

•, ,.,.AILED' ' , ,

" ,",

,• •••••••••••••••••,

\

••••

••,,

•STERILlZATlON TREATMENT

Fig. 4 The range of general lypes of survival curves.

isms bound together within films or slimes may be protected from penetration ofthe lethal effects of particular sterilization trealments. More imponantly.clumping may affect the number of survivors counted after treatment. Most sur·vival curves have been constructed for microorganisms exposed in aqueous sus­pension (including saline, inorganic buffers, etc.) or dried onlO a surface (e.g.,membrane fLlters, glass cover slips. capillaries).

Equal in importance to the condition of the microorganisms is the means ofestimating the numbers of microorganisms surviving incremental sub-processsterilization treatments. The characteristic approach to counting microorganismsover the whole general field of microbiology is the plate counl. Many survivalcurves have been wholly or partly derived from plate count daUl. However, thetechnique in the fonn of direct plating is only useful for concentrations ofmicroorganisms greater than about 20 per mL. Membrane filtration mayincrease the sensitivity of colony counting by about a factor of ten. Confidentextrapolation of survival curves to SALs of 10-6 or thereabouts would normallyrequire data covering lower probabilities of survival than can be achieved withcolony counlS. Data of lhis type is usually obtained from quanta) responseexperiments.

In quantal response experiments data is only recorded as good or bad. plusor minus. pass or fail In the case of sterililY. quantal response experiments setout to deleet nonsterility. normally by sterility test techniques. Replicate sam-

40 Chapter 2

pies are individually tested and. after incubation, scored as sterile or nonslerile.For a set of replicates, the proportion nonslerile (proponioo positive) is a mca·sure of the probability of occurence of a nonsterile item (the SAL). Thisapproach is nm economical with samples if low probabilities of nonslerility arebeing targeted. II is also subject to a lower limit of sensitivity set by practicalconsiderations. The frequency of spurious results in aseptic tronsfcrs attributedto aceidentallaboratory contamination (false posilives) is usually quoted 115) asbeing around 10-3 (Borick and Borick [16J reported supportive data: 30 ofapproximately 60,000 sterility Ie lS on materials known to be sterile were foundto be contnmin:l.ted in testing).

Figure 5 summarizes the limits of sensitivity of the methods used in !.heconstruction of survival curve. Plate count methods only yield meaningfulresults when the number of microorganisms per sample tested is greater thanabout ten; quantal response me,thods do not come into play until the probabilityof oceurenee of nonsterility falls below I (below 100%) and are limited by falseposilives to SALs greater than 10-3.

To make valid ex.trapolalions from exponential survival curves it is usefulDot only to obtain as much sensitivity as possible but also to cover as wide arange as possible. This should be at least through three. preferably four. log

~ 10 3

~a: 10 2WQ.

~ 10 'U

10"

\ COl.O~NT

\ REGION

QUANTAL RESPONSE

REGION

FALSE POSlTlVES... - -.. --_.--

EXTRAPOlATlOIt

REGION

-,-- •

STERIUZAnON TREATMENT

Fig. 5 Construction of a survival curve from various methods of obtaining data. subjectto their limitations.

Sterility and Sterility Assurance 41

cycles. With laboratory studies of pure cultures of microorganisms this shouldonly rarely be a problem. However. since the 19805 there has been some consid­erable interest in constructing survival curves for the innate contaminantmicronora on actual product ilems (often as mixed cultures). With some veryclean products. for instance hypodermic needles for which the average numberof contaminants per item prior to sterilization is usually less than I. it may not beeasy to cover a rearonable range of inactivation versus a lower limil of sensitiv.ity of an SAL of 10.3. To some extent this may be resolved by multi item quantalresponse experiments. i.e.• incubation of several items logether in one culture..

Eslimates of the probability of occurrence. of nonsterile items can beobtained from such data through an expression derived from the binomial distri­bution (17). Suppose a population of items is contaminated with from 0 to nmicroorganisms per item. A proponion of microorganisms will harbor one ormore mkroorganisms; therefore any random item taken from the iX>pulation canbe designated to have a probability q of being free from microorganisms (sterile)and a probability p of carrying one or more microorganisms.

Assuming that the nonsterile items are randomly distributed throughout Lbepopulation. then the probabilities of there being O. I. 2. CIC.• nonsterile items in arandom sample of n items drawn from the population 3!e given by successivetenns of the binomial expansion:

q" _L q"-l ,(q + pY' = q" + (n> P + (n> p . .. (2.7)

Thus if represents the probability of such a sample conroining no contaminateditems. Conversely. I - q" is the probability that a sample of II items contains oneor more contaminated irems.

For any series of quantal response data. the ratio of nonsterile items(growth) 10 sterile items (no-growth) after incubation is an estimau: of Ihe termI - if. This estimate in lum allows calculation of q and p. Imponantly. p is ameasure of the probability of an item being nonsterile.

An example of how this calculation may be used is given in Annex 2.A final laboratory issue connected with mixed cultures is how to handle

multiple media data r171. The compendial melhods of testing for sterility havelong advocated the use of replicate media and incubation conditions 10 ensurerecovery of as wide a range of microorganisms as possible. It is recognized thatanyone recovery condition is not absolutely selective for a particular kind ofmicroorgani!."11l. and it is to be expected that on occasions the same kind oforganism will be recovered in more than one condition. 00 the other hand. therewill be occasions when a single particular recovery condition will only be suit·able for the recovery of a particular kind of organism: if this condition were notpart of the test it could be falsely presumed that no viable microorganisms werepresen!.

42 Ch<ilpler 2

When it comes to determining survival curves there is no need 10 considermuhiple media unless the curve addresses naturally mixed cultures within whicheach component has nOI been or cannot be precisely identified. However. anystudies with naturally contaminated products ought to use multiple media.

Doolan and coworkers (18J used three compendia! recovery conditions toconsU'UCt survival curves for naturally contaminated medical devices: soybeancasein djgesl medium incubated at 2~2YC. the same incubated at ~35·C. andfluid thioglycoUate medium incubated at 3O-3S·C for 14 days. Three lesls forsterility therefore constituted the basis for observing the presence of viablemicroorganisms in three replicate conditions making up one sample.

Their problem was how (0 interpret the data. Table 4 illuslrates someresulls fTOm this type of experiment The data are scored as "net positives";media are coded as replicates before incubation. and lhen. after incubalion. onenel positive is scored for anyone. two, or three growths in the replicates makingup a single sample. In Table 4. the proponion positive obtained from nel posi­tives is 0.6.

Table 4 lUu5lrati\"e Tabulation of Data for Scoring Net Positives

Incubation condiltonS

SampleSCDMb,3O-3S'C

FTM'".3O-3S'C

I234S6789

10Proportion

positive

-+ ++ + ++ + + +

+ + +

- + ++ +

- -0.40 0.30 0.30 0.60

'Positive 1lO"..th in any ~. two. or thftt 0( thr rnediwn·~condilioru was 5COfnt as a l'ld

posilh'C..bscDM = soybc:an casein diJfil medium USPcFTM :z. nuid thiollycollatl: medium USP

Stenlity and Sterility Assurance 43

There are alternative ways of interpreting this dam:

(a) When the proportion positive is calculaled from each of the individualcolumns, each individual value is less than 0.6; Lhe value obtained from lhenet-positive approach must always be higher or equal to that oblained fromone medium alone.

(b) Another way of interpreting such data might have been to score eachmedium as positive or negative and express the total as a proponion of thetotal number of teslS. From this approach a proportion positive of 0.33would have resuhed from the data in Table 4. The proportion poSitivefrom lhe net positive approach must always be higher or equal to the valueobtained from this alternative interpretation.

(c) A mird way is to give equal weight to each positive and express this asa proponion of the number of samples. In Table 4 the number of samplesis ten and the proponion positive calculated by this method would be J.O(100%). This method maximizes the value of Lhe proponion positive. Itassumes that each medium is absolUlely selective for 3 particulor kind ofmicroorganism that is unable t'o grow in any of the other conditions. Italso assumes that contunination of a product item by one kind of microor­ganism excludes the possibility of contamination by anolher kind. Theseassumptions bear no relation to practical circumstances.

The net-positive approach provides a properly conservative means ofinterpreting multiple media data. and multiple media must be used in the con­struction of any survival curves that are intended 10 avoid false negative resultsfrom naturally contaminated product items.

2. Exponemial Survival CUIVes: Exponential survival curves are linear whenthe logarithm of the number of survivors (or the surviving fraction. N,/NoJ isploued against increasing SlerilJzation treatment on an arithmetic scale. Thisfonn of death has been noted for pure culture microbial populations since thebeginnings of the twenlieth cenlury. II follows the pattern of first-order chemicalkinetics and has prompted the assumplion that death of microorganisms is theresult of some molecule essential for the funherance of viability haVing under­gone some irreversible reaction.

A simple expression describing exponential survival curves i.s given in Eq.(2.3):

N, = No . .-kJ

1be practicalities of constlUCting exponential survival curves are not alwaysstraighlforword.

44 Chapler 2

(2.8)

(3) Some microorganisms are so sensitive to sterilization treatments th3' itis impossible 10 construct any pan of the survival curve except for the zeropoint from colony-count data. Therefore all data must come from quanta)response experiments. The number of replicates required to plot a reason­able survival curve may rapidly become limiting.

(b) Some sterilization treatments. particularly heat treatment, are notamenable to precise control of incremenlal delivery. Heat becomes lethalto microorganisms throughout the time taken to reach the specified steril­izing temperature ("heat-up time") and the time taken to cool down afterthe specified exposure period.

In research laboratories lhese difficulties are pan of the expected chal­lenge. In industrial control Jaborotories they are serious dislfactions fromObtaining information quickly, usually the D-value. There are several standardapproaches to obtaining D-values from simple quanta) response experimentsusing manageable numbers of replicates and without h3ving to draw out survivalcurves on semilogarithmic paper. These methods assume exponential survivalkinetics and should therefore be used with some caution if Ihere is any doubt.

Probably the most widely known method of calculating D-values fromquantal response experiments is the Stumbo (191 method. Each sample shouldconsist of at least ten (and rarely more) replicate items carrying the same numberof contaminants {No>. Samples should be exposed to increments of sterilizationtreatment, and then each replicate should be tested for viable microorganismsseparately in suitable recovery conditions. The number of sierile replicates q isscored after incubation. The D-value may be calculated from

tD = -:----::--:--__::_

log 10 No -)ogIOB

where I = duration of sterilization treatment (usually time, but for ionizing f'Jdia­tion , would be dose). NO = initial number of microorganisms on each replicate,and B = 2.3026Iog lO(nlq), where n is the number of replicates in the sample andq is the number negative (sterile).

A separate D-value is usuaUy calculated for each increment of sub-processsterilization treatment and an overall D-value laken from the arithmetic mean.

The same type of experimental design, allowing that each increment ofsub-process sterilization treatment d is equal, may be used 10 calculate D-valuesby the Spearman Karber method (20]. 1be total duration of sterilization treat­ment should be chosen 10 cover the whole of the quanta) region from 'I' which isdefined as Ihe longest time at which every replicate is still found [0 be nonsterile,to time 't> which is the shortest exposure at which every replicate is found 10 besterile as tested. The Speannan Karber equation allows estimation of a factor II

as

, I

Sterility .nd Sterility Assurance 45

U = Ii. -d

2(2.9)

(2.10)

where u = an estimate of the time of expo ure required to inactivate 50% of thereplicates. 'i. =the shonest time of exposure to yield all sterile replicates. d =the

1:-1time interval between exposures. n = number of replicates per sample. and~ r{= the sum of sterile replicates from I._ to (Ik - 1). - I

The D-value can be calculated from u through the expression

uD = -=--=--::=--,---,.,.­

0.2507 + loglONowhere No = initial number of microorganisms on each replicate.

Annex 3 iIIusuates how the Stumbo and Speannan Karber methods can beused to e rirnate D~values from quantal response results.

J. "Should~r~d" Survil'al Curves: Survival curves that inilially show no sen·sitivity to a sterilization lreatmenl but then respond in a typical exponential man­ner as exposure continues are referred to as "shouldered." They confonn to themathematical fonn

N,-,-'-= 1 - (I - e-k')" (2.11)

No

wbere all terms are the same as those in Eq. (2.3), plus n, which is called theextrapolation number, i.e., the intercept of the extrapol:lled exponential ponionof the curve onto the N,tNO axis.

"They are altributed to one or all of several mechanisms.

(a) Repair. In some microorganisms there may be cellul;lf mechanismsthat repair molecular damage done by sterilization treatments. While thesemechanisms are operative there will be no loss of viability (the shoulder)unfil they themselves are irreversibly damaged by the sterilization treat­ment.

(b) Multitargel concepts acknowledge the likelihood of loss of viabili!)'being caused only through more than one essential molecule being irre­versibly damaged in the cell.

(c) Muhihit concepts acknowledge one vilal molecule having to reac,1more than one time before it is pennanently lost {o its role in the continu­ance of life.

For the purposes of ex.trapolating SALs. shouldered survival curves can betreated as exponenlial curves after making an appropriate allowance for Ihe

46 Chapter 2

shoulder. In fact shoulders often become negligible with intense sterilizationU'ealments, for example at high temperatures with salUrated steam. However.there is some danger in applying the Stumbo or Spearman Karber methods toshouldered curves. D·values (and therefore the treatment recommended 10

obtain particular SALs) will be overestimated if the shoulder is not recognized.Whereas this is intrinsically conservative as far as assurance of sterility is con­cerned. unnecessarily excessive sterilization treatment may have deleleriouseffects on some other characteristic of a phannaccutical preparation or medicalproducl.

4. "Tailed" SI4rvi~'a/ Curves: Tailed survival curves cannot be extrapolated.They are characterized by a slope that diminishes with increasing exposure 10 lIlesterilization treatment. They are often described as ··concave."

Tailed curves are particularly interesting because they challenge the nOlionof exponential curves being extrapolated ad infmituDl to SALs of 10-6, 10-12,

10- 18, etc. How valid is extrapolation if the curve begins 10 tail off beyond Ihemeasurable region?

There is one basic question that oughl to be addressed about tailed survivalcurves: are lbey artefacts or are they genuine? They have certainly been DOled

in many experiments. Beside the undoubted occurrence of examples of genelicheterogeneity and helerogeneily of treaunenl, there are three theories [21] thatme,ril some consideration.

(a) The viUllislic theory has il that individual cells in a population (even agenetically homogeneous population) are not identical; IhereFore some in·dividuals would be very sensitive to a sterilization treatment, the majorityaveragely sensitive. and olbers quile resistant. Although plausible, there isvery little evidence to suppon this concept.

(b) 1be melhods of expression of survival data may create artefacluaJtails. It has been pointed out by various authors that Ihere is a systematicbias in the Stumbo and other melhods of inlerpreling quanrat response dataleading 10 calculated D·values increasing with increasing exposure tosteri1ization trealment.

(c) The confidence limits of survival curves must be understood to be farbroader at longer exposures to steriliz31ion treatments because (whereasthe inacljv31ion of a large population of microorganisms is a slalisticalphenomenon) lbe death of individual microorganisms al low numbersbecomes a series of discrete events [22J.

Tails have not been seen throughout the whole range of sterilization treatments.For instance. no genuine case of lailing has been auributed to inactivation byionizing radialion. This may be because of the nature of biochemical mecha­nisms of inactivation specific for nucleic acids. On the other hand, it may be

Slerility and Sterility Assurance 47

attributable to the greater ease and precision with which radiation can be deliv­ered to popuJations of microorganisms compared with other sterilization treat­ments. On the other hand, there are many instances reported for heat and chemi·cal methods of sterilization. In a review anicle on lIle subjecl of tails. Cen (21)concluded that it seems prudent not to ignore the possibilily of departures fromthe exponential order of inactivation of microbial populations if it is desirable 10

avoid the risk of heallll or commercial hazards.

5. Survival Curves with Mixed Cultures: Although most published survivalcurves have been obtained in the laboratory using pure cultures of microorgan­isms, most "real life" sterilization processes must deal wilh mixed cullUres.probably heterogeneously diSlributed over hems. There are comparatively fewpublished dala on the behavior of mixed cultures. except as they have arisen byaccident as conlamination in laboratory siudies. There is no reason to belie\o'clIlal the differenl components within a mixed cullure do nOI act completely inde·pendenlly of one anolher with respect to their responses to sterilization treat­ments. This teods to be a fundamental assumption in all approaches to validationof existing sterilization processes and of new process de\o'elopment.

REFERENCES

I. Tire Therap~ulic Subslonus R~gulations, 1952. London: Her Majesty's StationeryOffice.

2. McKay. A. M. (1992). Viable but non-cullurable forms of potenlially pathogenicbacteria in water. Lellus inAppJi~dMicrobiology 14: 129-135.

3. Byrd, J. J.• Huai-sbu. X.. and Colwell. R. R. (1991). Viable but noncuhurable bacte­ria in drinking waler. Applitd and En};ironm~nlalMicrobiology 57: 875-878.

4. Pearson. A. D.• Colwell. R. R.• Rollins. D., Watkin·Jones. M.. Healing. T.. Green­wood. M.• Shahamat, M.• Jump. E., Hood. M.• and Jones. D. M. (1988). Transmis.sion of C. jejuni on a poultry farm. In Campylobactu IV (8. Kaijser and E. Falsen.eds.). Goteberg. Sweden: University of Golcberg.

5. Sharpe. A. N. (1973). Automation and instrumentation de\o'clopments for the bacte­riology laboratory. In Sampling-Microbiological Moniloring of Environm~nIs fR.G. Boord and D. W. Lovelock. eds.). London: Academic Press.

6. Galante, L. J.• Brinkley. M. A., Drennen. J. K.• and Lodder, R. A. (l990). Near­infrared spectrometry of microorganisms in Liquid pharmaceuticals. AnalyricalCh,misrry 62: 2514-2521.

7. Knudsen. L. F. (1949). Sample size of pareOleral solutions for sterilily testing.Jourrl(Jl a/the American PJuJnnaceulical Association. 38: 332-337.

8. MaxweU Bl)'oe. D. (1956). Tests for the sterility of pharmaceutical preparations.Journal ofPharmacy and Pharmacology 8: 561-572.

9. TaucrsaJ, K. (1961). Control of slerility in a manufac,lUring process. In RecentlNl'~/opntenIs in tM Sterilisation of Surgical Morerials. London: PhannaceuticalPr=.

48 Chapter 2

10. Brown. M, R. W.. and Gilbert. P. (1977). Increasing the probubiJity of srerilily ofmedicinal products. Journal of PhnrmDC)' and PhQrmDcoJog)' 29: 517-523.

11. Canadian Standards Association (1979). Industrial sterilization of medical devicesby the steam process. CSA Sta!1dLJrJ Z314.4-MI979. Rexdale. Ontario. Canada:Canadian Standards Association.

12. Doolan. P. T.. Dwyer. J.. Dwyer. V. M.. Filch. F. R.. Halls. N. A.. and Tallentlre. A.(1985). Towards microbiological quality 3S..\urance in radiation sterilization pro­cessing: A limiting case model. Journal ofApplied Bacleriology 58: 303-306.

13. Bill. A. (1992). Bioburden: Sampling and remo 0.1. In Bioburd'n ;1/ MedicalDe)'ice and Surgical Dressing Manufaclur~. Procudings of EUCOMED Conjrr­met!, March 23 and 24. 1992. Brussels. Belgium: EUCOMED.

14. Halls. N. A. (I992). Bioburden: Determination. 1n Bioburden in Medical DI!'1iceand Surgical Dussing Manufacture. Proceedings of £UCOMED Confereflce,March 23 and 24, 1992. Brussels. Belgium: EUCOMED.

15. TaUentire. A. Dwyer. J., and Ley. F. 1. (1971). Microbiological quality control ofsterilized products: Evaluation of a model relating frequency of contaminated itemswith increasing radiation treatment. Joumal ofApplied Bacun'ology 34: 521-534.

16. Boriek. P. M.. and Barick. J. A. (1972). Sterility testing of pharmaceuticals. cos·metics and medical devices. In Quality Control in the Pharmaceutical Industry(M. S. Cooper. ed.) New York.: Academic Press.

17. Doolan. P. T.. Halls. N. A.• and Tallentire, A. (1988). Sub-process irradiation of nat·urally contaminated hypodermic needles. Radiati01l Chemistry a"d Physics 31 (4­6), 669-703.

18. Doolan. P. T.• Halls. N. A.. Joyce. T. M.• and Tallentire. A. (1983). Net positives:Conservative approach to measurement of proponions positive in substeriliz3tionprocess studies. Applied and Environmental Microbiology 45 (7): 1283-1285.

19. Stumbo. C. R. (I973). Thumobacteriology in Food Processing. 2d ed. New York:Academic Press .

20. Pflug. I. J. (1977), Principles of thennal destruction of microorganisms. In Disin­fectio", Sterilization and Pus~n'ation_ 2d ed. (S. S. Block. 00.) Philadelphia: Leaand Febiger.

21. Cerr, O. (1977). Tailing of survival curves of bacterial spores. Journal ofAppli~dBactuiolog)' 42: 1-19.

22. Fredrickson. A. G. (1966). Stochastic models for sterilization. Biotechnology andBj()('nginuring 8: 167-182.

ANNEX 1. CALCULATING THE PROBABILITIES OF ACCEPTINGBATCHES OF PRODUCT BY THE PHARMACOPOEIAL TEST fORSTERILITY

The probability of selecting If consecutive sterile items from a population con­taining a proponion p of nonsterde items is given by the expression

(l-pY' (12.1)

, I

Steritity .nd Sterility Assuronc:. 49

The phannacopociaJ TeslS for Sterility typically pass batches from which twentyitems have been tested and shown not to be nonsterile. There is no doubt lhat itwould be unethical to release a batch of product containing nonsterilc items in!be proportion of one in (say) two hundred.

In this example p would equal 0.005 and n would equal 20. The lenn

(I - p) = 0.995

Taking logarithms,

log,o 0.995 = -0.0022

Solve for (I - p)" by multiplying

20 x -0.0022 = -0.0435

Withdrawing from logarithms.

anlilog -0.0435 = 0.9046

TherefOR: we would find that even with a frequency of occurrence of one non­sterile item in two bundred. a balch would have 3. greater than 90% chance ofpassing Lhe Test for SteriUty.

ANNEX 2. CALCULATING THE PROBABILITIES OFOCCURRENCE OF STERILE AND NONSTERILE ITEMSIN MULTl.ITEM EXPERIMENTS

(3) In a multiilem quantaJ response experiment. 15 items were bulked in eachtest that could be scored as showing growth or no-growth. Of 20 lesLS (each of15 bulked items), one showed growth. Le.. the ratio of nODsterile tesLs to sterile",SIS was 0.05.

(b) According to Doolan et al. (17). thlS ratio is an eSlimare of the 1'enn (I - qn).

(c) If (I - '1") = 0.05. when n is equal to 15. the value of q can be calculated bysimplifying terms:

ql5 =0.95

Taking logarithms.

log10 0.95 =-0.0223

Divide this term by 15 to find log 10 q; this equals -0.0015. q is therefore !beanlilogarithm or -0.0015. i.e.• 0.997.

(d) The probability of occurrence of 3 nonsterile-ilem in the population of ilemsfrom which the sample was drawn can be calculated from (p + q) "" I, i.e.• pequals 0.003 or one in 333.

50 Ch01lpter 2

ANNEX 3. CALCULATING D-VALUES FROM QUANTALRESPONSE DATA USING THE STUMBO AND SPEARMANKARBER METHODS

The following dam was obtained [rom a hypothetical investigation into thermalinactivation of bacterial cndospores. 1be initial number of viable spores perreplicate was I x 1()6 (NoJ.

Time of Number of Numberexposure (min) replicates (II) sterile (P)

4 10 06 10 08 10 3

10 10 512 10 814 10 10

(a) Calculation of D-value by lhe Stumbo method.

2d

U = 'k-

t nD = - 2.30310810--

loglONo q

where t = time of exposure. NO = initial numbtr of microorganisms per replicate.i.e.. 1()6. and n/q = number of replicates per sample divided by the number ofsterile replicates.

(i) D-value from 6 '0 8 min = 216 -3.3 = 0.74 min.(ii) D-valuefrom 6 to 10 min = 4/6 - 2 = 1.0 min(iii) V-value from 6 to 12 min = 616 -1.25 :: 1.26 min(iv) D-vaJue from 6 to 14 min = 616 -1 = 1.2 min

Mean D-value = 1.05 min.

D-value calculated by the Stumbo method = 1.05 min.

(b) Calculalion of D-value by !he Spearman Karber m<'hod.

k-I

- : L rj

j .. I

where 't = soonest lime of exposure for which all replicates were found to besterile (14 min), d = time increments of exposure (2 Olin), n = number of repli·

• - Ical'es per sample (I S), and L rj :0: the sum of sterile samples from , I to 'A: - I

•• I

Sterility and Sterility Assurance 51

(0 + 3 + 5 + 8). i.e.. 0 = 14 - 212 - 2/10. (0 + 3 + 5 + 8) = 9.8. D = 0/0.2507 +I0810No = 9.8/6.2507 1.57 min. D-value calculated by the Spearman Karbermethod = 1.57 min.

3Sterilization by Gamma Radiation

l. Radiation Dnd RadioactivityII. Effects of Radiation on Microorganisms

A. Molecular and CelluJar Effects of RadiationB. Effects of Radiation on Microbial Populalions

m. ApplicationsIV. Industrial·Scale Cobalt-60 Gamma Irradiation Sterilization

A. CobaIl-60 Gamma IrradiatorsB. Control of Dose

V. The Choice of DoseA. The 25 kGy "Standard" Dose8. Irradiation in ScandinaviaC. Irradiation in North America

555657585966677276767777

Terminal sterilization of phann.aceuticaJ dosage forms by ionizing radiation iscomparatively rare. On the olher hand. ionizing radiation is used extensively forterminal sterilization of heal-sensitive medical devices and for hcal-sensjlivcph31'1tlllCeutical packaging components prior to aseptic processing. It is stricllyan industrial process; there is no hospiml-scale radiation slCrilization.

SleriJizalion by ionizing radiation typically uses gamma rays. There issome use of accelerated electrons, but to 3 far lesser extent than gamma radia-

53

, I

54 Chapter 3

tion. The pioneering work II) directed speciflcaJly toward evaluating the effec­tiveness of irradiation as an induslrial scale microbicidal process was done in the19405. The lelhal effeelS of gamma radiation on microorganisms were alreOldywell known. and gamma sources with practical industrial potential were becom­ing available as by-products of controlled energy release in nuclear reactors.Caesium-137 formed in spent fuel rods was the first isotope considered. butinsufficient quantities were available without introducing ex.pensive separationprocesses. Around the same time, coball~59 was beginning to be used as anahemalive to steel for neutron absorption in British gas-cooled reactors.Absorption of one neulmn per alom converts cobalt-59 to radioactive cooolt-60;sep:mnion from other radioactive isotopes was unnecessary. Thus cobalt-60became the predominant gamma source for industrial sterilization and hasremained so despile Ihe U.K. no longer being a significant source of supply.

In the early days of industrial irradiation it was the possibility of food irra­diation thai excited greatest interest. However. the first commercial applicationof the microbicidal effects of radiation was sterilization of surgical SUlures byEthicon Inc.. Somerville. New Jersey. U.S.A. I.n the period 1956 to 1964Ethicon was using a 2 MeV electron beam accelerator to sterilize the greater panof lhe U.S.A.'s requirement for surgical s.utures. The earliest induslrial-scalecobalt-60 gamma irradiation plants were opened in 1959 and 1960. The first ofthese. built 10 a British design. was opened in Australia in 1959 to eliminateanthrax spores from bales of imponed goal hair; the olher was buill for theUnited Kingdom Atomic Energy Authorily at ils Wantage Research Laboratoryand was eventually to become allied 10 the slerilil..alion of medical devices. Acaesium- 137 plam was opened for Conservatome Industrie. Lyon. France in1960. also deslined to be used for sterilizing medic.al devices. Progress wassomewhat slower in Ibe U.S.A where il was 1963 before the first industrial-scalegamma irradiator was commissioned. Ils purpose was 10 support a food irradia­tion program which was being initialed by Ihe Quanennaster Corps of the U.S.Anny. In 1964 Elhicon replaced their accelerator with a cobalt-60 gamma irra­diator.

Medical device Slerilization has become lhe primary applicalion of gammairradialion. wilh cobalt·60 remaining the predominant source. Food irradialionis still a controversial subjecl even in lhe 1990s. AI first. in the 19605. the fate ofexpansion of irradialion sterilization was rather slow. The most immediatedevelopments were in Europe. where a single.use medical device industry wasdeveloping in pace with Ihe new sterilization technology in a generally favorableregulatory climate. Not so in the U.S.A.. where Ihe FDA had initially classifiedirradiated medical devices as "new drugs" requiring lengthy validation proce­dures before approval for markeling. This silualion had eased by the mid· I970s.panly at least due to growing concern over the probable carcinogenicity of themain altemalive"cold" sterilization process with ethylene oxide. In 1975 there

Sterilization by Camma Radiation SS

were only 55 irradiators in service throughout the world; by 19&4 this haddoubled 10 110, nluch of this growth being in Ihe U.S.A.

I. RADIATION AND RADIOACTIVITY

All matter is made up of atoms, each atom consisting of a central nucleus circledby negalively charged orbiting electrons. Two IYpes of panjcle ex-isl withinatomic nuclei, positively charged prolons and uncharged neutrons. All s13blealoms are eleclrically neutroil, the number of protons equalling the number ofelectrons. Stability is also a funclion of lhe number of ncutrons in the nucleusbecause of lhe balance of forces necessary to bind lhe nucleus logether.

Radlooclive mal'enals are IDalerials in which the nuclei of the aloms ate

unstable. Unstable nuclei are subject to sponlaneous decay or disintegration byejecting a panicle in an endeavor to acquire a more stable configuration, thusforming "daughler nuclei." Radialion is emiued as panicles are ejecled. The 51unit of radioactivity is lhe, becquerel; a radioactive source has an activity of onebecque~1 if iC is disintegrating al a rate of one atom per second.

Consider the stable atom coball-59. It has 59 particles in ils nucleus. lisatomic number is 27, which means that 27 of the 59 particles are prolons; theremaining 32 are neutrons. The purpose for which coball-59 was used in the:early gas-cooled reactors was to absorb neutrons. Wben coball-59 is bombardedby neutrons. an extra neutron is added to the nucleus. thus forming Ihe unstableradioactive isotope cobalt-60. Radioactive decay of coball-60 is a single-stageprocess to stabilize to its daughter. nickel-60. with emission of one gamma mypholon (a pulse of electromagnetic energy of extremely shon wavelenglh andhigh energy) and one bela panicle (a rapidly OlOving electron).

Radioactive isolopes emit different types of rddiation:

(a) Alpha panicles. An alpha particle consists of two protons and Iwoneutrons. When alpha panicles pass through materials. they knock offelectrons from nearby atoms, gradually slowing down and losing energy asthey do so. Even very energetic alpha particles are broughl to rest in " fewcentimeters of air. Alpha decay of an isotope may, however. also produce11 rele3..1tC of gamma radiation (see below).

(b) Bela panicles. A beta panicle is a high-velocity el~IIon th:u loses itsenergy in the same way as an alpha particle. It is capable of penelDting farfunher through malerials than alpha panicles. for instance up to a fewinches of aluminum. This lypc of radialion is sometimes used for slcril­il.8lion, usually from electron accelerators rather than from radioactiveiSOIOpeS, but it imposes quite severe practical limitations on the depth ofproduct that can be presented to the source. This is nol because microbio­logical contaminants need 10 be killed within the depth of a material (they

56 Chapter 3

are usually a surface contamination) but because sterilants are required topenetr3r.e through packaging mar.erials and to surfaces that may beobscured by oilier depths of materials.

(c) Gamma rays. Gamma rays are photons of eleclromagnetic radiationwim energies in the range of I keY to 10 MeV. They are very similar 10 x­rays. They are very penetrative through malter. They only lose energywhen they collide with a nucleus, but they Jose all their energy in one col­lision. Thus gamma rays are able to pass through mallcr with the sameenergy as lhey had when they entered. just fewer of them emerge. Somealways penelrate any thickness of a barrier. but the numbers get fewer andfewer wilh thickness. This propeny of penetnnion is the first and mostimponanl propeny of gamma radiation as a robust process for induslrialsterilization.

As time goes by. the activity of all radioactive sources diminishes accord­ing to an exponential law characterized by its half-life. The half-life is Ihe limetaken for the aClivity to fall to half of its original value. Cobah-60 has a half-lifeof 5.3 years: caesium-13? has a half-life of 30 years. Thus a radioaclive isotopeis decaying and losing activity at all times regardless of whether it is being usedOf not. This factof has economic implications for induslrial-scale irradialion.

The alternative to isotope sources of radialion is the production of acceler­ated electrons. Accelerators have an economic advantage over isotopes in thaithey can be switched off when not required. Their disadvantage is in their poorpenetrative properties.

An accelerator is a device in which an elecuic field is developed by appli­cation of a vollage at opposite ends of a linear path. The greater the voltage, thefaster the speed of the electrons; the faster the speed of the eleclrons. the greatertheir penetrative power through maner. For practical sterilization purposes.accelemtors of less than 100 MeV are being used for products such as hypoder­mic needles in which the requirement for penetration is minimal.

II. EFFECTS OF RADIATION ON MICROORGANISMS

Radiation damage to biological systems and other materials is caused by theenergy absorbed from the radiation. The damage is proportional to the amountof energy absorbed. The amount of energy absorbed is referred to as dose(correctly, absorbed dose). and it is measured in unils of energy per kilogram.The SI unil of dose is the gray CGy), which is defined as an absorbed radiationdose of one joule per kilogram. Where time of slerilization Ireatmenl wasreferred to in Chapler 2, dose should be substituted when dealing with irradia­tion.

Steriliution by ~m;l R;ldi~tion 57

Prior to the introduction of SI unilS, !he unil of absorbed dose of mdiationwas me rad (also krad and Mrad). and Ihis unit still appears in some compendia,regulatory documents. and oLher sources of infonnation. For reference. 100 rodis equivaJenl to one gray. and 2.5 Mrad is equivalent to 25 kGy.

A. Molecular and Cellular Effects of Radiation

The absorption of ionizing radiation in matter leads to numerous phenomena ofhigh complexilY. In biological materials these can be categorized within fourstages:

(a) A physical slage in which extremely unslable ex,cited or ionizedmolecules (primary products) are produced from interaction of gamma rayphotons wilh orbiling elec:uons.

(b) A physicoc,hemical slage in which Lhe primary products react sponta­neously or in collision with each other. bonds are broken, and highly reac­tive ions.. radicals. and trapped cbarges are fanned. h is the reaction ofthese reactive groups that produces observed changes.

(c) A chemical stage. This is the reaction and interaction of the reactivegroups described in (b) above. It is basically a slage of acbieving thermalequilibrium.

(d) The biological stage comprising a series of biochemical reactions atdifferent levels of cellular organization, giving rise 10 observable biologi­cal effects. Loss of viability of the microbial cell is principally due lOdimerization of DNA bases and scission and cross-linking of the sugar­phosphate backbone of DNA.

Microorganisms of a given Iype may respond differently to radiationaccording 10 differeDI physical or chemical conditions existing within the cell orin irs immediate environment. Of these environmenlal facton. the moSI exten­sively srodied have been Ihe effectS of oxygen and the effects of hydralion.

Microorgani ms are more sensitive (0 radiation when oxygen is presentduring and after irradiation than in environments from which oxygen has beenexcluded. In other words, oxygen sensitizes microorganisms to radiation. Thisis likely due to reaction of oxygen wilh free radicals formed by ionization of tar­get molecules. One hypothesis poslulates radiation giving rise 10 lwo types ofdamaged molecule, R' and R". The firm of Ihese is a free radical wilhout effecton the survival of cells unless oxygen reacts with it:

R-w. R' + H+ + e- nonlethal

R' + 0, -> R02 lethal

58 Ch~pt~r 3

1lle second type of damaged molecule is lethal irrespective of the presence orabsence of oxygen:

lethal

Whereas oxygen sensitizes microorganisms. water (in the presence of oxy­gen) proteclS microorganisms from radiation damage. 1be drier ceUs are, !.hemore sensitive they arc to radiation-induced lethal damage. The reason for lhishas to do with irradiation damage thai occurs only when cells are irradiated inthe presence of oxygen (the immediate oxygen effect). This effect is believed 10

be due to interaction of oxygen with extremely shon-lived species produced atthe time of irradiation. Increasing cellular water COnlenl resulls in a subsulJuialdecrease in this type of damage. presumably due to competition from the watermolecules for these short-lived radicals.

B. Effects of Radiation on Microbial Populations

In a typical experiment designed for quantitative study of irradiation inactivationof microorganisms, a population of cells is exposed to a series of incrementaldoses of radiation. After each dose the numbers of microorganisms surviving arecounted and plotted as fractions of the original population on a logarithmic scaleagainst radiation dose on an arithmetic scale. Radiation inactivation of microbialpopulation is probably the best resean:hed of any microbicidal treatment Thisis primarily because of the precision with which doses of radiation can be deliv­ered to microbial populations. Radiation experiments do not have to introducecompensatory factors for heat-up and cool-down in the way that thermal inacti­vation studies mu t, nor are there any residues or traces of radiation left behindwhen the sample is removed from the radiation field. A secondary but highlysignificant factor is the comparability of radiation resullS. An absorbed dose of xGy has the same significance whereve.r il is delivered. and for whatever configu­ration the sample was irmdiat'ed in. There are no local differences caused byequipment design or irregularity. as long as the measurement of dose is done cor­reclly. Imponantly. the rate of delivery of dose (dose rale) has no effect on theradi:uion response of microbial populations.

Survival curves for irradiated pure cultures of microbial populations con­form to the idealized types described in Chapter 2. In radiation microbiology theD-vaJue is invariably caUed the DIO value. II is doubtful whether a UlJly -tailed"survival curve has ever been seen in a properly conducted irradiation study ofpure cultures. There are. however, major differences seen from microorganismlO microorganism and from condition to condition.

The extenl to which various microorganisms respond lo radialion is verymuch determined by different abilities to "repair" radiation-induced damage.Differences in Ihese abilities give microorganisms unique innate sensitivities toradiation, as was shown by TalJentire [2] w~n five different IYpes of micrt>Or-

Sterilization by Glomma Radiation

Table 1 Innate Sensith'ity of Microorganisms to Gamma Radiationwhen Grown Under Similar Conditions and Irradiated Under IdenticalConditions

59

Organism

Ps. aeruginosaStaph. aureusStrep. fut!ciumB. pwnilus sporesB. spJuuricus spores

Soun:'~: TaJlenlir'e (1980)

Shape of curve

ExponentialExponentialShoulderedExponentialExponential

DIO (Gy)

20100500

17004600

ganism showed appreciably different response to radiation (Table I) when grownunder as ncar identical conditions as possible and irradiated under exactly thesame conditions.

The limits of innate sensitivity are wide. typified by D 10 values of around20 Gy for Pst!udomonas spp up to 4.6 kGy for spores of BocUius sphaericuswhen irradiated in air. Typically. gram-negative bacteria are more sensitive torad,jation Ihan gram·positive bacteria. and gram-positive bacteria are more sensi­tive Ihan baclerial endospores. Danish workers have isola1ed some unusualgram·positive cocci identified with Streptococcus !aeciwn A2 and Micrococcusrodiodurotls. which are exceedingly resistant to radiation owing to eXlended"shouldered" inactivation curves.

The effecls of oxygen and water described above as affecting radiaLiondamage 301 the cellular level are reflecled in Ihe responses of microbial popula­tions to radialion. Other chemical factors may also proieci or sensilize microor­ganisms. The effects of aemperature on radiation response of microbial popula­tions tend to be complex. bUI in general. increases in lemperature during irradia·tion bring aboUI slight increases in radialion sensitivily. These effects may becomplicaled by the presence or absence of oxygen and water.

III. APPLICATIONS

The mosllime-proven melhods of sterilizalion have been lhe use of high lemper­atures and filtration. However, medical practice has always included objeclS thaiare neither till.roble nor able to withstand high lemperotures. which were··steriliz.ed" by methods of doublful effecliveness. such as liquid sterilizing fluidsor formalin cabinets. In the 1940s and 1950. this probIem began to be aggra­valed by rapid advances in surgery and anaesthetics. which demanded the use ofheat·sensitive plastic tubing. valves. and prostheses. The oulcome of l.hese

60 Ch~p'tr 3

changes was the provision of as many as possible of the cheaper plastic and rob­ber items as single-use disposable products presterilized by the manufacturerwithin individual sealed packs. This promoted the introduction of "cold" steril­ization methods. mainly gamma irrawalion and exposure to elhylene oxide,which are only practical on an indusLriaJ scale. The ndoplion of cold steriJiz.edsingle·use medical devices also obyialed many of the risks and costs to hospitalsthai arise from cleaning and reslerilizing complex devices.

The highesl product temperatures reached in induslrial·scale gamma irra­diators is usually in the runge 30-40-C depending on source strength. Manydevices or components made from plastics defom) at thermal sterilization tem­peratures but are quite capable of wilhslanding the lernperalures reached ingamma irradiation. For reference. the Vical softening points (the maximumtemperature to which a polymeric component may be subjected without deform­ing) of some commercially available plastics suitable for medical applicationsare gjven in Table 2. As early as 1964 the range of medical prodUCIS being ster­ilized by exposure to gamma radiation extended to plastic disposable hypoder­mic syringes and needles. surgical blades. plastic and rubber catheters. drainagebags. blood lancets. cannulae. diaysis units. infusion sets. gauze and conon wooldressings. and surgeon's rubber gloves. Within the pharmaceutical industry thepharmacopoeias acknowledge gamma irradiation as a sterilization process appli­cable (0 drug substances and final dosage forms. bur its main application issterilization of heat-sensitive containers for a5Cplic filling processes. The

Table 2 Vic31 Softening Temperatures of RadiaLion-Sleriliznble Medical GroKkMolding Polymers

Polymer

LOPE

HOPE

Polypropylene

ABS

PVC

Brand name

Resin 722

290 81

Tenite

Cyeola< 0 H

Alpha3006R-81Clear

VicDI softeningManufacturer lemperature CC)

Dow Chemical. 90Mjdland. Michigan. U.S.A.Amoco ChemtcnJs Corp.. 125Ch)cago.lliinois. U.S.A.Eastman ())emteal 143Products, !(jog port,Tennc:sstt. U.S.A.Borg W3JTIer. Amsterdam. 971bc NetherlandsAlpha Chemtcal'. 7540 Parham Dri\·e.Easlleigh. Ham~hirc.5054NU. Englaoo

----

St~riliution by Gamma R.adiation 61

x 100 = 22.5%

various effects of gamma radiat'ion on specific phannaceuticals were reviewedby Jacobs in 1985 131. but few general conclusions could be drawn except thatsolid preparations are generally more stable than liquids, and frozen solutions aremore stable than liquids.

Theoretically, it is unlikely that aqueous phannaceutical fonnulations aregoing to be amenable to radiation sterilization. Consider the drug roni,idine(molecular weight of 314) in I% aqueous solution:

The number of drug molecules per gram molecule (314 g) is equal to 6 x1()2.l (Avogadro's number).

1berefore 100 g of solution would contain 1 g of ranitidine or approxi­mately 2 x 1021 molecules.

Therefore 1g of aqueous preparation would conlain 2 X 10 19 molecules.

On irradiation, the greater part of the energy would be deposiled in thewaler to fonn reactive species; essentially none of lhe energy would bedeposited in the drug.

The yield of reactive species, from water can be calculaled from the energyequivalent (l kGy is equal to 6.242 x lOI8 eV/g) and the radiation chemi­cal yield G (the mean number of elementary entities produced. destroyed,or changed per 100 eV) for water (equal to 3). For 25 kGy the yield ofreactive species from water is equal 10 4.5 x 1018 per g.

The proponion of molecules of ranitidine in 1% aqueous preparation thatwould be changed as 8 result of irradiation at 25 kGy is calculable on thebasis of a chemical change occuning on every encounter belween drugmolecule and reactive species, thus

4.5 x IOt8

2 x IOt9

On the olber hand, irradiation could lheorelically do less damage to thesame drug in its solid fonn, thus

I g of ranitidine contains 2 x 1021 molecules.

The electron volt (eV) equivalen. of 25 kGy is calculable from I kGybeing equivalentto 6.242 x 1018 eV/g. to equal 1.5 x 1()20 eV/g.

Each primary ionization requires 30 eV.

Therefore 25 kGy would induce (1.5 x 1020/30) or 5 x 1018 ionizationsper g.

62 Chapter 3

The proponion of molecules of ranitidine in solid foml changed as a resultof irradiation al 25 kGy is calculable on the basis of each primary ioni7..a­lion producing a chemical change. thus (5 x 1018n x 1021) x 100 = 0.25%

On the caulionary side it was noted mal it is usually not possible to predictthe radiation stability of pharmaceutically active substances from previousknowledge of other related substances, as minor differences in chemical struc~

ture can apparently have significant bearing on radiation stability and on thera­peutic activily.

Compared with all other methods of sterilization (hot or cold), gammaradiation has the overwhelming advantage of penetration through materials. Theadvantages of penetration ex.tend lhrough all aspects of medical products. frominitial design to (he final presentation of Ihe products in (heir Shipping packs.Nature abhors steriHty; a sterile environment. whether 3 microbiological culturemedium or a medical device. can only be maintained sterile if separated from thenonslerile world by barriers thai are impermeable to microorganisms. For tenni·nal sterilization. these barriers must be designed wilh lhe consideration that thesterilant muSI penetrate to all parts of the device that might come into contactwilh lhe patient either directly or indirectly through a delivered therapeuticagent This is a major constraint upon all industrial-scale sterilization procc:ssesexcept gamma irradiation. Some panicular situations where this is a decidedadV30lage are considered below.

(a) lnterna! cavities. Many devices have sealed internal cavities; forinstance. syringe plungers are usually fitted with elastomeric tips that sealwith an interference fil to the barrel wall at two or three diameters sepa­rated by one or two internal cavities. A similar situation applies wheremanufacturers only cap or seal the fluid paths of devices for which thesterility of the internal lumina (e.g.. infusion selS, catheters. etc.) needs tobe guaranteed. If sleam or ethylene oxide is the chosen method of steril­ization. special provi.sions must be made for complete replacement of airby the sterilanl from these internal cavities. This may frequently requirerecourse to methods and design considerations that introduce other com­promises and connicts. for instance. acceptance of higher sterilizationtemperatures than necessary or introduction of vented caps fined withmicrobial filters or tonuous paths.

(b) Selection of packaging materials. Penneable materials are necessaryfor unit containers subjected to ethylene oxide sterilization or steam ster­ilization (unless the conlenLS of the container are liquid). Paper is widelyused for devices and surgical products because of its permeability. How­ever, it is opaque. it lears easily on devices with sharp edges. and it is notalways impenneable to microorganisms. Alternative penneable materials.such as spun-bonded polyolefins (Tyvek) are very expensive. The effec-

Sterilization by Gammi RidiJtion 63

tiveness of penetration of gamma radiation has allowed the introduction ofnumerous nonpenneable polymeric packaging materials to the manufac­ture of unit containers: their advantages over traditional materials includetransparency, durability, and cost effectiveness. Funhennore, with gammairradiation, seals are not subjected to potentially deleterious effects ofmoisture and pressure changes seen in other methods of sterilization.

Another advantage of gamma radiation over other terminal sterilizationprocesses is its reliability. The lethality of gamma radiation is dependent solelyon dose. It is self-evident that conuoUing one parameter must be simpler thancontrol of any interaction of two or more parameters, and that processes that areeasily controlled are inherently more reliable than those that are nol.

1lle greatest disadvantage 10 gamma irradiation of plastic medical devicesand phannaceutical containers is the deleterious effects that radiation has onsome polymers that might otherwise be preferred materials of manufacture.When radiation excites or ionizes the atoms or subunits of polymeric mate,rials,the two principal effects are chain scission and cross-linking. Chain scissionrandomly ruptures the bonds that link the monomeric subunits of the macro­molecule. On its own it reduces chain lenglh and leads to gas evolution andunsaturation. When it is accompanied by cross-linking between the resultinglow molecular weight fragments, the end results are crystalline structures andthree-dimensional malrices within the polymer.

TIlese effecLS are dose dependent and differ from one polymer to anolheraccording to their molecular structure. Polymers containing aromatic groups aremore resistant to radiation-induced chain scission and cross-linking ilian purelyaliphatic molecules. However, predictability is further complicated by commer­cially available polymers being only very rarely pure macromolecules; usuallylhere are other compounds included in their formUlations, e.g.. antioxidants,light.stabilizers, plasticizers, and fillers. Some of lItese. included as processingaids, may exacerbate radiation damage; others may have lite opposite effect andimprove radiation slability or disguise its deleterious effects. Some grades ofpolymer are specifically claimed to be nsdiation stable. From an end-use pointof view, the mosl serious adverse effects of these interactions of radiation withpolymeric materials are discoloration and weakened mechanical propenies. I.nmedical devices markets, while and blue are perceived as "clean" colors, yellowand brown as "diny" colors; radiation-induced discoloration in susceptiblepolymers ranges from slight yellowing to complete opacity. The induction ofbrittleness and fragilily in products intended to have strength and resilience arethe most damaging effects of radialio" on mechanical properties.

1bese issues have not prevenled a whole array of polymers becomingavailable for the manufacture of medical devices sterilizable within the doseranges usually recommended (fable 3). The two principal poJymers thai have

Chapter 3

Table 3 Examples of Polymers Used for Irradiation-Sterilized Medical [)(vices

StabilityApplication up to Stability

Polymer examples Processing 25 kGy > 25 kGy Comments-

ABS· Closure- Injection Good To Rigid. mostAcrylonitrile plercmg Molding 1000 kGy often trans-Butadiene devices. lucent/opaqueStyrene roller clampsAcrylics Luer Injection Good To Clear with

connectors. Molding 1000 kGy slight yellow-injection- ness l1f~r

sites irradiationCellulosics Ventilation Various Good To Repeated

filters 200kGy irradiationembrillies

AuoropJaslics TUbing Extrusion -' PI FE unstable,to FEP stablea,25 kGy andhigher

Polywnides Auid Extrusion Good To May harden on(Nylon) filters 500 kGy irradialionPolyethylene PrOleC[Or Injection Good To Translucenli

caps molding 1000 kGy opaque. soft!waxy feel

Polypropylene Hypodermic Injection -' -' Embril1lessynnge... molding over lime

Polystyrene Syringe Injection Good To Rigidplungers molding 10.000 kGy

Polyvinyl Tubing, Injeclion _. Discolorschloride drip chambers molding

extrusionPAN· Hypodennic Injection Good To Rigid. clearPolystyrene synnges molding 5000kGyAcrylonitrile

·Specific to I pastic:ular formulation.

proved problematic with regard to radialion stability are polypropylene andpolyvinyl chloride (PVC).

(a) Polypropylene is in many respects an ideal material for manufacture ofmedic,at devices. It is biologically inert, naturally trans)ucenl. resilient. andnexible. From a manufaclUring point of view it can be processed by blow-

Sterilization by Gamrm Radiation 6S

molding or injection-molding, and molded parts exhibit only minor shrink­age and accepl print fairly readily.

Polypropylene is the most widely used material for the manufacture ofslerile disposable hypodennic syringes. However. the suitability of irradi­alion sterilization for polypropylene devices was until the 1980s severelyrestricted by yellowing and insidious embrinlernent after irradialion.

Radialion-induced discoloration of polypropylene is due mainly to theformation of colored radiolysis by-products from phenolic compoundsincluded as processing aids in commercially available formulations.Embrittlement is iniriated through chain scission bringing about the reac.·tion

R-R-w.R· + R

which continues in the presence of oxygen over time:

R·+02 ..... R02·

R02· + RH RooH + R·

Ro,· + R· RooR

R02· + Ro,· ..... RooR + OzR· + R· ..... R-R

In polypropylene Ibis process of autooxida.ion is long-lived. leadingeventually to severe embriuJemenl. which may render a ronnulation Ibalcould have appeared to be acceptable (when assessed immediately afterimldialion) totally unacceplable a few months later.

Bolb yellowing and embriulement of polypropylene are dose dependentbut are ignificant Sl or around 25 kGy (the customary compendial dose forsteriHzing medical devices). The extent of radiation-induced discolorationcan be relieved by use of phosphite slabilizers. Stability against embril­dement ma), be improved by adding antioxidanlS. but these may in tumcreate problems such as surface ·'blooms.··

An aJtemalive approach to improving radiation stability has been lhroughcoupling tbe desirable processing propenies of polypropylene with Iheradiation stability of polyelbylene to form copolymers (polyallome,,).Block polymers in which the chains comprise polymerized segmenls ofeacb of Ibe monomers are as amenable to processing as polypropylenewhile having beller clarity and radiation stability.

(b) PVC has a wide variety of applications and potential applications as amaterial of manufacture for medical devices and packaging films. It is

66 Chapter 3

clear and lranSparenl and may be nexible or rigid according to its plasti­cizer content. It is widely used in the fonn of extruded tubing for infusionsets. catheters, elC. Deleterious mechanical effeelS are nol generally seenat sterilizing doses of radiation. but special fonnulations are necessary ifsevere discoloration is to be avoided. Discoloration is initialed by radia·tion-induced free· radical formation. usually by scission of the carbon-chlo­rine bond nf the PVC:

-CH - CHCI --> CH, - CH· + CI·

The free-radical reacts with the PVC chain at a methylene hydrogen alomto fonn conjugated double bonds within the PVC chain:

-CH, - CHCI + CI· --> -<:H = CH- + HCI

The polyenes fonned in these reaclions cause discoloration through theirabsorption in the UV and visible speclra. Conventional formulations ofPVC become perceptibly yellow at 5 kGy and deep brown at 25 kGy.Radiation~stable formulations of PVC usually incorporate free radicalscavengers 10 compensale for the reaclivily but orten still require the inclu­sion of optical brighteners or blue tiOls to disguise the discoloration.

Finally, gamma irradiation sterilized producls are safe products. Gammaradiation leaves no residues. and al doses used for sterilizalion it does not havesufficient energy to induce radioactivity in malerials it traverses. Furthermore,gamma irradiation does nol pose a radioactive waste problem. The radioactivematerial is completely contained. and when sources become too weak for indus­trial purposes there are contractual agreemenl~ to ensure their return to theirsource of supply. where they may either be disposed of or resold for inslallationin research irradiators.

IV. INDUSTRIAL·SCALE COBALT-bO GAMMA IRRADIATIONSTERILIZATION

A dose of 25 kG)' (2.5 Mrad) is quoled in all of the major phannacopoeias assuitable for sterilizing medical prodUCIS. This dose is also quoted in the Guide 10

Good ManujaclUring Proclice jor Medical Devices alld Surgical Producls. 1981(4). This document emphasises the need for the process specification to beaccompanied by high sland3rds of GMP in lhe manufacture of lhe product priorto irradiation. The USP elaborates on the potential for 25 kGy to have damagingeffects on some products and is prepared to accepc properly validated lowerdoses that achieve SALs of 10-6.

From the very early days of irradiation sterilization, the major regulatorybodies ha\'c not demanded pharmacopoeial sterility resting of irradiated fmishedproducts. This has been an endorsement of Ihe effeclivencss and the levels of

Sterilization by Gamma Radiation 67

control achievable in industrial-scale irradiators. At the intemationallevel thereis vinually universal agreement regarding standards of plant layoul, constructionand operation; measurement and monitoring of dose; assignment of responsibil­ity; and other physical aspects of the technology. The whole !>osis of successfulindusnial-scale irradialion sterilization lies in me fact thaI its technologyimposes cenain obligalOry safety measures, interlocks. fail-safe devices, back-upsystems, ele., that in tum make it difficult for dose delivery lO be compromisedby either mechanical or human error.

A. Cobalt-60 Gamma Irradiators

J. Shielding: No two industrial-scale cobah-60 gamma irradiators are identi­cal, but all share certain similarities arising from having been designed around aneed to have products exposed 10 radiation while at the same time preventingescape of radiation to the outside world. 11 is quite usual for lhe "shielding" to bethe most slrik.ing fealure of gamma irradiation plants. In a typical plant (Fig. I),irradiation takes place within a building (the "ceU") which has its roof and wallsbuill from poured concrete 2 m lhkk. This allows safe use of source

Fig. 1 Simplified represenlation of a coball~60 gamma irradiator.

68 Chapter 3

strengths of up to 0,75 x 1017 to 1.5 x 10 17 becquerels of cobah-60 «(WO to threemillion curies lei]).

Such massive shielding is necessary because there is no definite range topenetration of gamma radiation through malter. For passage through any partic­ular material the number of gamma ray photons diminishes in an exponentialmanner until they become insignificant compared to environmental backgroundradialion. This attenuation of gamma radiation as a result of passage through anabsorbing material can be described by the equalioD

1,=/0-" (3.1)

where I, = the intensity of irradiation after passing through a barrier of thicknesst; '0 = the intensity of irradiation if the barrier had not been presen!: and u = aconstant, the linear absorption coefficient of the material comprising the barrier.

The linear absorption coefficient u is a funclion of the type of material andof the energy of the incident photons. Gamma radiation from cobalt-60 is morepenetrating than gamma rJdialion from caesium-137, and so different thick­nesses of the same material are required to obtain identical levels of shielding.Equation (3.1) is most commonly used to calculate the thickness required fromshielding materials to altenuate radiation to one half (the half value layer, HVL)or one tenth (the tenth value layer. TVL) of its intensity. Because anenu.njon isan ex.ponential function, the HVL and TVL are independent of the intensity ofradiation for which shielding is required. Some HVLs and TVLs for gammaradiation and typical shielding materials are shown in Table 4. According tothese figures, the." concrete walls 2 m thick of industrial-scale cobalt-60 gammairradiators should reduce the intensity of irradiation by a factor of about 10 10; thesame degree of attenuation should be obtainable with about 6 m of water or 360mm of lead.

Table 4 Approximate Half Value Layers (HVL) and Tenth Value Layers (TVL) ofShielding Materials Used in Large-Scale Gamma Irradiators

Shielding thickness (mm)

Caesium-I)7 Cobah-60

Shieldingmaterial HVL TVL HVL TVL

Lead 7 22 12 40Steel 16 53 21 70Conc~tc 48 157 62 200Water ISO 540 200 700

Sterilization by Gamma Rildiation

2. The Source: The world's largest producer of cobalt-60 is Nordion. fonnerlyAtomic Energy of Canada Ltd.• Kanata, Ontario. Canada. The starting materialis nickel-plated slugs of chemically pure coball-59 sealed into zircalloy capsulesusing a tungsten-inert gas welding process. These primary capsules are held in anuclear reactor as neutron absorbers for one year or longer in order to reach therequired radioactive slrength of cobalt·60. The radioactive capsules are thenremoved from the reactor and double-encapsulated into stainl~ss steel "pencils"aboul 11 mm in diameter by 450 mm long.

In most ordion irrndiators the pencils of cobalt-60 are mounted verticallyin a source rack. The configuration is formally described as lami.nar. TIle sourcemek is suspended from a hoisl mechanism on the roof of the cell such that it ismovable belween two locations. One of these localions is in the cenler of thecell in a position where producr can be moved around the source during il irra­diation. 1be other localion is below noor level. This is for safe storage of thesource when produci irradialion needs to be interrupted for loading or mainte­nance work within lhe cell.

Irradiators may be of "wet" storage or "dry" storage types. In Nordionplants the storage location for the source rack is rypically al the bottom of a poolof water 6 m deep sunk. in the cell noor. For dry storage, a pit is provided in thecell noor and the source rack is fixed to a Shielding plug that fills the mouth ofthe pit when the source rack has been lowered into the storage location. Drystorage phs may be Jess than 2 m deep with the plug amounting to more than halfof the depth. 1be majority of modem irradiators are of the wet storage type. buteach method has its own advantages and disadvantages. Geological conditionsmay favor dry storage in the event of the subslratum being unsuitable for deepexcavation. On the other hand. with wet storage the source ruck is visiblethrough lhe water. This makes it possible to inspect. manipulate, and if neces·sary correct problems ;n S;IU.

For source loadings in excess of about 2 x 1016 becquerels (500 kCi) bothtypes of stor.lge are likely to require cooling. A hear output of I kW can beexpected from 2.5 x 1016 becquerels of cobalt-60. For wei storage. cooling usu­ally consists of external circulatioo of \be pool waler through a chiller; for drystorage. dtere may be a need for cooling pipes to be incorporat'ed into the con­crete shielding around the pit. Wet Storage cooling water mU5l be muintainedion-free t'o prevent corrosion of the stainless steel pencils.

3. C(m,,~)'or$: There mUSI always be some mechanism within industrialgamma irradiators for moving product around the source and in and oUI of thecell. On the basis of the type of conveyor used. gamma irradiators may bedesigned for continuous or batch operalion.

Most modem irradialors are of the continuous Iype with open loop con­veyor systems that allow the prodUCI to be loaded and unloaded outside the cell

70 Chapter J

without interrupting the process. Entry and exit of me prodUCI is usually via adouble-walled concrete passage incorporating right angle bcnds-the maze.Each of the two walls of the maze is usually about half as thick as the roof andthe other walls, such that together they offer the same 81lenuation as other pansof the shielding. TIle right angle bends and the length of the maze prevent mdia­tion from escaping 10 the outside world. because radialion, like light. travels instraighllines. The dose delivered to the product is conlrolled via the speed of theconveyor; at slow speeds a panicular product would be exposed to the source fora longer time and therefore receive a higher dose of radiation IMn it would if theconveyor were moving the product al a faster speed.

BalCh irradiators are far rarer, conveyors are of lIle closed loop type whollyronlAined within the cell. Loading is done by pcorsonnel who enler the cell withthe source in its safe storage location. The source is then brought to its opera­tional position in the center of the cell and the conveyor is set in mcxion. movingthe product around the source. When sufficient time has elapsed to ensure thatthe product has absorbed its appropriate dose. Ihe source rack: is relurned 10 ilssafe localion. and personnel enter the cell to unload and reload the conveyor.

There are broadly two types of product handling conveyors used in cobalt­60 ganuna irradialors. conveyor bed designs and carrier designs.

In conveyor bed systems the product is packed inlo lole boxes, tennedirradiation containers. 1bese are supported from beneath by rollers or traysalong which the containers are transponed into lhe irradiator. past lhe source.and back oul again. Figure 2 shows lhe Nordion J5 8500 continuous-Iypegamma irradiator. In this sterilizer the irradiation container is an aluminum talebox approximately 900 mm high. 600 mm in length. and 400 mm in depth. The400 mm dimension separates the two sides of the tote. which alternalely face Ihcincident radial ion as the comaine.r is moved from one stretch of the conveyor 10

Ihe next.The conveyor has two vertical prodUCl levels and two passes on each side

of the source. The conl.a,jners are indexed through 63 positions by discharge ofeighl pneumatic cylinders (one at each end of each stretch of the conveyor).assisted by cross-transfer cylinders that move the product between parallelsl:I'e(ches and an elevalor th.u moves Lhe prodUC( one conlainer 3t a time from thebouom to the top level. Some operators of this type of irradiator have achievedan economy by sub tituting cardboard shippers for aluminum (Oles as irradiationcontainers. The economy is from the manning required for one loading opera·tion instead of I'WO loading and one unloading operations. The cost can be inincreased downtime if the purchased cardboard shippers do not meet the sameclose dimensional tolerances of the aluminum IOles. A small dimensional orgeometric irregularity magnified through a line of eight totes may 100 easilycause conveyor jams and increased downtime.

Sterilintion by G~mm~ Radiation

TOTE BOX IRRADIATOR

/~~~tl?A~C~CI!5S h~tchSOlJrce hoist "" o>:- Roof plug

n:::::::::~""'~ ••mecn~ni5f (3 pll!ces)

71

-.'l"l"'adi~ onroom

Sto"~gl! pool ,

Shipping container

ControlconsolI!

Equipment room(ail'" filter5, Compf'l!550r. deionizer,

chilll!r)

Fig. 2 Nordion J5 8500 continuous-type gamma irrodiator.

In carrier-type irradiators, product packages are moved around the sourcein aluminum carriers suspended from an overhead monorail. One such irradiatoris Nordion's JS 8900 (Fig. 3), which has a 3 m high carrier and can be buill withtwo. three, or four passes on each side. of the source. The source rnck is termedan "overlapping source" because it is 4 m high versus the 3 m carrier. Product isloaded outside the cell, one circuit of the track is completed, and then the carriersare unloaded and reloaded with new product for sterilization.

Some carrier-type irrndiators have shelved carriers taller than the sourcerack. These are called "overlapping product" irradiators. This design is compar­atively unusual in newer irradiators. Movement of the product from shelf toshelf within the carriers is needed to achieve reasonable levels of dose unifor­mity. Each product package is loaded onto the lowest shelf of a conminer: itmoves through the cell showing opposite faces to the incident radiation as itpasses down each stretch of the conveyor. On completion of a complete passageon the bottom shelf. the product box is transferred automalically to the secondshelf by means of an elevator and a pneumatic ram situated oUlSide the cell.

72 Ch.1.ptrr 3

fig. 3 Carrier irradiator with venical and horizontal load/unload positions.

Each product box makes four complete transits through the irradiatorsl.arting on the lowest and finishing on the highest shelf.

B. Control of Dose

The lethality of gamma radiation is dependent solely on onc paramcter­absorbed dose. In practice. absorbed dose is dependent upon the slrCnglh of thesource. the npparcnt density (bulk density) of the product packages being irradi­ated. and the time of exposure. There is no fundamental relationship betweensource strength and dose absorbed in medical products; an empirical relationshipmust be determined for panicular irradiators and products by means of mea·suremcnt of dose (dosimetry). This is the key work that must be done in vali­dating a particular irradiation sterilization process. II addresses the question ofhow long a panicular product should be exposed in a particular irradiation con·tainer 10 a source of a particular strength to have absorbed 3 specified minjmumdose in aU parts of the container.

J. Validol;oll by Dose·Mopp;ng: All variable conditions mUSI be specifiedduring validation and controlled in routine practice thereafter. A loading patternmust be established for each type of product This should specify the number.position. and orientation of product packages within lhe irradiation container.The distribution of density within the loading pattern should be chosen to be asnear uniform as possible in order to minimize dose variations.

The so-called bulk density of the irradiation container is an important con·lIol paromcter in irradiation. This is lhe gross weight of the loaded irradiation

Sterilization by Gamma Radiation 73

container divided by its volume. It is used as the main criterion for decidingwhether two product types may be irradiated one after the other in a continuous·type irradiator without overdosing one or underdosing the other. Since the vol­ume of !.he irradiation container is dictated by the dimensions of the conveyor.many operalors are content to monitor the weight of loaded containers for thispurpose.

Following !.he establishment of a loading pattern. a dose mapping exerciseshould be done. A loaded irradiation container should have a large number ofdosimeters distribuled to a defined panern lhroughoUi its volume. The containershould then be passed through the irradialor. noting the date. the time of e.po­sure to the source. and the speed of lravel of the conveyor. At all times when the"dose mapping conlainer" is in the irradiator, the other containers falling theconveyor should be of the same bulk densiry as the investigational material. h isDonnal to do dose mappings in lriplic31e containers to account for any unex­pected variation. 11 is a mailer of choice whether they are run adjacent to oneanother.

The purposes of dose mapping are

(a)To delermine dose uniformily. The !'alio of the highesl dose 10 the low­eSI dose (the max/min ratio) can be of importance to successful irradiationof materials that may be adversely affecled by high doses of radialion. It isimportanlthat lhe specified terilizing dose is understood to be a minimumdose. in olher words. product in the low dose zone should receive no lessthan lhe specified sterilization dose; all other parts of the container aretherefore overdosed in relation to this product and to the specified dose.

(b)To deleonine the low dose point or zone. This is me significant pointor zone in the container as far as sterilization is concerned. All pans of theproduct being sterilized must receive the specified sterilizing dose as 3

minimum. Routine dosimeters need not be placed in the low dose zone.indeed it may be wholly impractical, bUI me relationship of the routinedosimeter location to the low dose zone must be known.

(c)To detennine the appropriate conveyor speed (exposure time) for thespecified sterilizing dose. With some prior knowledge it is usually possi­ble for a dose mapping to serve the purpose of confirming a conveyorspeed predelermined to be appropriale to lbe specified S1erilizing dose.

Dose mapping validations are necessary for new products. new irradiation con­tainer loading pauems, altered conveyor configurations. and changes 10 thesource, including source replenishments.

2. Routine Dose Control: Routine dose conlTOl is straightforward as long asreliable validation-dose mapping infonnation is available. The conveyor speedthat assures a dose of x kOy on a panicular dale need only be adjusted to com-

, I

74 Ch~pter 3

pens3te (or source decay. and lhereafter the required dose will always be deliv­ered and received. CobaIt-60 decays exponentially wilh a half-life of 5.3 years:for practical purposes lhi means that the conveyor speed should be slowed downby 1% per month 10 ensure that a p3rtic.ular product continues to receive a speci­fied dose over lime.

Irradiation is a continuous process: therefore it is inevitable that prodUCl~

wilh different bulk densities will have t'O be adjacent to one another al some timeor Olher on lhe conveyor. TIle conveyor speed to achieve a specified sterilizingdose differs between products of differing bulk: densilies. 'The greater thedensily. the slower the speed required to achieve a specific dose. Within lheconfines of some acceptable level of overdosing this siluation is manage,able.Every attempt must be made to minimize the density differences of producls thatmust be run adjacent to one another. and any errors must be toward overdosingrather than underdosing.

Although the controlling factor in industrial irradiation is the conveyorspeed. it is usual to supplement this by inclusion of dosimeters in the irradiator.The dosimeters are there as a confirmatory monitor that all factors remain undercontrol. Routine dosimelers are not used as a coolIol or a feedback system.

J. Do!imt!/ry: Dose delivery in industrial·scale cobah-60 gamma irradi.tlors isachieved lhroogh controlling me time of exposure of the product to the source.The initial relationship between source strength. product bulk densilY. time ofexposure. and absorbed dose has to be determined empirically by dosimetry.

Continued monitoring of dose as it might vary with adjustments to the timeof exposure in response to source decay Of product changes must also be doneroutinely using dosimeters.

DosimelrY is the measurement of absorbed dose. The unit of absorbeddose is the grJy (Oy). Because dose is a measure of absorbed energy. calorime·Lry is the fundamental method of measurement. However, calorimelry suffersfrom being insensitive. complex, slow and highly demanding in technical skillsand experience. Primary dose measurement is usually done with subslances thatare chemically changed quantitatively in response to the amount of radiationabsorbed. For most purposes the standard primary system is me Fricke or fer­rous sulfate dosimeter. In this system, which consists of a solution of ferroussulfate in dilute sulfuric acid. ferrous ions FeH are oxidjud by absorbtion ofradiation to ferric ions Fe+++. Fricke dosimeters lite usually presented in glassampoules: the yield of ferric ions is measured by UV spectrophotometry at 340om. They respond linearly to absorbed dose over the range 10 to 500 Gy. areaccurate to about 1%. and show no dose rale dependence. Fricke dosimelers donoI measure doses in Ihe ranges seen in industrial-scale irradialors. 1lley are,however, widely used for calibrating the radiation fields in national and interna­lional standards laboratories. which are in tum used to c:alibrale the types ofdosimetry systems used in industrial-scale irradiators.

Sterilization by Gamma Radiation 75

The types of dosimeter used most often for dose mapping and routine dosemonitoring are referred 10 as secondary systems. This is because their responseto radiation cannol be predicted from theoretical considernlions. Instead, theirresponse has to be calibrated from batch 10 batch and from rime 10 lime againsI3calibrated "standard" radiation field. The mOSI commonly used types are madeor dyed acrylic (mainly Harwell 4034 dosimeters). The overwhelming advan­Iage or Ibis type or dosimeter is its robUSlness. Table 5 highlights the differencesbetween conditions in standards laboratories and those in industrial-scaleirradiators. h is more imponant for dosimeters in routine use to give consistent~ uhs under variable conditions than 10 be highly accurate but environmentallysensitive and operalor dependent. Harwell 4034 dyed acrylic dosi.melers readout in the visible spectra al 640 nm; their range is 5 to 50 kGy. and their accu­racy is around 5 to 10%.

It should be appreciated that lhere is a considerable difference between thedose I'3Dges 011 which radialion fields are calibrated against Fricke dosimeters andlbe dose ranges for which the same fields are used to calibrate secondarydosimeters. These differences should be of no importance as long as neither ofthe dosimetry systems is dose rate dependent However. it is not uncommon (0

find dosimeters thai respond theoretically and quantitatively over wider r.mgesof dose being used in the commissioning of new irradiators and lO supplementsecondary dosimeter dose mapping after source repleniShments. These aresometimes called transfer dosimeters because they may (theoretically but nOIusually practically) be used 10 calibrate radiation fields and also to monitorindustrial-scale irradi:HofS. Ceric-cerous (10 to 50 kGy) and poIas ium dichro·male (Ito 100 kGy) dosimeters (bolh accurale 10 about 4%) are lhe most com­monly used transfer dosimeters. Both. however. are liquid systems; readout canbe potentiometric or by UV spectrophotometry. and a fair degree of skill isrequired in their use and handling. A solid-slate system, alanine. which is accu-

Table 5 Comparison of Radiation Conditions Be1'A'cc:nS~Sources and IndustriaJ Irradiaton

Foetor

Dose raleTemperatureSpatialdistribution

HumidilySlOrageofdosimel'crs beforeand after irradiation

ConstantConstanlDefined

ConstantConstant

Industrialirradiators

VariableVariableDependaot uponproduclloading

VariableMay be variable

76 Chapter J

rale over the wider range of I Gy to 100 kGy. has been quoted as having a lot ofpromise (5]. The melhod of readout.. eleclrOn spin resonance spectrophotometry.is neither straightforward nor cheap.

V. THE CHOICE OF DOSE

The question is. what is an appropriate sterilizing dose of radial ion? Clearly.once a dose has been decided upon, the same general principles govern its deliv­ery and its control irrespective of whether that dose is 10 kGy. 25 kGy or what~

ever. Are all products sterile if exposed [025 kGy? Are there other more appro­priate doses to achieve specified SALs? The answers [0 these questions are byno means simple. The underlying philosophies were reviewed in depth by Tal­lentire in 1983 (61. As wil.h most other sterilization technologies, the majorschools of thought have split between process specifications and validated SALs.Since ganuna irradiation is a fairly modem technology it is possible to (race theorigins and the reasons behind the various options.

A. The 25 kGy uSlandard" Dose

1lle first published reference to 25 L:Gy as an 3ppropri3te irradiation sterilizationdose came from Artandi and Van Winkle in 1959 [7]. II did nol come in thefonn of a general recommendation. It arose from technical considerations sur­rounding accelerated electron sterilization of catgut. These workers determinedthat catgut sutures could be irradiated to 50 kGy before showing properties infe·rior to those of heal-sterilized SUlUres. They also "established the minimumkilling dose for over 150 species of microorganisms," and chose 25 kGy as Lheirsterilizing dose because it was 40% above that necessary to kill the most resistantmicroorganism and well short of that which would damage the product. Justwhat they meant by a "minimum killing dose," was nOI explained within thetechnical content of the paper, but clearly the exponenlial nature of microbialinactivation was not taken into consideration.

By 1965, 25 kGy was quoted [8] as being the accepted dose of radiationfor sierilizing disposable medical equipment in most countries excepl Denmark,where 45 kGy was being claimed to be necessary. As a consequence there was aflurry of scientific activity to validale Ihe choice of 25 kGy relrospectively.Much of this work wali done in laboratory studies with the mOSI radialion-re.ljis­tant microorganism then known, the spore of Bacillus pumi/lis E601 and cen­tered on the detennination of inactivation factors. An inactivation factor is thenumber of decimal reduction values (DJO-values) delivered by a panicular dose.For these spores irradiat'ed in air the D IO was consistently found to be 1.7 kG)',hence the inactivalion factor was 15. This implies thaI an SAL of beller than10-6 can be achieved for items contaminated wilh up to 109 spores of thesemicroorganisms per item. and by inference for higher bioburdens of less radia-

Steri'iulion by Camm, Radi.tion 77

tion·resislant microorganisms. However, under different environmental condi­tions microorganisms respond differenlly to radiation. Under anoxic (absence ofoxygen) condilions of irradiation the D IO for spores of Bacil/us pumi/uf E601was consistently found 10 be 3.4 kGy. the inactivation factor for 25 kOy wastherefore about 7. and the maximum bioburden per item that would suppon a10-6 SAL was only aboul 10. Since anoxia is quite improbable in praclicalsituations il was felt that these studies supported the view of 25 kGy as being asatisfactory "standard" sterilizing dose. Most regulatory agencies outside of theU.S.A.•till prder the "standard" 25 kGy .laDdard dose with 'wo provisos:

(a) It can only be used in conjunction with implementation of GM.P (goodmanufacturing practices) in order to achieve a low bioburden prior lo.ster­ilizaHon.

(b)Lower doses may may be acceptable if there is technical need. e.g., withdose-dependenl delelerious effects on malerials, and if mere is evidence tovalidate the lower dose.

Somewhere around me confidence in a '·sWldard" dose are the practicali­lies of it being simple and easy to administer by regulalory aUlhorilies. and of ilbeing easy to justify by medical device manufacturers who may nol be prepared10 commit to the levels of microbiological effon required 10 val.idatc other doses.

B. Irradiation in Scandinavia

In the 1960s the Danish authorities held that there should be a common standardof acceptance for all sterile prodUC.IS irrespective of melhod of slerilization.1bey were not initially basing lhis on a SAL of 10-6 but on an inactivalion faclOrof 8. because this was their standard for thennal inactivaJion processes. Fur­lhennore. they maintained that lhe microorganism to which the inactivationfactor referred should be the most resistant type known. presented to the steril­ization process under worst-case conditions. For irradiation, Ibe referencemicroorganism was chosen 10 be a stnlin of Slr~pl{)coccusJa~cium A2 with ashouldered inactivation c,urve irradiated in dried serum broth. The standard dosewas set al 45 kGy.

1be situation in Scandinavia eased in the 19705. 1be Nordic Pharma­cOfKWio introduced the 10'. SAL, and .taodard sterilizing doses were modifiedto take account of the average bioburden per item prior to steriJization. 1beminimum terilizing dose was SCt al 32 kGy.

C. Irradiation in North America

Although a11 major pharmacopoeias now cite the 10-6 SAL for aU methods ofsterilization. the USP goes funher in its recommendalions concerning irradiationthan its European counterpart. Ao approach to validalion of lhe 10-6 SAL i.

78 Chapter 3

indicated. vi:llhc Association for the Advancement of MedicallnstrumenuLion's(MMI) Process Control Guidtlints for Gamma Radiation Sltrili:al;on ofMtdical Devias. /984 (9). Several methods are described here but only tWO arebeing much used. Th~ two methods share the same philosophy, thai the mostrelevant approach to validation of a sterilization process is to consider theresponse to treatment (in this case irndiation) of the innate bioburden in situ.Arguments about the choice of reference microorganisms are thus avoided, asalso are arguments about t.he relevance of labor:nory "worst case" conditions 10

practical situations. The two methods are contained in Appendix.es BI and 82 10

the Process Control C"idtlines.

J. The AAM/B/ Me/hod: The AAMI Bl melhod is based on inactivation of ahypothetical mixed culture of microorganisms. This hypothetical mixed culturecontains microorganisms of various 010 values present in different defined fre·quencies; lhis is referred to as the "standard but arbitrary" distribution of rddi·ation responses. Inactivation of populations of variou different initial numbers,but always conforming to the -standard bur arbitrary" distribution of radiationresponses. was simulllled by computer. From the simulation :1 series of tableswas derived relating initial number of contaminants 10 dose and SAL.

Practically, me method first requires an estimate of average numbers ofmicroorganisms on items. The contaminant population is assumed to respond toradiation in the same way as the "standard bUi arbitrary" distribution.

By reference 10 the lables in Appendix B I of the GJI;delifl~s. doses of rJdi·alion appropriate to SALs of 1<r2 and 10-6 can be determined. The second prac·tical consideration is to test the hypothesis by irradiating one hundred items atthe tabulated 10.2 dose. Unless the actual distribution of radiation sensitivilies ofthe microorganisms on the items is more resistant than the "standard bUI arbi·trary" distribution. then all one hundred items will be slerile when tested. Thislben supports the 13hulaled dose required (0 achieve a 10-6 SAL.

The method relies on four assumptions:

(a)The validity of the computer simulation. This is based on exponentialinactivation, and on each component of a mixed culture responding inde·penden~yof the o,he".

(b)The validity of the "standard but arbiuary" distribution of radiationresponses. 1l1e data used to develop this distribulion was taken from verylimited sludies. onelheless. the items from which the microorganismswere isolated were flI'St gi en "screening" doses of radiation 10 eliminatevery sensitive types. It is biased toward greater radiation resi tance thanone might be likely to encounter excepl in very unusual situations (forinstance dried in serum broth).

Sterilization by Cdimma Radiation 79

SAL

IIDOSE WINDOW

I'----".••••• •, , , ,

, ,". EXTRAPOlATION BASED

•,••..• ON 0 10 CALCULATED moM

", DOSEWINDOW•,

• , , ,, ,

•,

..'0

, , ,,,,

,o'bFFPDOSE DOSE------ .

10.2 DOSE + IEXTRAPOlATED DOSE

• PROCESS DOSE

Fig. 4 CoordinateS of the AAMI 82 dose selling method.

(c)The validity of the eSlim.,e of bioburden. This oughl'o be reliable, butin faCI the melhod has a conservative bias because an overly low eslimaleof bioburden would give a commensurately low tabulated 10.2 dose andlead 10 failure in lhe irradiation trial.

(d) The validity of Lhe mcLhod of testing the ilems for sierility after theirradiation trial. Adequate controls muSI be considered.

2. Th' AAMI 82 M"hod: The AAMI 82 me,hod assumes far less Iban lheAAMI 8) mcthod. It assumes cxponential inactivation. II does nol assume any"standard bUI arbitrary" distribution and h does not require estimation of biobur­den. II does, howcver. continue 10 require well controlled sterility testing melh-

, I

80 cmpterl

ods, and it requires more elaborate experimental irradiation equipment lhan theBJ melhod.

The 82 method makes use of a series of nine doses of radiation that areonly fractions of the probable process dose (sub-process doses) to determine twOsets of coordinates of a functional relationship that describes inactivation ofinnate contaminants on product items. The first set of coordinates is for the dosethai represents an average of three microorgani ms per Hem. This (called theFFP or first fraction JX>Sitive) is lhe lowest dose at which at least one item in aset of (Wenty is sterile. The second set of coordinates is for the dose at which a1(T2 SAL is achieved.

'The region between the two sets of coordinates is called the "dose win­dow" (Fig. 4). 1be response 10 radiation within the dose window is assumed tobe: exponential, and therefore a 010 can be calculated. 'The dose required toachieve an SAL of 10"-6 is then derived from the 10.2 dose plus as many calcu·lated D IO vaJues as are required to achieve the target The 82 method lhereforerecognizes thal there may be two componenlS 10 radiation sensitivities of a con­laminant population: a sensitive ponioo that is addressed lhrough determinationof the aclual 10:2 SAL dose and a more resistant "tail" population than isaccounted for by extrapolation.

REFERENCES

I. Dunn. C. G.. Campbell. W. L. From. H. and Hutchins. A. (l9-a8). Biological andpholochtmical effects of high energy elCCU'OStalica.Uy produced roentgen rays andcathode rays.. Journol ojAppli,d Physics 19: 605-616.

2. TaHen1i~. A. (1980). The spectrum of microbial radiation sensitivity. RadiationPhysics and Ch~nlis"y 15: 83-89.

3. Jacobs. G. P. (l985). A review: Radiation slerilizalion of phannaceuticuls. Radio­tioll Pllysics a"d Ch('mistr)' 26: 133.

4. Depanmenl of Heallh and Social Security (1981). Guid(' 10 Good ManufacturingPracti("t for MtdicallH"icts and Surgical Producu, 1981. London: Her Majesty'sSt.:l.liontry Office.

S. Miller. A.. Chadwick. K. H. and am, J. W. (1983). Dose assurance in radiationproces ing planls. RmJialion Physics and Ch~minry21: 31-40.

6. TaUcnlire, A. (19 3). Philosophies underlying sterility assurance of radiation-trealedproducts. M,dical De"ius and DiagnosliC's Industry S: 36-42.

7. Attandi. C.. and Van Winkle. W. (1959). Electron beam mrilization of surgicalSUIUres. Nud~onia 17: 86-90.

8. Ley. F. S.• and Tallenti~. A. (1965). Radiation sterilisation-The choice of dose.Pharmoceldical Joumal19S: 216-218.

9. Association for lhe Ad\'ancemenl of Medicallnslrumenlalion (1984). Procfss CO"·trQI Cuid~JiIll!S for Camma Radiation Sftrili::/JI;on of Medical Deviur. AAMI:Arlington. Virginia. U.S.A.

4Sterilization by Saturated Steam

I. Heat TransferU. Effects of Steam Under Pressure on Microbial Populations

Ill. Applications of Steam SterilizationA. Aqueous LiquidsB. Nonporous SolidsC. Porous Solids

IV. Aut'ocl:1ves and AUlodave CyclesA. Downward-Displacement AutoclavesB. High-Vac.uum AutoclavesC. 51'earn Quality and Superheating

V. Tcmpcratureffime Criteria for SterilizationVI. Validation and Control of Steam Sterilization

A. ValidationB. Routine ControlC. Some Autoclave Problems

8384878787888888899999

102102104105

Sterilization by saturated steam under pressure is the classical lime-proven andmost economical method of inactivation of microorganisms. When a require­menl for sterility arises for a new type of medical device or pharmaceuticalprodUCl, steam should always be given first consideration. This is because.

81

82 Chapter 4

importantly. steam of the correct quality leaves no residues. Regrettably. it isunsuitable for heat·sensitive and moisture-sensitive products and packagingmaterials.

For the most part, steam raises the temperatures of objects that come intoits contact by condensing on the cooler surfaces of the objects. In this way itloses latent heat to the object. This effect when applied to microorganismsresults in death through coagulation of cellular proteins. This biochemical effectbegins to be significant af moist heal temperatures above about 80·C and pro­ceeds rapidly wilhin the normal processing range of about llO-C to 140°C. Thisis quite different from the mechanism of inaclivation of microorganisms by dryheat. which is mainly oxidative; as a consequence, I.he temperature rangesrequired by the (wo processes are quite dislincl. Far higher lemper.llures arerequired from dry heat sterilization to achieve the same lethalilies as thoseobtained with steam sterilization.

M.icroorganisms differ in their respon..~ to high temperatures. The innateor inherent resistance 10 thermal inac.tivation of baclerial endospores is consider·ably higher than iliat of vegetative microorganisms. There are also considerablespecies-to-species differences within the spore-forming genera themselves.Much of the eJliperimental work on thermal inactivation of microorganisms haslherefore been done on lhe more heat·resislant spores because the)' have mea­sureable variations in their responses to heat as well as being Iroublesomeorganisms with respect to their potential to survive heal processing. Althoughthere are some distinct differences, the overall enzyme complements of heat­resistanl spores and vegetative cells are broadly similar. This implies Ihat theremust be some faclors within lhe spore to stabilize. protect, or repair essentialheat-labile proteins that would not survive high temperatures jf present in thevegetative cell. These factors are complex and subtle. but they clearly involvelow waler conlent. the presence of chelated metal ions. panicularly Ca++. Ihepresence of dipicolinic acid, and enzymatic repair activity.

TIle first application of Sleam under pressure 10 microbial inactivation isusually attributed to Appert's food canning process. The principle of Ihatmethod. namely the displacement of air by Sleam within a pressure vessel.usually lenned an autoclave (or a reton in the canning indu51f)'). has remainedsubstantially the same for over 100 years. 1lle classical autoclave was sel tooperate al an internal pressure of 15 psig (approx.imately I aim above normalatmospheric pressure), affording a temperature of 121·C. There is nothing par­ticularly significant about the pressure of I bar or the temperature of 121·C otherthan the availability of technology that can achie'ie these condilions and a con­servative tendency to standardize thermal inaclivation to conditions of knowneffectiveness. Modem autoclaves may be specified and constructed to operaleover a range of internal pressures, providing different temperatures determined

Sterilization by Saturated Steam 83

.5,.-----------------------,

.0

25

wCJ~20-~ 15

10

5

oLL. ..l...-__--' ...J.... l......__-l.J110 115 tOO 125 130 ,.5

TEMPERATURE ("C)

Fig. 1 Temperature/pressure equilibrium for steam.

by the temperature/pressure equilibria for steam (Fig. I). Small-scale and large­scale technologies are available.

I. HEAT TRANSFER

The effectiveness of thennal inactivation rests wilh heat transfer from the steamto the microorganisms. Provided thai there is a temperature difference betweentwo pan.o; of a system. heat flows from one part 10 the other by one, two or allthree of three mechanisms:

(a) Conduction. In this mechanism heal is transferred from one substanceto another by vibrational energy of atoms or molecules. There is no mi;ll;,­ing of the two substances. Transfer of heat from an autoclave through thewalls of a container or packaging material occurs by conduction. Forliquids. funher heat transfer by conduction is minimal.

(b) Convection. Convection only occurs in fluids. Heat transfer is bywann fluids mixing with cooler Ouids. This mechanism is of significantimportance to sterili7.ation of fluid loads in autoclaves.

84 Chapter 4

(c) Radiation. Radiant heat energy moves through space by means ofelectromagnetic waves. If radiant energy comes into contact wilh anobject. heat is absorbed by the object or conducred through it. Radiant healdoes not make a significant contribution to heat transfer in autoclaves, butit is important to dry heat sterilization (see Chapter 5).

Heal penetration into items being sterilized by saturated steam begins wilh lheoutside of each item (consider me items to be fluid containers) having a layer ofcondensed stearn adhering (0 it Transfer of heat is by conduction from thes(eam 10 the condensate, to the walls of the conlainer. and on into the fluid. Eachstationary boundary layer presents its own resistance to heal penetration. Thisresistance can be minimized by technologies lhat improve heat traMfc,r, such asmovement of the fluid within the containers or turbulence within the autoclave.Liquids have low thennal conductivities, but convection currents caused by localtemperature gradients lead to continuous movements within the fluid, thusreducing Ihe thermal resistance of the innennost boundary layer of the syslem. Itis not usual to find sterilizer loads being agitated exccpt in lhe case of rolal)'washer/aUloclaves. It is quile usual on the other hand to find lurbulence withinthe steam in the autoclave being achieved by fans or recirculation.

II. EFFECTS OF STEAM UNDER PRESSURE ON MICROBIALPOPULATIONS

Thennal damage to biological syslems is caused by absorption of heal energy.Well-controlled laboratory studies show that when pure cultures of microorgan­isms are held in saturated steam al a constant sterilizing temperature lhere arelinear relationships betwecn the logarithm of the number of survivors and thetime of exposure (eltponential inactivation).

Figure 2 illustrates the terminology used for inactivation of microorgan­isms by saturated Sleam. The thennal resistances of particular microorganismsare expressed via the slopes of the expontnlial relationship. The D-value is thelime in minutes (DT) at a temperalure T required to reduce the number of sur­vivors by 90%. This relationship is described by an equation lhm is directlyrelated '0 Eq. (2.3) in Chapter 2:

log Nt =-I

o + log NO (4. I)

where No = number of microorganisms prior to treatment, N, = number ofmicroorganisms surviving afler time 1 expressed in minutes. and D = the D-valueexpressed in minutes.

It should be borne in mind that Dr-values relale specifically to lhe condi­lions under which they were determined. The immediate environmenl or sub-

Steriliution by Saturated Steam

6

2 4 8 8 '0

HOLDING TIME (mIn)

AT TEMPERA11JAE T

Fig. 2 D-values.

85

strate on which micrOOfganisms are healed can have a significant effect on themicroorganism's beat inactivation characterislics. There is a wealth of researcbdata on this topic. for instance in connection with the physiological state of themicroorganisms. in connection with pH. and in connection with the ionic com~

position of the environment. Some generalizations may be made. for instancethermal death rates are lowest at neutral pHs; proteinaceous materials and facsand oils are protective against heat inactivation. Nonetheless. in practical sleril­ization situations this type of infonnalion can only be supplementary 10 empiri­cal data determined for particular products. packaging components. elc. Table Ishows differences in D~values that can arise between slX>res of three differentspecies of Bacillus on various substrates that might be associated with medical orpharmaceuticaJ products.

The eXlX>nentiaJ relationship belween numbers of surviving microorgan­isms and time of exposure at a particular temperature pardllels first-order chemi­caJ kinetics. In these circumsl3Jlces it should follow that equivalent lethalities atdifferent temperatures of exposure should be predictable. When D-values havebeen determined for pure cultures of the same microorganism at different tem~

peratures (Fig. 3), it has been shown that there is a linear relationship betweenthe logarithm of the D~values and the temperatures at which they were deter­mined (T). T2> T3• etc). 1be teon z is given to the slope of this line; l. is thenumber of degrees of temperature change necessary to aher the value of D by a

86 Chapler 4

Table 1 Some DIll"Values Versus Saturated Steam of Bacterial Endospores MoulI1cdin or on Various Substrates

Dill-value (min)

Spore Distilled water Glass Rubber

B. 51earolhennophUus ATCC 7953 1.99 1.32 1.84B. coagu/ans 0.99 0.80B. subri/is 0.61 0.56

Steel

1.47

(4.2)

factor of 10. A z-vaJue of lO·C is widely quoted for bacteria] spores and iscommonly used as a constant in computations of [hennal lethality. An alterna­tive to the z-value. most often quoted in academic texts, is the temperature coef­ficient or QIO-value. This is defined as the change in rate of reaction broughtabout by a change in temperature of IO-C

Q_ Dr + 1O

10-Dr

where DT = D·vaJue detennined al rc and, DT+ 10 = D-value detennined al Tplus woe.

10

LOG OFD·VALUE

(mins)

1

0.1

120

,!l-- --_., z

130

Fig. 3 z-Values.

TEMPERATURE ('C)

Simliution by Sllurlted Stelm 87

QIO-values in the range of 5 to 20 have been found for bacterial sporesagainst saturated steam sterilizing temperatures. These ralher high temperaturecoefficients show that for steam sterilization. a discrepancy of only I or 2-Cfrom the specified temperature may have significant effeclS on the processlelhality. For inounce, a deficiency of 2 to 3·e could easily require a doublingof abe exposure lime to achieve the same lethalily as the specified proce s.

A D-value accompanied by the temperature T at which il was delcnninedand its z-vaJue provide a complete definition of the heat inactiv31ion characlerls­tics D; {or any panicular microorganism.

III. APPLICATIONS OF STEAM STERILIZATION

For an item to be suitable for steam sterilization it must be sufficiently beat-sla­ble 10 with tand process temperatures. Funher to this. it cannot be allowed 1,0 besusceptible to moisture damage. because sterilizing conditions will nol beachieved in regions of the product thai are not pemleated by steam. Subject wthese two constraints, produclS that are sterilizable by saturated steam fall iRlothree categories. (a) aqueous liquids, (b) nonporous solids. and (e) porous solids.

A. Aqueous Uquids

There are several types of sterile aqueous pharmaceutical pnxlucts-single-doseand multi~seophthalmics, and small~volume and large-volume multi-dose andsingle-dose parenlerals. From necessity, all of these types of prodUCI are filledinto hermetically sealed nonporous containers. Those prodUCIS thaI are termi­nally sterilized by saturated steam may be contained within glass ampoules. glassvials. glass syringes. glass bottles. or flexible film infusion bags. There is nonece ity for steam to penetrate into lhese containers nor come imo contael withthe prodUCI. As IODg as \he sterilizing lemperature is obtained, the water contentof the product itself will be suffICient to ensure thaI microbial inactivation is dueto moist heal protein coagulation mechanisms.

B. Nonporous Solids

Most solid dosage forms of parenteral phannaceulicaJs are unsuitable for lenni­nal steam sterilization. Many medical devices are nonporous solids, for in tancescalpel blades and rubber CalhelCfS. and may. panicularly in hospil3l.scaJe oper·ations, be steam Slerilized. Moisl heat is usually the method of choice in labo­ratories and in induslrial aseptic filling facilities for instruments and machinepans that are required to be sterile. This may apply even 10 equipment that iswell able to withstand dry heat sterilization temperalures. for th~ sake of reduc­ing Lhc lime involved in processing. In all cases il is critical that the items 10 besterilized be hermelically sealed into conlainers Ihal are capable of serving IWOfunctions. to allow steam to penetrale and come into contact with all pans of the

88 Chapter 4

product. and to provide effective barriers to microbial ingress while still intact.Papers made to specifications appropriate for steam sterilization are available forwrapping items and as made-up bags.

Rubber closures for vials or olher containers being filled aseptically on anindustrial scale are nonporous but share some of lhe problems of porous loads.They are most frequenlly steam steriJized by passage through double-endedrotary washer autoclaves. which by agilalion of the product ensure effectivesteam penetration to even lhe potentially occluded pans of the closures. Steamcomes into direct contact with these bulk items; they are not usually wrapped norpacked into hermetically sealed containers. Special precautions must be takenfor unloading to avoid compromising their sterility. Static autoclaves may alsobe used for prewashed versions of lhese Iypes of components. They should thenbe loaded inlo me 8U1oclave in shallow layers in perforated trays or boxes.

C. Porous Solids

Slerile dressings, filters. and cellulosic materials in gener•.d are porous materials.It is generally problematic to ensure sleam contacl with all parts of porous mate·rials because of entrapped air. Sterilizing temperatures will not be achieved inthe presence of air. The mechanism of microbial inaclivation in the presence ofair will be that of dry heat. whkh is mucb slower lhan that of saturated steam.Avoidance of entrapped air is addressed through specific porous·lo.1d sleriliza·lion cycles ramer than lhrough any special packing or loading provision olherIhan those constraining the sterilization of omer solid products.

IV. AUTOCLAVES AND AUTOCLAVE CYCLES

The simplest autoclave is the domestic pressure c.ooker. Steam is generatedwithin the pressure cooker. Air is alJowed to discharge through the vent until"pure" steam only is seen 10 be emerging. The vent is sealed by a weigbtedvalve. Pressure and temperature increase within the pressure cooker until theweighted valve begins to lift. The heat is turned down and the whole setup is leftto simmer at the lemperalure dictated by lhe pressure within the cooker. All lab·oratory and production aUloclaves. and washer/auloclaves, branch from thisstem. There are five specific cycle stages seen for lhe domestic pressure cookerthat must be addressed in the technology of all autoclaves: (3) air removal. (b)heat·up. (e) hold lime. (d) drying. and (e) cooling.

A. Downward-Displacement Autoclaves

Many aUioclaves will be of the downward-displacement (gravity-displacement)type. They operate on the principle of air being more dense than steam. As

Sterilization by saturl.ted Steam 89

sleam is admitted to lhe sterilizer. air is displaced downward and out of the ster­ilizer via a drain at the bottom.

Figure 4 represents a longitudinal section through a downward.displace·ment autoclave nnd its associated pipework. II consists of an inner steel chambersurrounded by a stearn jacket Jackets are optional. Good insulation is an aller­native. The obje<:tive of jacketing andlor olller forms of insulation is to speed upthe cycJe by minimizing the need to heat up lIle mass of metal of the vessel itself.to pre\'ent condensation fooning on lIle vessel's internal walls. and to help speedup drying of the load at the end of the cycle.

Sleam i upplied at high PIOSSUIO from a steam generato' to !he jackel anddirectly to the rear of me chamber via 3 reducing valve. Tbe incoming learn hits3 baffle placed to prevent wetting of the load. Because it is hotter and lighlerthan air. the learn RlO\'eS immediately upward and stratifies above lbe air in lhechamber. With increasing pressure me steam forces me air outthmugh the drainat the bonom of the chamber. A temperature sensor is stralegically located al lhecoole t poim in the drain line. The exposure period should be timed from whenthe equilibrium lemper.llure that is correct (or the pressure lit which the auto·clave has been set to operat'e is achieved in lIle drain line. It is quite usual to findmodem autoclaves equipped with "floating" temperature sensors that can belocal'cd in the chamber or in lhe load itself. Nonetheless it is the drain sensorttmf is normnlly slowesl to reach the set sterilizing temperature and is thereforecril:ical for control purposes.

Modulation of lite supply of sleam to me chamber during heat-up andexposure may be achieved by thermostatic feedback from the drain line orlhrough pressure transducers in Ihe chamber.

B. Hish-Vacuum Autoclaves

The main factor accounting for lack of reliable 3.uainmenl of sterility in auto­claves has been entrapped air acting as a barrier to direct contact between stearnand product. Thus the main thrust for technological advances in steam steriliz.o.·tion has been concerned with improved effectiveness of removal of air from thechamber and ia:s contents. Advanced high vacuum autoclaves have evolved fromdownward-di placement types by the addition of condensors. vacuum pumps.and ejector s)' terns to assist in air removal.

1be first stage in a high-vacuum cycle is to evacuate the loaded chamber.Some cycles may require a single evacuation. oLbers may require a pulsing cycleof evacuation followed by steam injection repeated several dOles. The nature ofthe load dictates the cycle. Evacuation may be controlled through a pressureswitch or transducer or simply by a limer governing pump running time. II isusual to find a condensor positioned belween lbe chamber and the pump for pur­poses of pump pro,cction.

a

90 Chapter 4

~• •HB..

'r1' •

! ~ •~ ~

! •,• !• •; • ~

• "03 • ~

~ l<~ c-

o "l ~w ••~

.-gii • ~

"•"c•

h~• u

~u~>• • • ! o • • , ••• I •• -.- g-0•-w 0u

..J E

~u:: ~0 •

~ ia: Q.

~

~Q. ~

~,-.. ..J ".. ! ~ 'E

" i •• '" a: ~

~0: W CW u i!= ~

w a> 0

~ ~Cl

% ...0 , ..~

.-...0 ,U~

i~•~ w

~u

X~5 i ~u •

a

Sterilization by Saturated Steam 91

High vacuum autoclaves are also usually equipped with air detector probeslocared in the pipework adjacent to the autoclave and linked to the cycle controlinstrumentation, During the cycle the temperature of the steam/air mixture isconstantly monitored against a preset lower tolerance indic3ling unacceptablelevels of air.

A IypicaJ high vacuum cycle for a nonporous solid load is illustraled inFIg. 5.

J. Porous Loads-Pulsed Cycles: Of alilypes of au(oclave load. porous mate­rials present the most serious problems of air removal. Air acts as an insulatorand therefore impedes the condensation of stearn on microorganisms and reducesprocess lethalily. A further problem is that it is necessary that porous dressingpacks and other cellulosic materials be completely dry at cycle completion.

These problems are usually addressed lhrough pulsed cycles. Figure 6shows a typical porous load pulsed cycle in which air removal lakes place overfive pulsed evacuations, each evacualing the chamber to a negative pressure ofminus one bar (gauge). The first evacuation removes a large percentage of theair from the chamber. Evacuation is followed by injection of sleam to atmo­spheric pressure; the steam mixes with the remaining air and to some extent pre.heats the load. Most of the remaining air is removed from the load by fourrepetitions of tbis vacuum pul!ile. Following the vacuum pulses, lhe cycle is set(or four pulsed injections of Sleam to a preset positive pressure. The first pur­pose of lhese "'positive" pulses is preheating. bUI they also contribule to mopping

PRESSURE (GAUGE) TEMPERATURE (OC)

'00

12'

E

,......_.._....•-......_.._......_._ .............._.. ,I .

' . II •••••.•

I ................ I.• •••••••I

................

'...ATMOSPHERIC PRESSU

I

IOllAR

.1lAR

• 100mb

llME

Fig. S High-vacuum cycle.

, I

92

.-

.-

.-

--Fig. 6 Porous load pulsed cycle.

Chilpter 4

•,I,•,

I,••;,

,

Ij,

I!....

up any air remaining in the chamber or in the load. Cycle exposure is timedfrom when i1 "floating" load temperature sensor and Ihe air detector probe reachthe specified sel temperature. The imponance of the air detector probe in porousload cycles cannot be overemphasised.

At lhe end of the exposure period the chamber is evacualed to facilitatedrying. During the drying phase, heal from the jacket helps to "nash" off con­densate as the pressure drops.

2. Fluid Load.<;-BalLasled C~'Clt!s: Heat penelration iolo volumes of fluidlends 10 be slow. This is because aqueous products in primary containers usuallyhave a very small surface area relative 10 their volume. Heat must be conductedlhrough the walls of containers that are mOSI otlen made from malenals likeglass or plastic. which are intrinsically poor conductors of heal. Thereafter. uni·form lemperatures in the fluid are dependent upon convection of heat within thecontainers.

The consequence of slow heal penetration is that the greater part of fluidload autoclave cycles is spent in bringing the load up to Ihe exposure tempera­ture and cooling it down again. For a particular autoclave. producl. productcontainer. and load configuration there is not much Ihat can be done: about thehcat-up time. If the product is at all heal sensitive it is often practical to takeaccount of the conlribution made toward microbial leLhalit)' during heat-up as

, I

Sterilization by Salurated Steam 93

well as at the ex;posure t'emperalure ralher than risking product deteriorationfrom overlong exposure. In some instances, panicularly for large-volume par­enteral products. the total cycle lime can be reduced by acceleraled chilled watercooling of the product afler exposure. Alternatively, some autoclaves may beequipped wilh heal exchangers and forced draft fans wilhin Ole chamber to assiSl.rapid cooling.

Some types of container for liquid prodUCLl\, for instance glass ampoules.are sufficienIly robust to witllsland the rigors of steam sterilization under pres­sure. Sterilization cycles for ampoules are generally uncomplicated. However.other types of container are not as robust as ampoules and require rather moreelaborate st'erilizatioD considerations. With stoppered vials. screw-cap Ixmles,nexible bags. and filled syringes there may be a significant increase in internalpressure within the headspace above the fluid during autoclaving, These internalpressures may result in closures and plungers being blown out. When a closureis completely blown out a prooucl item becomes obviously unusable; perhqpsmore dangerously. a panially displaced closure may compromise the sterile bar­rier and go unnoticed. These difficulties may be avoided by use of air-ballasted(air overpressure) sterilization cycles.

(n ballaslcd cycles the increase in pressure within the headspace or thecontainer is counterbalanced with an internal chamber air overpressure. Figure 7illustrates an idealized type of ballasted cycle; the autoclave operates as for aDonna) fluid load cycle up to the point al which Ihe exposure temperature isattained (in this case 121-C or one bar [gauge] sleam pressure), and then com-

TEMPERATUFlE (CC)

.,,, ~PR5SSURE (mBAR)

STEAM I'I\£MUI'I£

~ ~ •• ~ ~~... TOtAL PAES9UAE lo'IA + ST&J,lJ_••••_ ••__• T£MPEM1\IRE

JACKET

FRONT B.EVATlON

FAN

C...

•,I",80

••

:.-::~.~. ~.~.;;~:::.~.;:..• • •...: ~ ...,

.. I , .,.. , " ..• • •! , ,.. . .

• • •.. . ... I I.. . ,.. . ... . .

... " ,.. , ... , .. '.. ; .., ... ; "

"..

•Fig. 7 Ballasted autoclave and cycle.

94 Chapter 4

pressed air is injected into the chamber to a final pressure of 2.6 bar (gauge). Atme end of the exposure period the pressure of 2.6 bar is maintained with com­pressed air only unlil cooling is complete. Ballasting pressure is usually main­mined with air until all temperature probes register no more than sooe; this is toensure thaI closures will not be displaced in some pans of the load as a result ofuneven cooling.

In practice. ballasting must take place before lhe exposure temperature isreached. It happens throughout the healing phase to compensate for theincreases in internal pressure thai accompany lhe increases in temperature. Dif­ferent technologies address this differently in tenns of the number of incrementoccurring during heat-up. lberc may be only as few as two or three steps, ineach of which air is injected first. followed by steam to predetemlined interme­diate temperature and pressure equilibria. On the other hand. the modulation ofinlernal pressure with increasing temperature may be vinually continuous inmany tiny increments.

The pressure required to counterbalance internal headspace pressures maybe determined empirically (Figure 8) or calculated from Iheoretical modelling.If done empirically. some care should be taken in relation 10 the amount of deadspace in the system connecting the vessel under study to the pressure gauge. iferroneous results are to be avoided. This is particularly important for smallheadsp.ces and large dead spaces.

124114 116 118 120 122

IJIAL CONTENTS TEMPERATURE (oq

"21'0

2

1.6106

2.2

IJIAL HEADSPACE PRESSURE (BAA)2.8

2.4

2.6

fig. 8 Measurement of vial heads~ pressun:'.

Sterilization by Saturated Steam 95

(4.3)

A theoretical model was described by Allwood rII lhat proposes an equa­tion for determining the internal pressure within a container in psia:

PoV,T,PT2 = + Vapor pressure within the container

V,T,

where Pn = internal pressure at exposure temperature Tz rAJ; Po = assumedstandard atmospheric pressure, 14.7 psia; T1 = assumed temperature of liquid attime of filling. 20'C or 293'K; VI ~ (Headspace volume at T1 + Volume of H,oin headspace at T1 - water vapor in air at T1): and V2 = Headspace volume al T1- Volume of expansion of liquid at T2.

The important constraints within this model are the headspace volumeabove the liquid in the container and the volume of liquid in the container itself.Typically. larger ratios between headspace volumes and fluid volumes lead tohigher internal pressures.

Joyce and Lorenz [2J presented a theoretical model based on similar prin­ciples. Table 2 summarizes the theoretical conclusions from this model over atemperalUre range of 116 to 121'C against fill volumes of I to 90%.

Both theoretical models and Lhe empirical dala shown in Fig. 8 reachbroadly similar conclusions with regard to the pressure developing within closedcontainers raised to steam sterilizalion temperatures.

Table 2 Calculated Internal Pressures (psia) for Joyce and Lorenz (2)

Temperature re>Fill (%) 116 117 118 119 '20 12'

Pressure (psia)

I 44.4 45.2 46.2 47.1 48.1 49.025 44.6 45.5 46.4 47.3 48.3 49.350 45.3 46.2 47.1 48.1 49.1 50.'75 47.7 48.7 49.7 50.7 51.7 52.890 60.9 62.3 63.8 65.3 66.8 68.3

3. Rotary Drum Washer/Autoclaves: It is nOnTIai practice in large-scale asepticCllling of pharmaceutical products for vial closures to be sterilized into the asep­tic filling room via double-ended washer/autoclaves. Figure 9 is a represcnlalionof a rotary drum washer/autoclave and its processing cycle. The setup has someresemblance to 3. domestic aUlbmatic washing machine except thai the drumrotates mther more slowly. The whole chamber fills with water dwing lhewashing phase. the water is pumped out after the final rinse, and autoclavingproceeds as DOnnal.

96 Chapter :I

0.00.0 •

•••••••0 ... 0 •••........• 0 •••• 0

•••••••0 •••• 0

•••••••••••••••00 ••• 0

DRJVEWHEEl

RlJBBEfI--t­Pt.UGS

ROTATINO ORUM-t--I

••

r-

.f

fI,/'" "" ........ '\

••

•••

••THERMAl. PROFILE

• . "-- • • •

Fig. 9 ROlary drum washerJaulOclave.

II is not possible within these autoclaves 10 operate with temperatureprooos within the load because of damage to the probes caused by the rotation.Since vial closures are sterilized in bulk and because it is undesirable 10 holdthem in aseptic areas for lengthy periods of lime before use. the possibility ofusing biological indicators as routine controls is also unacceptable. This placesgreat emphasis on proper validation of these cycles and indeed on the design ofvial closures. Vial closures of some designs can all 100 easily nest within oneanother, thus creating surfaces to which steam cannot penetr.1te. Autoclaves ofthis type should be equipped with mechanisms that ensure that they cannot oper­ate unless the drum is rotating. or at the very least alarm signals should bedisplayed on the autoclave and on the permanent record in the event of drumfailure.

4. Double-Elided AUlocltH'es: In many industrial applic:ltions autoclaves willbe "bridges" between areas with different cleanliness classifications. The twomain scenarios are that of an autoclave being loaded in a nonsterile preparationarea for its sterilized contents to be unloaded in an aseptic filling room. and of.1Oautoclave being loaded in a clean filling room for its sterilized contents to beunloaded into an uncontrolled packing area. In either example the risk is com-

Sterilization by Salurated Steam 97

promising the status of the clean side if both doors are open at the same time.Clearly, lhese double-ended autoclaves must be equipped with special interlocksand precautionary devices to prevent this from happening.

The two doors of double-ended autoclaves should be interlocked in such away that it is impossible to have both open atlbe same lime. If there, is no needto bridge areas, it is cheaper and simpler to purchase single-ended autoclaves. Ifpossible. double-ended auloc,laves should be unidirectional. lr should only bepossible to open the doors at one end of the autoclave by operaling controls atIhat end. This mean, thai a loading operalor should nol be able to open lheunloading doors by some remote means. and the same applies to the unloader. Inother words. the equipment should dictate that the staff in each area be whollyresponsible ror their own access to the autoclave in their respective areas.

There should be automatic signals at both ends of the autoclave indicatingdoors locked when both doors are locked and secure. Other signals at both endsshould include a device indicating sterilizing during the whole cycle and cyclecomp/ele when the cycle is over. This is to allow staff on both sides to know theSlatuS of the autoclave and prevenl any misguided attempts at entry. Once theloading door has been closed, it should not be possible to open the unloadingdoor until the autoclave has satisfactorily completed a sterilization cycle. This isto prevent an autoclave being loaded with nonsterile items and then beingunloaded without them having gone through any form of processing.

5. Autoc/ave Control Syslems: Modem production-scale autoclaves are usuallycomputer controlled. Older models may be electromechanically controlled(hardwired). Manual control is rare. and much of the knowledge and many ofthe skills required in the past to operate autoclaves successfully are being lost toturnkey operation. [n many respects Ihis is a giant leap forward to more consis­lenl assurance, of sterility. A genuine concern is that some will think that com­puter-controlled autoclaves are foolproof~ indeed. they are not.

h is important to know how particular autoclaves work and to know howparticular cycles are controlled. The software in compuler-controlled autoclavesshould be invi.olable; it should have been validated. and it sbould respond in apredictable manner to signals received. sending OUI only the correct responsesignals and verifying thai what was meant to happen acluaJly did happen. How­ever, somewbere in the loop there will be sensors. switches. valves, and pieces ofmiscellaneous plumbing. Murphy's law, which states that anything that can gowrong will go wrong. applies to these systems. and unless they are well under­stood by sterilization scientists as well as by engineers, electricians. and me­chanics.lhe risk is nonsterililY.

Autoclave control systems respond 1'0 three Iypes of signal. time. tempera­ture, and pressure. By far the most reliable of these is time. because of the in­trinsic reliability of quanz timing devices. Temperature sensors are quile reli­able but respond more slowly than pressure transducers. Pressure may be con-

98 Chapter 4

trolled by pressure switches that are activated (or deacriv3led) at onc specificpressure only. or by pressure transducers that respond and send signals ovec cali­brated ranges of pressures. Pressure sensors are electromechanical devices de­

.pending upon movement of nex.ible diaphragms to cooven pressure signals intoelectrical pulses.

Usually only one phase of a sterilization cycle is controlled by time. andthat is the exposure or temperature hold phase. Once me sterilizing temperatureis reached and registered by the computer, lIle purpose of !.he timer is to signalstop to any further steam injections after the appropriate elapsed time. Inessence this timed phase has a one-tailed error. If the signal is sent, the worstfrom the slandpoint of sterility that can happen is that it is ignored and lIle sler­ilization phase will be protracted. This is not a problem from the sterilizationstandpoint unless the effect of heat damages the hennetic enclosure of the itemsbeing sterilized.

Time signals. because of their reliability, may be used to suppon otherphases of autoclave cycles. So-called "phase time excesses" may be built intopreliminary vacuum phases, heat-up phases, and cooling phases. A phase timeexcess is an alarm condition raised in response 10 a panicular phase of the ster·ilization cycle happening faster or (more typically) slower than expected. Phasetime excess alarms may provide essential indications of vacuum pump failure orsticking air or steam valves. The more complex the cycle. the greater the need 10confirm that the validated condition is being repljc,ated in routine use.

Temperature signals are used for several purposes. First. they are alwaysused to signal that the sterilizing temperature has been reached. Two or moresensors should be used for this purpose. and the timed exposure phase should notbe staned until all of the sensors reach specification.

In some autoclaves. temperature signals are used to modulate the temper­ature within its specified band during the exposure period. In such cases the sig­nal via the computer is directed to a stearn valve that opens in response to a lowtemperature signal and closes in response to a high-temperature signal.

It is quite usual to find a high internal pressure maintained in aUloclaves atthe end of their sterilization phases until some cooling has taken place. This istypical for nuid load cycles, which may be held under air pressure until Ihe sig­nals from all of the sensors indicate that the temperature is lower than SO·C. InIhese cases the signal is to close the air injection valve and open a pressurerelease valve al the appropriate temperature. Cooling may be subject to a phasetime excess.

Many phases of an autoclave cycle may be controlled through pressuresignals. Pumping down to preliminary vacuums continues until pressure signalsisolate the pump and open a steam valve. The sterilil.ation hold phase may alsobe controlled by pressure signals. The pressure al wltich the specified tempera­ture was reached may be maintained within limits of pressure (and hence of tern·

Sterilization by Saturated Steam 99

perature) by pressure signals to the steam valve. With current technology, pres­sure signals afford finer control of temperature within the sterilization phase ofautoclave cycles than temperature signals.

C. Steam Quality and Superheating

Since microbial lethality is a function of the latent heat of steam being dis­charged during condensation. it follows that the steam itself should be of 3 suit­able quality to ensure that maximum energy is available. Ideally, this is "dry"saturated steam supplied from a steam generator located close to the point of use.A less than ideal siluation is for the boiler house 10 be a long way from the aUlO­

clave, thereby allowing the steam to condense and pick up excessive amounts ofwater droplets before reaching the aUloclave. This makes it necessary to incor­porale moislure traps in the steam lines and ensure that all pipework is well in­sulated or tmeed if wet loads resulting from the condensation of water in thesteam are 10 be avoided.

A second area of concern associated with steam qua lily is superheating.This is a phenomenon related to the phase equilibria of steam under pressure(Fig. 1). In some circumstances it is possible at a fixed pressure to increase thetemperature of steam above its equilibrium temperature. It is then referred to assuperheated or supersaturated steam. Superheated stearn is not as effectivelylethal 10 microorganisms as saturat.ed steam-the biochemical mechanisms oflethality are similar to those of dry heal. If supersaturated conditions prevail, thelethaJity of the process will be much lower at any specific:d temperalure than thaiwhich would be expected from saturated steam. Supersaturation rnay arise fromautoclave problems or load problems or bolh. For instance, the sleam in thechamber may pick up heat from a jacket running al 100 high a temperature orpressure, or condensation of the steam may be impeded by very dry cellulosicmaterials in lhe load.

V. TEMPERATURE/TIME CRITERIA FOR STERILIZATION

Microbial lethality from stearn sterilization is a function of two parameters, tern­peralure and time. The choice of a cycle is ultimately dependenl upon the heat­stability of the product, the type of primary container, the k.nowledge of heatpenetration into lhe product, the aUloclave lechnology available, and the k.nowl·edge of the microbiological contamination prior to sterilization. However, (hepbarmacopoeias (particularly the USP) offer some sound general advice to theselection of criteria.

The classic pharmacopoeial approach to steam sterilization has been lhecompendial cycle. CompendiaJ cycles are described in terms of process specifi­calions, for insrance 15 min at 121'C (USPIEPIBP) and 134'C for 3 min (BPcompendial cycle for dressings). The specified temperatures and times are for

100 Chapter 4

the exposure period and make no allowance [01" lethality contributed by heal·upand cool..oown phases of the sterilizatjon cycle. Historically. a claim 10 sterilitycould be made for a balch of items Lhat could be shown to have undergone acompendial cycle. and from which an inappropriately small number of items hadbeen shown to pass a phannacopocial sterility lesl. This is no longer the case,and although compendial cycles remain a viable option for steam sterilizationcycles. most responsible sterilizers and all regulatory agencies will require Cur·ther supportive evidence that even the compendial cycles are really capable ofassuring sterility.

Beyond lhe compendial cycle. the next level of sophistication is theoverkill cycle. Overkill cycles are based on inoctivation factors and thereforeshare a common origin with the "botulinum cook" of the canning industry. TheUSP Slates. "a JelhaJity input of 120 may be used in a typical overkill approach:'Clearly some knowledge of D-vaJues is necessary to apply this approach: for ref­erence and in (he absence of further information a 0 121 for spores of Bacillussrearolhermophilus may be used. The USP quotes a D121 of 1.5 min. Using thisinformation. an overkill cycle .....ould be set as 18 min exposure 31 121-C. Withlocal detailed knowledge. a D-vaJue for the most heat-resisl3Jlt microorganismsIIctually concam.inaling a particular product may be substituted for thai of B.slt!orolhumophUus in establishing an appropriately longer or shoner overkill cy­cle.

Overkill cycles, however. take no account of the numbers of microorgan­isms cOOiaminating product items. Consequently. an overkill cycle for a heavilycontaminated product will not differ from thai for a similar but far more lightlycontaminaled product In 1980 the USP first formally recognized the now-stan­dard sterility assur.mce level (SAL) of 10.6 (i.e., assurance of less man onechance in 1.000.000 that a sterilized item is microbiologically contaminated) as alegitimate target for sterilization processes. However, application of the SALapproach to determining sterilization cycles requires much more microbiologicalinformation than the compendial cycle approach or the overk.ill approach.namely an actual k.nowledge of the numbers of microorganisms conlamimuingproduct items. This practical infonnation may then be coupled with aD-value(assumed for a reference microorganism or detennined for aClUal heat-re istantcontaminants) and used (0 detennine an appropriate cycle. For instance. productitems contaminated at an average level of 100 colony-forming units per item.with all contaminants assumed to have a Dl21-value of 0.75 min. would be as·sured of an SAL of 10-6 by an exposure of 6 min at 121·C.

TIle mm"e away from compendial cycles to overkill and SAL cycles wenthand in hand wiLh an increasing interest in the concept of equivalent lethalitiesbeing oblainable from different temperatureJtinlC combinations-the Foconcepl.

1be Foconcept originated from laboratory studies of the type summnrizedin Fig. 2 and Fig. 3. which show that a particular lethal effect can be oblained

Slerilization by S.lurated Sleam 101

through different combinations of time and temperature. The F0 value is a refer­ence point to lethality al 121·C. FO values are expressed in minUles. An Fovalue of however many minutes tells you thai the particular combination of tem­perature and time being used has equivalent lethality to that number of minutesal 121"C. The Fo concept allows valid cycles based on times at 121'C to betranslaled iDle other temperature and time combinations equally valid.

At one time. the pharmacopoeias were specifying a standard Fovalue of 8min for Sleam sterilization regardless of load type. presterilization microbiologi­cal contamination levels. or product stability. The last editions of lhe majorpbarmacopoeias in which an Foof 8 appeared were the 1980 USP and Ihe 1988BP. An Fo of 8 means that the sterilizing cycle being used has an equivalentlethalily to 8 min al 121'C.

The focus of recent editions of the phannacopoeias is that an F0 should besufficient to assure an SAL of 10-6. The FOvalue may be calculated for thetimed exposure period only, but it is more commonly computed from the tota!accumulated lethal heat input above 80·C.

The following equation may be used to calculate simple Fovalues:

FO= Dm (log A - log B) (4.4)

where Fo= minimum lethality, assuming z to equal IO·C; A ;;; number of viablemicroorganisms per item prior to sterilization; and B = SAL.

Equivalent times at other temperatures may be calculated from the fol­lowing equation [3J:

,F ' _ FmT - (4.5)

L

where Fi = the equivalent lime at temperature T to achieve a specified lethalityfor contaminants with a specified z-value, Ff21 = the equivalent time at 121·Cto achieve the same specified lethality (when z is equal to IO·C, Ft21 = FeY.

L=lethalrate(10[T-12IVz) (4.6)

Fo values for the accumulated heat input during heat-up. exposure, andcool-down ponions of a sterilization cycle may be obtained by plotting the lelhalrates obtained al successive time intervals during the sterilization cycle againsttime, and determining the area under the curve.

Mathematically this is expressed as

FO= Ldt (4.7)

Various methods are available for performing this integration (see Pflug(41), but nowadays Fa values are invariably integiJ.ted automaticaUy with stan-

102 Chapter 4

dard sterilizer monitoring equipment (for instance the Kaye Digistrip). Suchequipment usually assumes D 121 to be equal to I min. and :::: to be equal 10 to°e.

VI. VALIDATION AND CONTROL OF STEAM STERILIZATION

A. Validalion

Validation of a steam sterilization process must cover the series of actionsrequired to establish that the process is capable of doing what it is intended to do(i.e.. supporting a claim of sterility) and must define a plan for main,aining thevalidated state of conLroI. An overall scheme is described in Table 3.

In an ideal world. sterilization validation begins even before the purchaseof an autoclave. Ahhough Ihis would appear to belong mainly with the engi·neering side of sterilization. it is also of major imponance to the operational andcontrol side too. A steriliuuion cycle must be tailored to each product and itscomainer/c1osure system. so it is best to have first defined the range of potentialapplications. (0 be sure- that new equipment is correctly specified. On installa­tion. the autoclave should be checked out to ensure thai it meets its specification.that it has been properly installed. and that its instrumenlation is within calibra·tion.

Thereafter. the new aUloclave must be validated and scheduled for routinerevalidation. maintenance. and recalibr31ion 3t appropriate inlervals. Since (he

Table 3 Essentials of Steam Sterilization Validation

SPECIFICATION Must define chamber size. conslrUction quality. control systems.operating parameters. cycle operations. requiremenl for ser­vices. safety systems, machinclbuilding interface.

INSTALLAnON Must eruuu that the autoclave is located in a bounded area tocontrol water leaks. has good access for maintenance. ha.~ anair-break in the drain line. has sample ports for steam and watertesting. is located in an area that allows for heat dissipati n.

COMMISSIONING Must demonstrate that all operating systems are working. thatall safety systems are working. mat the autoclave has bc:entested for leaks and other malfunctions. that men: is cycle to cy·ele continuity of perfonnance. Mid! ;nc1ud~ calibration of allcontrol equipment.

VALIDAnON Mldt del1lQnstrat~ good heat distribution in empty chambers forall proposed cycles and good heat penetration sludies for allproposed loads. Must delermine ongoing operating specifica­tion. Must t!!tablish a caJibr:ttion program for all equipment in­volved in controlling or monitoring performance. a progrorn forroutine maintenance.

Slerili..tion by Sotorated Steam 103

critical parameters of sterilization by saturated steam are known. and the effectsof saturated steam on microorganisms in general are well documented. the mainthrust for autoclave validntion is to ensure that the specified temperature isachieved in all pans of the autoclave and throughout the load. Biological vali­dation is secondary to thenna! validation ex.cept in circumstances where it isconsidered that !hennal data is unreliable.

ThennaJ profiles should demonstrate uniformity of temperature throughoutempty chambers and from cycle to cycle. Any cold spots should be identifiedand if possible corrected at this stage of validation. Since revalidation mustbecome a regular occurrence, new sterilizers should be specified wilh accessglands for the thennocouples used for this purpose. Da.. from the thennocou­pies should be logged by a suitable multichannel recorder such as the KayeDigistrip. which also computes cumulaLive F0 values when temperatures exceedSO·C. Significant advances are being made in the field of computerized datalogging systems. panicularly with regard to information presentation and datastorage.

Panicular lood configurations must be specified in detail and documented.The loading paltern of an autoclave can have significant effects on heal distribu­tion and pcneuation. The second phase of thennal val idation is to demonstraleuniformily of he3t penetration throughout specimen loads (thennal load pro­files). For Lhis purpose temperdture sensors must be located within the itemsbeing slerilized. All loading patterns should be evaluated, and good replicationshould be evident from cycle to cycle for the same pattern. Once again coldspots (if any) should be identified and loading patterns modified 10 minimizethem. Specified Fovalues for particular products must be achieved in the cold­est pan of the load.

In the case of porous loads it may be valuable to perfonn heat distributionstudies on fully loaded autoclaves to provide additional assurance with regard toair removal and thermal uniformity.

These thermal siudies are me basis of validaLion and ongoing revalidalionof aUloclaves.

In some instances they may need to be supplemented by microbiologicalvalidation. but Ihis should be seen as the exception rather Ihan the rule. Theremay be reason 10 believe thatthcnnal data from load profile studies are ovcrop­timistic~ perhaps steam may be penetraLing into the load along channels createdby the thennocouple wires. Moreover. there may be reason to believe thai dryheat conditions are prevailing in some pans of the load: for inslallce, it is not un­known for some types of vial closure plugs to nest one inside another in rotarywasher/autoclaves. Sleam cannOl penetrate to the occluded surfaces, and non­sterile plugs may be discharged. In these cases biological studies are the onlymeans of ascertaining whether true sterilizing conditions exist.

a

104 Chapter 4

Spores of B. slearOlhennaphilus ATCC 7953 (or CIP 52.81 or NCTC10007 or NCIB 81 57) or Clostridium sporoge.es ATCC 7955 (or NCTC 8594or NCLB 8053) are the recognized biological lest pieces for sterilization by satu­rated Steam. They may be purchased as spore suspensions that can be inoculatedonto test pieces of one's own choosing, or as ready-made test pieces on paper oraluminum strips. An incubation temperature of 5S-C should be used 10 lest forviability after exposure.

B. Routine Control

Academic texts on Sleam sterilization often stale thai rouline control of steamsterilization processes should concenlrale on the measurable delcnninanlS oflethality. temperature and time. Temperature should be monitored at the coldestpoint. usually in the drain line, but if Lhis is not Ihe coldest spollhere should be a"floating" probe allhe coldesl spot. or the relationship between (he dr-din line andthe coldest spot should have been well established and documented. A perma­nent record of the temperature throughout the sterilization cycle should be acompulsory feature of all production-scale autoclaves, and lhis should be in­spected in dec:ail for batch release. In practice this is not sufficienl to confirmiliat sterilizing conditions have been attained.

Sterilization by saturaled steam is only effective for particular combina~

(ions of temperature and time in the absence of air. Permanent records of pres­sure should also be inspected as pan of routine balch release. Deviations fromthe known equilibrium that exists for stearn between temperature and pressuremay be indicators of superheating. Inspection of the pressure Iraces of lhe pre~

liminary phases of slerilization cycles may reveal faulty steam or vacuum valves.which could impact upon sterility assurance. Presterilization temperature tracesare often too messy to be meaningful in lhese phases of sterilization cycles. Thisis due to differences between hot and cold starts. differences in temperature be·tween probes in one pan of Ihe load and anolher. elc.

Where modem autoclaves can provide analog and digital records of tern·perature and pressure against time. both should be provided in the permanentrecord and inspected diJigenlly before product release. Manual transcription ofprocess conditions from dial or digital gauges is not an acceptable approach tocontrol of autoclaves.

Routine biological monitors are still being used as routine controls in theU.S.A.. but very rarely in Europe. With modern technology these should not benecessary.

For porous loads, two olher monitoring techniques are in use to give assur­ance of sterility where there is a risk of air enlrainment. superheating. etc. Theseare the Bowie-Dick test pack and the Lantor Cube. II should be emphasized thatthese are used in rather special applications and would be of Iiule significance tofluid loads or nonporous solid loads.

a

Sterilization by Salurated Steam 105

J. The Bowie-Dick Ttst Pack: The Bowie-Dick standard test pack consisis of aseries of folded stacked towels. Strips of heal-sensitive tolpe are placed fromcomer to comer 10 Conn a Saint Andrew's cross pauem at various depths withinthe pack. The towels are fairly tightly wrapped, sealed with tape and placed inthe autoclave along with a routine load.

The indicator tape changes color from light to dark when moist heat steril­izing conditions prevail. In the presence of air the tape does not darken. Well·outlined unchanged areas can be seen on the tape in areas where air has been en­trained.

2. The Lan/or Cube: The Lantor cube was developed as a reusnble and morereliable allemalive to the Bowie-Dick towel pack. It consists of a 15 em cube ofIaminaled polypropylene supplied in two halves. A sheel of paper carrying stripsof heat-sensitive indicator t.1pe is pJaced between the two sections of the cubeand clamped in place. Interpretation is the same as for the Bowie-Dick test pack.

C. Some Autoclave Problems

The autoclave. like any other piece of industrial equipment, suffers problems thatare not wholly addressed by preventative maintenance programs. Some of lhesemay affect production. some may affect sterility. some may have an effect onboth. Some of the difficulties that most autoclave operators periodically en·counter are, listed in the next few sentences. The consequence of most of thesecan be avoided by regular inspection and constant vigilance.

Sometimes drain oullets get blocked with broken glass or other such de­bris. thereby preventing free removal of air and accumulation of condensate in.he chamber.

Door seals perish. The consequence is that pressure cannot be maintainedand sterilizing temperatures are not reached. If not diagnosed through othersymptoms, perished door seals should be detected in periodic leak rate perfor­mance checks. These checks should be perfonned with a greater regularity thanthermal valid:uion but are arguably not part of routine control of the 3uloclave.The leak: check is an indicator of lhe ainighmess of the chamber and associatedpipework during a cycle. The lest involves pulling a deep vacuum; the pump i.sthen turned off and the vacuum held for a set time. usually 15 min. The pres.~ure

increase in me vessel should not exceed a specified small amount over the wholeof the hold period.

Temperature probes are damaged. Records become unavailable. Feedbackis interrupted.

Wei loads arise from poor steam quality or progressive pump deterioration.resulting in slow evacuation or inadequate vacuums during cooling.

Operator error results i.n products being loaded in the wrong configuration.or the wrong cycle is selected for a particular product load.

, I

lOb

REFERENCES

Chapter 4

1. Allwood. M. C. Hambleton. R.. and Beverley. S. (1975). Pressure changes in bottlesduring sterilization by aUloc!aving. Journal ofPharmnceuticaJ Sciences 64 (2): 333­334.

2. Joyce. M. A. and Lorenz. J. W. (1990). Internal pressure of sealed containers duringaUloclaving. Journal ofParenteral Sci~tlce and TechIJ%gy 44; 320-323.

3. Parenteral Drug Associarion (1978). Technical MOlWgraph No. I. Validation ofSu~am Steriliwlion Cycles. Parenleral Drug Association: Washington. D.C.. U.S.A.

4. Pflug. l. 1. (1973). Heal sterilization. In /tadustriaJ Su:riJi:.alion (G. Briggs Phillipsand W. S. Miller. cds.). Duke University Press: Durham. N.C.. U.S.A.

ANNEX 1. CALCULATING CONTAINER INTERNAL PRESSURESFOR A FILLED SYRINGE TO BE STERILIZED IN A 11S"C AUTO.CLAVE CYCLE

POV,T2PrJ = + Vapor pressure within the container

- V2T,

where PT2 = internal pressure at IISoC (38S"K); Po = assumed standard atmo­spheric pressure. 14.7 psia: T. = assumed temperature of liquid al time of filling.20·C or 293'K; V) = Headspace volume al T) + Volume of H20 in headspace at

T, - Water vapor in air at T1•and V2= Headspace volume 3t T, - Volume of liq­uid expansion at T2•

For lhe syringes in question. headspace volume was calculated as 0.164mL.

The volume of air in water at 20·C is known to be 18.7 mg!L; for a 0.5 mLfill volume, the volume is 9.35 x 10-3 mL

Waler vapor displacemenl of air 81 20·C is known to be 1.9 x 10.2 mlJmLof headspace; therefore for a headspace volume of 0.164 = 3.25 x 10.3 mL.

Increase in volume of H20 from 20·C to 115·C is known to bc: 5.37%(5.97% for an increase from 20'C to 12I'C); therefore 0.5 mL fill volume willincrease by 0.027 mL.

Pus = 14.7 x [0.164 + (9.35 x 10.3) - (3.25 x 1O.3)J x 388293 x (0.164 - 0.027)

= 24.2 psia or 1.67 bar

Counterbalancing the internal pressure developing in filled syringes meet­ing the specifications given above would require an air overpressure of 1.67 barabove the pressure required 10 oblain 115·C during exposure (0.7 bar). Thepressure during exposure would therefore be 2.4 bar (gauge).

, I

Sterilization by S~tu,.tedSte~m

ANNEX 2. CALCULATING A SIMPLE Fo VALUE

Po values can be calculated using Eq. (4.4):

FO= D tll (log A -log B)

107

where D121 = D-vaJue of a heat-resistant microorganism at 121-C. say 1.5 min;A =number of microbiological contaminants per item, say 100; and B =SAL.usuaUy 10";. Therefore

FO= 1.5(2 - [-0]) = 12 min.

ANNEX 3. CALCULATING EQUIVALENT LETHALITY AT 134·C TOAN Fo OF 11

The Parenteral Drug Association equation, Eq. (4.5), for calculating equivalentlethalities is

where F'r= equivalent time allemperature T to achieve a specified lethality forcontaminants with a specified l-vaJue; P:121 = the equivalent time at 121-C toachieve Ihe same lethalily (when 2 equals lO'e, F'12I =Fo); and L =lethal ra,e(IOIT- 121]/2).

F't21 := Fo= 11 min.

L=10[134 - 121]

10= 101.3 = 20·

IIF'T = = 0.55 min al 134-C

20

.1013 is calculaled by taking the logarithm of 100JogiO of 10 is one) and mulliplying by lhe power 10which il is raised. in IJ\is case 1.3. 'The answer. 20 is the antilogarithm of U.

Copyrighted all a

5Dry Heat Sterilization and

Depyrogenation

I. Effects of Dry Heat on Microorganisms and Bacterial Endotoxins 110A. Inactivation of Microbial Populations 110B. Destruction of Bacterial Endoloxins (Pyrogens) 11 JC. Other Methods of Bacterial Endotoxin Removal J 12D. Measurement of Bacterial Endotoxin' (LAL Method) 113

D. Applications of Dry Heat Sterilization J 15A. Ovens and Tunnels ] 15

m. Temperaturelfime Criteria for Sterilization and Depyrogenation 119IV. Validation and Routine ConlIOl of Dry Heat Sterilization 120

A. Validation 120B. Routine Control 120

Dry beat sbould be the method of choice for sterilization of heat-stable items thatare damaged by moisture or are impervious to steam. It can in facl serve one orboth of two functions; it may serve as a method of sterilization or as a melhod ofsterilization and depyrogenalion (destruction of bacterial endoto.llins). Ab~nce

of bacterial endoroxins is a biological quality of equal or greater imponance tosterility for phan1l8ceutical products and medical devices intended for parenleral

109

110 Chapter 5

application. None of the other large-scale methods of sterilization have thecapability of destroying eodoloxins and therefore depend to some extent on otherprocesses for elimination of endotoxins pnor to terminal sterilizalion.

TemperalUres required for dry heat sterilization (over l6(rC) are in a farhigher range than those necessary for saturated steam sterilization (110 to140'C). Depyrogenation requires even higher lemperatures (200 to 4OO'C) ifvery long periods of exposure are to be avoided. Temperature/time combina­tions lhal are sufficient to meet requirements for eodotoKin destruction are morethan sufficient to sterilize the most highly conlaminated items. Temperatureltime combinations that are only sufficient to meet requirements of sterility arenOI sufficient 10 meet pharmacopoeial and regulatory requirements for endotoxindestruction.

Heat transfer from air to product items in dry heat processes is not effi­cient. The main mechanism is conduclion: good insulalors like air are by defini­Lion poor conductors.

l. EFFECTS OF DRY HEAT ON MICROORGANISMS ANDBACTERIAL ENDOTOXINS

A. Inactivation of Microbial Populations

As with sterilization by saturated steam. thermal damag~ to biological system~ asa resull of dry heat sterilization processes is a function of absorbtion of heatenergy. Inactivation of microorganisms is by oxidation. The kinetics of oxida­Lion and population death approximate 10 first-order reaclions. but they are sig­nificantly different from the processes of coagulation of cellular proleins foundwith moist heal sterilization in that they require far higher temperatures and pro·ceed more slowly.

There are the usual species-la-species variations in response 10 dry heal.Spores are more resistant than vegetative cells. It does not follow thai lhosemicroorganisms that are unusually resistant to moist heal sterilization processesare also resistant to dry heat conditions. Spores of 8. st('arothem,ophilus forinstance are not as resistant 10 dry heat as spores of Bacillus subtilis var niger.This is exactly opposite to saturaled steam. Numerous natural factors may pro­tect spores against dry he.3t inactivation. notably antioxidants and reducing sub­stances. which are capable of impeding the availability of oxygen to the oxida­tive site or larget in the cell.

The terminology used in association with dry heal inactivation of microbialpopulations exactly parallels that of sierilizalion by salurated steam (see Chapler4). The Drvalue is the time required to reduce a population to 10 of its initialnumbers when held at a constant temperature T. The D-value of spores of B.subtilis var niger 3t 170'C (D 170) has been variously reponed from around 8 S 10

1.5 min for dry heat conditions.

, I

Dry Hut Sterilization and Oepyrogenalion 111

The z-values for dry heal, Le., the change in temperature required to effecta lQ..fold change in D-value. are around 20·C, i.e., twice those nonnally quotedfor satumted steam (I0'C). Qw-values of 1.6 have been quoted I IJ for dry heatsterilization in Ihe range 170 to ISO·C. considerably lower than lhose quoted inChapler 4 for s:nurated sleam sterilization. This goes some way to justify thehigher t:emperatures required from dry heat processes to meet the requirementsofsterJlity.

B. Destruction of Bacterial Endotoxins (Pyrogens)

Pyrogens are substances thal, when injected in sufficient amounts into the humanor animal body. will cause a variety of symptoms of which the most recognizableis a rise in body temperature. They are therefore significant to sterile parenteralpbamtaceuticals and to medical devices used for their administration. In phar­macopoeial terms. substances are categorized as pyrogenic or nonpyrogenicaccording to the response of injected rabbits versus specified temperatureincreases. The phannacopoeial definition tends to be tautological bUI onlyreflects the limitations of the only analytical technology available at one time.Recent editions of the major pharmacopoeias are now recognizing an alternativeto the mbbit pyrogen test. This is the LAL (Umulus amoebocyte Iysale) test, atest specific for bacterial endotox..ins.

There has been very little doubl for many years that by far the most sig­nificant source of pyrogens is microbiological. All microorganisms appear 10 becapable of producing pyrogens, and the most potent ronns are associated withgram-negative bacteria.

All gram-negative bacteria are surrounded by a loosely structured envelopelocated externally 10 the peptidoglycan cell wall. Much of the enzymatichydrolysis of nutrient macromolecules takes place within the cell envelope. Theouter layer of me envelope is a permeability barrier effective against diffusion ofexoenzymes into me greater environment; this outer layer is made up oflipopolysaccbarides linked to phospbolipids and proteins. The pyrogenicresponse has been shown to be stimulated by lipopolysaccharide fmctions of thegram-negative cell envelope. These substances are termed bacterial endolox.ins.Purified endotoxin is pyrogenic in lower doses than naturally occurring endo­toxins in which the pyrogenicity is presumed to be modified by associated pro­teins and phospholipids. Bacterial endotoxity is nol lost with loss of viability.Saturated steam. gamma radiation. and elhylene oxide sterilization processes thatinactivate microorganisms are not capable of destroying bacterial endotoxins.

PlJrified endotoxin consists only of lipopolysaccharide. This ilself hasthree distinct chemical regions; an inner core called lipid A. an inlermedintepolysaccharide layer, and an outer polysaccharide side chain. Lipid A, a highlysub lituted disaccharide of glucosamine, is responsible for pyrogenicity andother immunological and biological properties associated with endotoxin.

a

112 Chapler 5

For most practical purposes the term endotoxin can be regarded assynonymous with pyrogen, depyrogenation with endotoxin destruction. andpyrogen-free with endotoxin-free.

Dry heal destruction of bacterial endotoxins is complex and poorly under­51000; much of the research data is contradiclOry (2). Most experimenlalevidence has shown that destruction follows second-order chemical kinetics witha high initial rate of decrease of endotoxin followed by a much slower tenninalrate. These second-order models give a belief estimate of the kinetics of endo­toxin destruction al temperatures above 250'C Ihan in the 170 to 250'C temper­atuce band. Impure endotoxin may account for these anomalies. There is practi­cally no endotoxin destruction at temperatures below 80·C, and D-values for dryheat sterilization temperatures of around 170·C are as high as 20 min [2]. Theseobservations are in agreement with published z-values of around 4O"C for endo­loxin destruction. Evidence of endotoxin destruction in practical silU<uions isusually laken from empirical observation because of the uncertainty over its the­oretical basis.

C. Other Methods of Bacterial Endotoxin Removal

Agents other than dry hear that have been shown to be capable of inactivatinglipid A and hence eliminating lile pyrogenicity of bac-Ierial endoto"in includeacid and alkaline hydrolysis, oxidation by hydrogen peroxide, and alk')'lationwilh strong alkylaling agents (some depyrogenalion can be expected 10 occur inethylene oxide sterilization cycles). None of these methods is used on an indus­trial scale.

Removal of endotoxin from manufacluring equipment or materials can bedone by appropriate washing and rinsing procedures. Pyrogen-free water can beoblained by several means. The oldest and most effective method is distillation-.he heavyweight lipopolysaccharide molecules (MW of around 106) are leftbehind when water is rapidly boiled in a still. Reverse osmosis can remove 99.5to 99.9% of water's endotoxin load in a single pass. For lhese reasons, distilla­tion and reverse osmosis arc the only two mclhods of preparing Water for injec­lion allowed in the pharmacopoeias. The pharmacopoeial requirement for Warerfor injection is that there should be no more than 0.25 EU (endotoxin units}/mL.

Removal of endotoxin from low to medium molecular weight drugs andraw materials can be done by ultrafiltration, membrane filtration. depth fihration,or treatment with activated carbon [3 J. These processes are complicated by thecomplex fonns thal can be adopted by bacterial endotoxin under differingcircumstances. In its smallest fo.nD. bacterial endotoxin is likely to be lipopoly­saccharide arranged in a micellar structure wilh hydrophilic side chains on theoutside. Retention by hydrophobic membranes requires these aggregates to bebroken up. These aggregates are retained on hydrophilic membranes with 0.025J.lm pore size ratings hut pass through 0.2 J.lm membranes.

a

Dry Hif~t Stifri'izalion and Depyroge.nalion

D. Measurement of Bacterial Endotoxins (LAL Method)

113

In older editions of the pharmacopoeias. pharmaceutical products and medicaldevices could only be categorized as pyrogenic or nonpyrogenic by the ill VillO

rabbit test. This type ortesl'jng is being progressively replaced by an ill "ilro lestspecific for bacterial endotoxin. This is Ihe LAL (Limulus amoebocyte lysate)test.

The LAL test depends upon a reaction between endotoxin and a "c1ottable"protein conlained wi!.hin Ihe amoebocyte cel.ls of the blood of the horseshoe crab(limuluJ polyphemus). The specificity of the rea<.ion has been allribOled [4] toa complex cascade of enzyme mediated reactions.

TIle "standard" LAL test is based on !.he formation of a semisolid gelbetween LAL and baCleriai endotoxin and is conducled on the end·point princi­ple. LAl reagent is supplied with an identified sensitivity. e.g.. 0.03 endotoxinunits (EU) per mL. This means that when mixed with an equal volume of thematerial under lest a gel or clot will form if the material cont:tins 0.03 EU/mL orgrealer. When it is necessary to quantify endotoxin concenlration in a material itis usual 10 test a series of doubling dilutions against the reagent in temperature·controlled conditions. The greatesl dilution that gives a positive (formation of agel thai withstands inversion of the reaction tube) is the end point. and theconcentration of endotoxin in the malerial can be calculated by muhiplying thedilution faclor at the end point by the sensitivity of the LAL reagent

Valid assays require careful internal standardization. The first USP oolchof reference standard endoloxin (RSE) had a potency of I EU per 0.2 ng. but il is10 me potency of RSE and not to weight thai secondary standards (eSE. cenifiedor calibrated standard endOioxin) are related. Within each series of end-poinlassays it is usual to sel up a series of dilutions of CSE in concentrations of twotimes. equal to, half of, and one-founh of the labelled claim of Ihe LAL reagentbeing used. Valid assays require clOiting from the two higher concentralions ofCSE and absence of clotting from the two lower concentrations. A negalivecontrol and a positive conuol, in which the material under test is spiked withCSE al two times the labelled claim of the LAl reagent. are also required foreach series of tests.

With doubling dilutions the accur.1CY of the melhod can be no better lhanplus or minus one twofold dilution; in other words. a derived value of 3 EU/mlshould properly be reponed as greater Iban 1.5 EUlmL bUI less than 6 EUlmL.When average values from replicate series are required. the exponential nature ofthe dilution series (Le.• 1: I, 1:2, I:4, etc.) obliges the use of geometric means.Geometric means are calculated by multiplying the individual estimates andfinding .he nih rool. lhus

n..JaJ xu-z ... xan

114 Chaptet 5

In practice this means determining the logarilhm of each derived end pointand dividing the sum of these by the number of end points. The geometric meanis the antilog of the mean log end point.

Other LAL methods are available based on turbidimelty and colorimetry.Reaclion mixtures become turbid as gels. or dots foml between LAL and bactc·rial endotoxin. Turbidimetry (sensitive to 0.001 EU/mL) is more sensitive fordetecting bacterial endotoxin than gel clot assays (sen ili\'c to 0.03 EU/ml)because turbidity is discernible at low concenLr81ions of endotoxin at which firmgels do not form. The rate at which turbidicy increases is proponional 10 theconcentralion of endotoxin in the material under test. This principle may beapplied in end-point or kinetic assays.

Wilh turbidimelric end-point assays, turbidity is measured afler ~ fixedincubation period. By inclusion of standards aJongside dilutions of the malerialunder test. a standard curve can be created and the endotoxin concenlration canbe read off this curve for the material under lest. Equipment using microtiterplates is commercially available to minimize the amounts of LAL reagent neces­sary and 10 maximize the number of test selS per set of standards. The period ofincubation over which turbidimetric end-point assays is conducted is critical.All samples will be equally turbid with overlong incubation: if incubation timesare too short none of Lhe samples will be measurably turbid It is not possible toSlOp the reaction to take the readings.

The turbidimetric principle and automated microliter plate reading appa­ratus are more commonly used in the kinetic rather than the end-point mode.Turbidity readings of each reaclion mixture are taken at frequent intervalsthroughoul an incubation period. The logarithm of the time (the onset time)tak.en to reach a specified level of turbidity is inversely proponiQnal and line~lrly

related to the logarithm of the concentration of endotoxin in the material undertest. Standardization is necessary with each series of assays. This approach isonly practical for routine application using microprocessor-controlled equip­ment.

Chromogenic methods rely on the fact Ihat there are enzymalic reactionswithin the process of gel fonnation. Gel fonnalion in simplest terms is 3 Iwo·stage process. First there is the activation of a previously inactive cloningenzyme by reaction with endotoxin. and second there is the formation of a c101by a reaction between the activated enzyme and a coagulen substrate. In chro­mogenic methods the coagulen substrate is replaced by a synthetic analogcontaining a chromophorc. When attached (Q Ihe synthetic substrale the chro­mophore is colorless. but when cleaved by the activated clotting enzyme it turnsyellow. TIle rate of color production is proportional 10 Ihe concentration of bac·terial endotoxin in the material under test. The reaction is pH sensitive and canbe stopped by addition of acid.

Dry Heat Sterilization and ~pyrogenation 115

The methods of chromogenic LAL testing are very similar to those for tur­bidimetric LAL testing. Their advanl'ages over gel e101 and turbidimetric melh­ods are in relation to the range of products with wh.ich Ihey can be used. In allthree melhods. there are broadly two siages Ihat are potentially subject 10 inter­ference or inhibilion from the material under lest The firsl is the aClivation ofthe cloning enzyme by reaclion with endolox.in; this is common 10 aU Ihreemethods because il is this reaction that is specific for eodoloxin. The secondsrage of the methods has only to do wilh Ihe deveJopmenl of the first-stage reac­rion such thal the progress of Ihe first-slage reaction can be delected and moni·lDted. The second stage reactions in\'olved in Ihe fom181ion of Ihe three-dimen­sional matrix of the gel upon which gel dOl and lurbidimelric assays are basedare far more complex and lherefore far more subjecl to interference Ihan thecomparatively simple cleaving of the chromophore in Ihe chromogenic methods.

II. APPLICATIONS OF DRY HEAT STERILIZATION

Dry heat sterilization is available as both large-scale and small-scale processes.Oven steril,ization of labonuory equipmenl and medical instrumenLS is orten themost reliable and economic method regardless of scale but is usually found inlaboratory-scale and hospital-scale openuions. Larger-scale industrial ovens areused in pharmaceutical manufacture, more often in associalion with sterilizationof int'ennedialeS than with slerilil.8.tion of finished products, for instance. thebulk sterilization of petrolatum bases for sterile (mainly ophthalmic) oinlrnent.liiand sterilization of powdered excipienls in shallow trays (e.g., sodium cilrate) forsubsequent aseplic blending. 1be larger-scale processes usually operale to ster­ilize only, because Ihe higher temperatures required for the dual steriliza­tionldepyrogenation funclion would be prohibitively long.

Dry heat tunnel sterilizers are only found in large-scale processes. Theirmain applicalion is in the sterilization and depyrogenation of glass primaryproducl~nlact containers (boldes. ampoules, vials) prior to aseplic mUng andsealing.

A. Ovens and Tunnels

Slerilizing ovens are nOl intrinsically complicated. Figure I is a diagrammaticrepresentalion of a fairly Iypica) sierilizing oven. HEPA-fillered air (see Chapter8) is heated by passage over eleclric heating elemenlS~ heal is U1UIsferred fromI.he air to the product by forced convection.

There are four stages involved in oven sterilizalion. They are (a) drying.(h) beat-up, (e) exposun:, and (d) cool.<Jown.

In the drying stage, moistun: is driven off lhe producl 10 the atmosphereuntil the air temperature in Ihe oven is approximately SO·C, at which poinl a baf-

116

AIATCATMOSPHERE HEPA FilTEAS

TEMPERATU~E QSENSORS

HEATINGELEMENTS

Chapter 5

AlA IN

•

THERMAL PROFILE

~ --- .~TIUE ABOVE 200"C ...;• •• •• •

• •

fig. 1 Typical sterilizing oven.

III 'N ,,. 'Ill 1'" ~~ flO XI)

TIME 1m00000000luJ

fle closes to :lilow the temperature within me sealed oven 10 reach its operatinglevel. A positive pressure is maintained within the oven throughout the cycle.

Exposure is timed from the moment the sterilization thermal ~nsor locatednear the lOp of the oven reaches the set temperature. At the end of the timedexposure period the heating elements are switched off and cool-down begins. Insome ovens. cooling may be accelerated by forced passage of cold HEPA­filtered air over lhe product during the cool-down phase.

Each stage of oven sterilization has some considerable drawbacks associ­ated with imrinsic low heat transfer rates from air (0 product. Ineviwbly thismeans slow heating up and cooling down, a problem !.hat can extend cycles froma nominal 2 h to more than 3 h for practical purposes. A second drawback has todo with lack of unifonnity of temperature within !.he oven. Hot air has a ten·dency to stratify and to penetrate only poorly around masses of cooler materials.

, I

Dry Heat Sterilization .and IRpyrolenation 117

/- 'I',,, ,, ,, ,, ,, ,, ,, ,, ,, ,, ,, ,, ,, ,, ,, ,, ,, •, ,, ,, •, ,, ,, •, ,, ,, ,, ,., l •. , ,

~

.50

300

'50

100

50o 10

FIRST SERIESOF PROBES

20 30 ..TIME lminutes)

SECOND SEAlES

50

lHlRD SERIES

Fig. 2 Thermal profiles through LAF tunnel (progressive).

Compensating for these difficulties often forces the actual operating lempe-ra­tures of oven sterilization processes to considerably higher levels than the nomi­nal temperalUre. 1be potential for heal damage and even charring of Ihe materi­als being sterilized from a combination of these drawb3cks needs no funherelaboration.

Dry heal tunnel sterilization is a continuous process in conlTast to the balchprocesses in ovens. Sierilization and depyrogenalion in tunnels exemplifies theadditive nature of thermal processes; for most of Ihe time spent in Ihe tunnc,1 theproduct is being subjected to rising temperatures (Fig. 2) ralher than being held81 a constanlly maintained exposure temperature. The temperature of the tunnelis held canSlan', with the temperature of the product changing as il proceedsthrough. Energy is not being lost to healing and reheating between cycles as it iswith ovens.

Figure 3 is a roughly to scale comparison of the two main types of steril­izing tunnel in industrial use, radiant-heat sterilizing tunnels and the morerecently developed laminar airflo\\ (LAP) lunnel sterilizers.

Radiant-heat sterilizing tunnels have had e1(tremely wide usage in pharma­ceutical manufacture. Infrared heaters. located in the roof of Ihe tunnel, heat sur­faces of product items passing through the tunnel and internal surfaces of thetunnel ilSClf. The heat is then disseminated throughout the produc,t items by

118 Chapter 5

RADIANT HEAT TUNNEL

:::::..~..:.: : : : :..... :'F·:··:J2.::O~..~ YW.$"

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+ "' ..

D D DSTEAU2I'..o 10f.l: DR_

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lAF TUNNEL

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Fig. 3 Sterilizing tunnels.

radiation. conduction. and turbulent airflow. They are, however, quite large. beltspeeds are slow. product heat-up may be less than uniform. and they may createparticulate problems. That is not 10 say lhatlhese problems cannot be controlled.

Excessive numbers of particles may be generated in radiant-heat sterilizingtunnels from deterioration of the heating elements and the moving belt. and fromabrasion between moving product items. Panicles are not generally effectivelyremovable from this type of lunnel. although performance can be improved byinsrallation of HEPA filters at the tunnel entronce and at ilS junction with thecooling zone. It is of course quite standard to find the cooling zone equippedwith HEPA filters to prolecl me sterility or l.he ilems mal have passed throughlhe lunnel en roule 10 an aseptic filling room.

In LAF tunnels air is healed by passage over heating elements prior (Q

HEPA filtralion. so at least one source (deterioralion of the elements) is excluded

Doy H..t Steriliulion ..... OtP\'1'og....tion 119

from the sterilizer at source. The sweeping action of the laminar airflow thenconuibutes (0 the removal of omer panicles generated within the tunnel. Heatlnlnsfer is also much fasler. conlributing to a lower risk of product conlaminationfrom particles purely as a result of shoner throughput times (around 50% of thelhroughput time expected from radiant-heat tunnels).

III. TEMPERATUREfTlME CRITERIA FOR STERILIZATION ANDDEPVROGENATION

Sterilization Icmperalureltime combinations for dry heal are directly analogousto those for sterilization by saturated steam. The options are compendial cycle •overkill. or a validated 10-6 slerility assurance level (SAL). The USP is curi­DUsly ambiguous aboul SALs for dry heat processes; although accepting 10-6 as astandard it stresses that the approach is arlen (0 achieve SAL.s of 10-12. This isbecause so many dry beat sterilization treatments are also intended to inactivatebacterial endotoxins (see below).

Compendia) cycles are only applicable to ovens because there are toomany variables in tunnels (belt speeds. stoppages. preceding malerials. elc.) 10 beencompassed in simple generalized process specifications. Compendial cycles inthe Brill~h Pharmocopoeia require 160'C for 2 hs. 170'C for I h. or 180"C for30 min. The USP refers rother obliquely 10 temperatures in excess of 250"C.

In the absence of precise knowledge of the dry heat inacliv31ion Ch3r'J.cler·istics of the actual microbiogical contaminants of the product. Lhe referencemicroorganisms for overkill (twelve D-values) and SAL largels are spores of B.sub/ilis var nigtT. A D170-value of al least 1.5 min may be assumed for Ihesespores. For dry heat temperatures olher than 170·C. mere is a concept compara·ble to the F0 concept for steam sterilization. h is lermed the F II coneepl and ref·efences lethality to equivalent times al 170·C. TIle units of FI-rvalue are minulesor seconds. Calculations of FHuse the same equalions 3.'0 calculations of F() bUIthe l·value of 20'C is substilUled in the case of dry heat.

Consider the BP compendial cycle of 170'C for I h versus 3 D 17o-value of1.5 min for spores of B. sub/iliJ var niger. Clearly some fony D-values areobtainable exclusive of the lelbal input during heat·up and cool·down. This isfar in excess of the twelve D-values necessary to claim overkill and would per­mit a 10-6 SAL 10 he obtained for ilems conl3minaled by up 10 1()34 colony­forming units prior to sterilization. The FH values of the other two compendialcycles are 9S min for the 180·C cycle and 38 min for the 6O'C cycle-aU far inexcess afthe minimum nece sary.

There are no compendia! cycles recommended for endotoxin reduction.The standard quoted in the USP and being enforced by the FDA is that a claim todepyrogenation should be supponed by evidence thai endotoxin presenr on IheprodUCI prior 10 trealmenl has been inactivated to no more than JIlOOO of the

120 CNpler 5

original amount (three log 10 reductions). The minimum time required [0 obtainthree loglO reductions of endotoxin at 170·C has been variously reponed to befrom 3 h to over 66 h. Six )oglo reductions have been demonstrated at 250·C. Insummary, depyrogenation cycles have to be developed and validated empiri­cally. However, the USP recommends a temperature of at least 250-. Consid­ering that the FH value of a S-min cycle at 250·C (regardless of any heal-up orcool-down lethality) is an unbelievably long lime. it is safe to assume that anydry heat cycle thai achieves depyrogenalion will more than achieve any contem­porary sterility assurance slandard.

IV. VALIDATION AND ROUTINE CONTROL OF DRY HEATSTERILIZATION

A. Validation

Beyond the usual engineering aspects of validation with regard to specifIcation.installation. commissioning. qualification, and calibration, the two importafllaspects of validation of dry he,at processes are themlal validation and endotoxinvalidation. Biological studies with spores of 8. subliliJ var niger are rarelynecessary.

The purpose of thennal validation is to demonslrate that heal is uniformlydelivered to aU pans of the oven or tunnel load. With ovens the principles areexaclly the same as for autoclaves. The exercise of thermal validation of tunnelsis usually more complicated due to having to place themlocQuples in productitems. which are then allowed to be carried on the moving belt through the tun·nei. The difficulties however are not insunnountable. The lowest temperaturesin the load should be determined rigorously from one side of the tunnel to theother. from the leading edge of a new load of product items to me trailing edge.It is important Ihat lhe coldest spots be identified for subsequenl endotoxin inac·tivation studies.

For endotoxin destruction. items must be spiked with bacterial endotox.inand passed through preferably shortened (for lunnels this means faster bellspeeds than specified) or c,ooler cycles than the proposed process specification.The spiked items should be placed in previously idemified cold spots. They arethen retrieved and assayed by the LAL method for remaining Lraces of endo­loxin. The key to doing this successfully is to be sure that each item is spikedwith at least I(X)() times as much endOloxin as the minimal quantity detectable bythe available recovery and assay techniques.

B. Routine Control

Routine conlcol of ovens should be by temperature and time. A floating probeshould be placed in lhe load and recognized as the critical measure. Forced air

Dry Hut Steriliution and ~py,olenation 121

fans and over-pressurization systems should be equipped Wilh fail-safe warningsysaems 10 ensure lhcir continuing function.

Tunnels ~uire Ibe bell speed '0 be specified and controlled. Wben linkedwith other fixed-speed equipment such as washers and fillers. changes in tunnelbelt speed often become self-evident. Temperature monitoring should renect thecoolest zone in the tunnel and include a reference sensor related to the tempera­ture conditions recorded in the product during validation. Aoating probes in theproduct are not usual (for reasons of practicalily).

Rouline use of biological indicators is nOI necessary. Routine endoloxincontrols using spiked samples have never been seriously proposed.

REFERENCES

I. Emsi. R. R, (1977). Sterilization by heat. In Disinf~cl;on, Sl~rili:afion and Prrsu­vafion (5. S. Block. ed.). Philadelphia: Lea and Febiger.

2. ludwig, J. D.• and Avis. K. E. (1990). Dry hem inactivalion of endoloxin on the sur­face of glass. Journal ofParenfuaJ Sci~na and T«hnoJog)' 44: 4-12_

3. Weary. M.. and Pearson. F. (1988). A manufacturer's guide to depyrogenation. 8;0­pharm (April 1988): 22-29.

4. Dawson. M. (1992). Endotoxin Il:sling of medical devices. In B;oburd~n in M,.dioaJDe\'k~s and Surgical Dressing Manufach"e. Proceeding.1li of a EUCOMED Confer­ence. March 23f24. 1992. Brussels. Belgium: EUCOMED.

, I

6Sterilization by Ethylene Oxide

I. Inactivation Effects on Microorganisms and Microbial Populations 124A. Effects of Gas Concentration on Microbial Response to

Elhylene Oxide 125B. Effects of Temper31Ure on Microbial Response to Ethylene

Oxide 125C. Effects of Humidity on Microbial Response to Elhylene Oxide 125D. Other Factors Affecting Microbial Response to Elhylene Oxide 126

n. Applications of Elhylene Oxide Sterilization 127m. Ethylene Oxide Sterilization Processes 129IV. Validation and Rouline Control of Ethylene Oxide Sterilization 133

A. Validation of Ethylene Oxide SteriliL1tion Processes 135B. Routine Control of Elhylene Oxide Sterilization 145

v. Health and Safety 147

Of lhe several methods of sterilization that rely on inactivation of microorgan·isms to meet their objective, sterilization by exposure to ethylene oxide is by farthe most difficult to conltOl. The main advantages of ethylene oxide as a sleri­lant do nOllie in speed. simplicily. or reliabilily of control bUI r:llher in Ihe rangeof malerials lhat can wilhstand treatmenl wilhout damage. It is not used forpharrrulceuticals. It is. however. an extensively used alternative 10 irradiation as

123

124 Chapter &

a method of cold sterilization for heat-labile medical devices. and pharmaceuticalpackaging components. InduslriaJ-scaie and laboratory-scale technologies areavaiJable.

Ethylene oxide is a cyclic ether (C2"40) with a boiling point of lO.re atatmospheric pressure. II is colorless and virtually odorless. In its pure formelhylene oxide is highly flammable in air; in pankular circumSlances it may beexplosive. Its first practical application as a biocide was as recently as the )940swhen it began to be used for disinfeslalion of food crops. It is still used toreduce me microbial contamination of bulk spices. As with gamma radiation. itsdevelopment as a sterilization process for medical devices went hand in handwith the increased availability of biologically inert plastics, Until radiation ster­ilization became competitive in the 1960s and 1970s. it was lhe single mostimponant method of industrial-scale cold sterilization. In temlS of volume ofitems sterilized it bas now been overtaken by irradialion, partly because of sim­ple economic reasons and partly because it has been found 10 be carcinogenic.

I. INACTIVATION EFFECTS ON MICROORGANISMS ANDMICROBIAL POPULATIONS

Inactivation and death of microorganisms resuhs from alkylating effects onsullbydryl. amino. carboxyl. and hydroxyl groups within the cell. Ethyleneoxide replaces labile bydrogen aloms in these groups. Lethal effects are throughblockage of reactive sites on metabolically ac.tive molecules. Comparison ofactivation energies has shown that DNA and RNA are the most likely targetmolecules. Unlik.e most other chemical sterilanlS. which are several thousandtimes more active against vegetative cells than spores, the resistance of spores toethylene oxide and other alkylating agents is less than ten times greater than theresistance of \'egelal'ive organisms. For instance, spores of BadllllS stearolher­mophi/us and Clostridium sporogenes have been shown (0 show a very similarresponse to vegetative cells of Slreploccus faecium when exposed under identicalcondiLions (I].

The kinetics of inactivation of microbial populations exposed 10 ethyleneoxide are exponential [2] when the logarithm of the number of survivors is plOl­ted against time wilh all o!.her factors (e.g., gas concentration, humidity, temper­ature) held conslanl. Shouldered curves have been occasionally nOled; instancesof "tailed" inaClivation kinetics have been ascribed 10 clumping or environmen­tal protection. Good experimental data arc not easily obtained. Experimentaldesign should concentrate on rapid attainment of the gas concentration intended.Inactivation from residual sterilant may also lend 10 misleading results.

D-values have only a very Limited \'alue for comparing !.he resislance ofvarious microorganisms and various ethylene oxide processes. This is becauseinactivation characteristics are subject to the influence of numerous olber vari-

Sterilization by EthyJene Oxide 125

abIes that are unavoidably part of ethylene oxide sterilization technology, Thecomplexity of inleraclion belween gas concenlTation. lemperature of exposure,humidity during exposure, pressure during exposure. and the condition of themicrobial populalion prior to exposure has not been adequalely described byIheory.

A. Effects of Gas Concentration on Microbial Response to EthyleneOxide

The effects of lhe concentration of elhylene oxide on inaclivation of microor­ganisms are quite straightforward. Within limiting concentrations. the effect ofdoubling the gas concentration doubles the rate of inactivation. Concentrationsof less than 300 mgIL are insufficienl to achieve sterility wilhin practical processtimes. Very high gas concentrations imply very high pressures, and the gas lawsdictate that an increase in temperature is necessary within a sl'erilizer at constantvolume to mainmin equilibrium conditions at increased pressures, possibly toohigh a temperature to meet the intended cold sterilization purposes. In practice.gas concentrations are usually within a range of 500 to 800 mgfL. which is apractical compromise among s1erilization effectiveness and process time and forthe constrainls imposed by available technology.

B. Effects of Temperature on Microbial Response to EthyleneOxide

Alkylation reactions respond to temperature in the same way as normal chemicalreactions. Under constant conditions and within ahe range of limiting gas con­centraLions., ethylene oxide sterilization follows firsl-order chemical kinetics, i.e.•Ibe rale of inactivation is approximately doubled (the D-value is halved) forevery 10·C rise in rempe-ralure. The lowest temperature al whkh ethylene oxidesterilization is theoretically possible is the temperature at which the gas liquefies.which is lO.7·C at atmospheric pressure. Upper IimilS of temperature are of lessimponance, because the whole point of using ethylene oxide is 10 achieve coldsterilization. Other restricting factors at high temperatures are polymerizationand the pressure rating of ethylene oxide sterilizers. Industrial-scale processesnonnally operale within a cold sterilization range of 5010 6(YC.

C. Effects of Humidity on Microbial Response to Ethylene Oxide

Humidity is the single most important factor influencing the effec1s of ethyleneoxide on microbial populalions. Ethylene oxide is quile simply ineffectiveagainst dehydrated microorganisms in a dry environment.

Ethylene oxide is a chemical sterilant and mus11herefore come inlo conlaetwith target molecules (DNA and RNA) that are physically located within the"heart" of the cell, or within the core of bacterial spores. Waler acts as a carrierof ethyJene oxide Lhrough permeable barriers. The water activity of the micro-

126 Ch~pler 6

biaJ cell and the relatin humidity of the environment in which it finds itsclf areof critical imponance to water movement and to lhe penelJation of chemicalSleritants to their target sites. 11le siruation becomes complex. when it is under­stood that ethylene: oxide can itself increase the permeation of water throughpermeable barriers.

Most experimental work on the effects of humidity has been done onbacterial spores. Bacterial spores can survive over a greater range of wateractivities and moisture contents than vegetative cells. In particular they canwithstand considerable degrees of desiccation. Although spores are nOI octivelydividing microorganisms. they are not completely metabolically inactive. Ernstand Doyle [3 J postulated !.hal dynamic equilibria exist between spores and theirimmediate environment. determined in the main by the number and types ofactive sites on the surfaces of the spores. Active sites become physically with­drawn from the surfaces as the spores dehydrate. Spores with higher moisturecontents and therefore greater numbers of exposed active sites exhibit higherrates of exchange of molecules with lheir immediate environment than do dryspores with low water activities.

TIle equilibrium between the spore and me environmenl can operdte in twodirections, i.e., with water moving predominantly from the environment into thespore or conversely predominantly out of the pore inlo the environment. 1bedirection of the equilibrium as it affects water movement is for the mosl pan afunction of the relative humidity of the environment. Wilh relatively high envi­ronmental moisture, waler will move into the spore. The concenlration gradientbetween the moisture content of the spore and the moisture conlent of the eovi·rooment acts as a driving force in accord with Fick's laws of diffusion. At lowenvironmental relative humidities water will move Qui of the spore inlo the envi­ronment.

The significance of this model is that it describes Ihe optimal situation forwater permeation into the spore and therefore ethylene ox.ide permeation to itstarget sile as a function of the moisture contenl of lhe spore and the relativehumidity of the environment during exposure. The rate of microbial inactivationlherefore increase (a long as all other factors are held constant) with increasedrelative humidity during exposure. Kaye and Phillips (4) demonstrated a 33%RH optimum for microbial inactivation as a result of exposure to ethylene oxide.tn practical SilUslions it is bener to err on the side of too much rather than tooliute moisture. With industrial-scale ethylene oxide sterilization. humidity IC\'clsare usually in the range of 50% to 60% RH. 'The upper limit is usually dictatedby deleterious effects on packaging.

D. Other factors Affecting Microbial Response 10 Ethylene Oxide

Any factor that prevenLS penneation of ethylene oxide to its target site withinthe cell is capable of adversely influencing the rate of inactivation. Such factors

Storilizilion by Elhylene Oxide 127

may include organic matter or inorganic cryslalline material. Reduced sterilantpenetration bas also beeD noted Wilh clumped cells. These effects are probablydue as much to physical factors as to chemical ones. Allhough ethylene oxidebas a history of use as a crude fumigant. it is suilable as a sterilant only for cleanitems. which components for pharmaceulica) products and medical devices canalways be supposed to be when manufactured according (0 the Good Manufac­turing Practices.

Dadd and Daley [5"] observed thai some microorganisms may have a lim·ited ability to overcome lhe effects of ethylene oxide but Ihal Ihis did not to anygreat extent confer resistance. The spore coal did not cootribute to (he resistanceof resislant bacterial spores except as it constituted an increased number of aller­native target sites for alkylation. Spores did not become as sensilive to ethyleneoxide as vegetative cells until they had fully emerged from inside their sporecoalS.

II. APPLICATIONS OF ETHYLENE OXIDE STERILIZATION

Ethylene oxide sterilization is suitable for both small-scale and large-scale appli­cations. It is primarily a melhod of cold sterilization and has so many associ::uedcomplicalions that it is never used in preference to thennal steriliunion for heal­stable materials. Sterilization by gamma radiation is more rehable than ethyleneoxide for cold sterilization. and it is simpler to control. It is, however, limited bysuitability of materials and only operates on a large scale.

Ethylene oxide is penetrative (but less penelrative than gamma radialion).On an industrial scale this allows devices sealed within primary containers to bepacked into shelf pxks or shippers and paUetized before sterilizarion. Product isnormally sterilized on pallets.

In order to inactivate microorganisms. elhylene oxide must come int'ocontact with target sites in the microbial cell. Even though ethylene oxide isvery penetrative, this is a major complicating factor for any method of tenninalsterilization. Free movement of the gas 10 all parts and internal cavilies of eachitem being sterilized is an essential prerequisite of lhe process. This imposescenain constraints (arguably restrictive conslraints in the case of individuallypacked single-use medical devices) on the design of product items. Ihe design ofpackaging malerials, !.he choice of packaging rnalerials. and the manner in whichprodUCIS are packed in boxes. stacked. palletized, and loaded inlo sierilizers.

A basic prerequisite of produci design is that sealed il1lemal cavitiesshould be avoided for produclS inle.nded for tenninal steriJiz;uion by exposure toethylene oxide. Disposable hypodemlic syringes were among the first medicaldevices to be sterilized in large numbers by ethylene oxide. Syringe plungers areusually fitted with elastomeric rips that seal with an interference fit 10 the inler·nat barrel wall al two diamelers separared by an internal cavity (Fig. 1). The

128 Chapler 6

Fig. 1 Internal cavilies in hypodennic syringes.

region between the two "lands" of the plunger tip is usually considered the mostdifficult to sterilize because of uncenainty concerning gas peneuation. Althoughthis region is smelly a sealed cavity, ethylene oxide may gain access if theplunger tip is made from a gas-penneable material While natural rubberremains the commonest malerial for manufacture of plunger tips, the readyabsorption of ethylene oxide into this material prevents poor penetration from

Sleriliution by Elhylene OXide 129

restricting gaseous sterilization of syringes. However. other elastomeric materi·als, whicb may not be as pemleable to ethylene oxide. are becoming competitivewilb rubber and .... begiMing to offer adv3llIBges in the .....s nf malerials coSlSand consistency of quality.

Penelration is important to the selection of packaging materials wherethese are the primary barrier to microbiological contamination of the device afterteriUzalion and before use. Most often. cold-sterilized medical devices are

packed in flexible rather than rigid primary containers. Permeable materials arenecessary when ethylene oxide is the method of sterilization. Paper is com­monly used because it has a history of successful usage, because Ibe lechnologyfor printing on paper is readily available, and because it is cheap and recyclable.On the Olber band. it is not always impermeable 10 microorganisms~ it is opaque.and it may tear easily on devices with sharp edges or when subjected 10 pressuredifferentials or when carelessly handled or transponcd. Altemative permeablematerial that avoid the disadvan13ges of p3pe.r, such as spun-bonded polyolefins(Tyvek), are very expensive. Almost any material can be sealed 10 any othermaterial, and therefore composite pack:s made from more 1.h3Jt one material an:common. One part of the pack may be gas penneable, the olher impenneable.one pari opaque and prinmble, the other transparent. The tec.hnology of elhyleneoxide sterilization involves high humidities and pressure changes. The poten­tially deleterious effects of lhese aspects of lhe sterilization processes on sealintegrity are nol insignificant

With some olher medical devices. only the sterility of the internal lumina(fluid palh slerililY) is being claimed. Typically these devices .... sealed 3Ildself~ntained wilbnul 3Ily primary packaging. Some drug delivery systems. forinstance, are sealed at lheir ends, and fluid path sterility only is being claimed.Self.·contained sterile insulin syringes are one of the largesl bulk volume sterilemedical devices in the world. Wim I.hese devices it is necessary 10 vent the capsor seals to allow acce s of elhylene oxide. This creales 3 clear conniel wilh lIlerequiremenl for hermetic sealing or me slerilized device against entry ofmicroorganisms.

One way of resolving this has been 10 ensure mal any venting is achievedonly through ,erilanl-permeable antimicrobial fillers. Allemalively, venl capsmay be designed to have a small unfiltered tonuous passage to the eXlerior matcan be demonstrated empirically to make enlry of the microorganisms inlo theOuid path improbable. Before adopting such designs. manufacturers shouldaddress lhe choice of ethylene oxide versus olher methods of cold sterilization.With radialion sterilization. for instance, venting is not necessary.

III. ETHYLENE OXIDE STERILIZATION PROCESSES

E1hylene oxide is a product of the petroleum industry primarily produced as astaning malerial for polyelbylene glycol (antifreeze) and other related suh-

, I

130 Ch~pter6

stances. For sterilization purposes it is commercially available in the pure formor as mixtures wilh fluorinated hydrocarbons or carbon dioxide. Pure ethyleneoxide is highly flammable in air. lo sterilization it is normally used in conjune·tion with an inen gas such as carbon dioxjde or nitrogen from a separate source.Commercially available gas mixtures are nonflammable under normal operatingconditions of temperature and pressure. Mixtures of 12% elhylene oxide: 88%dichlorodifluoromelhane. and 20% elhylene oxide : 80% carbon dioxide havebeen commonly used. Environmental issues relaling to the use of fluorinatedhydrocarbons are seriously restricting the use of so-called 12:88.

Pure ethylene oxide is cheaper than gas mixtures. At one time it was usedundiluted. bUI it is no longer possible to have this practice underwritten forinsurance purposes. AU existing processes. whether using pure ethylene oxideplus a diluent or using a gas mixture, operate at a positive pressure to the atmo­sphere. Any leakage of gas from the chamber must therefore be toward dilutionin the external environment rather lhan toward fonnation of an explosive mixturein lhe chamber. Gas mixtures with nuorinated hydrocarbons or carbon dioxiderequire higher operatlng pressuR:s 10 achieve the same sterilant concentrations asdiluted pure elhylene oxide systems.

Industrial·scale elhylene oxide slerilizatlon usually takes place in steelpressure vessels (Fig. 2) equipped with waler or steam jackets to maintain theoperating temperature withln reasonable tolerances throughout the sterilizationprocess or cycle. lnlfinsically the equipment is no more elaborate than that usedfor stearn sterilization excepl lhat the vessels are often considerably larger.Ethylene oxide sterllizers with capacities of greater than t.OOO ft3 (eight or tenpallets) are nOl uncommon. Essential features include some means of evacuatingthe chamber 10 facilitate the introduction of ethylene oxide and steam, and somemeans of vaporizing (usually a heat exchanger) the ethylene oxide mat exists inits liquid phase in pressurized cylinders or drums.

Pure ethylene oxide for use in conjunction with a diluent gas and 20:80mixlUres of ethylene oxide are polentially explosive; all electrical equipment.switchgear. and monitoring and measuring systems used in association withthese fonns of the sterilant must be sparkproof. Serious conslderation should begiven to the location and design of gas stores and sterilization suites in relationto other areas within a factory. in relation to other factory buildings, and in rela·tion 10 the locaJ community. Blow-out roofs, windows. and walls are commonJyinstalled with the intention of channelling the shock waves from an explosion inthe direction of least harm.

The inlernal construction of ethylene oxide steriJizers is uncomplicated anduncluttered. There may be some form of forced air circulalion to prevent slrali·ficalian of the various types of gas prescnt in the chamber during sterilization(sterilant, diluenr.. moisture). There should be the devices or sample ports forcontinuous monitoring and recording of temperature and pressure within ,he

Steriliution by Ethylene Oxide

All

I-S1EAM

~

--

L

'-

APOlA2ER

EIO INERT VACUUMGAS PUMP

Fig. 2 Schematic representation of B typical ethylene oxide sterilizer.

131

chamber. There may be associated equipment to monitor gas concentration. Allelectrical equipment used in association with pure ethylene oxide sterilizers mustbe sparkproored.

In some instances there may be an ancillary chamber in which the productload is equilibrated to a specified temperature and humidity prior to its inlfoduc·­tion into the sterilizer. The intenuons of preconditioning are threefold:

(a) To equilibrate the microorganisms contaminating the product 10 con­ditions of temperature and water activity that are optimal for their inacti­vation by exposure to ethylene oxide.

(b) To equilibrate Ibe packaging (primarily cellulosic materials) '0 Ibeconditions of the slerilizer in order to prevent deleterious equilibria arisingin Ihe sterilization chamber.

(c) To optimize lhe utilization of the slerilizer. If equilibration is nol doneelsewhere it muSI be done in Ihe sterilizer. The residence time of the prod­uct in the sterilizer must lhen be longer than it need be. Preconditioning

132 Ch.-.pter 6

chambers thai do not operate under pressure are cheaper to build and (0

oper.lle than sterilizers.

It is critical to ensure that the length of lime between the removal of a loadfrom the preconditioning chamber and the beginning of its steriliution cycle isrigorously controlled. Il is all too easy, panicul:uly in dry climates with \'t:rylow humidities. for equilibrium (0 be rapidly lost.

A typical sterilization cycle (Fig. 3) begins with evacuation of the loadedsterilizer to a predetermined level. It is most important that sufficient vacuum isachieved. not only because it may affect the seuings for subsequent phases of theprocess but also because adequate air removal must be assured (0 avoid the fur­mation of explosive mix lUres with oxygen.

A known amount of steam is lhen inlroduced and the sterilizer is left 10"soak" for 3 short period. This is followed by injection of the sterilizing gas 10

ilS specified pressure. The sterilizer 3nd its contents are then held under theseconditions for the specified time of exposure, At the end of this period the gas isremoved by evacuation and replaced by air that has passed Ihrough a bacleria­retentive fiher.

,I WK

ATMOSPI EkC PRESSi"R;--_._-_.- .~- _._-----_._.- ._.._...... ..-_._----_.------ ._---_. -_..

RiEJEl)...Ell)

Fig. 3 Typical elhy~ne oxide slerili1..ation cycle.

Sterilization by Ethylene Oxide 133

[n routine sterilization. gas concenlrations of 500-800 mgIL, temperaturesof 50--6(rC. and relative humidities of at least 50% RH are used. The pressurewithin the sierilizer is importanl only as it relates to obtaining adequate elhyleneoxide concentration as dictated by the gas mixture being used. These operaringcriteria are as much a funcrion of the technology of Ihe process as Ihe-y are arencction of the optimal conditions for sterilization. To a great eXlent. the gasconcenLrations. lemperatures. and relative humidities used for ethylene oxidesterilization are dicL:ued by the relationships that govern the pressure and tem­perature of gases at fixed volumes. At temperatures lower than 50·C there maybe difficulty in maintaining the vapor stale of the ga~s in the chamber. Highergas concenlrations achieved through increased pressure may result in condensa­lion of water or ethylene oxide unless accompanied by increasing temperature.

The most important phase of the sterilization cycle is the introduction ofsteam under vacuum. Initial evacuation of the sterilizer is completely unavoid­able. 11 is also potentially deleterious to microbial inactivation. During evacua­tion the load in the sterilizer loses heal. and more imponanlly it loses moislure.Steam injection restores the lemperature and moisture conlent of Ihe load to itscorrect equilibrium. This must be achieved before the sterBant gas is added.There are two main reasons for this:

(a) Ethylene oxide has a greater capacity 10 penelrate through matenalsthan waler has. If Ihe two substances were to be injected inlo a sterilizer althe same time. the ethylene ox.ide molecules would pernleate and diffusethrough the load fastest. leaving the waler molecules behind. Ethyleneoxide is a comparatively poor stenlant in the absence of moisture.

(b) Water reacts with ethylene oxide. If the two substances were to beinjecled at the same time. the effeclive concentration of bolh systemswould be reduced. This would have a deleterious effeci on the rate ofmicrobial inactivation.

IV. VALIDATION AND ROUTINE CONTROL OF ETHYlENE OXIDESTERILIZATION

Sterilization by exposure 10 ethylc.me ox.ide is bounded by at least four variables:

gas concentration. time of ex.posure. temperature, and humidity. II is alsoaffected by product design. packaging design. and lbe composition of packagingmaterials. The shape. size. and materials of construction of individual sterilizers.the location of gas entry pons, and the presence or absence of forced circul3lionmay all influence sterilily assurance. There is no theory lO describe these inter­actions. Validation and rouline control of ethylene oxide steriliZ3tion processesboils down finally to the integration of a1l of Ihese variables by reference 10 bio­logical monilors.

134 Ch.tp'tr 6

Before validation even, very serious consideration must be given to lIlenature of the product that is intended to be sterilized and the type of packaging itis to be presented in. Again. it is only experience and experiment mal can indi­cale and confirm the suilability of particular presentations for ethylene oxidesterilization.

Most of the product-related faclors need only be evaluated on a broadbasis; products compatible with one ethylene oxide sterilization process are gen·erally compatible with mOSI others. Some of the factors that ought to beaddressed include

(3) Choice of materials. Although ethylene oxide is probably compatiblewith a wider range of materials than any other major sterilizalion process,Ihe choice of materials is not without pitfalls. Some polymers show signsof chemical degradation as a result of chemical reaction during steriliza­lion. Some polymers craze in response to gas mix.tures containing fluori­nated hydrocarbons but are satisfactory for other ethylene oxide processes.Physical properties may aller, and dimensional lolerances may be changedIhrough shrinkage or expansion. Materials should be chosen to allowdegassing (aeration) in a reasonable lime frame.

(b) Product design. The product must be designed with the considerationthat the sterilant musl be able 10 penelrale to all pans. Functional toler­ances should allow for the intense (and often abrupt) pressure and temper­ature changes thai arise in ethylene oxide sterilization.

Consideration of packaging materials should be specific to panicular sterilizationcycles and should extend beyond primary packaging materials 10 include thepotential effec[s of secondary packaging (shelf packs or shippers) and pallets onsterility assurance. In some instances these Jatter faclors may need to beaddressed on a sleriJizer.specific basis. where variations have been suspected ofbeing the cause of erratic and unpredictable biological test failures. The impor­tant packaging factors 10 consider are

(3) Choice of materials. There is a wide variety of materials available forprimary packaging (i.e., me packaging that is intended to provide the her­melic environment for the sterilized product} of ethylene oxide sterilizablemedical devices. The permeability of me pack to elhylene oxide and watervapor is of utmost impol1ance. but lhis does not mean that the completepack has to be made from permeable materials. The pack must be able towilhstand pres.sure changes without materials rupture. This may often leadto composile packs made from penneable and nonpermeable webs; otherconsiderations, for instance price. "printability." and appearance, accountfor the large number of composite packs currently being sterilized.

, I

Sterilization by Ethylene O"ide 135

Secondary packaging malerials are usually made from corrugated card­board; the grade and the direction of fluting can have serious effeclS onsterility assurance and on sterility maintenance. Absorption of moisture insecondary packaging may divert the 3vailabilily of elhylene oxide from ilStarget sites. lnsufficienl structural rigidity of secondary packaging maylead 10 damage to primary packaging during or after sterilization with con­sequent loss of sterililY.

(b) Seals. Primary packs must be sealed in a manner that wiU preventmicrobiological ingress. withstand the rigors of the sterilization cycle. andstill be easily openable by the customer at point of use. Within some packsthere may be more than one type of seal. some intended to be opened.others intended to be permanenlly closed. Syringe packs usually direct theuser to the openable seal by a printed instruction. Often this is disregardedin clinical practice, where users have found it easier to burst the packetsopen. The characteristics of good microbiological seals and of equipmentthat is capable of producing consislenl seal qualily can only be evalual,edempirically.

A. Validation of Ethylene Oxide Sterilization Processes

The overall scheme of validation of ethylene oxide sleriliztllion is no differentfrom validation of any other process. With new equipment it requires carefulconsideration of design before specification (Design Qualification), confirmationthat received equipment conforms 10 its specification (Installation Qualif'ic.alion),and continuation that received equipment can perform its specified functionswhen assembled. plumbed in, and linked up to local services (Opemtional Quali­fication).

Process qualification of ethylene oxide sterilization is both slerilizerspecific and product specific. II is particularly directed toward the measurableparameters of the whole sleri,lization cycle. If external preconditioning is used.validation should address the measurable parameters of aU combinations ofpreconditioning chamber and sterilizer. Where one preconditioning chambermay serve two or more sterilizers. or where one sterilizer may be served by twoor more preconditioning chambers. cycie-to-cycJe variability should be minimal.The essence of successful ethylene oxide sterilization lies in Ihe establishment ofa complete process specification with very light lolerances. The integral effeclSof me specified conditions on assurance of sterilily can only be validated byreference to biological monitors.

J. Load SpecijicQlion: The product and its primary and secondary packagingmust be specified. The loading pallem of primary packs ;n'o shelf packs shouldbe specitied. Occlusi()[) of gas-permeable surfaces one against lhe other shouldbe avoided if slerility is to be assured.

a

136 Chapter 6

The loading pattern of shelf packs on pallets mu.. also he specified. Pal­terns thai allow good circulation of gas to all pans of the load. designed with freespace and "chimneys." are best from :1 sterilization standpoint but may conniciwith commercial considerations. Sterilit,y assurance, cycle duriltion. sterilizercapacity. and throughput rates are inextricably linked to validated loading pat­lems for ethylene oxide sterilization. A further factor involved in det~rmining

loading pauerns is the rigors of ethylene oxide sterilization, which can affect thestrenglh of corrugated cardboard. Panial or lotal collapse of a stacked palletmay involve some compromise of sterility. The use of "dividers" 10 give greaterslfUctural suppon to Slacked pallets may impede gas penetrmion. All of thesefactors c::m only be addressed empirically; any significant change to a successfulloading pallem mu t be validated thoroughly.

If more lhan one product is to be sterilized at the same time in the samesterilizer. process validat'ion should be completed for each combinatjon. 1lle lotsize in many manufacturing operations is "tailored" to sterilil..er capacity. and insuch instances it should not be difficult to avoid mixed load. This may be moredifficult for conlmet sterilization operations. Ethylene oxide sterilil..3tion can·tracts should address the validation of mixed loads or prohibit them. Both rouleshave cost implications.

The types of patlets should be specified-wood. aluminum, or some othermaterial. Ethylene oxide is nOl absorbed by metal pallets. but the: same cannotbe said for wood. Wood is commonly used but difflCuh 10 control. Hard woodsmay absorb differently from soft woods. and some pallets may be water satu­rated, while others may be quite dry. Some of the unexpected and unpredictablesterilization failures seen in ethylene oxide sterilil.lllion may be associated withuse of wood pallets. Once validated. change from one IYpe of material toanother should nOl be permiued.

2. Equipment Specification: Any change in process equipment or any intro­duction of new process equipment should be considered for validation. Biologi.cal validation may not always be necessary if there is sufficient physical orchemical evidence 10 demonstT:lte equivalence. All equip~nl should be identi·fied: peciflC:l1ions, drawing. and instruction and maintenance manuals houldbe obtained and referenced in validation docu~nlation. No list of equipmentrequiring validation can profess (0 be comprehensive. All functional equipmentshould be subject to scheduled inspection and prevenlative mainlenance pro­grams. All measuring devices must be calibrated and scheduled for regularrecalibration.

a. External Preconditioning Chambers. External preconditioning cham·bers are nOI required to be as elaborat'e as sterilizers. This may in some caseslead to the belief that they are of only minor significance 10 the assurance ofsterility. This is not correct. Due care and attention must be given 10 the designand specification of external preconditioning chambers.

Steriliution by Elhylene Oxide 137

They may be built to hold more Iban one sterilizer load. but if all loads arenoI to be removed al the same time some alann or device should control thelenglb of time that access doors are left open. This should be minimal. Precon·dirioning chambers should be of sufficient size to allow free circulation of airand moisture to all pans of Ihe loads. The criteria for temperature and humidityspecified for precondilioning rooms should be identical or very close to thoseobtaining in the sterilizer; in practice that means somelhing close to those foundin a sauna but less pleasant. The finish of preconditioning rooms should berobust enough 10 tolerate these conditions. Forced air circulation is ncee.~sary 10

lhe atlainment of uniform conditions; fans should be equipped with alarms toindicate any failure.

Temperature and humidity in the preconditioning chamber should bespecified and continuously monitored and recorded. These criteria should berelated to the temperature and humidilY obtained within the load. The object ofpreconditioning is 10 equilibrate the load to the conditions of the sterilizer; suffi­cient holding time in the preconditioning chamber should be specified to allowthis (0 happen.

A maximum lime limit between removal of a load from Ihe precondition­ing chamber and the commencement of sterilization should be specified. Thismay be particularly impJr1ant in low·humidity locations.

b. Sterilizers and Ancillary Equipment. Allowing for all the normalcriteria that apply to pressure vessels used for sterilization, lhe mOSt imponantconsiderations to be specified for elhylene mdde sterilizers are the devices thatcontribute to the reproducibility and uniformity of control. Forced drculationmay be necessary, and any such devices, as in the case of preconditioning cham­bers, should be specified 10 have alarms LO indicale failure.

Gas concentr.lIion must be specified and demonstratcd 10 be achicved.There are three methods for control and moniloring available. There are direclmethods involving infrared analysis or gas chromatography. However. indirecimeLbods are more robusl and for that reason ought to be specified either on theirown or alongside direct methods. Use of a sensitive. ,"utnerable. direct methodof gas concentation withoUI indirect backup in largc·scale ethylene oxide steril·izers is usually seen to be an unacceplable commercial risk.

1be mOSI commonly used indirect method is by measurement of differen·lial pressure within the st'criliz.er by means of pressure recorders. This melhod ispermissible and valid only when using gas sources that are cenified by the sup·pUer. as it depends torally upon lhe differential pressure arising from ethylenemdde and nOI from some other gas. The gas concentration can be relaled topressure and temperature through Ihe formula

KxMWxPc=---,::---

T

a

138 Chapter 6

where c = gas concentration (mgIL); MlV = the molecular weight of the gas (MlVof ethylene oxide = 44); K ;: a constant, 732.2; P = the increase in pressure ininches of Hg: and T= temperature in -Rankeine (460 + -F).

The second indiceel melhod is by measurement of the weight of ethyleneox.ide delivered from the feed containers to the sterilizer. This method assumesno leakage. liquefaclion, or polymerization of ethylene oxide in the gas linesconnecting the gas source (0 the sierilizer. It is recommended thai the two indi­rect methods be used in conjunction with each other. The weight loss melhodensures that the increase in pressure is in faci due to gas from the ethylene oxidefeed container and not from diluent gas or some other source; whereas the pres­sure method provides an ongoing index. of gas concenlration during the exposureperiod. Gas makeups should be automated and specified to be drawn from theethylene oxide feed tanks. nm from the diluent gas. Gas makeup may be con­trolled by pressure switches or by pressure transducers connected to solenoidvalves controlling gas now.

Direct methods of measuring gas concentration include gas chromatogra­phy and infrared spectroscopy. Both methods are dependent upon small sam­ples. and the problems of drawing these samples should not be underestimated.The location of multiple sample pons should be such that a representative sarn­pie of a hopefully homogeneous gas mixture is obtainable. With most sterilizersoperating at positive pressures, it is nm usually necessary to have any specialmeans of withdrawing the gas. but it is essential to have sample lines heated andinsulated to avoid condensation of gas and water between the sterilizer and theanalytical instrument.

Both gas chromatography and infrared analysis are dependent upon theinstrumentation being calibnued against certified ethylene ox.ide supplies. Gaschromatography results are obtained as mol %. values which must then be con­verted to specifications defined in mgfL. Infrared results are directly correlatedto rngIL.

Chamber temperature should be controllable and monitored Ihroughout aUcycles. The temperature oblained in a load is a function of the initial producttemperature and its specific heat. the amounl of sleam injected. and the effec·tiveness of the insulation or the jacket at prevenr.ing heat loss. Temperatureduring the exposure phase of ethylene oxide sterilization cycles is nOI conlrolledby steam injection into the chamber as occurs in thermal sterilization. Loss oftemperature may be compensated for by steam injection into the jacket. Thecontrol probe is usually located within the chamber rather than within the jacket.and control of temperature is a good deal less fine than in steam sterilizersbecause of the slower response through the jacket. Ethylene oxide sterilizersshould be equipped wilh both jacket and chamber temper:nure indicators. andwith chamber temperature recorders. Sterilizers should be specified with access

a

Sterilization by Ethylene OJdde 139

pons (0 allow chamber and load temperature profiles to be obtained for the pur­poses of process validation.

Measurement of the humidity in the chamber and in the load has the leastsatisfactory lechnology of all the critical parameters of ethyle,ne oxide steril,iza­tioo. Direct measurement with gas chromatography or infrared analysis may notbe reliable in the presence of ethylene oxide. Specification of the pressurejncrease obtained from steam injection is normally thought to be a satisfoctorymean..'i. of conlrolling humidity, bUI it does nOI offer a monitoring option.

The time of exposure may be manually or automatically controlled. Othertime factors must also be specified and alarmed because they may affect sterilityassurance in an unpredictable fashion or they may be indicative of other processproblems. These time factors include the rate of evacuation at the beginning andat the end of the cycle. and the rate of increase of pressure as a result of gasinjection.

For air ingress at the end of the cycle, emylene oxide sterilizers should beequipped with sterilizing fillers. Ahhough the primary packaging material oughtto have been chosen to be a barrier to microbial ingress, there remains the possj.bility that the significanl pressure differentials and air flow rates that areobtained al the end of Sleriliz3tion may be beyond validated tolerances.

The condition of the gas vaporizer is a funher important consideration toethylene oxide sterilization. All cyUnder supplies of ethylene oxide present thegas in liquid form under pressure. which must then be vaporized before admis­sion to me sterilizer. lnadequate temperatures in vaporizers may lead to meinlJ"Oduclion of liquid ethylene oxide into the sterilizer. This is undesirablebecause it will not fulfill its purpose and because of staining and damage toproduct and packaging. Overly high temperatures may lead 10 degradation of theethylene ox.ide with resultant polymer buildup restricting gas flow in the feedlines.

3. Process Va/idarion (Physical): The equipment and the proposed precondi­tioning and operating cycles must be carefully specified in detail and with tighltolerances before starring process validation studies.

Multiprobe temperature and humidity distribution profiles should beobtained for empty preconditioning chambers. and temperature penetration pro­files should be obtained with probes localed within loads. The purpose of thesestudies is to demOnSlrale thai the load is being unifonnly equilibrated to the lcm­perature (and by inference to Ihe humidily) of Ihe sterilizer within the proposedpreconditioning lime frame. Any serious Jack of unifonnity detected duringvalidation sludies should be investigated and corrected (even if only by extend­ing the time of the preconditioning cycle).

It is normal practice to run large preconditioning chambers under opera­tional conditions at all times irrespective of whether they are in ure or not. If it

140 Ch~ptrr 6

is intended 10 use them on an ad hoc basis. validation slUdies should also coverthe time taken for the chamber and the load to attain specified operating condi­tions.

The sterilizer and its associated pipework should be tesled 10 ensure that ilis adequate to maintain lIle positive pressures and vacuum pressures proposed inthe cycle specification. The main thrust of physical validation is through tern·perature profiles.

Replicate temperature distribution profiles (from 3 to 24 sensors per cycleaccording 10 the size of the sterilizer) should be obtained for empty sterilizersoperated to the proposed cycle specification. All parameters should be withintheir specified tolerances throughout validation cycles. For economic reasons aninen gas may be substituted for ethylene oxide in these studies. The AAMIGuideline for Jlldwurial £thyl~lIt! Oxide Sterilization of Medical Devices (6)allows a variation of ± 3·C about the nominal specified tempemture; sterilizermanufacturers (7) claim ± 2·C to be possible. this being a fum:tion of the typesof controller used and of jacket design. At least one empty chamber tcmpcrJturedislribution profile should be run using ethylene oxide to discern whether Ihereare any signifi<.:ant chang.:s when the sterilanl is being injected. Problems withvaporize~ may be revealed by this means.

Empty chamber studies are sterilizer spt."Cific. Further process validationwork is product specific (load specific) and may be done wilh dummy produci ifrequired. If this option is exercised. care must be taken to ensure thai dummyloads are truly representative of genuine product. ScrJp produci is ideal for thispurpose but may not be readily available for high cost products (e.g .. cardiacpacemakers). If good product is used in validation, it should not be released assterile until the validation program has been completed and signed off as satis­factory.

Load configurations and pack.aging materials have a significant effec.t onthe rate of heat transfer into the product. Since it is microorganisms within theproduct that are to be inactivated, it is to the product that the critical processc,onditions musl be delivered. Muhiprobe temperature penet'Tation profiles overreplicate cycles are necessary. A wider lolerance of ± 5-C can be expected 16)due 10 slower response limes. Cold spors (if any) should be identified and cor­rected. or specifically examined in Ihe biological phase of process validalion.

4. ProcesJ Validation (Biological): Biological validation is the most criticalelement of process validalion of ethylene ox.ide sterilizalion. It is the onlymethod of integmting the inleraclion of gas concenlration. humidity, tempera­lure, time, sterilizer effe<;ts. load effeclS. etc. h must demonstrale that the SALbeing obtained is no worse than 10-6• and it muSt demonstrate uniformity oftreatment and it must provide the basis for routine monitoring. It is the onlyguarantee that Ihe specifications for product and for process achieve stcrilily.Some preliminary work done. in laboratory-scale sterilizers may provide favor-

, I

5lorili"'lion by Elhyle... Oxide 141

able indicafions. but in the end lrials must be done wilh actual product in a prtrduetion-scale sterilizer.

1be microorganisms used for biological validation of ethylene oxide Sler­ilizalion are the spores of Baci/lr4s subtilis var niger (Bacillus globigii). Thesespores are quile resistant to ethylene oxide though not the mosl resistant known.They are indicator organisms. The spores are stable over time and mnges ofstorage temperatures, and they are easily recognizable in cullure because theirgrowth has an orange pellicle. However, exactly what constitutes a s3lisfaclorybiological monitor using these spores and what constitutes an unsatisfactorybiological monitor continues to be a subject of debate.

1be (opic of biological validation of ethylene oxide sterilization processescan be subdivided under twO headings, microbiological monitor systems andprocess challenge systems.

3. Microbiological Monitor Systems. The most contentious issue sur·rounding the use of biological monilors for validation and routine control ofethylene oxide sterilization is that criteria for standardization h3ve never beendescribed sufficiently well for there to have been international acceplance in themanner that physical and chemical standards have been accepted. Standardiza·tion has been allernpled by various organizations, for instance the USP and theU.K. Depanment of He.lth.

All attemplS at standardization have agreed that cenain general character·istics of biological monitors should be specified: these are to use a recognizedstrain of microorganism. to specify lhe number of microorganisms per monitor.to specify a D·value. and to specify an expiry date.

Only the first of these characteristics (use of spores of B. subrilis var nigerATCC 9372 or NCfC 1(073) is without some form of complication with regard10 erhylene oxide monilors.

Even the standardization of numbers of spores per monilor is nol straight·forward. The numbers of spores recovered from monitors is not Iik.ely tocorrespond to Ihe numbers inoculated. The choice of carrier, the method ofloading the spores onto the camero and the methods of spore removal andrecovery may influence differences from the specified number. For instance.because of the fibrous nalUre of papers, it is easier (0 remo\'e spores fromaluminum carriers than from paper carriers. This does nOI necessarily mean thataluminum camers are better than paper carriers for the purposes of monitoringsterilizalion cycles. Other variables relaled to adherence include "wcttability"and the speed and manner in which a spore suspension spreads across thesurfaces of a carrier, perhaps spreading evenly, perhaps fonning clumps. Thismakes il difficult to translate a specified number of spores per carrier into aprocess for preparing consistenl biological monitors.

CommerciaUy available spore strips are usually intended 10 have 106

viable recoverable spores per strip. They may be loaded onto carriers by indi-

142 Chapter 6

vidual inoculation or by running a carrier Slrip lhrough a bath of spore suspen­sion at a controlled fate.

The response of particular microorganisms to panicular sterilization pro­cesses is usualJy expressed through the D-vaJue. However, the D-value of sporesused to monitor ethylene oxide sterilization processes is of less relevance topractical sterilization tban D-values of microorganisms versus other sterilizationprocesses. If biologic,al monitors were to be used in conjunction with gammaradiation sterilization (DOling that this is nOI necessary), the D Hrvalue versus thesingle parameler of absorbed dose would be directly relatable across all coball­60 gamma irradiators. Dr values for thermal sterilization processes are trans­ferrable from one autoclave, oven, or tunnel to another. With ethylene oxide thisis not the case; the D-value of a spore population is relevant only to the condi­tions of gas concentration. temperature. humidity, time of exposure. and gassingup and degassing times for which it was delennined.

Auempts have been made to standardize at least lhe conditions in which D­values may be detennined. AAMI (8) have specified a sterilizing vessel. lermeda biological indicator evaluator resistometer (BIER vessel), which allows rapidattainment and termination of exposure conditions in a precise and accuratemanner.

The time 10 reach target gas concentration must be less than 60 s. and gasconcentration is specified as 600 mg/L ± 30 mg/L at 54'C + I'C and 50 10 70%RH. 11:te time to exhaust a BIER vessel must be less than 60 s and accurate to± 10 s. It would be an unusual production sterilizer that could confonn to thesecriteria.

In addition to these fundamental problems of translating ethylene oxide D·vaJues from one situ31ion to another. the D-value obtained even in the best­controlled BIER vessel may be affected quile significanlly by Ibe melbods usedto prepare the monitors and the methods used to detennine resistance.

Most significanLly, biological monitors prep.u-cd at different levels ofdryness do not respond in the same manner to elhylene oxide inactivation.

In other words, the D-vaIue is a function of the previous history of themonitor as well as of the sterilizer in which it was determined. The compositionof the fluid from which the spores were dried may also influence D-vaJues bycoating Ihe spores with a layer of material through which moisture or ethyleneoxide may have restricted permeability. This also gives importance to the con­ditions in which the spores were grown (complex or defmed media) and washed.Graham [101 quores D-values of 2.4 min and 3.5 min for the same sirain of B.subtilis var niger grown in different liquid media and ex.posed to ethylene oxideunder identical conditions.

Finally. the requjrement to specify an expiry date implies that something isgoing to deteriorate or change over time. The most obvious characteristics arenumbers of spores per monitor (do the spores die over time? do they lose adher-

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144 Chapter 6

TIle number of spores per strip and the number of trips per load should forvalidation purposes be related in some way to the bioburden of the product.What type of relationship this should be is Jess obvious. lllere are (wo broadappro3Ches to this: the first is 10 relate Lhe microbiological challenge on eachspore slrip to the average bioburden on individual products and the required levelof slerililY assurance (SAL); lhe second is 10 relate the total microbiologicalchallenge in the sterilizer load 10 the total bioburden within the sterilizer.

80th approaches require some estim.l11ion of product bioburden. 11 is notadvisable for elhylene oxide sterilization processes to be validated without at

least some c5timates of numbers of product contaminants. This is irrespective ofwhether cycle development is by the so-called overkill or the so-called bio­burden method (see below).

If the number of spores per load is 10 be relaled to the total bioburden ofthe load. lhe average bioburden per item must be multiplied by the number ofitems in the load to give an estimate of the t0l31 bioburden in the load. Thenumber of spore strips to be used in validation may be c.alculaled by dividing thisnumber by the number of spores per biological monilor thus

mNumber of spore strips per load; No x-­

C

where No = the average number of contaminants per ilem prior 10 sterilization. m= the number of items per load. and C = the number of s(X)res per biologicalmonitor.

If the number of spores per spore strip and the number of spores per vali­dation load is to be related to the individual product item and a sierilily assur­ance level of 10-6. it is first necessary to know the average number of contilmi­nants per product item. In the intereslS of conservatism Ihis number may berounded up or upplemented by a safely faclor. The target is to use as manyspore strips as is necessary to provide assurance that this bioburden is beinginactivated to an SAL of 10.6. This can be calculated from

INumber of spores per load; No +-­

SAL

where No = the average number of contaminants per item prior to sterilizationand SAL = the required sterilily assurance level.

The number of spore strips per )oad can be obtained by dividing this figureby the number of spores per slJip.

Given that these methods can provide a means of deciding how many bio·logical monitors should be used for validation purposes. there are twoapprooche to cycle validation. These have been temled overkill and bioburden.

An overkill cycle is quite simply one Ihat inaclivatcs Ihe microbiologicalchallenge plus an additional safety factor. As a rule of thumb. the shortesl expo-

Sterilization by Ethylene Odde 145

sure time obtained in validation runs that will consist'eotly inactivate all of lhemicrobiological challenge organisms should be doubled to specify an adequateovc[kill cycle.

Bioburden cycles normally require resistance data as well as estimates ofnumbers of microbial contaminants. In Ihis respect AAMI are using the termbioburden in ilS broadest sense, almost as a synonym for microflora.

Resistance work cannot be done in large·scale production sterilizers,because the gassing and degassing times are too long. These must be done inlaboratory-scale equipment or BIER vessels. They should be done preferably formicroorganisms actually isolal'ed from the product and with microbiologicalmonitors. A comparison should be made of the resistance (D·valucs) of bothlypeS of microorganism in simple situations (say on strips held in glass petridishes) and in product items or simulated prodUC1 ilems_ 1be cycle should bechosen on the basis of the determined resistance of the bioburden being sum.·cien~y treated within the speeified paramete" to provide a 10-6 SAL. Finalproof of this must be obtained with microbiological monilors in the productionsterilizer. As an example. assuming Ihal the average bioburden per item is equalto n microorganisms of D·value Db' the exposure time for a 10-6 SAL may becalculated from

Exposure time = Db . (log n + 6)

The number of spores per biological monilor required to be inactivated 10

verify tbat Ibis cycle is effective can then be calculaled from

(log n + 6)Log number of spores per monilor = Db . -'-""::'_-'--

Dm

where Dm = D-value of BaciJ/u$ subliJis var niger.For validation purposes, microbiological monilo~ are inlended not only 10

provide an index of achievement of SAL. but also 10 provide (among otherthings) some index of process uniformity. They must lherefore be placedthroughoul the load in pallet positions close to and far from the enlry ports forgas and ste::tm, near the surfaces and deep within stacked pallets. Microbiologi­cal monitors should be placed alongside lemperalure sensors during validalion.bUI it should be DOted thalUle sensors may introduce a route for easier access ofgas than mighl occur in their absence. The choice of locations for biologicalmonilOrs is in the long run arbilrary_

B. Routine Control of Ethylene Oxide Sterilization

The emphasis for routine control of irradiation sierilizalion and thenna) steriliza­tion has been toward tight conlrol of the physical parameters that lead 10 micro­bial inactivation ralher than toward control through biological testing. Alonelime there would have been an emphasis on biological methods for conlrolling

146 Chapter 6

lhese olher sterilization processes.. but as the scientific basis of these processeshas become better known. and 'heir technology h3S become bener con,rolled,biological monitoring has diminished in impon'ance. This is not yet lhe casewith ethylene oxide sterilization. Both biological and physicaJ methods of mon­itoring are absolutely necessary to provide reasonable assurJ.oce of sterility.

The first requirement of a routine monitoring program for ethylene oxidesterilization is specification of the critical parameters. Any excursion beyond Lhespecified tolerances [or anyone of these critical parameters must stimulaterejection or resterilization irrespective of whether biological monitoring criteriaare met. This acknowledges the fallibility of biological monitoring methods nndthe limitations on the numbers of biological monilOrs that may be used practi·cally. The insltUmentation used to monitor these characteristics must be inde­pendent of the instrumentation used for control.

Other excursions beyond less critical specifications and tolerances shouldbe OOIcd as a maUer of routine in lhe event of these providing an early warningof some progressive deterioration or loss of control that may in the long runimpact upon sterility or safety, for instance wear and tear on vacuum pumps.

TIle second critical requirement for routine monitoring is a biologicalmonitoring system. Biological monitors should be placed in the product lood.retrie\<ed promptly aner sterilization finishes. be len for 3 conuoUed period 10

lose any residues or lnlCes of ethylene oxide, and then cultivated in appropriaterecovery media.

Fewer biological monitors are needed for routine monitoring than wouldbe used for validation. There should be some concentration on cold spots (ifany). but at the same time there should be some attempt at random or represen­tative covering of all pans of the sterilizer and of the load. The history andreproducibility of pan.ieular ethylene oxide sterilization technologies may innu­enee Ihe confidence that can be placed on a panic:ular proc~ss. Precise andreproducible technology does not merit as much biological moniloring as lessreliable equipment A sound sterilization history of a panicular sterilizer cou·pled with a particular cycle lind a panicular prodUCI does 001 require as muchbiological monitoring as a new sterilizer. new product. or new cycle. How manybiological monitors is a matler of professional judgement.

Most biological monitoring is done on a quantal response (growth/nogrowth) basis. and if anyone biological monitor shows growth the load shouldbe rejecled or resterilized. Once again this decision should be made indepen­dently and irrespective of whether physical proc:c s specification have been mel.This acknowledges the empiricism of the ethylene oxide sterilization process.With so many variables it can be quite possible to fail to achieve sterilizing con­ditions at some point or points locally in the load. If there is biological evidenceof this having happened the decision can only be the conservative one.

Sterilization by Ethylene Oxide 147

Spores of B. subtilis var niger form an orange pellicle in simple slandardrecovery media. so failure is easy to recognize and simple to distinguish fromincidental contamination. Laboratories with high incidences of incidental con­tamination should seriously review their procedures for recovering biologicalmonitors from the sterilizer and from the product. They should also review Iheiraseptic techniques. This emphasis acknowledges that microorganisms compete,for nutrients such that fasler growing contaminants may obscure survival of thebiological monitor.

One of the major commercial difficuhies with biological monitoring is the

incubation of the biological monitors. Various sources may recommend 7, 10, or14 days. With reliable technology and a satisfactory sterilization history it isquite reasonable to divorce the incubation of biological monitors from the ship­ment of product from the sterilization site, as long as the product is not actuallyput into use and is accountable and retrievable in the event of a subsequent bio­logical failure. This is nOl parametric release, bUI a commercial risk, and itshould be exercised with considerable care and only when processes subsequentto sterilization but concurrent with incubation. e,g., degassing. packing, andlabelling in shippers. transportation. and warehousing. operate over a time scalethat ensures that nonsterile product is unlikely to reach the final user beforecompletion of incubation.

In addition to the use of biological monitors it is also advisable with ethy­lene oxide sterilization processes to include a rouline batch-by-batch pharma­copoeial sterility tesi. lls slatislicallimitations remain as a barrier to ils value forconfinning sterility. However, it should nol be discounted as a funber means ofinvestigating the possibility of failure to achieve sterility (i.e" as a test for non­sterility) in a poorly predictable situation,

V. HEALTH AND SAFETY

Ethylene oxide damages biological systems. Ils effects are oot pecuUar tomicroorganisms, bUI apply to aU biological systems. Ethylene oxide and its by­products are toxic. mutagenic, and carcinogenic to animals and humans. Ideallyethylene oxide residues should not be present on medic,at prodUCIS. In practicethere arc tcchnologies available to ensure that the use of ethylene oxide need notresuh in an unacceptably high risk to human heahh.

Acute effeels of elhytene oxide and ils by-produci ethylene chlorhydrin(CH2ClCH20H). formed by re3ct;on with chloride ions. include symptoms ofnausea,. dizziness, and signs of mental dislurbance. Ethylene chlorhydrin mayalso cause kidney and liver degeneration. The most serious effects of both sub­stances may lead to cancer. Exposure lO ethylene oxide induces irreversiblechromosomal aberrations (sister chromatid exchange) and other precancerouschanges in the peripherallympbocyres.

148 Chapter 6

There are two broad groups at risk. The first of these comprises ethyleneoxide sterilizalion worke,rs who are subject to exposure by inhalation and per­haps to skin contact with liquid ethylene oxide while changing gas cylinders.The second group comprises the recipients of ethylene ox.ide sterilized products.including patients who may be receiving lreatmenl and medic-a! or nursing staffwho must come into regular and frequent caDlact with ethylene oxide sterilizedproducts.

The group at highest risk are sterilization workers. patients on hemodyaly·sis equipment. and persistent users of hypodermic equipment (e.g.• djabeticsusing insulin syringes) and habitual users of ethylene oxide sterilized gloves.There is a middle risk category of patients on intravenous therapy using ethyleneoxide sterilized infusion sets over a comparatively shon period of time. In num­bers this is the largest group. The lowest risk is to patients who receive one limeimplants (e.g .. cardiac pacemakers). one single dose of e.thylene oxide absorbedcompletely.

The most stringent altitudes to worker protcction have been those of theOccupational Safety and Heallh Administration in the U.S.A. There are threelimit standards to be considered. The permissible exposure limit (PEL) is a mea­sure of good indUSlrial hygiene practice expressed as an 8-h time weighted aver­age (fWA8). The PEL sel by OSHA is I ppm TWAS measured in a manner rep­resentative of the employees' brealhing zone. Personnel are subject to medicalsurveillance; areas where greater ethylene oxide exposure may be expected mustbe identified. respiratory protection must be provided for particular tasks. andrisks should be identified through appropriate signs and labels. If an action limitof 0.5 ppm nvAS is being consistently achieved. then the frequency of medicalsurveillance and monitoring may be reduced. The third limit is an excursionlimit (El) not to be exceeded over a IS-min period. TIlis has been set at S ppm.

In Europe PELs for ethylene oxide range from I ppm TWAS in Belgiumand Denmark through 5 ppm TWAS in the U.K. and France up 10 50 ppm TWASin the Netherlands. France ha~ a IS-min EL of 10 ppm.

Surrounding these limits are a series of complications. In general PELscan be attained by use of segregated areas for ethylene oxide sterilization andethylene oxide storage. Ventilalion is of utmost importance, and. since ethyleneoltidc is heavier than air. low-level exhaust systems are to be preferred. Otherengineering measures may include interlocks and remote control consoles.Shon·term excursion limits (ELs) may necessitate positive pressure self-con­tained respiratory pfOlCCtion to be used for particular tasks. for instance unload­ing sterilizers and changing gas cylinders. PersonaJ protection should not. how­ever, be considered until it can be shown conclusively that ELs cannot beachieved by engineering controls or changed workplace practices.

Ethylene oxide residues remaining on products present a more complicalcdpicture. Limits should properly be based on risk. assessment. Satisfactory risk

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Sterilization by Ethylene Oxide 149

assessment involves an analysis of a multitude of complex factors concerningparticular medical products, their frequency of usage. and the characteristics ofthe recipients. Some stiU unfinalized residue limits provided by the FDA in1978 provide n guideline to appropriate targets. These range from 5 to 250 ppmby weight of device depending on the type of device (III. By and large theserecommendations follow the risk categories described previously. There is someconjecture over ppm by weight of device being the most appropriate measure todefine limits. In many cases surface area in contact with the recipient's tissue orin contact with fluids being infused may be argued to be more appropriate.

Methods of extraction and analysis have been published by the Associationfor the Advancement of Medical Instrumentation [12]. Water or olher aqueoussystems are most commonly used for extraction. Two extraction methods arerecommended, exhaustive extraction and simulated use. In fact both methods. ifused correctly. represenr simulated use. Exhaustive extraction is recomme,ndedfor devices such as implanls. which by merit of their prolonged contact with tis­sue over time can be expected to transfer all of lheir residual ethylene mdde 10

the recipient Simulated use extraction of a less exhaustive nature might includeOuid path extraction over a simulated maximum hold period for infusion sets andhypodennic syringes. Analysis is by gas chromatography.

1bere are four ways in which elllylene oxide may be retained in products atthe end of sterilization cycles. These are ao;; gaseous ethylene oxide. as elhyleneoxide dissolved in water. as elllylene oxide within but nOl attached to the productor packaging material, and as molecularly adsorbed or absorbed ethylene oxide[13]. The lOlal amount of residual elhylene oxide and lhe balance among thefour forms of residue at the end of sterilization are functions of the sterilizationprocess conditions. the composition of the product, the size of the product~ thepackaging materials, and the packing density. Dissipation of residues afler ster­ilization is a function of tbe product- and packaging-related factors listed aboveplus the conditions in which the product is being held (aeration).

The arnOUDI of residual ethylene oxide in a product can be significantlyinflue,nced by sterilization process condilions. Gas concentrations and exposurelimes within the exposure period of the cycle should be sufficient to achievesterility. but their effects on residues should be considered before prolonginglhem unnecessarily. Importanlly. free gaseous ethylene oxide is easiest toremove from product loads. and this is best addressed by postexposure evacua­tion and aeration. Multiple evacualions and forced circulalion aeration at tem­peratures around 30·C have been found to be effective. The effects of increasedtemperalures extend beyond the removaJ of the free gas (0 (he removal of otherforms of bound ethylene oxide.

Aner removal of the load from the slerilizer, funher dissipation of residuesis a function of time, temperature. and ventilation. Generally. the rate of ethy­lene oxide dissipation doubles for every w·e rise in temper:1ture (the QIO is

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150 Chapter 6

equal to 2). and ventilation should be sufficient to maintain a concentration gra·dienl between the ethylene oxide in the product and the ethylene oxide in theatmosphere, Movement of elhylene oxide from the product into the atmosphereis governed by Fick's laws of diffusion. Given that there are technological andcommercial restrictions on the conditions in which ethylene oxide sterilizedproducts can be held after sterilization and before release to the market. seriousconsideration should be given to the ways in which product and packaging com·position and design can affect the dissipation of ethylene oxide.

II is paradoxical lhal the abilities of ethylene oxide to penetrate malerialslhat make it an effective sterilant are (he same abilities that cre-ale residues.Polymeric materials are very penneable to ethylene oxide. Penneability isaffected by the solUbility of the gas in the polymer and the diffusivity of thepolymer to ethylene oxide. Emylene oxide is less soluble in polyethylene andpolyeSlers (around 10.000 ppm) than in say cellulosics or PVC (around 30,000 to40.000 ppm according to the level of plastici1.ers present in me fonnulation); softplastics and natural rubbers have higher diffusion coefficients for ethylene oxidethan harder polymers such as acrylics and slyrenes [14). Polymers with high dif­fusion coefficients will reach saluration solubility quicker Ihan Ihose with lowerdiffusion coefficienl'i. A polymer thai takes up residues only slowly wiU releasethem only slowly. Since devices may often be manufactured with several differ·ent Iypes of polymeric material, il is difficult to predict or quantify overallresidue levels and prdctical rates of dissipalion. A component such as th~ rubberplunger lip may as a result of ils high diffusivity and thickness amount for mosl

of the residues in a hypodennic syringe. although it is in ilself only a minorcomponent.

Much of what has been said about the movement of ethylene oxide in andout of products also applies to packaging materials. Primary packaging materi·als are only very rarely of a significant thickness. and should be chosen to allowrapid penetration of ethylene oxide to the product during the exposure period ofthe sterilization cycle. They should therefore present an insignificant barrier 10

the dissipation of residues. Molecules released from the product dissolve in thepermeable primary packaging materials on the side with Ihe.higher concenlrationand diffuse in the direction of the concentration gradient toward the side with Ihelower concentration of ethylene oxide. The residues desorb to the atmosphere onthe side of lower concentration.

In the long run. corrugated cardboard boxes and wooden pallets may allowresidues to hang around the load and the product longer than necessary. Loadsshould not be left to aerate in condilions where ventilation may be reslricted bytoo great packing densities or where some pallelS may occlude air movementaround olhers.

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Copynghled aena

7Sterilization by Filtration

I. Membrane FiltrationA. Pore Size RatingB. Microbial Panicle Passage

U. AppUcarions of Sterilizing FillrationA. Construction of Sterilizing FiltersB. Filtration of LiquidsC. Filtration of Gases

ill. Validation and Routine Control of Sterile FiltrationA. Validation by the Filter ManufacturerB. Validation and Routine Testing by the Filter User

IV. Summary of Reasons for Filtration Failure

154155158159160164164165165167176

Microbial inactivation is not the only way of achieving sterility. A major aher­nalive to killing microorganisms contaminating medical and pharmaceuticalproducts is to remove lhem from the products. Filtration is one means of doinglhis for Ouids.

Filtration does not rely on chemical reaction to inactivate microorganisms:it is thcrdore suitable for heat-labile and radiation-sensitive fluids. Filtralionshould leave no lr.Jces nor leach chemical conlaminants into lhe producl. As a

lS3

154 Ch~pter 7

continuous or semicontinuous process it is suilable for sterilization of volumes offluids Ibal migbt be probibitively large for oIher melbods of balcb sterilization.

Modem melhods of filtr3tion can be as reliable a mechanism of steriliza­tion as methods relying on biochemical inactivation of microorganisms. Fibra­tjon in its simplest sense of removal of panicles from a fluid by passage of Lhefluid through a medium lhat has pores large enough to allow the fluid to pass buttoo small to allow the particles to pass (sieving) is only one comp<ment of abighly complex process.

I. MEMBRANE FILTRATION

Sterile filtration of liquids and gases is DOW vi.rtually always done using mem­brane fillers. The first U.S. patent for membrane filters was filed in 1922 andpertained to cellulose acetate membranes. A wide range of membrane fillermedia are now commercially available to suit various applications: celluloseesters, polyvinylidinefluoride, polytetrafluoroelbylene (PTFE), and polyhexam­elbyleneadipamide (oylon 66), separately or as laminates witb polyethylene.polypropylene, and polyester for more robust physical characteristics.

Membrane filters arc thin. uniform porous sheets that in the older scientificliterature were described to be acting as screens or sieves that trapped on theirsurfaces all panicles larger in size than lhe pores in the membrane or larger thanthe interstices of !.he mesh of the membranes. This type of panicle removal isabsolute in the sense of it being independent of the fillr3tion conditions. Sievingis nOl influenced by pressure differentials nor by the chemical or physical prop­enics of the nuid unless lhese are sufficient to deform, damage, or otherwiseaher the size of the pores. Particle entrapment by sieving alone is now known tobe an excessively simplistic interpretation of what happens to particles duringmembrane filtration.

Physical sieving or surface retention of particles larger than the pores inthe membrane is only one of several mechanisms of panicle retention that mayoccur in membrane fillers. In reality. membrane filters are also capable ofrelaining particles that are dimensionally smaller than their pores.

These olher mechanisms of retaining particles within the deplh of mem­branes may be mechanical or physicocbemical. Mechanical means of entrap­ment apply equally to liquid and gas filtration; they include inenial impaction tothe walls or surfaces of the pores and lodgement in crevices and "dead ends:'Theoretical m<XIels and other illustrations of membrane filt'ers often ponraypores as being cylindrical in shape and as passing directly from the top surface ofthe membrane to the bottom surface by the. soonest straightest roule. In factpores are rarely cylindrical. they do not have smooth walls. and their passagemay be extremely convoluted even through very thin (typically 0.015 em) depthsof membrane. Physicocbemical interactions witb tbe filter medium can be very

Sterilization by Fihration ISS

complex and differ according 10 the Iypes of particles. Ihe nuid in which they aresuspended. and the dynamics of the fillration system. Electrostatic forces are ofmore importance in gas filtration than in liquid filtralion. Adsorption of surfac­lants from liquid suspensions 10 both panicles and pore surfaces may result inmutually repulsive ionic forces thai encourage the passage of panicles. On theother hand. retention of small panicles may be favored in liquid suspen ionsLhrough covalent bonding, and lhrough attraction by van der Waals forces whenzeta potentials of both panicles and pore surfaces are low (usually less than 30mY). Mosl particles carry a negative zeta potenlial in WOller; for Ibis bondingmechanism 10 be operative in filtration it is necessary therefore for the mem­

brane to have a positive zela potential. Commercial membrane manufacturersoffer charged media for specialized applications.

These other mechanisms mayor may nOi occur in particular circum·stances. All are reversible and all may reach saturation levels beyond which theyare ioeffective for particle retention. In Ihe long run, reliance can only be placedon surface collection for absolute removal of panicles from fluids. For steriliza·lion purposes. the particles that must be retained by filters are microbiologicalones. The FDA defines a sterilizing filter in terms of microbiological particlepassage: "a sl'erilizing filter is one which, when ch[lUenged with me microor·ganism Pseudomonas diminuta. at a minimum concentration of to7 organismsper cm2 of filter surface, will produce a sterile effiuenf' [I]. The possibility ofmechanisms other than sieving contributing to microbial retention is acknowl·edged by the recognition that (he number of panicles in the challenge may beable to saturate the rcten(ion mechanisms of the filter. The FDA goes on. how­ever, 10 relate satisfactory sterilizing membranes (0 a rated "porosity" of 0.22 J.1mor smaller. chosen because Ps. diminllta ATCC 19146 has an inherendy smallmean diameter of 0.3 J.1m. Regardless of whclher membrane filtration's mainmecbanism of panicle retention is sieving or some other means, pore size isclearly seen 10 be of significanl importance to the choice of membranes to beused for sterilization.

A. Pore Size Rating

What is meant by lhe pore size ratings of commercially available membranes?Pore size rutings of commercially available membranes are not indices of maxi­mum pore opening diameters obtained from direcl microscopic measurement orby panicle passage methods. Ratings are almost always determined by indirectmeans based on theoretical considesalions and on nondestructive lest technology.

Theoretical aspects of pore size rating have been addressed by Schroederand DeLuca [2J. WeI membranes are impermeable 10 Ibe flow of gas until apressure is reached Ihal is sufficiently bigh 10 dislodge the I.iquid from the pores(Fig. I). The diameters of cylindrical pores can be calculaled from this differen-

I ,

Sterilization by Filtration 157

Some examples of how theoretical pore diameters may be calculated frombubble point pressures are given in Annex I.

The two functions yand e relating to the wetting liquid indicate the sped­tidty of the method to panicular test conditions; for instance. if methanol wereto be used as a wetting liquid, bubble points would be usually about one-third ofthose found with the same membrane jf water were to be used as the wettingliquid.

For practical applications, an empirical nonideality factor K should beapplied (0 correct for pores that do not have perfeclly circular unbroken perime­ters. thus

d=K·4y·cos 8

p

Emptieal pores may be addressed lheoretically. For ellipses wil.h differing diam­eters it is lhe smallest diameter that is of significance in filtration because thesieving mechanism of the membrane can only be effective for panicles Largerthan this dimension.

The surface tension forces that mUSl be overcome to allow displacement ofa liquid [rom a membrane are mainly a fUDclion of the size of the perimeter (thecircumference) of the pore. The hydrostatic forces that promote displacement ofthe liquid are mainly a function of the area of the pore. At lhe bubble point,when these two sets of forces are in equilibrium. the smallest diameter d of anelliptical pore can be calculated from the equation

4y· cos 8 .J (1 + £2)/2d =--=----"..:----'-­

£P

where £ = the ratio belween the largest and smallesl diameters of the ellipse.This equation shows that.. at any panicular bubble point pressure" E is

invc.rsely proponional to d. Therefore lhe assumption of a pore being cylindrical(£ = I) will provide a highe, estimate of d than any estimate obtained by co,­reeling fo, the emptical shape of the pore. To assume lhatthe pore is cylindricalintroduces an inaccuracy in the estimation of lhe pore's diameter; it is. however.a conservative inaccuracy.

Pore size ratings of commercially available membrane filters are usuallybased on bubble point pressure calculations. This is not 10 say thai they arepurely based on these theorelical considerations. Practical knowledge of theprocesses of manufacture of membranes. detailed knowledge of pore size disLri­bution from electron microscope studies, correction factors to compen.sate forirregular pore peripheries. and other considerations are likely 10 be taken into.:accounl in determining ratings. Pore size ratings of say 0.22 }.lm from differenlmanufacturers are not necessarily derived by the same method and should nottherefore be assumed to be describing identical membranes.

158 Chapter 7

Factually. one can expect to find membranes with specified pore size rat­ings to have a proportion of pores with larger diameters than the rating wouldsuggest. yet quite capable of retaining microorganisms physically smaller thanlhese pores. Electron microscope studies [3J of commercially available mem­branes that had pore size ratings of 0.22 lim and that had been shown 10 be capa­ble of meeting the FDA's microbiological particle passage criterion revealedpores with diameters larger than 0.3 ).1m on their surfaces and throughout theirdepths. In summary. pore size rating cannot be correlated precisely to the size ofthe smallest microbiological particle thai me membrane is capable of retaining.

B. Microbial Particle Passage

Pore size is clearly nOI the only factor influencing me retention of microorgan·isms on or in membrane filters. Olher mechanisms in addirion to sieving arfectmicrobial relention. The FDA's microbial particle passage standard, Ihat a steril­izing filter should be capable of retaining a challenge of at least 107 microor­ganisms of a particular type per cm2. is an end-resuh crilerion that does not byitself help to quantify measurable charac.teristics of membranes or membranetypes that contribute to retention.

The design of microbial particle passage experiments is not without itsmicrobiological complications (see Seclion m below), and since these tests aredestructive of Ihe material being tested lhey are not well suiteO to routine appli­calion. Microbial particle passage experimentation has. however. assisted inidentifying some important faclOfS involved in microbial retention. In principle,removal of bacteria from fluids by retention on or in membranes is simple 10

determine by challenging a filtration unit with a suspension of a known concen­tralion of a specified microorganism, usually Ps. diminUIa. An index of thefilter's retention properties can be oblained by comparing the concentration ofmicroorganisms in the challenge to Ihe concentration of microorganisms reeov·ered downstream.

In practice there is an upper limit to the number of microorganisms thatcan be used 10 challenge a membrane. This is delennil)ed by the open pore vol­ume of the membrane and the pore size. The numbers of microorganb,mstropped on the surface of a membrane affect the amount of open porr volumeand lherefore diminish flow (clogging). Particle size is also a factor. becausepores are moSI effectively blocked by particles of about the same size as thepores. The most discriminating experimental conditions exist when all pores arebeing challenged by potential penetranlS, i.e .. when the challenge is sufficient 10

cover the membrane surface completely but below the clogging concenlration.Scanning electron micrographs of this situation (4) show a double layer of cellsof Ps. diminllfo from a challenge concentration of 108 per cm2 on the surface of0.22 )J.ffi pore Si7.e rated membranes. When membranes become clogged. exces·sively high differential pressures are required to mainlain a constant flow rate.

, I

Sterilization by Filtr~tion 159

and furthermore the proportion of lhe challenge population penetrating the mem­brane diminishes [3].

The performance characteristics of the membrane. flow rate and applieddifferential pressure, may also affect microbial panicle passage experiments.The rate at which a liquid is able to pass through a membrane is defined by lheequation

Q=c.AP

V

where Q =flow rate, A =filtration area. P =differential pressure across themembrane, V = viscosity of the liquid, and C = resistance to flow by the mem­brane.

This equation indicates that at a constant differential pressure across lhemembrane, flow rale will decrease as the effective filtralion area becomesclogged by particles. Experiments are normally performed. therefore, at constantdifferential pressure (usually in the range of 30-50 psig) but with a dintinishingflow rate, or at a constant flow rote obtained by progressively increasing the dif­ferential pressure ac.ross !.he membrane.

For membranes with pore dimensions larger than those of Ps. diminuto,higher pressures pose a more severe challenge than lower pressures [4]. Thissuggests lhat emrapment of microorganisms in the tortuous passages of the poresis one of the main mechanisms of retention. Pall (5] describes panicles as"stupid," blundering passively through pores, potentially deformable, squeezingthemselves or being squeezed by pressure lhrough narrower passages than theymight otherwise traverse.

Pall [3] has also demonstrated that the proportion of the challenge popula­tion of Ps. diminuta passing through a membrane is approximately constantregardless of the initial size of the challenge. This implies thai if a number ofmembranes of Lhe same type were 10 be set up in series, each would remove thesame proponion of its incoming challenge, and therefore that microbial retentionby membrane fIlters is an exponential function similar to meLhods of sterilizationinvolving microbial inactivation. The importance of this is in relation to theetreel of membrane thickness on microbial retention; given two membranes withidentical pore sizes (as a rating from bubble point pressure measurements. or as adirect measurement by some other means), the thicker of the two membranesprovides greater assurance against microbial penetration. Membrane Ihicknessmay lherefore be placed alongside pore size rating as an important index of theeffectiveness of a slcrilizing filter.

II. APPLICATIONS OF STERILIZING FilTRATION

There ace two main occasions for the use of filtration in the sterilization of Ou­ids. The first is when nuids are damaged or destroyed by exposure to other ster-

Sterilization by Filtration 161

1. Disc Filters: Traditional types of large disc filter holde". if used at all forproduction purposes. are usually about 300 mm in diameter. The direction offluid flow is from above the filter to below. The membrane is sandwichedbetween metal inlet and outlei plates equipped with the sanitary connectionsnecessary to operate the filler. Because of the fragility of disc-type membranes.there must always be a support plale direclly beneath the membrane. Supportplates must be porous, often photoetched, chemically inert, and have minimaleffects on flow rate. This often means an uneasy compromise. Prevention offlow restriction requires extensive void space; this acts against the plate'smechanical suength. Usually there is also a loose mesh drain plate beneath Ihesupport plate and resting on the outlet plate (Fig. 2). When serially stacked discfillers are used. each membrane requires its own support pJate but there will onlybe one drain plate.

The whole unit is sealed by means of pressure on one or two O-rings.Where the design of the holder includes only one O-ring it is intended to servetwo purposes, fIrst to seal the inlet plate (0 lhe outlet plate and second 10 seal themembrane by downward compressive forces against the support and drain plates.Where the design includes two O-rings, one seals the inlet and oullel plales andthe other. of a smaller diameter. sits between Ihe membrane and the inlet plate.

Small disc rulers and small single·use stacked disc filters in plaslic bous·jngs are available and have the appearance of cartridge filters bUI consisl of aseries of separate membranes rather than of one continuous plealed membrane.This added complexity may make sterilization by saturated Sleam more difficuh.Deep vacuums intended to ensure effe<:tive air removal from the filter media

........./.

SUPPORT P1AlE

::---;", OAAIN P1AlE

0U11.ET P1AlE

Fig. 2 Schematic representation of a disc filter.

Steriliution by Filt,.Uon 163

are quite brittle and lherefore unsuited to aUloma'ed pleating. The pleatedsuppons give a small radius of curvature to the pleated membrane rather than asharp fold that could lead to membrane dam3ge. The outer suppon layer actsalso as a prefilter. protecting the membrane in another way.

The plea.. are posilioned between a perforated hollow lUbe. which com­prises the inner core of the cartridge. and another perforated hollow lUbe (Fig. 4)or "'cage," which k.eeps the pleats in place and rorms the ouler cylinder or thecartridge. The whole assembly is held logether by two ,erminal end caps. All ofthe various components are bonded together in order 10 avoid any possibilily orfluid bypassing lhe membrane; bonding is achieved by use or low melting pointthermoplastic sealants.

WELOEDL.QCA1"IIIG FINS

HEl!- SEAL~ECy//~EHC CAPS

~LEC--­SUO SEAH

CLOSE Pt£ATN;FOR OPTtt4UM AREA

OOJElE a·RlNGs FOR-='HTEGRrTY a: SEALING

1fJtJ1. LAYEREDMEDIUM WITH

:..---- SlI'!'CRT ANaDRAINAGE LAYERS

STIlONG OUTER__C.OGE 10 RED CE

RISK OF 1WQ.t<GIlAMAGE

SlR(N; ClJ'lE 10--RESIST HIGH dp

T"MST • LOCK FORs.oFE RETEN11ClII

F".. 4 ConstruCtion of a filter canridge. (Coones)' of the Pall Company. Glen Cove.New Yodel

164 Chilpter 7

The movement of nuid across the membrane in canridge filters is fromoutside (0 inside. When cartridges are fitled into their housings, sealing surfacesare bet.....een the end C3ps and adapters on the housings.

Housings may be singJe-cartridge housings or mulli<anridge housings.They arc cylindrical. they are made of stainless steel. and all connections. elc..are designed (0 consider minimizing the possibility of microbiologicaJ contami·nation. Where the inlet and outlet portS are located at opposite ends of thehousings. the term "slraighHhrough" housing is applied: where the inlet andoutJet ports are located at the same end. the lerrn '1"·type'· housing i applied.The T-type housing is most suiled for incorpormion into rigid pipework syslems.

B. Filtration of Liquids

The general case for pharmaceulical products includes slerile filtration of small­volume parenteral either in aseptic manufacture or prior to terminal ~h.:rilila·

tion. sterile filtration of ophlhaJrnic products. and filtration of large-volume par­emerals prior to tenninal sterilization.

For sterile filtration of ophthalmics and small-volume parente,.....1 productsit is not unusual to find several filters mounted in series. For instance a com­pounded bulk. product may be fihered "through the wall" from a clean area intoan aseptic filling room. In these cases there: art: usually two filters mounted inseries. one on either side of the wall. The filtrate may be fed directly to ;1 fillingmachine, alternatively it may be collected in an intennediate vessel. held for 3

while, and then filled out. Intermediate vessels should be equipped with sterilevent filters to prevent pres!i:ure increases leading to "blow-backs."

Most large-volume parenterals are tenninally sterilized, but regardless ofthis it is quite usual to find them being passed through 3 sterilizing filter prior toautoclaving. This is because of the risk of endoloxic shock from parenteral infu­sion of large volumcs containing even only small concentrations of nonviablebut still pyrogenic microbial material.

C. Filtration of Gases

Canridge-type hydrophobic membrane filtration has largely replaced depth fil­tration as a means of sterilizing gases. Collection of panicles from <1 gas streamby membrane filtration is, as ",'ilb liquid filtration. a function of both sieving andother means of retention. Adsorption and e.lectrostatic attraction are far moreimponant to retention of panicles in gas filtration than in liquid fillfalion.Because there are more mechanisms and intcractions betw,-"en pore surfaces andparticles. removal of p3rticles is more easily accomplis~ from gas streams thanfrom liquids. Gases are quite satisfaclOrily sterilized using 0.45 J.lm pore sizerated membranes.

Sterilization by Filtration 165

Sterile ftllration of gases has three main applications: first. when sterile airis required as an ingredie,nt gas in fennenlation processes; second. when gasesare used for service purposes in ~terile manufacture. for instance to actuatevalves and to stabilize head space conteOle;; and third. to protect the veming ofsterile enclosures such. as storage tanks.

Gas filters may be evaluated prior to usc by the same methods used for liq­uid filters (see Section mbelow), allowing that they can be effectively dried outand sterilized without loss of the qualities being tested. Alternatively they maybe evaluated by exposure to panicles ~n a gas stream. for instance by the sodiumnarne test, which is also used for HEPA filters. Microbiological tests have to beconsidered in a somewhat different lighl. Leahy and Gabler 17) describe anaerosol challenge lest of 107 Ps. diminUla bacleria per mL over a four-day periodas an appropriate manufacturer's validation tesl. It would not be practical forrouline use.

In-process verification of the sterility of filtered gases may be done byconstantly "bleeding" off a trickle of gas through a pressure reducer on thedownslream side of the slerilizing filter. The bleed may be filtered through agela.tin membrane. which should be removed daily or at other suilable inlervalsfor incubarion and examination for evidence of microbiological cont'aminadon.

III. VALIDATION AND ROUTINE CONTROL OF STERILEFILTRATION

The prinh~ purpose of slerile fillralion is to produce a slerile effluent that has notbeen altered as a result of the process of sterilization. Wililin these considera­tion • validation musl addre 5 the penonnance of both me Hiler media and Ihewhole fiJu,uion unit including housings, seals. connections. elC., versus its prac­tical application. As with any other sterilization process. the continued effec­tiveness of sterile flltration CaDnOi be assumed without confirmation from routinemonitoring; end-product sterility resling (or testing for nonsterility) is unsuiledfor tbis PUlpOse.

A. Validation by the Filter Manufacturer

Validation of membrane charac.teristics nonnally requires specialized techniquesthat He wimin the expenise and experience of the membrane suppliers.

I. Extraclablrs: Membrane filters and lheir housings should be chemically andbiologically inen. Filter manufacturers are therefore obliged to provide evidencethat minimal amounts of chemical substances are released from the materialsgoing into lheir products.

Since the membrane usually presents the largest surface area of malerial incommercial fiJlers. it is from membrane contamination during manufacture thai

166 Chapter 7

most "extractables" arise. Dust panicles, chemical pore formers. and solventsmay be left on membranes. Wening agents may be released from hydrophilicmembranes. Indeed. Triton X·lOO was at one time widely used as a wellingagent for sterilizing membranes. until it was found 10 be cytotoxic.

Extractables testing may be done 00 each individual component or materialof manufaclure. or on me composite assembly. lbe purpose of eXlractablcstesting is to detennine the amount of material that can be eXt:faCled in water andin the range of solvents for which the manufacturer claims suitability. Extractionconditions should include a simulation of those processes that may affect thematerials prior to use; the most obvious of lhese for sterilizing filters is sleamsterilization. Typically. filters are auloclaved and then immersed in water orwhatever solvent at 20T and agitated for a defined period. Some extractionswith volatile solvents may be done at higher temperatures and may includereOu),.ing.

TIle extracts are quantified. This may be qUOted directly as the weight ofnonvolatile malerials per filler cartridge or per unit weight or per unit of surfacearea. according 10 which is most appropriate. Alternatively. extraclables may bequoted in relation to a pharmacopoeial oxidizable substances test The primarypurpose of this test is to monitor water quality. the inf~rtnce being that (heamounl of materials extractable from Ihe filler is no w~ than (he pharma­cOJXleial tandards for water.

Biological tcsting may require separate extraction. AU malerials shouldmeet phannacopoeial biological safety standards. These standards for plasticsrequire that each materiaJ be separately extracted in saline. alcohol diluted insaline. polyethylene glycol. and vegetable oil for specified conditions of timeand temperature. Extracts must be tested against mice for acute systemic loxic·ity. against rabbits for intracutaneous reactivily, agains( rabbits again for pyro­gens (or nowadays more likely by LAL testing for bacterial endolOxins). ::mdagainst microorganisms for mUlagenicity by the Ames test.

The most stringent biological test is by implantation of filter media or car­lridge materials into the muscle tissue of rabbits.

None of the testing done by the manufacturers of filters can guarantee thaiunacceptable substances or biologically acli ..'t subslances are nol going to beextracted into a panicular pharmaceutical fonnulation. It is therefore incumbentupon all users of sterilizing filters 10 perform validation trials panicular to theirown applications. This usually means nonvolatile utractables and lAL testing.but it may include other methods if these do not gi\'e adequate assurance.

2. Microbial Relenlion: It is not common for filter users 10 perform microbia!retention tests. In principle. microbial retention testing is quite simple; the testorganism is Pse"domofUJS dimillUlo ATee 19146 and the challenge is 107 bacte­ria per cm2 of effective filtration area. The enlire filtrate that has passed throughthe filter under teSt is collected on an analytical membrane: thi is then incubated

Sl";~ution by Filtration 167

on an appropriate medium, and any bacteria that have passed through the testmembrane are counted as colonies. The ability of I.he test filter to retain thechallenge organism is expressed as the log reduction value (LRV) of lhe test fiI·ler. The LRV is defined as Ihe loglo of the ralio of the number of organisms inthe challenge 10 the number of organisms in the filtrate. Most commercial filterstested by this method yield l:l sterile fillmLe; in these circumstances I is substi­tuted in the denominator of the equation required to calculate Lbe LRV, and theresults are reponed as "greater than" the LRV calculated.

Ps. dim;nulQ is an inherenlly small. moIile. gram-negative bacterium thatwas originally isolated as a contaminant of filter "sterilized" fluids. Its size.however. is not independent of nutritional and pity iologicaJ faclOrs thai may beencounrtred in its cultivatjon. Smallest cell sizes occur only in the stationaryphase of the organism's growth cycle. Regreuably for microbial relenlion lesl­ing, microbial cuhures in this phase of growlh also contain significant numbersof dead cells and debris. These particles Ihal are nOl discemable as part of lheviable microbial challenge are capable of clogging the JX)res of lhe fillers underlest and effectively reducing the challenge. In otncr phases of lhe growth cycle,the size of Ps. diminuta is larger than ilS quoted 0.3 ).1m.

Saline lactose broth is most often used for the organism's cullivation. At30·C sialic incubation, Ps. dim;IJuta grows as small separate single cells. Inricher media, Ps. dim;nma may grow as a pellicle; with agitation, groups of fouror five cells may bind togelher in rosette-shaped clusters. Both of these situa­tions are less challenging than inl:ended.

The Health Indusuy Manufacturer's Association (8) has described detailedmethods of perfonning microbial retcntion leslS on disc- and cartridge-type ster­ilizing filters. In both cases it is firsl necessary to pass a volume of peptonewater, saline, or other appropriate fluid through the filler, for two purposes: firslto wei the test filler thoroughly and second to ensure Lhat the leSt filler is itselftelile. 1be test is DOl considered valid unless this negative conlrol is sterile.

For disc.lype fillers. the challenge suspension is then passed Ihrough the memobnlne while maintaining a constant differential pressure of 30 psig acres the:membrane. for cartridge filters the standard is to maintain a now rate of 3.86 Lper min per 0.1 m2 of effective filler area.

B. Validation and Routine Testing by the Filter User

There are cenain Ihings that must be done by filler users as pan of validationbefore using a particular type of filler. other tests have to be done before and/orafter use of each individual filter: and yet other indicators ought to be manHoredthroughout use of individual filters. The distinction between validalion and rou·tine monitoring is less clear for sterile fLltration than for any other sterilizationprocess.

168 Ch~plt'r 7

As mentioned above. it is an obligation of lhe fiher user to validate the

sterilization processes applied 10 lhe filter. Most oftcn sterilizing filters will beautoclave<!. 1be cffcclS of a particular autoclave proces on !.he quality andquantity of extr.lctables obtained from filters should be evaluated usin~ thepharmaceutical fluid lhal is intended to be fihered. The filtrate should bedemonstrated to be p:u1ic1e free and biologically inert: chemical utrnctabJesshould not exceed the levels of contaminants allowed in the product qualityspecification.

The integrit)' of sterilizing filters is most often validated and roulinelymonitored by nondestructive methods. The U.S.. European. and U.K. guidelineson sterile tihration refer (0 four methods of integrity mea.~urement: filtrationnow rate, bubble point tests, diffusion (forward now) tests. and pressure holdtesls. Each of these has its uses in detennining that roulindy used filters are per~

fonning to the same standards as those validated for the paniculur producls :tndprocesses.

I. Filtration Flow Rate: In some cases the time raken to filler a specified vol­ume of product under constant pressure may be taken as an index of fillerintegrity. Unduly fast filtration may be indicative of a major loss of physicalintegrity. Filtration flow rate is nOi sufficiently sensitive to detect defects in fil­ters, which al!.hough physically quite small could contribute ignificantly to lossof sterility. A pinhole large enough (0 allow microorganisms to SlTeam through 3

filter might only increase the rale of flow by as liule a" one mL per min: com·pared to, for example, an ove,rall now rate of 10 L per min, the effect of the pin­hole on the overall lime of riltr,.uion woukl be insignificant.

Furthennore, filU'ation now rate is only useful when fluids are being lit­tered into intennediate vessels before filling. 11 is impraclical where the rate offiltration is governed by the operating rate of on·line filling machines.

2. Bubble Poinr rtS': Theoretical a"pects of !.he bubble point lest have beenaddressed in Section I above. The bubble point tcst predicts !.he perfonnance ofa filler by detecting the differential pressure at which a fluid is displaced by gasfrom !.he pores of a welted filter, !.hus allowing capillary flow of the gas throughthe filler.

The classic laboratory bubble point test (Fig. 5) looks crude from a tech·nological slandpoint but i in fact very sensitive for determining the bubble pointfor a sample of membrane. A welled filter element is held between supportscreens, and the differential pressure across the filter is gradually increased bymanually adjusting a supply of compressed air. 11le pressure at which the linttbubble appears downstream of the filter is read off from an upstream pressuregauge. Everything is dependent upon successful visual detection of the firstbubble. This i.s not readily ac,hievable for commercial filters because disc hous­ings. cartridge housings, Clc.. prevent bubbles being seen.

170 Chaptet 7

REGION 3CAPILLARY FlOW

REGION tDlFFUSIONALFlCHI /

~_=::::::':::::~==,REGION28UBIllE OR mANSITlON POINT

L.JPSTFEAM GAS PRESSURE •

Fig. 6 Behavioc of a weued membrane with increasing gas pressure.

all manufacturing processes. perfect unifonnity of pore ize is not achie able inthe manufacture of membrane filte~. 1n region 2 of Fig. 6. gas is beginning tonow through the largest pores in the membrane, and flow increases as progres·sively smaller pores become evacuated of nuid. Automated bubble point testingequipment utilizing the principles of Fig. 6 10 determine the bubble point fromthe transition pressure may not be effective in dislinguishing membranes Ih:uhave the same mean pore size bUI wilb different distributions of pore sizes (Fig.7). Third, pores are rarely neally cylindrical passing through the membrane bythe shortest pas-sible roule. In actuality. (X>res follow lanuous roules throughmembranes and do not have unifonn diameters throughout their lengths. so thatpores with identical surface diameters may require quite different pressures todisplace their nuid contents.

For routine control. it is unlikely that the bubble point is ever detennined.It is qu.ite usual for automated equipment to test a fiher at a single pressure lhatis. if the validated condition is being maintained. known 10 be lower than thebubble point. A failure condilion is nagged if the bubble point is reached at Of

below that sel single-point pressure.Pall and Kimbaucr {J] have identified an alternative expression of the

bubble point pressure. Al pressures below the bubble point. diffusional now ofgas through the membrane is proponional to the applied pressUfe_ If the rate ofgas flow per unit pressure is plotted against pressure (Fig. 8). flow will be con-

Sleriliulion by Filtr.tion

MEMBPA"E WITH TIGHTPORE 0ISTAIllUT10N

/

171

PORE OIA.'EI ER~

Fi&- 7 Possible variation in pore size distributions.

CAFUAAY FLOW1HRClUGH OPENPORES

UfFUSIONAL FLOW

Fig. 8 Behavior of a weued membrane with increasing gas pressure.

172 Chapter 7

Slant while only diffusional now is occurring, and the transition to capillary nowwill be effectively abrupt. This transition pressure is referred 10 as K L.

1be deficiencies and nonidealities that occur in bubble point testing shouldnol be laken as contmindic3tions of the usefulness of the melhod for in-processnondestructive filter integrity testing. It is in raer an extremely valuable in-pro­cess test. Its limitations hould be understood and its value confined to compar­ing one filter with anomer of the same type.

J. Diffusion T~st: Filter integrity may be evaluated u.sing the fact thut gas isable to now through welled filters by diffusion at pressures lower than the bub­ble point pressure. In practice. diffusion tests are aUlom:ued: equipment is avail­able 1'0 measure now rate across fi hers by upstream or downstream now mettrsor by upstream pressure gauges. Mea.~urement by downstream flow meter istermed "forward flow," a leon originally used by the Pall Corporation todescribe their own variant of diffusion testing, now widely used to describe anytype of diffusion testing regardless of location of the measuring de\'ice. Diffu·sion testing is done in-plocc. in-process. and nondestruetivcly.

1be principle of the lest is that of diffusion of gases from locations wherethey are in high concentration (upstream of a filter) to locations where lhey arcin low concentration (do",'ostream of a filter). Gas will pass through a weltedmembr.::me thus: it first dissolves in the liquid at the upstream side of the mem­brane. it then diffuses through the liquid phase. and it finally leaves the liquiddownstream of lhe membrane because of the lower partial gas pressure on thatside. In simple lenns. we can consider membranes to be impermeable to gas,and therefore any movement of gas through a tiller must be only through theliquid-filled pores. Where there is a large equivalentllrea of pores there will be agreater amount of diffusion than when there is only a small equivalent area ofpores.

The liquid aCls as a barrier to free migration of gas. The rate of diffusionof gas is brought 10 a steady state when the rate of diffusion of the gas inlo &.heliquid on the upstream side is just equal to the rate 31 which the gas is diffusingout of the membrane on the downstream side. Various steady-sute conditionsmay exist according to pressure (the solubility of gases in llquids increases withpressure), temperature (the solubility of gases in liquids decreases with lemper­ature but the rate of diffusion increases). and other controllable foclors. Theinterrelationships of these factors can be predicted from Fick's laws of diffusionand the gas laws.

Fick:s laws allow the rate of diffusion al steady state to be calculated from

D·H·p·eJ=----

L

174 Chapter 7

O.22um

;f

j

Ij

•!/O.45um

;';,

f•

f

/•,f

~ ..•./

•

•;•;

./•,

/a.Bum•

!;

;•

i

•,;

UPSTREAM GAS PRESSURE •

Fig. 9 Bubble points and diffusional char.lclcrislics of mcmbrdllcs with similar voidfractions.

5. Wclter Penetration Test: Hydrophobic fillers are in the main tested by bub­ble point, diffusion. or pressure decay methods based on wetting with solventswith lower surface tensions than those of the membranes. This is because thevoid space of the membrane will not be completely penetrated by waler to allowthese tests to be applicable. The main alternative solvents used in associationwith hydrophobic filters are aqueous solutions of isopropanol. This practice.ahhough perfectly legitimate, introduces other problems. namely the problem ofremoving all of the solvent, to ensure that product coming into contact with fiJ·lered gas or air is not to be adulterated. and the problem of flammability ofsolvents. The water penetration (water intrusion) test is an alternative methodapplicable only to hydrophobic filters, which by using water rather than solventsavoids the problems described above.

The principle of the water inlIusion lest derives from the mercury intrusiontest, which (applicable to both hydrophilic and hydrophobic membranes) isresuicted to laboratory conditions. The membrane is placed in conlact with thefluid (water in the case of Ihe water penetration tcst. mercury in the case of themercury intrusion test). and the pressure is increased. with the purpose of forcingthe fluid into the pores. The volume of fluid forced into the pores is a measureof pore size and void space volume and thus of filter integrity.

In practice the test is done as almost a mirror image of the bubble poinltest The filter housing is filled completely with water. and pressure on theupstream side is increased incrementally until water now is seen on the down-

Sterilization by Fillt.ation 175

stream side (breakthrough pressure). Pore diameter is related 10 breakthroughpressure in me same way as it is to bubble poine pressure:

cos 9d=-4y·

p

This is the same equation as the bubble point equation except for the inclusion ofa minus sign to compensate for the fact that the contact angle between nonwel­ling liquids and hydrophobic filters is greater than 90- and the cosine has there­fore a negative value. In deUliI. d = the diameter of the pore; y = the surface ten­sion of lhe liquid filling the pore; P = the breakthrough pressure; and 9 =theconlact angle belween the liquid and the pore wall (greater lhan 9<t forhydrophobic membranes).

In practice it is not usual to test filters 10 the water breakthrough pressurebut. as with diffusion lests, only 10 some point below a breakthrough pressurederived by the filler manufacturer; breakthrough at these lower pressures may beindicative of loss of filter integrity or seal integrity failure. alternatively falsefailures may be caused by air pockets in the housing or residual water lefl inpores after sleam sierilization. AI pressures below breakthrough. water is forcediDla the pores of the filler at a rate proponionalto the increase in applied pres­sure. If pressure is held constant below breakthrough, water penelralion siabi­Hzes to a COnSlafll rate with time, usually measured in terms of mUIO min. Thisrate is typical for particular types of filter and can be used as a comparaliveindex of filter integrity with manufacturer's values and values obtained beforeand after use. The rather curious denominator of 10 min is because of the reallyvery small volumes of waler that penettale hydrophobic filters below break­through.

Water peneuation rates are usually calculated according 10 lhe gas lawsfrom measurement of pressure decay upslream of the filter over the whole periodof testing with the gas (air) volume above the fluid held constant. They aretherefore subject ro temperature variations. A1lhough the principle of Ihe waterpenetration tesl is sound, and the avoidance of the use of JX>lentiaily adulteratingsolvents is attraelive. the low cales of water penetration calculable from onlyvery small pressure drops within lest systems have raised doubts about therobustness of the method for routine application in its conlfibulion 10 the deci­sion-making process.

6. Microbiological Moniloring: Microbiological methods of monieoring theemuent from a sterilizing filler are 100 insensitive to discern any but the mostserious failures of the filtration system. Such failures are better de,teeled by thenonmicrobiological in-process integrity tests described above. It is, however,nonnal in the applicalion of sterile filtration 10 ensure that the microbiologicalchallenge to the filter is well wilbin the Iilter's sterilizing capability. Microbio­logical limits should be sel therefore for the Donsterile challenge material; if

c ,

176 Chapter 7

measured numbers are high or exceed justifiable limits. prefiltration or someOlher means of reducing the microbial challenge should be introduced.

IV. SUMMARY OF REASONS FOR FILTRATION FAILURE

In cenain circumstances sterilizing filters may allow the passage of microorgan­isms. It may be of value for preventing this to summarize the main reasons forthe occurrence of nonsterile effluents.

(a) Incorrect assembly. Sierilizing filters must be assembled aseptically.It is difficult to legislate this except by thorough training and good supervi­sion. The number and frequency of aseptic connections should be mini­mized as far as practicality dictates.

(b) Passage through defects. Filters may be flawed or defective. Thedefecls may be within the membrane. or in the housing. or between themembrane and the housings. Rouline integrity testing should be capable ofrevealing these types of defects through consideration of all data frombubble point. diffusion, and pressure hold tests.

(c) Passage through the membrane. The mechanism of panicle retentionby membrane fi1ters is not confined to sieving. h is nO( therefore out of thequestion thai there may be a coincidence of a microorganism at the b<momend of its size range and a pore at the top end of its size ronge. Pan of theanswer to this possible failure mode is for filter manufacturers to controltheir pore sizes within bands of only narrow variation, From the user'spoint of view. the challenge should be kept well within the capability ofthe filter.

(d) Grow-through. Microorganisms retained on a wet membrane for anextended period of time at ambient tempenuures may be able to reproduceand grow through to the downstream side of the filler. This is probablymore likely with air filters than with liquid ftlters or other gas filters. Inpan this is due to the time over which air filters are kept in place; in pan itis due to (he likelihood of moisture being carried in the airstream. II can beavoided by prefiltration and by controlling the dural ion of use.

REFERENCES

I. Food and Drug Administration (1987). GuideJint on Sltrile Drug PrcxJucl.f Pro­duced by Aseptic Prou-s,sing.

2. Schroeder, H. G .. and DeLuca, P. P. (1980). Theoretical aspects of sterile filtrationand inlegrily tc::.ting. Phan"auuricaJ Tt'chnology 4: 80--85.

3. Pall D. B.. and Kimbauer. E. A. (1978). Bocleria removal prediction in membranefilters. In Procudings o/52nd Colloid and Suiface Scienct! Symposium. Universityof Tennessee. Knox\'iUe. Tennessee.

Steriliution by Filtration 177

4. Leahy, T. J., and Sulli ....an, M. 1. (1978). Validalion or bacterial retention capabilitiesof membrane fillers. Phannaceutical Technology 2: 65-75.

5. Pall, D. B., Kimbauer. E. A.• and Allen. B. T. (1980). Pa.rt.icul&le rtltntioo by bacte­ria rtlC:nuve membrnne filters. Col/oids and Surfaces I: 23S-256.

6. Meltzer. T. H. (1987). Filtration in the PhamlQceulicol I"dustry. New York: Mar­cel DekJ<er.

7. Leahy, T. 1.. and Gabler. R. (1984). SlC:rile filtration or gases by membrane filters.Biotechnology and 8ioenginuring 26: 836--843.

8. Health Industry Manuracturer's Association (1982). A,ficrobioJogicaJ £"uluar;QIl ofFiltus for SIe.riJizing Uquids. H1MA Documenl No 3. Volume 4. WashingtonD.C.: Heallh lndustry Manufacturer's Association.

9. Rtli. A. R. (1977). An assessment of le§1 crilCria for tvaluating the perfonnance andintegrity ofsterilizing fillers. Bul/nin o/the Pann"ral Drug Association JI: 187­199.

ANNEX 1. CALCULATING THEORETICAL PORE DIAMETERSfROM BUBBLE POINT PRESSURES

If the conlaCt angle between liquid and membrane lS zero. lhe pore diameler canbe c.lcul.1ed from

4yd=~

P

where d : pore diameter, y = surface tension of the wetting liquid. 3nd P = bub~

ble point pressure.Wlth water as the wetting liquid, tbe bubble point obtained with a particu­

lar membrane was 4.800 mm Hg. The surface tension of water is 72 dyn/cm.Thi is convened into compatible units by mulliplying by a c()I'Rction factOf of7.5: therefore

d = _4_X..:.(7",2:-:X_7_.5:..}4,800

d=0.45 11m

With isobutanol as a welting liquid. the bubble point obtained with anothertype of membrane was 4.5 psig. The surface tenslon of isobutanol is 1.7 dyn/cm.This is convened into compatible unilS by mulliplylng by a correclion factor of0.145: 'hcrefore

4 x (1.7 x 0.145)d = -.:.....,-,,------'­

4.5

d = 0.22 ""'

8Aseptic Manufacture

L Applications of Aseptic Manufacture 180n. Systems of Aseptic ManufaclUre 181

A. Contamination-Prevention and Conlrol 182B. Typical Facilily Design 195C. Specific Aseptic Systems 209

III. Sterility Assurunce from Aseptic Filling 215IV. Validation and Control of Aseptic Manufacture 217

A. Validalion 218B. Routine Monitoring of Aseptic Manufacturing Facilities 229

V. COnlartlinalion Control for Tenninally Slerilized Products 236

All pharmaceuticaJ products are expected to be clean in !.he sense of being freefrom viable and nonviable panicles. Aseptic manufaclure is a system lhat has atilS core 3 situation (aseptic filling) whereby the dosage fonn. product contactcontainers. and closures that make up the final presentation are brought logelherafter previous stages of cleaning and sterilization. There are no cleaning or ster­ilization processes subsequent to aseptic filling. II is imperative therefore lhalthe conduct of aseptic fining is such that the cleanliness and a.fisurance of sterilityof the finished product is no worse than the cleanliness and assurance of sterilityof Ihe individual components.

Current regul.tory thinking (panicuJorly wilb !be FDA) holds lbat asepticmanufacture is a process of last reson. If 3. finished presentation is suitable for

179

Aseptic M~nufadu,e 181

be multi-<lose. There are many heat-stable dosage forms in heat-slable presenta­tions thai are aseptically filled rather than being lerminally sterilized. Past regu­lalory thinking has peonined this practice. a1lhough it is now severely frownedupon.

In some circumstances the decision to fill aseplicaJly or to sterilize termi­n:dly must address more complex sterility-related issues than product stability.Consider. for example. the transfer and admixture of a small-volume phanna­ceutically active dosage form from a glass vial into a large-,'olume bagged infu­sion Ouid. The most widely used approach is to use a sterile syringe to withdrawthe dosage Conn from the vial and inject it into the infusion fluid via a septum.Some presentations are now available whereby the conlents of a vial may bedirectly injected into an infusion fluid bag wilhout the need for the twO syringe­mediated aseptic transfers. These involve specifically adapted infusion tluidbags and plast'ic-modified vials. The modified vials cannot be terminally steril­ized. The question to be addressed is which is of greater risk or benefit to thepatient. a terminally sterilized product that must be aseplically Iransferred twiceat point of use or an aseptically manufactured product that need only be trans­ferred once?

Ophthalmic products, eyedrops in single-dose and multi-dose presenta­lioos, and eye ointments in multi-dose presentations are aseptically manufac­tured. So also are some other topical products. Glass eyedrop bottles are nowless common than plastic ones; aluminum ointment tubes are still more com­monly used for sterile products than plastic lubeS. Plastic caps are alma tuniversally used.

Many aseptically manufactured presentations are fonnulated to includepreservatives. These may serve one or both of two purposes. Firsl. they con­tribute an active antimicrobial process fonowing on from aseptic filling. Thiscan be important to affording maximum patient protection. For a prodUCI t'o bepotentially infective. microorganisms must nOI only contaminale it. they mustalso be able to survive in it. Aseptic manufacture can never provide a 100%guarantee against contamination. but preservatives may prevent the imperfec.lions of aseptic manufac,(ure having serious consequences. The second purposefot including preservatives is particular to multi-dose presenlalions where theirpurpose is to prevent proliferation of any microorganisms that might contami­nate the product over repeated inlrusions in use. II must be emphasized Ihalpreservatives are not included as an alternative to product protection duringmanufacture. The standards of protection and control during ascplic manufac­tute of preserved and nonpreserved formulations musl be the same.

II. SYSTEMS OF ASEPTIC MANUFACTURE

An integrated system of controls and prolective measures is essential 10 the suc­cess of aseplic- matlufacture.

182 Chapler 8

It is generally agreed that the most critical operation within aseplic manu­facture is the aseptic filling stage. At the point of fill, the dosage foml is at mostrisk of contaminatjon from environmental air-borne microorganisms while it isbeing transferred from one fonn of closed containment to another. This stage ofaseptic manufacture must be located in a clean room and should have some fur­ther form of localized protec-tion from air-bome contamination.

The pnxluct may also become contaminated by surface-la-surface transferof microorganisms from personnel and the manufacturing equipment itself.Cleanliness of manufacturing equipment is therefore imperative. Effectivecleaning and decontamination procedures are therefore a necessary pan of inte­grated aseptic manufacture,

It is also essential for equipmenl to be maintained in a slate of cleanlinessand sierili(y belween cJean-downs. In unprmccted environments, contaminationby air-borne microorganisms is inevilable. It is nonnal lherefore to house asep­tic filling equipment in areas that are protected from the general contaminatednatural environment (clean rooms).

Movement of personnel, components, equipment. and services in and outof protected areas needs also to be controlled if the protective barriers are to beeffective in preventing contamination. Many of these necessary intrusions canbe brought into me prolected area via some form of decomamination or steril­ization process. for instance via a double-ended autoclave. Personnel cannot beeffeclively decontaminated in these ways bUl must be dressed in sterilized gar­ments that prevent skin-borne and hair-borne microorganisms and respiratoryand digestive activities from contaminating the prolected area, the manufacturingequipment, or any component of the finished presentation. Personnel musl beeducated and disciplined in Ihe specific techniques of working in aseptic areas,and specific localized protection should be given to manual operations to reducethe dependency on knowledge. experience, and skiHs.

The separate and independent components of an effective system of aseplicmanufacture overlap mlher than abul against one another. Momenlary failure ofany onc system does not necessarily compromise product slerilily. Nonetheless.good assurance of avoidance of contamination can only be obt.ained from knowl­edge Ihal each system is capable of perfonning as it oughl to perform (vali­dation) and thai it continues to operate effectively in routine use (routine moni~

toring).

A. Contamination-Prevention and Control

1. Envir01lmemal Air: Microorganisms are ubiquitous in unprotected envi­ronments. Air is nOI a milieu Ihat supports Ihe growth of microorganisms. but ilalways carries a contaminanl microOora of mainJy desiccalion-resistant microor-

Aseptic Manufacture 185

; ,WAY tOA"lEAE._f toO'..Fl.ME J

£ ...'"'( IOClIN f'\Nlj( )

TERMiNAlHEPAFILTERS

II:1,il

SUPPLYFAN 1'\'\'\

FRESH AIR PRETREATEDTO DEHUMIDIFY

PRE. HEAT­FJlTER EXCHANOf:R

~"'"

FIWNGAOOM

EXHAUST AIR

O)W,llffAH

AETURHAIRHEPAFILTER

fig. 2 Layout of a typical air handling system.

Housings for tennjnal HEPAs should have ainight seals to the ceilingbehind lite grilles or diffusers. The sealing of the HEPA filter should be on theair-inlet side of front-withdrawal HEPAs and on the air-outlet side of back-with­drawal HEPAs. Filler housings should be equipped with an access port behindthe filler 10 3110w access of smoke for validation purposes (see below).

Allhough environmental air is ilSelf a source of potential conmmination, ilcan also. once tihered. be used to prevent contaminalion. Microorganisms arenot equipped with any means of moving in an airstream; signiticantJy, Ihey can­not "swim upslream" against a positive air pressure.

Pressure differentials have widespread use in aseptic manufacture to pro­tect critical operations and critical areas from adjacenl areas of lower criticality.The second proteclive effect thai can be exerted by air is a "sweeping" one.High-vdocilY filtered air (greater than 90 ftlmin). lermed laminar flow. has thecapability of protecting critical oper.uions by sweeping panicles and microol­ganisms away from the sensitive area. Vertical and horizontal laminar flow unitsare available, classified according 10 the direction of airflow. Laminar flow iswidely used for localized protection in aseptic manufacture. for instance OIl pointof till in aseplic filling. over aseptic manipulalions involving personnel andnumerous olher situations.

186 Chipter 8

2. ManufacI14ring Equipment, Facililies and Services: Microorganisms cansurvive. and indeed proliferate. on the surfaces of seemingly inert objeclS likewalls. floors. and machine pans. The presence of even small amounts of organicmaterials. oils. or greases may increase lheir resistance (0 naturally inimicalforces present in the environment Unprotected manufacturing facilities andequipment inevitably become contaminated by deposition of microorganismsfrom the environmental air. and by surface 10 surface transfer from personnel. Infacilities protected from the general environment by the provision of filtered air,pressure differentials. and perhaps laminar flow, surfaces that are decontami­nated ought not to become reconlaminaled. Cleaning and decontam.ination ofequipment is a primary and major system of conlaminalion control.

Demounlable prodUCI contacl machine pan. for aseptic filling are bestcleaned outside the filling room. It is not a good idea to situate wash-bays incritical areas. Water is always a potentially major source of microbiologicalcontamination. and waler-borne microorganisms. panicularly Pseudomonas spp.are notable for !.heir ability (0 increase in numbers in circumstances where nutri­enlS are in vcry low concentration. and 10 be able to use Ihe most unlikely com­pounds to suppon growth. Returning these pans to the filling room shouldpreferably be via a terminal sterilization process. or after decontamination withan appropriale disinfectant via an interlock. air lock. or hatch designed to preventloss of air pressure in the protected area and to faciliulle disinfeclion.

Fixed equipment. walls. floors, and ceilings can only periodically be thor­oughly cleaned down and deconlaminated. There are only two approaches tocleaning acceptable for aseptic manufacturing areas. wet wiping or a vacuumcleaner equipped with a HEPA filtered exhaust

Vacuum cleaners. however. are only suited to areas outside aseplic fillingrooms because of their relative inefficiency of collection of smaller particles II).leaving wet wiping wilh disinfectants as the "standard" method used for cleaningfilling rooms.

TIle choice of disinfectants is never easy. Cross-contamination of theproduc-t with disinfectant traces is to be avoided at all costs. All disinfectantsbrought into aseptic filling rooms either for major clean downs or for routine useshould be filtered into sterilized containers via 0.22 ....m pore size sterilizing fil­lers. It is also imponant for disinfectants to be rotated to prevent selection ofresistant populations of contaminants. Alcohols (ethanol or isopropanol) arewell suited lO aseptic filling room use, as also are some proprietary disinfectantssuch as chlorhexidine that are specifically manufactured for such purposes.Periodically the whole aseptic manufaclUring area may be fumigated; formalde­hyde has been in common use bUI is declining due 10 its potential risk to theheahh of personnel who muSI work in fumigated areas.

Services should be provided from outside aseptic filling rooms. either byrouting within the fabric of the surrounding walls. noors. or ceilings. or from

Aseptic Manufacture 187

adjoining less critical areas of rne aseptic manufacturing facility. Light fiuingsshould be fitted flush to walls or ceilings to prevent unnecessary uncleanable sur­faces. Water should be avoided. Compressed air should be filtered through 0.22J.UTl pore size hydrophobic sterilizing filters.

3. Dosage Forms and ProduCI-Conract Components: The object of asepticm:muIacture is to bring a sterile dosage form and the finished presentation'spresterilized pnxluct contact components together without contaminating them.Confidence of their sterility is of major imponance. This means validated ster­ilization processes and low levels of microbiological contamination prior to theirsterilization.

Parenteral solid dosage fonns, e.g.. antibiotics. are most usually deliveredas lhe sterile drug substance from primary fennentation and conversion opera­tions to secondary aseptic manufacturing facilities. They mayor may not beblended aseptically wilh other sterile eJtcipienlS before aseptic filling. Blendingshould be considered as an aseptic process and be subject to all of the nonnalconsuaints. Entry into the aseptic filling room should be via an interlock. airlock. or hatch designed to prevent pressure loss in the protected area. Interlocksmay incorporate some fonn of decontamination of the sealed container in whichthe dosage {onn is delivered. for instance ultra-violet irradiation. disinfectantbathing. or peracetic acid decontamination.

Liquid formulations should be prepared in an area that is as clean as possi­ble. Water foc parenteral products should be of pharmacopoeial \Valerfor Injec­tioll quality. Mixing vessels and orner equipment should be cleaned and disin­fected. II is normal for these areas to be provided with filtered air from HEPAfilters of somewhat lower efficiencies lhan those providing protection to asepticfilling rooms. Sterilization of liquid products should be by filtration through0.22 .... m sterilizing filters directly into the aseptic filling room. Two filters inseries are often used; the first of these is nominally to protect the inlegrity of theaseptic filling room. and the second to sterilize. tn practice one filler is suffi­cient for sterilization; two mounted in series simplifies lhe decision-making pro­cess in the event of one but not both filters failing post-use integrity testing.

Filtered sterile dosage fonns may be collected in sealed vessels in theaseptic fLlling room for subsequent connection to the filling machine, or the fLlleroutJet may be connected direcl1y to the filling machine.

Product conlaCt components should be washed and sterilized inlo asepticfilling rooms. Glass vials are commonly passed through a combined washer/dryheat tunnel connecling unprotected receiving areas to the asep1ic filling room. Inolder or low-volume operations. glass components may be dry heat sterilized inovens. Rubber stoppers and aluminum ointment tubes are preferably sterilizedinto aseptic filling areas via double-ended washer/autoclaves. Radiation orethylene oxide sterilized prodUCI contac·t components like ointment tube caps andeyedrop caps should be introduced via an interlock as described for solid dosage

, I

188 Chapter 8

forms. Product contact gases for sparging or over-pressurizing should be sleril­ized by passage through 0.22 J.l.ffi pore size hydrophobic sterilizing filters.

Any storage of sterile dosage fonns or sterilized components in asepticfilling rooms should be in seaJed containers, llnd these should be afforded addi­lional localized filtered air or laminar now protection to prevent _heir contentsbecoming contaminated when they are opened.

4. Personni'J: A Donnal heallhy individual sheds about len mill.ion or 6- 14 g ofskin scales daily. About 10% of these carry microorganisms such as Sruphy/o·coccus and Propionibacterium. Some of these may be pathogenic. In a normalpopulation. 5 10 30% of individuals may carry SlaphylococCliS a"re"s in theirnoses and 2 (0 5% may carry S,reptOCOCcuJ pyogellcs in their throats. Microor­ganisms may also contaminate wounds and skin infections. Poor personalhygiene may permit transfer of faecal organisms on the hands.

There are five considerations given to prevenlion and control of contami­nation from personnel sources: (a) training (knowledge and pr.tctice). (b) medi­cal screening. (c) control of access. (d) containment of the individual. and (e)protection of critical operations.

Untrained individuals should not be allowed 10 enter aseptic filling roomsnor auempl 3S4:ptic manipulations associated with product manufacture. Train­ing should address 3n understanding of the sources. routes. and control systemsrelating 10 microbiological contaminalion and should concenlrate on developingdexterity with respect to the specific techniques of aseptic operatjons. Personnelcan disseminate microbial aerosols even when standing or sitting slill. but therate of dissemination increases dramatically with movement. More people in anarea means more dissemination.

Individuals with medical conditions that may lead to abnonnal1y highshedding or dissemination of microorganisms should not be chosen 10 work inaseptk filling rooms. Conditions include excema, psoriasis. styes and boils.coughs, colds. and hay fever.

It is personnel with symptomalic conditions who musl be screened out ofaseplic work; lhe large proportion of the population who are nonsymplomalicnondisseminating carriers of pathogenic microorganisms are no more a risk thanother healthy individuals. Some aseptic manufacluring operollions bar non­symptomatic carriers of Staphyhx;oc(;uS and Streptococcus from critical opera­tions. This is slrangely illogical, since nonsterility is a condition thai COlD resultfrom contamination by any microorganism. and pmhogens are no more signifi­cant than nonpathogens.

Access of personnel 10 aseptic filling rooms should be via a defined route.through a series of al least lwo changing rooms progressively meeting higherstandards or cleanliness. and againsl a pressure dirferemjaJ.

The principal method of protecting aseptic filling rooms from conlamina­lion by personnel is through Ihe provision of effective containmenl of their

190 Chapter 8

sures are obtained by externally mounted pumps delivering and exhausting airthrough HEPA fillers. Access for manipulations may be through glove ports orthrough "half-suits'" The difficulty with isolators is how to gel the product andcomponents intended for aseptic filling in and out of the isolator. With flexiblefilm manual work stations this can be resolved by bringing materials 10 theisolator in special containers thaI can be aseptically connected to entry pons,The situation is rather more difficult wilh high-speed industrial filling lines;some compromise of inlegrily of the isolator is unavoidable.

By and Large the method of choice for localized protection during asepticmanufacture is by laminar flow fihered air. The principal of laminar flow is tomaintain the sterility of a work. area by continuously purging it wilh unidirec­tional filtered air moving along parallel flow lines. Laminar flow technologyand aseptic manufacture have become so closely identified that the two termshave almost become synonymous. Since its introduction in 1961. laminar flowtechnology has led to a progressive expansion of high-speed aseptic manufactureand of the available range of aseptically filled phannaceutical products.

Very small panicles such as those that carry microbiological contamina­tion (usually in the range of 810 14 11m in diameter) are nol heavy enough to fallto ground level under gravitational forces alone. Instead they remain in air cur·rents and move around as the air eddies.

Particles generated upstream of exposed products in aseptic filling rooms.or particles caught in regions of turbulent air, may be swept onto and into theproduct. The action of laminar flow (usually understood to mean an air velOCityof around 90 ftlmin) is 10 sweep across an area withoUl creating turbulence orregions of dead air. In the event of a microbial or paniculate contaminant beingintroduced into lhe laminar flow protected area, whether by having been carriedin or having been generated there. the high-velocity unidirectional airflow carriesit quickly away.

The big advantage of laminar flow over containment is access. Personnelcan easily gain access to the area in which their intervention is necessary. Prod·uct and components can be conveyed into laminar flow protected areas withouthaving to pass through complex barriers. Nonelheless these advantages can onlybe achieved lhrough very careful application. Figure 3 illustrates a horizontallaminar now work. station. This Iype of application may be used for localizedprotection of manual operalions like making and breaking aseptic connections orstorage of machine parts or vial stoppers after sterilization but before loadinginto the filling machine hopper. This type ofwark station is also commonly seenin laboratory applications. Quite simply. the work. stalion draws air from thegeneral environment and forces it through a HEPA filter and out over the workarea via a diffuser. The diffuser serves two purposes. first 10 protect the HEPAfilrer face from physical or chemical (spraying with a disinfectant. for instance)

Aseptic Manufacture 191

~ INTAKE nRJUGH PREFk.TER ..,......D~

'''GNEom" GMJOE..... (!) "'OCITY

""""'"-

- -----.... -------....1-

""

lj'.UHOW

'"""""----------------

tEPA'I.ttR

Fig. 3 Horizontall3mill3r flow work station.

damage by lhe operalOT, and second 10 ensure laminarity of air movement (Fig.4).

Figure 5 illustrates a venical laminar now canopy such as mighl be usedfor localized protcclion of a tilling macttine or the off-loading station from asterilizer. Fixed or portable versions of Ibis type of unit are available and have awide application in aseptic manufacture.

The precise location of portable units mus( be defined. Laminar flowunits. although creating a protected cnvironmenl wilhin lheir confines, maythemselves create lUrbukmce and dead air in lhe room in which me)' are shualedand inlo which me)' exhaust This may create a secondary source of contamina­tion. 1be airflow patterns around laminar now units relative to fixed air exitregisters, sc.reening. and other equipment in a room cannol be predicted.Empirical evidence from smoke panern studies is necessary to be confident oftheir effecliveoess.

In the carly days of laminar flow. lhe idea of enlire rooms being protccledby laminar now wali greeted with enthusiasm. They are now rarely found inconnection wit.h aseptic filling. 1lley are far more expensive to construct .hanconventional turbulent now clean rooms, because of Ibe need for more filte.rsand greater enginee.ring capabilities to move a lot of air at high velocity. More­over, a horizontailaminar flow wall provides unidirectional air flow only as faras Lhe first work sLation. 1bereafter it is no different from 3 conventional clean

Aseptic Manufacture 193

••DEAD :•SPACE

, ,

, . DEAD: SPACE

~:

DEADSPACE

, ,1-------1, ,":"" "":"• •

, """, , ., ,"", '"§", ~P~"§

fiB- 5 Venicallaminar flow canopy (showing smoke patterns for full- and half-lengthcunains).

The phannacopoeias restrict the methods by which Water for Injectionmay be prepared 10 distillation and [0 reverse osmosis. The nature of the distiJ­lation process dic1ates Ibat freshly disLillcd water must be sterile and (except inthe case of some systems failures) pyrogen free.

1be molecular dimensions of bacterial endoloxins are 100 large to passthrough reverse osmosis membranes. and therefore Ihe process is permitted bythe pharmacopoeias for production of Waler for Injection. However, the outletwater from reverse osmosis units can easily become microbiologically contami­nated by formations of films or slimes downstream of the membranes. AI leasttwo microb.iological problems may be encountered wilh reverse osmosis. Firsl,microbiological films may slough off into the water at unpredictable intervals or

, I

194 Chapter 8

as a resuh of disinfection; second. lhese films are peculiarly resistant to disinfec­tion and once fanned may be very difficuh to eradicate.

Ingredient waler should be used immediately or slored under c,ontrolledconditions. Large sterile production facilities use stainless steel storage and dis­tribution loops that keep water in continuous turbulent motion at temperaturesabove SO·C. All water contact surfaces should be smooth and continuous to pre­vent accumulation of nutrients, and lhe system should be designed 10 allow fre­quent draining, flushing, and sanitizing (preferably by steam at 120·C). Mainsshould be sloped to allow complete drainage, and "deadJegs" should be avoided;where this is impossible they should be kept as shan as JX)ssibJe (as a rule ofthumb, less ilian six times the diameter of the piping). Stored water should bekept under a blanket of inen gas such as nitrogen. and tanks should be providedwith air filters to allow venling as water is drawn off.

Water of Water for Injection and Purified Water qualities has other appli­cations in addition to its use as ingredient water. For instance. the final rinsewater for washed product contact components and machine pans should be of nolesser quality than ingredient waler. Purified water from W"e loops is best usedfor machine washing. but SO'C is an unacceptably high temperature for humancontact in manual operations. For these purposes water may be drawn from thehigh-Iemperature circulatory loop via local heat exchangers. However. the.>ie areprone to microbiological conramination; an ahemative approach is to run a lowertemperature loop (e.g., at 4O·C) for these purposes. It is advisable to raise thetemperature of 4Q"C ring mains 10 80·C overnight each night to avoid buildup ofmicrobiological contaminants.

At any moment all of the waler in an aseptic manufacturing facility mustbe in the product, in the distribution system, or passing through the drains. Seri­ous measures must be taken 10 ensure that drainage systems do not becomesources of microbiological contamination and proliferation.

All equipment drains should have air breaks of a few inches separatingthem from floor drains. Larger air breaks do nOI necessarily give more assuranceagainst drains causing contaminalion. Splashing from water falling gre,ater dis­lances may lead to the formation of microbial aerosols. which may then con­taminate exposed product or manufacturing equipment. Further avoidance ofsplashing may be facilitated by use of conical lun dishes 10 channel falling walerinto floor drains.

Floors of preparation areas where waler spillage is inevitable should beconstructed "true" to avoid puddles and should have a slope of about I:50 to aflush filling floor drain. Floor drains are be.st kept as dry as possible to avoidmicrobial proliferation. The moSI effective method of sanitizing drains is byheated traps. These are heating elements capable of boiling water located in theV-bend beneath floor or sink drains. They are nonnally operated by limingdevices to work only when there is water in the drain.

I ,

Aseptic Manufadure 195

8. Typical facility Design

At first glance it might seem that the lerm aseptic manufacturing facility is syn­onymous with lhe [erm dean room. h is not. Although aseptic filling is alwaysdone in a clean room, the clean room is only one compooenl of a complex of bar­rier systems that make up an aseptic manufacturing facility.

At the hean of aseptic manufacture is the filling machine or filling stalionitself; the point of fiU is thought to be the mOSI critical location to exercise con­taminalion control. The US? recommends a double barrier concept of contami­nation control, with the primary barrier toward prevenlion of microbiologicalconlamination being some son of localized prOiection around this region. Theclean room (aseptic filling room) in which the filling machine is located shouldconstitute tbe secondary barrier. In practice lhere are usually far more layers ofproleClion offered 10 Ihe poinl of fill.

Figure 6 diagrammatically represents the successive series of barriers tocontamination that exisl in Iypical aseplic manufacluring facilities.

(a) The first layer of protection is outside the plant itself. The condition ofthis area may contribute to contamination control. h is usually not thoughtdesirable for eanh banks to abut directly against the external walls of thefac.ilily nor for pools of stagnant WOller 10 lie in the close periphery. Manyaseptic manufacturing facilities prohibil broken soil created. by buildingactivities to be left uncovered close (0 the eXlemal walls.

(b) The incoming warehouse is the first layer offering proleclion by aphysical barrier. Ihe facility waJls. Apan from the fac-IS that warehouses

EXTERNAL ENVlAONMENT

WAREHOUSE

\-.-1:-rGENERAL PRODUCTION

~~GAAYAREA..=// WHrrE MEA

POINT Of' ALl.

Fig. 6 Layers of protection to aseplic filling.

, I

196 Chapter 8

can be kepi reasonably clean. and mal pest control systems can be exer­cised in warehouses. the protection against contamination afforded by Ihislayer is minimal.

(e) Even in facilities dedicated wholly to aseptic products there will besome form of general production. for instance packing sterile sealed con­lainers for shipping. The third layer of protection is this general produc­tion area. Pest control systems must be more effective than in open-bayedwarehouses. All staff can be routed through the general production areavia firsl level changing procedures that require external street clothing tobe replaced by simple factory overalls. which may range from a simplesmock over street clothes 10 a complete change into company uniform andshoes. This is the lasl region within Ihe facility that should be consideredas a public area. Further progress toward the aseptic filling room shouldonly be allowed to trained per;onnel.

(d) The first layer offering significant protection to aseplic manufacturingshould only be enlerable via a changing area requiring pc:rsonllel to pUI on

shoe covers. head covers and dedicated ovemlls. This part of an aseplicmanufacturing facility is often, but not universally, referred to as a "gray"area. Typically it is the pan of the facilily where actives and excipienls aredispensed and mixed. and where machine parts and components arecleaned and prepared for entry into Ihe aseptic filling room or "white"area. Importantly, the gray areas must be maintained at a higher air pres­sure than the surrounding parts of Ihe facility, Air pressure must be care­fully balanced in aseptic manufacturing suites to ensure that there is acascade of pressure differentials from the most to the least critical oper:l­tions (Fig. 7). Best practice is for all personal clothing or genenil factoryuniform clolhing to be removed on entering the gray areas. Aseplicunderwear may be provided to wear beneath gray area nonsterile overalls.

(e) The while area, clean room, or aseptic filling room should be seah::doff from other areas. for access of product, components and personnel.Direct access thai is not via some form of inlerlock is not permitted. Per­sonel access to the white area mayor may not be via a gray area. If it isnal via a gray area. personnel should not be permitted 10 change directlyfrom general factory attire into aseptic area clothing.

(I) The final zone of protection is the localized protection given to point­of·fill and to manual processes conducted within the filling room.

The inlerrelationships between standards of cleanliness and activities needed forgray areas and those of white areas are critical to the successful operation ofaseptic manufacturing facilities.

198 Chapter 8

poneOI washing and sterilization into one process. Irrespective of lhis. the load­ing ends of autoclaves, ovens. and tunnels leading into the aseptic fining roomare located in gray areas. Access to sterilizers for maintenance and repair is notallowed from inside lhe white areas.

Personnel wishing to gain access to lhe wbile area must pass lhrough adedicated changing room. These should be supplied with air of the same quaJityas while areas, but they are strictly speaking gray areas. It is preferable for I.hewhile changing rooms 10 vent air to gray corridors or some other gray areaphysically separated from the gray areas in which product and components areexposed. The white changing room should not be accessible directly from thegeneral factory environment.

These changing areas are the most likely route of entry for microbiologicalcontamination into aseptic filling rooms. The doors leading into changing roomsand those from the changing rooms to the aseptic filling room should be intcr­locked in such a manner that only one set of doors can be opened at one time. Insome societies safety considerations preclude this type of interlock and warninglights or alarms may be used to supplement personnel disciplines. Other fealUresof white changing areas are step-over benches (boot barriers). hand·wash sta­tions. and hot-air hand driers. The purpose of the step-over bench is to segregatethe areas in which only designated sterilized (or sanitized) footwear may be wornfrom the rest of the aseptic manufacturing facility. which has less stringent foot­wear requirements. The hand-wash station should be provided with elbow- orfoot-operated laps. Water should be provided in the temperature r.lnge of 45 to5S·C, and polentiaJ contamination from water drainage should be given specialconsideration. In some aseptic manufacturing facilities the risk of contaminationfrom this source has led to a total ban on water from aseptic filling roomchanging areas. This is probably best practice but places high demands on disci­pline with regard to hand washing at earlier S13ges in the progression of person­nel from the general factory through gray changing areas.

Last but not least. best practice is for aseptic changing rooms to beequipped with horizontal laminar now protection. providing a gradient of aircleanliness from the filter bank situated at the cleanest end (access to the asepticfilling room) of the room to exit registers at the entrance.

Provision of aseptic area garments to changing rooms must be given seri­ous consideration. Unless presterilized garments are being provided throughtransfer hatches, laundries should be situated in the gray areas adjacent. Wash­ing machines should be of double-ended barrier types and should incorporatefacilities for integral disinfectant rinsing and drying.

2. lVilile Areas: The pressure differential between the white area and adjacentareas should be no less than 1.5 rom (0.05 in) water gauge. There must be noless than 20 air changes per hour. and the balance between recycled and fresh airmakeup should be around 9: I.

Aseptic Manufacture 199

Aseptic filling rooms arc invariably required to be Class 100 clean rooms.M.o t ofitn they are turbulent now (conventional) clean rooms, but some facili­ties may operate laminar now clean rooms. With conventional clean roomsHEPA filtered air is introduced into the room Ihrough registers sitml1ed in Iheceilings or at high levels on the walls and leaves the room through low level exicregisters (Fig. 8). The effects of conventional clean rooms are dilUl'ion and filtra­lion. The oir becomes thoroughly mixed within the room, and particular aUen­tion must be paid to avoid areas of dead air. Laminar now clean rooms(described above) are more cosily and are not frequently seen in connection withaseptic manufacture.

Within the filling room, all aseptic manipulations and process stagesrequiring exposure of the dosage form or product contact components should beprotected by localized laminar flow drawing air from the Class 100 environment.1be importance of this protection is not in the sterility of the air bUI in thesweeping effecl of lhe air.

3. LoyouJ: The design of aseptic manufacturing facilities and the layout of thevarious functional operations relative to the need for proximity and the need tomaintain asepsis is rughly complex and product specific. The simplest case isone of a single aseptic filling room with wholly dedicated gray areas and otherservices; more often several filling operations may be clustered together in onefilling room. or several filling rooms may be serviced through one combined

gray"""

...........~nl!llll1illll '--------,

••on AE_ftR

fi'WM!!

.........

I

/ XlV\

Fig. 8 Turbulent now (conventional) clean room.

, I

200 Chapler 8

= Laminar Flow = Door

WASHER

••

FLUNG_E

AUTOClAVE

Fig. 9 Roor plan-a5epl:k ,,'i31 filling line.

Figure 9 illustrates n liquid dosage form vial filling line with dedicatedservice areas. The now of vials from empty 10 full follows a U-sh<lped tr.td "ina washing/LAF sterilizing/cooling tunnel positioned parallel 10 an inspectionmachine. The two arms of the U are connected by a localized laminar flow pro·Iceled filling machine toc~ned in a Class 100 aseptic filling room.

In this facility there are two major gray areas, and the filling room liesbetween ahem. The dosage foml is compounded and mixed in the same majorgray area into which vials enter empty and leave filled but sepamted from theseactivities by a partition. Closures are processed through a double-ended rotary·drum washer/autoclave located in the second gr.ay area on the olher side of thefilling room. The aseptic changing area and the laundry are located beside oneanother: both are protected by horizontal laminar flow.

In Fig. 10, two solid dosage fann aseptic filling rooms share common grayareas. Once again the flow of vials from empty to full follo ....'S a U-shaped track.and again there are two major gray areas. Within each gray area the same per·sonne! can service both lines with ,'ials or closures.

1llere is no compounding or mixing in this proces. The dosage fonn isreceived sterile from primary manufacture and is passed into the sterile areathrough a lamjnar flow protected interlocked transfer hatch. lbe dosage formconlainers are thoroughly decontaminated befon: bc:ing placed in the transfer

204 Chapter 6

Classification of manufacturing environments in Clea" Room Standards isbased on numbers of physical particles of specified sizes present in unit volumeof air. Panicles of six size ranges (equal 10 or greater than 0.2 Jlm. 0.3 J.lrn.0.5 J.lnl. Illm. 5 1l0l and 10 )lm) are cited. Unit volume is described as per n3 inthe 1973 F~deraJ Standard, per m3 in the British SlGndard and in the 1992 Fed·era/ Standard. Classes are defined al; numbers in the Federal SWfldards(Classes 100. 1.000. 10.000, and 100.000 in FS 209B. and M 3.5. M 5.5. and M6.5 in FS 209£) and BS 5295. /976 (Classes I. 2 and 3); in BS 5295. /989 classesare defined by letters of the alphabet (Classes E. F. G. H. J. and K). Table 2compares the vanous classifications.

The lrnditional applicatjon of physical particulate standards 10 the pharma.ceutical and medical devices induslrie has focussed on particle sizes equal 10 orgreater than 0.5 J.1rn and equal to or greater than 5 ~m. Monitoring equipmenthas been specified to this end. Progressive revision of the Clea" Room Sum·dards has led to an increasing emphasis on smaller sized particles requiring morehighly specified instrumentation for monitoring. FS 2090 and FS 209£ forinslance now include class limits for panicles of equal to and greater than 0.2 J..IOland 0.3 ~m within Class 100 (Class M 3.5).

Considering the size of microorganism and the si7.es of the panicl~s

upon which air·bome microorganisms are likely to be carried (Fig. 12), it is verydoubtful whether contamination from exceedingly small panides of less than0.5 ~m diameter are of sufficient relevance to the pharmaceutical industry to

l- - -~J

10 ~m PARTICLE TYPICAL DIMENSION Of YEASTS

0.5 ~m PARTICLE. TYPICAL DIMENSION Of BACTERIA

Fig. 12 Relative sizes of particles.

Table 2 Comparison of Panicle Count RequirementS in Oean Room Standards Applicable to Aseptic Filling (FS 209 Class 100 (M 3.5). !BS 5295.1976 Class I, OS 5295.1989 Classe, E and F) ii'

~

Routine monitoring..

Validation ~c

BS 5295 FS209 BS 5295 FS209 ~1976 1989 B(1973) E(I992) 1976 1989 B(l973) E(I992) ~

Minimum number o( 3 NS 2 2 3 NS AS ASlocal ions per room

Recommended noor 20-25 m2 to m2 Area in ft2 Area in ft2 20-25 m2 10m2 AS ASarea per sample divided dividedloc:uion by 10 by 10

Locations to Close to Equal Uniformly Unifonnly Close to Equal AS or where ASbe sampled walls work subareas spaced spaced walls work subareas cleanliness

stations & stalions & is criticnl. orwhere control where control high countsis important is important in validalion

Replication 81 NS x5 NS NS NS x5 NS NSeach location

Recommended NS Al least once Inilial & AS Daily Weekly AS ASfrequency per year and periodic

after clO!lureceeds 7 d-

NS • not specified; AS = as OIherwisc specilied. '"0'"

206 Chapter 8

merit wholesale replacement of instrumentation unless the revised Standards areimposed by regulatory pressure over-enlhusiasm for the "latest revision" conceplcould too easily lead to unwarranted emphasis on very small panicles and over­emphasis on air-borne contamination to the detriment of the greater risk of sur­face-lo-surface contamination from personnel working in aseptic facLlities.

The FDA's Guideline on Sterile Drug Products Produced by Aseptic Pro­cessing [2} requires Class 100 conditions of FS 2098 for aseptic filling rooms;the Orange Guide [3] asks for Class I of BS 5295./973 or Class 100 of FS 2098.Classification of clean rooms to these Standards does not require measurementof panicles smaller than 0.5 J.lrn. The EEC Guideline (4) however refers to FS209D, which additionally requires measurement of panicles equal to or greaterthan 0.2 llm and 0.3 IJm. There is no evidence of regulatory insistence on mea~

surement of these particle sizes. It is probably reasonable 10 assume that anyfuture revision of the FDA Guideline is likely to follow suil and change its Stall­dard from FS 2098 to FS 209£. which coolains the same provisions. It is to behoped that any such revision should specifically exclude the measurement andmonitoring of panicles smaller than 0.5 IJm.

a. Sampling RequiremenlS for Clean Room Classification. In the CleanRoom Standards. sampling is considered for two circumstances, for validalion(termed certification in the Standards) and for routine monitoring. Further dis­linctions may be made on the b.'lSis of the. operational condition of the cleanroom when samples are taken. The Standards of the 19705 are quile explicit FS2098 states thai "counts are to be taken during work aClivity periods." BS5295.1976 requires validation to be done in the unmanned slate: and routinemonitoring in lhe manned state.

The Slwu/ards of the 1980s and 19905 allow classificatjon to be: claimedfor"as built:· manned or unmanned condilions (85 5295.1989), or"as built:· "atrest." operalional, or "as otherwise specified" (FS 1090 and FS 209E). Thisincreased scope in me Clean Room Slalldards is nOI helpful to interpreting thepharmaceutical regulatory IileralUre thai predates lheir publication. The docu­ments offering guidance on aseptic filling make sparse reference to the oper­ational state under which clean room classification should be claimed.

Some types of aseptic filling are not suited to particle counting while oper­alional at all; for inslance, solid dosage fonns generJte particles from the productitself; classification to FS 2098 has always been interpreted freely to accommo­date mis. Other types of manufacturing equipment are particle generators whenin operation. It is partly to prevent comamination from lhese sources that local·ized proteclion is so widely advocated for pharmaceuLicaJ manufacture. A foot­DOle to FS 2090 states that Ihe "as otherwise specified" condition should refer tothe degree of control specified by me user or contracLing agency. This may beregarded fairly as an in\'italion 10 specialized industries such ali aseptic pharma­ceutical manufacturers to derive their own requirements for clean room c1assifi-

, I

Aseptic Manuncture 207

cation while remaining within lhe overall coverage of the C/~a" Room Standard.Normal practice is for classification to be done in the unmanned nonoperationalcondition.

Classification and monitoring to the Clean Room Standards requires l.hatparticular numbers of samples of air be laken per unit floor area. that samples betaken in specified locations. and that samples be laken at appropriate frequen­cies. Table 2 summarizes these requirements. It is evident lhat the evolution ofthe US standard from FS 209B to FS 209£. both for validation and for routinemonitoring. is toward lhe "as otherwise specified" condition, which bounces theonus for decision-making back to the user and his ··customer." This is not thecase for lhe Brirish Standard. To validate a clean mom. the British Standard 8S5295./989 asks for more locations and more replicate testing at each locationthan BS 5295./976 or eilber revision of lbe U.S. mndard.

In BS 5295.1989. conditions for routine particle monitoring are no lessdemanding lhan lhose specified for validation. The frequency for routine moni­toring is weekly versu monlhly for FS 209D.

This means that lhe amount of testing required to claim compliance withBS 5295./989 is far higher than the amount of tesling required to claim compli­ance for lhe same clean room with any other standard.

Figure 13 illustrates sampling locations to BS 5295./989 and FS 209D(and FS 209£) for an aseptic filling clean room approximately 7.3 by 11.3 m (82

37 ft

• 24 ft

2FLOOR AREA = 680 ft

~

••

".~

SAMPLE LOCATIONS

=68

FS2090

~ 7.3 m• ,

1 i2 '3 4,! •

8.,

-._.._._~_. --_.._...;.-..__.._-j 7 i6 5; ,

~

! 1

, ~

'9 ,10 11' __~_"M"'_.+""_""""_"

ji

I,,!,

15 114 !13 12

as 5295;1989

1.3m

Fig. 13 Sampling locations for compliance with Clean Room Standards 85 5295.1989and FS 2090.

208 Chaptrr 8

rol} FS 209D requires the largest number of locations. 68; as 5295. /989 speci·fies 15 locations. but with 5 replications lhis amounts 10 about lhe same numberof samples. 70. as FS 209D. It is only when it comes to rouline monitoring thatthe full impact of selecting compliance with one or other of the two 1980s stan·dards becomes apparent. FS 209D asks for monthly monitoring and allows theuser and "customer" 10 agree on the number and location of monitoring poinls.FS 209£ allows the frequency of routine monitoring to be "as specified:' BS5295./989 demands a weekly repetition of the particle counting exercise donefor validation.

Clearly there are issues here that the phannaceutical industry needs 10

address. Particles of the mailer sizes specified for measurement in !.he CleMRoom Standards are not of direct concern to particulate contamination issuesfacing manufaclUrers of sterile phlUlt'laceuticals. FunhemlOre. it is the quality ofclean room technology that is the factor of greatest importance to the pharma­ceutical industry. of which lhe attainment of clean room classification is merelyan indirect indelt, In these circumstances a major increase in the amouDI oflesljog required to claim compliance wilh a particular c1a.'lsificntioo offers nobenefit to the industry. and may indeed unnecess..uily incrc::asc: testing costs,downtime. CIC.

b. Tests on Filters. The two most impon3nl specificalions for HEPAfilters are the specified efficiency of the filter media and the specifted integrilyof the filter as installed (the leak test).

Table 3 compares the minimum filter efficiencies cited in the guidancedocuments on aseptic manufacluring and in the Clean Room Sta,ldards. It iscurious !.hat the specification for filter efficiency in the "Orange Gu;d~" istighter than the specifications given in the two C/~an Room Standards it refer-

Table 3 Comparison of Minimum Filler Efficieocies Referenced inGuides to Aseptic Pharmaceutical Manufacture and Clean Room 5landards

Document

"Orange Guide" (3)FDA GujJ~lin~ 121EEC Gujd~fjn~ 14)FS 2098 [5J - Class 100FS 209D (6)- Class 100F5 209E 122)- Closs M 3.5855295 - 1976(71· Closs I855295 - 1989(8(- ClosS<5 E and F

Minimum mler efficiency

99.997%a99.97\1,bAppropriate99.97\\\bNSNS99.995%'95%

NS "" Not sJX=C1fiC'd: aTcsced according 10 8S 3928: bTeskd acroIding 10 MIL·f-51065.

Aseptic Manufacture

Tabl~ 4 Comparison of Maximum Acceptable Percentage Recoveries orUpsu:eam Challenge P3lticles in FilleT Installation Integrity Tests Referencedin Guides to Aseptic Pharmaceutical Manufacture and Clean Room Standards

209

Document

"Orange Guide"(3JFDA Guideline (2JEEC Guid~lim £4]FS 2090 (5]- Class 100FS 209D (61- Class 100FS 209B (22J - Class M 3.5OS 5295 - 1976(7J - Class IOS 5295 - 1989(8J - Classes B and F

NS:;;; Not spocifted.

Maximum % recovery

NS0.01NS0.01NSNS0.010.001

ences (FS 2Q9B and BS 5295.1976). The FDA Guideline does nol require such ahigh slandan! of fliler efficiency (99.97% versus 99.997%).

Table 4 compares lhe maximum acceptable percentage of the upstreamconcentralion of a challenge population of particles that can be recovered down·slI'eam of Ihe filler. BS 5295./989 is exceptional in requiring no more than0.001% for Classes E and F (nearest classification to Class 100 of the Federalstandards); the nearest specification in the other documents is 0.01%. Theimplications of BS 5295_1989 to test methodology are significant; instrumenta­tion mat is sufficienlly sensitive to meet other stand3rds is not necessarily of suf·ficient sensitivity to meet BS 5295.1989.

Overall. the standards being adopted worldwide for classification of asep·tic ph3Jm3ceutical filling rooms are those of FS 209. This is bec3use of thepractical Oexibility of this SlaOdard. The British Standard has diverged fromstrict equivalence with its introduction of levels of testing activity thai may bemore appropriate (0 the electronics industry. The availability of Clean RoomStandards has been importunt to the development and responsible control ofaseptic manufacture over the past 30 years or so. In Ihe immediate future ilremains to resolve how the same Standards can address the needs of the elec­tfOrucs industry and pharmaceutical industries without compromising one or theother.

C. Specific Aseptic Systems

Vial tilling has been used to exemplify typical aseptic filling processes. It is, ofcourse. an imponant application, bUI there are other applications peculiar to spe-

, I

210 Cholpter 8

cific product Cont3C( conlainers that merit separate lre3lJ1lenL These includeglass ampoules. form-fill-sealed plastic ampoules, and lyophilization.

J. Glass Ampoules: Glass ampoules are narrow-necked single-piece containerssealed by fusion of lhe glass. Most ampouJes are puU-Staled. The neck of theampoule is healed by high lemperature burners directed at an area close to the lipbut leaving enough of lhe lip to be held finnly in mechanical jaws. The ampouleis rotated in the name 10 ensure even healing. When !.he glass has softened suffi­ciently. the tip is pulled ropidly away from the body of the ampoule. Theampoule continues 10 roLate, twisting closed any unfused capillary.

Alternatively. ampoules may be tip-sealed. The ampoule is rotated whilebeing heated in the burners or it is healed by means of a pair of burners. one oneach side of the ampoule. Sealing is by fusion of a bead of glass at the tip of lheampoule.

The absence of separate closures dic1.3tcs different aseptic systems to vialfilling, for instance, there are no closures 10 wash and sterilize. The ampoulesLhcmselves require to be trealed in il very similar way to vials with respect towashing and sterilization prior to fiUing. Two types of ampoule may be pur~

chased for aseptic filling; they may be purchased as ·open" or pun:hased as"sealed by I.he supplier." It is necessary to wash open ampoules in gray areasbefore dry heat sterilization and filling_ "Sealed by the supplier" ampoules areDOt necessarily washed if Lhcre is sufficient confidence in the supplier's pro­cesses. bur they must be cut open before sterilization and filling. One of themajor product and patient related problems associated with ampoules is glassspicules. which can allegedly be causative agents of pulmonary embolism.These spicules may arise during manufacture from opening "sealed by the sup­plier" ampoules or at point of use from opening by the user. Positive pressureswithin ampoules may reduce the amount of particulate contamination arisingfrom opening.

2. Form~Fi/I~S~al: The form~fill-seaJ process is one in which the primarycontainer for the dosage form is fonned from a thermoplastic. aseptically filled.and then sealed in one integrated system. The technology has spread from thefood industry into sterile pharmaceuticals via products for respiratory therapy(nebules for nebulizers). which are required to be single dose but which are notrequired to be slerile. Many of the fundamental malerials concerns regardingpermeation. leaching. chemical reactivity. toxicity. and extractives were resolvedfor these prodocts_ Pharmaceutically inen formulations of polyethylene. poly­propylene, and various copolymers and polyaJlomers are commercially availableand suitable for pharmaceutical form-fill-seal applications.

At the bean of the process is the blow-molding machine. This technologyis not unique 10 aseptic processing. In the machine, molten plastic is extrudedunder high temperolures (around 200"C) and pressures (350 bar) as a hollow

Aseptic Milnufacture 211

/

p

\'-..-/

7~

"./ \.

f"'- I,

/ •

•

•

•

" "

lr. If"'-

Fig. 14 Blow-fill-seal process.

tube (Fig. 14). The extruded material (the "parison"), while still molten, is thenfed between two mirror image halves of a die in the shape of !.he container to beformed. The two halves of the die close around the parison and seal it at its base.A mandril is inserted automatically into the neck of the panially formed con·tainer to blow higb-pressure air into the mold thus forming the body of thecontainer.

1be process becomes somewhal more elaborale for aseptic filling (9). Inorder to combine the processes of molding and filling. the mandril comprisestwo concentric lubes. 1be outer tube delivers the air (0 form the container-foraseptic filling the air must of course be filter sterilized-the inner tube deliversthe aseptieally filtered dosage form.

The final stage of the system is the removal of the mandril followedimmediately by closure of the Iwo halves of the pan of the mold which forms theneck. of the container. The whole process takes no longer than 10 to 15 S, andseveral containers can he formed. filled. aod sealed in parallel molds within thistime f.rame.

This type of tecbnology has given rise 10 some considerable amount ofdebate within Ute pharmaceutical industry and the regulatory bodies. ConcernsaOOm the cleanliness of the machinery and contamination of the polymer gran·ules have been largely resolved: so also have issues surrounding the quality ofthe services (cooling water, hydraulic fluids, etc.). The main area of contenLion

, I

Aseptic M~nurKture

Pressor.

lJQUID PHASE

213

,-SOlID

PHASE................. ._ .

VAPOR PHASE

100

T....ponolul9 ('C)

Fig. 15 Phase diagram for waltr.

there are four shel .....es. which can be refrigerated or heated according to the needsof the proce>s. The chamber can be evacuated by a lwin pump selUP and is pr0­

vided with air. nitrogen, and $learn lines.In essence the process comprises six stages:

ta) Filling. The dosage form is compounded in concentrated aqueous[onnulation, sterilized by filtration lhrough 0.22 J.lm membranes. andaseplically filled into unsealed vials. Protection from gross con18minalionis often afforded by lhe final closures being loosely inserted in [he ..... ialnecks.

(b) Freezing. This is done with the ..... ials loaded into trays designed to fitinto the refrigerated shelves in the cbamber of the lyophilizer. Freeziogmay take se.....eral hours.

(c) Evacuation. The chamber of lbe lyophilizer is evacualed 10 low pres­sure.

(d) Sublimation. Waler .....apor subliming from the frozen dosage form iscondensed on heal exchanger surfaces held at or around -50·C within lhelyophilizer.

, I

214

CHAMBER

Chapter 8

L-_ STEAM

CONDENSER

DRAIN

CHILLER

VACUUM PUMPS

COMPRESSORSCIRCULA110N

PUMP

Fig. 16 Schemali(~ represenlalion of a lyophilizer.

(e) Heating. The rate of sublimation increases with increasing tempera­ture at temperatures below the eutectic temperature. Controlled healing isa nonnal feature of commercial lyophilization. Care must be exercised 10

avoid heat-induced product degradation. The complete cycle to less thanI% moisture may lake as long as 24 h. depending on the surface-la-volumeralio of the dosage fonn.

(t) Sealing. AI the end of the process. sierile air. nitrogen. or other appro­priate inen gas is bled inro the chamber and the c10slUeS are forced homeby rams or by movement of one shelf against the one above.

Lyophilization has several sLages and subprocesses beyond aseptic vialfilling thai increase lhe potential for contamination and introduce additionalrequirements for sterilization. First. vials for lyophilization are usually lr.lyed

216 Chapler 8

opposite eCCee.r on microorganisms to thaI which most pharmaceutical dosageforms are formulated to have.

The acceptable standard to be achieved in simulation trials is for there tobe no more !.han J contaminated item in 1000 ilems. This limit, which has beenestablished on the basis of the probability of false positives arising in microbia­logicallIansfers being in the region of 10-3• is frequently interpreted to mean thatSALs actually achieved for aseptically manufactured products are no beller than10-3. This interpretation is not correct Media fill resuhs apply to the asepticsystem, not to !.he products being passed through the system. SALs in productsare a function of the probability of items becoming contaminated and of thosecontaminants surviving. The media fill provides only the first factor in such anequation. such that even in lhe unlikely evenl of all contaminants surviving(probability of survival equal to 1) the product SAL would be no worse than10.3.

In 1981 Whyte [13J proposed a model 10 describe the potential probabilityof microbiological contamination of the contents of containers originating fromthe air in the environment of aseptic filling rooms. The model incorpordted threeadditive effects:

(a) The first factor in this model assumed that the rate at which particlesselile out of the air onto horizonlal surfaces is largely dependent upon thesize of the particles. Assuming Slake's law and an average contaminatedpanicle diameter of 12 JJm, the number of particles deposited from air intoan open coominer can be described by the expression

Number of particles deposited = 0.0032 . d'- . C . A" . r

where d ::;: equivalent particle diameter (IJm). C ::;: airborne panicle con­centration per cm3, An ::;: area of the opening of the neck of the container.and I:::;: time the container is open (s).

(b) Another mechanism which he considered was that contamination couldarise from particles from an airstream being thrown into the open neck ofthe comainer. Whyte presumed that the greatest risk of contamination ofthe contents of a container by impaction from this source would be fromair flowing parallel to the neclc. of the container. He quantified this risk as

Number of panicles impacled::;: C· An' V· E· t

where V ::;: air velocity parallel to tbe neck of the container (cm/s), and

JO·6. V.d'E = 3.27 x ---­

I

where 1::;: diameter of the neck of tbe container (em).

218 Chapter 8

teotly fulfills its intended purpose. A degree of overkill cannot be buill in 10 apassive target couched in absolute ternts--lolal protection from conlaminmion.

Thorough and well founded validation is therefore imperative. Routinemonitoring is merely an extension of validation. and ongoing conlroll.:ornprisethe systems of response not only 10 excursions beyond predetermined monitoringtolerances but also to potentially deleterious trends that may become evidentwithin those tolerances.

A. Validation

The first stage in the design of a validation system for an aseptic manufacturingoperation is a detailed analysis of the process into its unit operations, In this wayit is possible 10 identify those systems and subsystems that have an effeci on thepotenlial microbiological quality of the product. This exercise mu I includethose systems intended to reduce contamination by washing. by disinfection. orby sterilization. which have independent validation requirements. as well as Ihespecific asepric systems intended to prevent contamination of the dosage formand product conlaet components. The validation program should address anddocument each of these systems. Consistency over lime should be addressedthrough periodic revalidation. By and large, routine monitoring only addressesthose systems thal can be monitored wilhout causing major disruplion to manu­facture. Many aseplic control systems do nOI faJl into Ihis category and musttherefore be revisited for validalion purposes al intervals well within theirexpecled shelf life.

The supposed biological integration of the individual effeclivenesses ofthese systems is by completion of satisfactory media filling trials. This does nolmean that filling trial should be considered as a substitute to validation of eachindividual process Ihal may impact upon product contamination. As Slatedabove. filling trials are in fact a poor measure of the degree of prolection fromcontamination that is expected and indeed obtainable from aseplic manufacture.Regardless of Ihis, the filling trial is recognized to be the only approach 10 iOle·gration currenrly available. Current regulatory thinking is that it is an absolutenecessity.

The following pages identify the principal individual aseptic syslcms thatmust be ....alidated and appropriate standards to be met.

J. JIIslmmentation: All instrumentation. measuring devices. and alarm systemsinstalled on equipment and in facilities intended for aseptic manufacture shouldbe ascertained to read or react over appropriately pccified ranges. Complexequipment should be designed to allow access to measuring de.... ices for calibro·lion or removal for calibration. Calibralion of Ihe signal from the measuringdevice is nOI sufficienl wilbout the measuring device itself being checked.

220 Chapter 8

able changes made to fabric or fitrings as a result of modification or maintenanceactivities. Services in panicuJar hould be: ascenained to being provided fromoutside aseptic filling rooms. new electrical fittings should be flush to walls orceilings. and any incidental damage to walls. noors, or ceilings should have beenmade good.

The major system of contamination control is the air handling system. Thevarious exercises thai make up the validation of air handling systems are com­mon to txuh while and gray areas. It is only lhe standards that differ (Table 5).

Table 5 Compar1soo of Air Siandards for White and Gray Areas

Number or particles per mJ (Approximate figures per ftJ in pa!t"nw54=s)of size equal to or grealer than . ..

White area standards 0.3 ~m 0.5 J1Il' I~m 5~m 10 ~m

855295: 1976 Class I 17] 3.000 0 0855295: 1989 Class E (8] 10.000 3.500 0 0

(300) (100)855295: 19 9 Class F (81 3.500 0 0

(100)FS2098: Class \00 (5] 3.500 0 0

(100)FS209D: Cia 100 16] 10500 3.500

(300) (100)

Gray Area Standards

855295: 1976 Class 2 (7) 300.000 2.000 30( 10.000)

855295: 1989 Class J 18] 350.000 2.000 0( 10.000) (70)

855295 : 1989 Class K (81 3.500.000 20.000 450(100.000) (700) (150)

FS2098 : Class 10.000 (5] 350.000 2,500(10.000) (65)

FS2098: Class 100.000 /5) 3.500.000 25.000(100.000) (700)

F5209D: Class 10.000 (6) 350.000 2.500(10.000) (70)

5F209D: Class 100.000 (6) 3.500.000 25.000( 100.000) (700)

Aseptic Manufacture 221

3. Validation of HEPA fj,lIers. There are two imponant specificationsfor filters intended to produce high-quaJity. microorganism-free air. These arethe specitied effic.iency of the filter medium and the specified integrity of thefilter as installed (lbe lenk test 0' DOP test).

The former is specified by the manufacturer, determined according to lhepeneU'alion of part'icles of sodium chloride wilh mass median diameters of0.6 t1m and need nOI be verified as pan of lhe validation program.

The leak test or DOP test (n:t.med afier dioctyl phthallatc, Ihe original bUInow rarely used oil source) is an in situ test to verify that fillers do not leak oninstallation. The leak test is not a second efficiency test. It is intended 10 dis­close leaks around the frames and damage to the filter medium. An aerosol ofoil particles wilh mass median diameter of 0.3 J.lm is used to challenge the tiller:detection is by aerosol photometry on the downstream side. Standards of integ­rity are specified as maximum pennissable percenlages of me upstream concen­tration of particles that can be recovered downstream of the filler.

b. Validadon of Air Circulation. There are three factors relating to aircirculation that are importanlto the validation of aseptic manufacture. These areair velocities. airflow patterns. and air exchange rales.

Achievement of salisfactory air velocities is imperalive to laminar nowinsl3.lJations. The laminarity and the sweeping effects Ihat are essential to theireffective operalion are functions of air ..:elocity. The standard within the phar­maceutical industry is an average velocity of 90 ftlmin with a plus or minus 20%tolerance.

The problem is where to measure the velocity? The options are close tothe filler face. in the working area. or at (he extremities of Ihe protecled area (airexiting from. say. the curtains of a venical laminar flow canopy). The resolutionof where to measure may not be easy. For instance the fiher face of a large ver­tical flow canopy shrouding a filling machine may be more than 1 m above thefilling zone. By and large the pharmaceutical industry has chosen to measure airvelocity dose to the filter face.

The basis of modem aseptic manufacturing technology is airflow protec­tion. Airflow panems determine the effectiveness of airflow protection. Wheredoes the air from input regi!Uers or from laminar flow installations go? Is itsweeping ove·r lhe areas Ihal require protection? Is the airflow causing eddiesand dead space? None of this can be accurately predicted. There arc 110 stan­dards to be met. This is purely a quaJililal.ive exercise Ihal must be done empiri­cally. 1be rncthod is to use smoke pencils or 10 use a smoke generalor andobservation. None of Ihis work can be done with an area sierile and opel1ltional.Records for referral and inspecdon are best maintained on film or video. Videocameras intended for underwater use can be deconlaminated using disinfecLantsto minimize the polential for carrying microorganisms inlo clean areas.

222 Chapter 8

The microbial and particulate quality of aseptic filling rooms is maintainedby dilution as well as filtration. The rate of exchange of room air is imponam tothe contribution made by dilution. Room air should be changed at least 20 timesper hour. Around 90% of the air should be recycled through the fillers. Thereshould also be a satisfactory pressure differential (minimum 0.05 inches \VG or15 pal;cals) between clean rooms and adjacent less protected areas.

c. Room Classification. Classification of aseptic filling rooms and grayareas to clean room standards is an imegral pan of validation. The key technol­ogy is (he measurement of concentrations of nonviable panicles per unit volumeof air. Classification has been addressed in some detail above.

In some instances the tenninology of room classification may be usedwhere it is neither specifically necessary nor specifically correct The FDAGuideline (2) refers to a "per-cubic.foot panicle count of no more than 100 in asize range of 0.5 Jim and larger (Class 100) when measured nol more than onefoot away from the worksite and upstream of the air flow'" The samplingrequirements of lhe Clean Room Slandards may be omitted when superfluous tothe purposes of aseptic manufacture.

White areas (Class 100 clean rooms) should clean up to their c1assific3lionwilhin 20 min of starting up air handling systems unless there is some majorparticle generator present in the room or leaks in the ducting.

d. MicrobiologicaJ Characteristics. All of the microbiological char-Jeter­istics of aseptic manufacturing facilities described below under Routine Moni­toring (Seclion IV.8) should also be completed during validation as a base linemeasurement of how lhings are when aU syslems are known to be operating intheir intended manner.

3. Aseplic Filling Room Gannenu: Operators and their garments are poten­tially the most significant source of contaminalion of aseptic filling rooms. Itshould be ascertained that garments for use in aseptic filling rooms are madefrom materials that shed virtually no fibers or particulate mailer and that theyshould retain microbial particles shed from the body. Fabric edges should besealed and seams should be all enveloping. Validation records should includemanufacturer's infonnation on the barrier characteristics of the garmem fabric,i.e.. air permeability, pore size. and particle removal.

Garments should be laundered (or cleaned) and sterilized in an effectivemanner. Validation of the laundering process should demonstrate thai bothlaundered and unlaundered garmenls are contaminated by no more lhan 5.000particles of length greater than 0.5 J.lm and no more lhan 25 fibers (particleslonger than 100 Ilm with a length·to--width ralio exceeding 10:1) according 10

ASTM F 51/68 115).Radialion is suitable for sterilizing aseptic area garments, with three provi­

sos. First. plastic studs and zips should be avoided to prevent embrittlement cre-

Aseptic Manufacture 223

aling sources of particles and loss of integrity. Second. radialion may acceleratethe fading of some colored garments. This could be helpful to controlling thenumber of slerilization cycles through which the garments can be passed but isprobably not the best way of doing this. Third, irradiated garmenlS may carry anaroma of ozone to which some personnel may be sensitive. Types of steam ster­ilization cycle used to sterilize gowns and scrub suits in hospitals are unlikely tomeet me same criteria for sterilization validation applied in the phannaceuticalindustry. Validatable cycles may require very long poststerilization drying toguarantee against dampness.

4. Disinfectants and Cleaning Processes: Disinfectants are an integral part ofcleaning processes for aseptic manufacture. They are usually purchased as can·cenr:rated stock solutions; for use they should be diluted in water of PurifiedWater or WOIer for Injection quality. They should be filtered into white areas,usually. because of their viscosity. through 0.45 ~m slerilizing fillers. Eachdiluted balch should be allocated a shelf-life. and differenl disinfeclants shouldbe used in documented rotation.

Validation of disinfectants should be concentrated on two aspects of theirpotential 10 create problems in aseptic manufacture. First, they may themselvesbe sources of microbiological contamination; second, Ihey may nol be effectiveagainst microbial contaminants.

Samples of disinfectants should be taken as close to their point of use aspossible and examined for microbiological contamination by passage through0.22 flm membrane filters. Great care must be taken to nush the membranes (0

ensure removal of any residual traces of disinfectanl before they are placed in oron microbiological recovery media.

The effectiveness of disinfectants should also be evaluated. The approach10 this is not as simple as it mighl seem. Very obviously, the simplest method ischemical analysis of the active ingredient. However, it is usual to find Ihat thisis supplemented by some form of microbiological data. The principles of micro­biological .esting of disinfectants go back to the Rideal-Walker method of 1903and probably e:1Clier. Even with this long history. disinfectant testing is not free.nor evcr bas been free, from criticism and controversy.

Whereas the real test of a disinfectant is in its practical application. theresponsibility for evaluation lies in the laboratory. The resistance of microor­ganisms to disinfeclanlS may vary widely, influenced by the presence of organicmaterials, the wetting capability of the surface being disinfecled. the temperatureat the time of application, and species-ta-species. strain-to-strain. and phase-of­growth characleristics of target microorganisms. Laboratory tests attempt tocontrol these factors within tighter limits than would ever be encounlered inpractice.

The basic concepl is to compare the effectiveness of the proposed disin­fectant with that of phenol by reference (0 a slandard microorganism. The Asso-

a

224 Chapter 8

eiation of Official Analytical Chemi IS is probably the best source of detailedslate-of-the·an methodology. For validation of di infectanlS for purposes ofaseptic manufacture. the relevance of the lest may be impro\'ed by includingmicroorganisms representative of local contaminalion in lhe lest.

5. Liquids and Lubricams: All automated filling equipment must require peri­odic lubrication. Wherever possible. lubricants hou1d be sterilized prior tobeing brought into aseptic filling rooms. bul in practice this is nOI always com·patible with lubricating characteristics. Only sterilized lubricants should be usedfor any pieces of equipment that come inlo direct contact with the dosage famtor product contaci containers; for other applications within aseptic filling rooms,lubricanls containing bactericides should only be used. These should be evalu·aled against reasonable standards of microbiological cOnl'aminalion (for instance.no more than one colony.fonning unit per mL or gram).

6. Media FUling Trials: Media fills are inlended to simulate the risk of con­taminalion that may arise from the aseptic a sembly of sterile produCI elementsby Subslitulion of a slerilized placebo for the dosage form. The placebo shouldbe chosen to be of similar now/filling pe:rfomlanCe characterislics to the steril·ized dosage fonn to mimic accurately the normal conditions of the produclionprocess.

For aqueous liquid produclS the placebo most commonly used is a liquidmicrobiological growth medium (broth). For solid dosage form. placebos suchas lactose. mannitol. and polyethylene glycol may be filled and microbiologicalgrowth medium added afterwards.

a. Conditions of Simulation. Wherever possible. filling trials shouldsimulate the wocst case. However. for inilial validation of a new or modifiedfilling machine or a new or modified facility. filling trials should be conductedindcpendenlly from rouline production. with filling lines set up specifically forthe (rial. For revalidation, trials should be done at the end of a normal fillingoperation when the risk of contamination due 10 accumulated errors is greatesland when operalors are weariest. This is not possible for anlibiolic prodliCb. Inthese cases the filling machine and the filling room must be cleaned free ofantibiotic 1Caces before conducting the filling trial to prevent bias in the results.

Before conducting a trial for inilial validation purposes. all a peelS ofevery aseptic control system should have been verified as meeting their valida·tion standard Microbiological monitoring above and beyond that ootTllallyexpeeled should be dooe in tbe filling room before and ofter !be filling trial.

The filling equipment should be set up according 10 ilS documented Stan·dard Operating Procedure and run under normal operating conditions. Fillingtrials should be conducled by those personnel who are normally employed in thefilling process. Ongoing revalidalion filling trials should be seen a an opponu·nity to reinforce correcl aseptic praclices. Personnel should be dressed in nonnal

Aseptic Manufadure 225

aseptic filling room gannents and perfonn routine production manipulations inresponse to events (sampling. check-weighing, etc.).

For liquid filling trials the containers need only be filled with sufficientmedia to wet all of the interior surfaces in a manner that simulates exposure innonnal production. In other words. it is not necessary to simulate the exact vol­ume of liquid filled in routine production. Indeed. exact simulation could createa quite impossibly large demand for sterile media beyond the capacity of labo­ratory sreriliters. While suggesting Ihis. it is imponant to recognize that everyinternal surface must come into contact with the medium. This may necessitatethe incuootion period being split into two halves. the first half with the contain­ers incubated in their nonnal position, the second half with the containers incu­bated invened.

Some companies do not sterilize media in the laboratory for filling trials.Instead they pass the media through the normal stages of sterile filtration applied10 products being manufaclured on the filling line. In this way they caneconomize on aUloclave capacity while at the same time evaluating Ihe asepticmanipulations associated with filtration. Although evaluation of these asepticmanipulations is essential to successful aseptic manufacture. the nonnal applica­tion of the filling trial is to the fiUing process alone. Aseptic manipul8lionsassociated with filtration and lransfer are belter evaluated independently. bring­ing greater focus on appropriate corrective action,

For solid dosage form fiUing uials. the final concentration of placebo inthe microbiological growth medium sbould satisfy normal (pharmac.opoeial)criteria for absence of growth inhibition. There are two approaches to the subse~

queDt addition of microbiological media The first is to have a liquid filler online. This is the: simplesl and easiest of !.he two approaches. but il does run lherisk. of exposing solid dosage form filling personnel to an unfamiliar and poten­tially contaminating operation. Personnel must be specifically trained in runninga trial in addition to their normal training required for solid dosage form filling.The second approach is to have the pJacebo.filied containers taken to a laOOra­lOry to be filled with media. This of course increases the risk of laboratory con­taminalion and must be catered for by large-scale use of sterilized controls (oftenin a proportion of one control 10 two lesl containers).

Each filling trial should consist of at least 3000 items (it is normal praclice10 exceed the minimum to account for breakages. elc.). The medium of choice isSoybean Casein Digest Broth USP. Where necessary. for inslance in antibioticfilling processes where. no maUer how effective the cleaning. residues cannot beexcluded. an inactivating agent (e.g.. penicillinase) should be added to themedium at an appropriate concentration.

b. Interpretation of Results. After incubation. filled containers musl beinspected and scored as sterile or nonslerile. Any contaminating microorganismsshould be identified to al leasl generic level and where possible related 10 their

226 Ch.plrr 8

most likely sources and to events occurring during the filling trial. TIle value offilling trial results is greatly enhanced by reliable knowledge of the lime theitems were being filled and the order in which they were fiUed.

The SlandanJ to be achieved is no more than one contaminated item in oncthousand items. The FDA adds the proviso that this should be achieved to a 95%confidence level. What exactly does this mean? How should it be interpreted;!

Th< PDA Technical Monograph on 3Stptic filling [19] quotes the follow­ing equation to describe the probability of finding one or more contaminateditems in a sample of siu N taken from a universe with a contamination rate of0.1 %:

P(x>O) = l-e-NP

where P(6 > 0) :;: the proixlbility of detecting one or more conlaminated items inthe sample (for the purposes of the FDA's interpretation of filling trials this isequal to 95%), N =lhe number of items in the sample. and P =lhe probability ofoccurrence of contaminated items in the universe.

This equation describes an "operating characteristic" curve-thc reJalion­ship between the percentage of conlaminated items in the unh'erse and the prof>..ability of accepting or rejecting the univene on lhe basis of finding OIl least oneconmminaled item in a sample of a specified size (N).

What this equation means in relation to filling trials is thai if a series ofsamples each comprising 3000 items were tak~n from a universe that actuallycontained contaminated items at a frequency of one in one thousand (0.1%). wewould find one or more contaminated items in 95% of our samples. It effec­tively defines a minimum sample size of 3000 (precisely 2996). because 95 %confidence cannol be achieved with any smaller sample size. It also. by theinclusion of the expression p(x> Or detennines the pass/fail criterion as acceptthe universe with zero contaminated items in the sample. reject one or morecontaminated items.

There are alternative interpretations of pass/fail criteria for liIling trialresults.

For instance, the proponion of contaminated items in 3 sample taken froma greater universe is not an exoct measure of the proponion of contaminateditems in the universe. It is only an estimate. For instance. one contaminateditem in a sample of 3000 items provides an estimalC: (Pesc) of 0.03% of the actualfrequency of occurrence of contaminated items in the universe (P). which maybe higher or lower than 0.03%. The reliability with whjch Pest can be claimed 10reneet P can be ca1culaled from the confidence limits of Pest

Confidence limits may be calculated at 90% or 95% or whatever. Forinstance we can be 95% confident !.hal P will lie between the lower and upper95% confidence limits of PC'st' ele.

228 Chapter 8

individual aseptic control system has been valichlled and confirmed to be satis­factory. At least two afthe three replicate lrials that are normally thought neces­sary for initial validations should have zero contaminated items; the third shouldbe allowed (0 have no more than one contaminated item in the sample. Anyother pattern of resuhs should demand a thorough investigation. corrective actionand, if necessary. revalidation before the facility is released for aseptic produc­tion.

With revalidation filling trials, zero contaminated items is once again acautious go-ahead. and one contaminated item in 3000 should demand an inves­tigation and appropriate corrective action where necessary while allowingproduction to continue. Two or three conlaminated items should require anyproduct manufaclUred on lhe filling line since the date of the failed trial to bequarantined until an investigation and a successful repeat trial has been com­pleled.

More than three contaminated ilems in 3(x){) means a significant problem,in all likelihood compromising product sterility. The line or facility should beclosed until all investigations. corrective actions. and satisfactory repeat Irialshave been completed. Product manufactured afler the lrial and before discoveryof the failure should be considered for rejection unless there is a strong balanceof evidence 10 support its release. U necessary. product manufactured before thetrial should be considered for recall.

The translation of failed filling trial resulls IQ action againsl product is notan easy one for scientific. commercial, and pragmatic reasons. 11 may be six.months since completion of the lasl successful filling trial and a great deal ofproduct may be perceived 10 be at risk. The failure may be an isohHed evenl dueto an operator error; alternatively. it may signal a repealed problem that has pre­viously gone unnoticed. Systematic equipment faults are easier 10 confirm andconfine Ihan systematic people faults.

I.nvesligations should address lhe type of microorganisms isolaled from thefailed filling trial, their likely sources. their sensitivities to the antimil.:robi31characteristics of products that have been filled on the line, and most imponantlytheir history of past occurrence. For instance. microorganisms thai have beendetecled and represented as laboratory conlamination in past Tests for Sterilityshould be considered as genuine product contaminants in those bau::hes passedon retest. Batch withdrawal should be undenaken if they are also capable ofsurviving to any extent in the product. Identities should be at least to specieslevel in failed filling trials.

7. Frequency of Rt'va[idarion: New aseptic manufacturing facilities and fillinglines must be validated before they are allowed to be used for routine production.Thereafter. routine monitoring is a form of conlinuous abbrevialed revalidation.However. the major validation activities are lotally impossible when lines andfacilities are operalional. Revalidation and. if necessary, replacement of deterio-

229

rating equipment should be done at sufficiently frequent intervals to ensure con·tinuing control. In practice the revalidalion frequency is mOSI oneo an opera­tional compromise. typically lWice per year during shutdowns.

B. Routine Monitoring of Aseptic Manufacturing Facilities

The purpose of routine moniloring of aseptic manufacturing facilities is to obtainsome measure of the level of control being achieved. The ideal is that monitor­ing should be done in a way that wiIJ promptly reveal any failure of lhe controlsystems to meet their intended purpose. As often as not. practice falls somewhatshort of this ideal.

The topics of how to monitor aseptic ntanufaclU.ring environments. where10 focus monitoring activities, how ofleo to monitor. and what 10 do with (here ults are frequently debated. The regulatory literature is somewhat barren onthose questions. For instance. the FDA's Guide/illt! 011 Slrrile Drug ProductsProduud by Aseptic Processing [2) deals wil.h Ihese lopics in only slightly morethan three pages of typescript; the "Orange Guide" (3] does so in four para·graphs. InduSlrial inlerest is reflecled in two documenlS. one published by IheParen1eraJ Society in Ihe U.K. in 1989 [161, .he other in the U.S. by the Par·enteral Drug Associalion in 1990 [17). Whereas these lasl two documents differquite substantially in their t.reatmenl of environmenlal monitoring, their basiccon iderotions remain as how. where. how oflen, and how to respond 10 envi·ronmental monitoring.

I. Methods for Environmental Monitoring: The methods available for moni­loring aseptic manufacturing facililies are so well worn dUll even the regulatorydocumenls are confidenl enough to recommend them. Microbiologkal air sam·pIing (active and passive), microbiological surface sampling (swabs and conUlCtplales), microbiological touch pla1es (finger dabs), physical particle monitoring,and monilOring of pre ure differentials are Lhe recommended approaches. Evenso, there are many issues surrounding their use.

a. Measurement of Numbers of Microorganism" in Air. The "standard"active meLhod uses a slit sampler. The principle of Ihe slil sampler is mal ofinertial impaclion; panicles moving in an airstream have an individual ineniaand may be deflected onlO a surface wbere Ihey may be trapped by impaclion.Slil samplers (Fig. 17) are provided with pumps thaI draw air from the area heingsampled through a narrow slit. The effecl of the slit is to increase the velocilY ofthe ajrflow and hence Lbe inertial velocily of any panicle being carried in theairstream. 1be accelerated panicles are direcled onlo Ihe surface of a plale ofnulrienl medium Ibm is rotaled continuously or progressively. Pumps providedwith slit samplers usually operate at fixed rates, and the volume of air sampled isconlrolled b)' an on/off timer. Equipment suitable for use in connection with

230

SLIT / "PLATE -I" -'

AIR IN

...~. ".. ... ., , .

Chapler 8

AGAR PLATE

FlEXIBLEt-. . .~ - .

TUBEPUMP

AIR OUTFig. 17 Slit sampler.

o.

aseptic filling rooms should consider pumping rates that allow 3,0 to 3.5 01 3 ofair 10 be sumpled in a reasonable time frame.

An alternative active air sampler is the centrifugal sampler (Fig. 18).Unlike the slit sampler, the centrifugal sampler is a nea.lightweight sterilizeabJeinstrument This type of sampler uses centrifugation to remove particles from anairstream and trap them on agar strips. Centrifugal samplers have two technicalproblems. First, their sampling rates are low, too low to be sensitive enough tobe of value in Class 100 clean rooms; second. the volume of air sampled is notaccurately known 118), so they cannot legitimately be claimed to be quantitativedevices.

The final method of air sampling is a passive one. This is the sell Ie plateor fallout plate. Quite simply this is an agar plate len open and exposed for aspecified period of time. Arguably this is as much a method of quanrifying sur­face conl.aminalion as one of measuring air-borne contamination. As with manyapparently simple methods. the senle plate represents a significantly complexequilibriu.m and results should be interpreted with great care.

Panicles settle by gravity on 10 sellle plates. Large and heavy particlestend to seule out due to gravitational forces; with increasing air movement onlythe very heaviest particles settle out. This Limits the value of the method inlaminar now proreeted areas or other clean rooms where still air is not intended.Of course it can be argued that dead air is the main concern in clean rooms, and

, I

/'AIR IN

Aseptic Manufacture

\ \ AGAR STRIP

AlROVT

Fig. 18 Centrifugal air sampler.

231

settle plates arc an effective method of detecting it This argument depends onsitting the settle plate in the correct area. and it invites the question of why thatlocation should be monitored with setde plates rather than corrected and thenmonitored by some better device.

The quantitative aspects of scule plates are debalable. What does onecOlony-fonning unit represent? One viable microorganism? Or several microor­ganisms. perhaps up (0 a few hundred. carried on one large nonviable particlethat has settled out? This rather restricts the quantitative value of sellie plates.and some agencies concentrate only on their qualitative value, Le .• microorgan­isms identifiable as being of human origin, or microorganisms from <tir or dust orwater. etc.

Some microorganisms may seule on seule plates and Slay on the plates.while others may bounce off. What therefore is the seHle plate an index of?Some microorganisms may remain viable on lhe plate but find the mediumunsuitable for growth.

This is of course a problem common 10 all microbiological methodology.Other microorganisms may die on the surface of the plate. This introduces along-standing debate concerning how long settle plates should be len exposed.Leaving them open too long leads to desiccation and death of the trappedmicroorganisms; if the plates are not exposed long enough the sensitivity ofdetection is diminished.

The Parenteral Drug Association [19} recommends 30 min for aseptic fill­ing rooms; the Parenteral Society [16] recommends 4 h. Whyte and Niven 1201have argued lhat the viability on agar plales of microorganism commonly foundin room air is little affected by desiccation and quore allowable exposure periods

232 Chapter 8

of up to 24 h. There is no right answer to these problems. The time of eXJX)suremust be len to the discretion and professionalism of the microbiologist operatingthe monitoring program.

b. Measurement of Numbers of Microorganisms on Surfaces. There aretwo methods of monitoring surfaces. swabs and contacl plates.

Swabbing is a technique in which a surface i lightly scrubbed with amoistened "collector" (swab). most often a collon or 3)ginate "bud." The swabis then rolled over the surface of an agar plate. Alternatively the swab may beimmersed in a liquid and agir3ted to suspend the microorganisms collected. Theliquid is then plated. or passed through a sterile membrane filter and plaled on anutrient medium.

The contact plate involves pressing an agar plate against the surface beingmonitored. The Rodac plate (diameter 55 mm) is a commercially available petridish designed for use as a conlacl plale.

An imponant distinclion is th3t the swab provides an index of the numberof viable microorganisms contaminating an area of surface. whereas the corUnetplale provides an index of !.he number of contaminated sites within a p:tniculararea of surface.

The choice of whi h method to use. and where to use them. is debalable.Contact plates require thoU Ute surfaces that have br-en teslcd be thoroughly disin·

feeted after testing to prevent traces of nutrient provok.ing microbiologicalgrowth where none might otherwise have occurred. Many workers believe thatthis is an unacceptuble risk in connection with aseptic fiUing rooms but acceptthat the technique is useful in gray areas. Olhers reject contaci plates com·pletely. arguing that lhey are only suited to smooth plane surfaces. which bylheir nature are easily cleanable. Swabs have many advantages in this respeclbecause they can 00 applied to surfaces that pose cleaning problems. for instancepans of machinery that are difficult to access bUI that may lead to significantproduct contamination.

c. Microbiological Touch Plates (Finger Dabs). Finger dabs are done byaseptic filling room personnel being required to pres the tips of their fingers onto the surface of an agar plale. This is not really a method of monitoring theaseptic environment; it is either a method of monitoring the effectiveness of per·sonnel discipline or 3 cosmetic exercise intended to reinfon:e aseptic practices.

d. Measurement of umbers of onviable Panicles in Air. The mea­surement of numbers of nonviable panicles in air i an integral pan of roomclassification at ,·alidation. and of course classification to clean room standardshas a requirement for routine monitoring. ~ most widely used method is baM:don light scattering.

The principle is thai an air sample is drawn inlo a sensor and passedthrough an area of inlense light. Any particles in the airstream scatler the lighl.Thus Ihe moving panicles appear as bursts of light and can be detected wilh a

I

Aseptic Manufacture 233

phOlOmultiplier or a photodiode. Each burst of light is converted into a pulse ofelectrical energy. The height of these pulses is proponional to the amount ofUghl scanered by the particles. A digital counter sorts and counl's the pulsesaccording to their heights.

Two systems are available. polychromatic white light and lasers. Lasersare able to concentrate more energy in a smaller spot and are therefore able toregister smaller panicles. The lower limits of sensitivity for white light systemsare in Ihe order of 0.3 IJffi. and for lasers about OJ 5 1J111. For aseptic manufac­lUring facilities it is nm nonnally necessary to measure particles smaller than 0.5).1m. Instruments should have the capability of sampling at least one fi3 perminute.

Panicle counters are not easily disinfected for taking into aseptic fillingrooms. They may be dedicated '0 particular areas. or remote instruments withmobile or fixed sampling tubes can be purchased.

2. Sample Wcalion Selection: Uniform contamination over a complete asepticmanufacturing facility is remotely improbable. It would require a lotal break­down of systems and would be evident in many ways other than environmentalmonitoring. The selection of locations for environmental monitoring becomestherefore a maller of professional judgement that should lake account of twomajor considerations: locations where. if contaminated. product quality wouldbe most seriously affected; and locations that due 1'0 some vicissitude of designor control are susceptible to microbiological contamination or proliferation.

Monitoring of air for both viable and nonviable particles should be done inlocations that reflect exposure of the dosage form to potential air-borne contami­nation. This usually means at or around table-top or machine-bed level with thesampling probes pointing venically upward. II is not, however. necessary tomonitor at the precise locations where dosage fonns are actually exposed. [nmost instances it is better to choose indicator locations within filling roomswhere the possibility of monitoring leading to product contamination is minimal.Air samplers themselves create air disturbance and movement that may disturb.detlec,t. or divert laminarity. cause momentary localized dead space. or unbal­ance pressure differentials. There is a major dilemma (0 be resolved in everychoice of air sampling location: the choices are between the combination of themost relevant monitoring location with potential compromise of control. and thecombination of an indicator location with less relevance to product contamina­tion coupled with no compromise of control. Most pragmatists choose the lauer.

If surface-tQ-sUTface contamination is potentially more significant than air­borne contamination. as many aseptic fi.lling operators now believe. then touchplates (finger dabs) become a critical pan of environmental monilOring. Not somuch does l.heir value lie in whether they indicate contamination or absence ofcontamination but in the identification of contaminants to their most likelysource. Touch plates should be seen as a route to identifying systems failure and

234 Chapler 8

can be lrUly relevant 10 product contamination. Wilhin aseptic filling rooms,personnel should be required to disinfect their hands thoroughly after touch plateimpressions have been laken 10 ensure that traces of media remaining do nOI lead(0 microbial proliferation in the filling room.

Surface samples should nol be so concentrated on direct relevance thaithey lead to unnecessary intrusions into critical filling zones. It is best lhat with­in while areas they concentralf' on locations that are difficult 10 access for clean­ing and locations where manual operations afe routinely c.arried out. The pur­pose of these choices is again to expose systems failures.

Surface monitoring can be very imponant in gray areas. Identification offailure of control systems implies investigation and correction. Once a whitearea has been cleaned and fumigated. with mooern HEPA filtration systems themost likely route of entry of contaminants is via the gray areas. Discipline is notexpected to be as severe in these areas. There may be water. sinks. clean ratherthan sterile equipment, clean nUher than sierile clothing. etc. In changing roomsthere may be opponunity for cross contaminalion of dedicaled clothing wilhstreel clothes. The possibilities for introducing routes of cOnlamination arelegion. The choice of locations for routine environmental monitoring in theseareas should concentrate very heavily on locations that may lead to microbialproliferation.

The choice of locations should be documented but should not be 100 rigid.There are several reasons for this. First. environmental monitoring assistantsshould not feel so confined to a mechanical task that they fail to notice vulnera­bilities or lapses in control. The lrained and discriminating human eye is a benerenvironmental monitoring method than any of the fonnal systems describedabove. Second. tilling personnel should not become conditioned 10 cleaning anddisinfecting monitoring locations at the expense of other locations. 1l is all toeasy in rOUline environmental monitoring 10 condition employees to bad habitsrather than good ones.

3. Frequency of EnvironmenIaI Sampling: Environments become sterilethrough being cleaned. decontaminated. and sterilized. The mainhmance ofsterility is achieved through a series of conlrol syslems Ihal overlap and back upone another. These systems for maintaining asepsis operale continUOUSly. Theireffectiveness at mainfaining asepsis is monitored on the same basis as any otherconlinuous process. The aseptic environmenl cannol be batched into neat day­sized or week-sized chunks Ihat can be sampled. tested. and classified asaccept/reject on the basis of the results. The frequency of environmental moni­loring should not therefore be set as a simple function of time. daily. weekly. etc.

In the absence of reliable data indicating a history of control for an asepticmanufacturing facility. it is good sense 10 lisl all those events that might have adeleterious effecl on the provision of an aseplic environment (batch changes.shift changes. process interruptions. etc.). The environment should at first be

Awptic M~"uf~dure 2JS

monitored at a frequency that wilJ confinn or contradict the hypothesis thaichange has a significant deleterious effect on the aseptic environment

Particular attention should be given to any buildup of contamination thatmay arise between major clean·downsldeconlamimllions. If buildup should ariseit may be necessary to improve the cleaning melhods. improve the environmenlalcontrols. or shonen the interval between c1e3n-downsldecontaminalions.

When there is enough reliable data 10 discriminate between significantchanges and insignificanl changes arising from scheduled. routine. prediclableevents. and to support the interval of time belween c1ean-downs/decontamina­lions. the environmental moniloring program can be addressed as a function ofIime-.

The assumption that is being made when it comes to interpreling results isIhal the quality of Ihe environment between two successive monitorings does nOIdiller from the quality measured al each time of monitoring.

4. Respcmoing to Monitoring Data: Data from routine environmental moni·loring programs are not sufficienl on their own to cenify thai all processes andconditions thai might influence the sterility of producLS manufactured in theaseptic facility are satisfactory and under control. They are only one pan of theoverall system of sterility assurance.

Data should be evaluated to discern any change (improvement or delerio­ration) to the level of control exisling in an area or at a localion. Limils shouldbe set and any c"cursions beyond those limils should demand a fonnal response.Each 10C31ion should be: addressed separately for these purposes. Progressivechange within these limits should also be reviewed in an attempt 10 prediciimprovement or deterioration.

Limit.s should be set carefully. Table 7 idenlifies some limits that havebeen proposed. Microbiological tesl methods are generally less sensitive thanchemical or physical methods and are vulnerable 10 limits being exceeded byaccidental conmmination that does not necessarily relate 10 the aseplic manu­facturing environment. Almost every textbook. laboratory manual. and melhodsguide 10 microbiology conmins a statement that plates for colony countingshould contain when possible 30 to 300 colonies. Environmental moniloringlimits for aseptic environments are typically well shon of these numbers.

For most microbiological purposes plates with counts within proposedenvironmental limil's would be rejected on the basis of chance contamination(were it to have arisen) having an unaccepmbly high proponional e(fecl on IheresulL

Microbiological methods are separately too insensitive to detect fluctua­tions in environmental control that could be significant to the sterililY of asepli­cally filled products. In combinalion they are a more powerful tool. because theprobability of two or three independent metl10ds being subject 10 simultaneouschance contamination becomes rapidly more rem01'e.

236 Chapter 8

Table 7 Proposed 116.17) Target Le\'els for Environmental Monitoring of AsepticManufacturing Facilities

Test method

Scnle pl:nes

Active air samples

Surface samples

Touch pl:lI.c..IO

White areas

< 5 du per 90 mm plate;exposure 4 h< 10 du per mJ

(equjvslcOl to < 3 pet 10 ft l )

< I cfu per 10 rt3

(equivalent to < 3.3 per ml )< 5 du per 0.0024 mJ

(equivalent 10 a Rodac Plate)< I cfu per 5 fingers

Gmyareas

< 40 du per 90 mm plate:exposure 4 h

< 80 cfu per mJ

(equi...alent 10 < 25 per 10 fll)

< 80 du per 0.0024 Ill)

(equivalent to 3 Rodac Plate)< 5 eru per 5 fingers

v. CONTAMINATION CONTROL FOR TERMINAllY STERILIZEDPRODUCTS

Some or all of the principles of aseptic manufacture are used in (he manufaclUreof tenninally sterilized products. There are two main rellsons for this. The firslis microbiological. TIte numbers and types of microorganisms present in or on 11

product item prior to sterilization make up a major determinant of sterility andmerit serious consideration. TIle second reason is the avoidance of large nonvi­able panicles in parenteral products.

11lere is substantial e\lidcnce (much from the literalure on drug abuse)indicating that particulate matter (undissolved substances) is a health hazard inparenteral products (211. The precise hazards depend very much on the physicaland biological propenies of the panicles, and their site of lodgem~l1t in the VIlS­

cular system. Phlebitis and pulmonary infarctions are the most significunt prob­lems associated with particulate maUer. Particulate maller is an unwanted andunnecessary addition to parenteral therapy.

Terminally sterilized parenteral products are therefore usually manufac­tured under conditions very similar or even identical to those of aseptic manu­faclure.

The sources of particulate matter are packaging components, manufactur­ing conditions. and fonnulation components. ~ mechanisms of controllingpanicles in all of these categories have already been alluded 10 earlier in thischapler. Their control is through the same mechanisms described for the controlof microbiological contamination. 'The washing processes for glass producicontacl containers and rubber closures are JT'KH't: related to removal of spiculesand elaslomeric fragments than to abe removal of microbiological conlamination.Pressurized air and laminar flow control both microbial and particulate contami­nalion from the manufacluring process. The choice of materials for manuf3c-

A5eplic Manufacture

•

237

turing equipment should consider their potential to contribute particles to theproduct. Filters capable of removing microbiological panicles are also capableof removing JX>lentially hazardous larger nom iable particles.

Pharmilcopoeial limits for nonviable panicles in large-volume parenteralprodUClS have heen long established. The USP introduced standards for small­volume parenterals in 1985.

This standard for small-volume parenterals was sel al 20% of thai for largevolume parenleeals on the unfounded assumption that the average patientreceives five small-volume parenlerals for each single large-volume infusion.

There are no compendial standards to limit panicu]ate maHer in thedevices used for delivery of parenteral producLS to the patient. In-line filters arebeing increasingly included in infusion sets and devices.

Microbiological control and panicu)ate control go hand in hand in thetechnology of aseptic manufacture. Microbiological control of medic,aI devicesprior to terminal sterilization rarely requires such intense dfon or as much capi­tal investment.

Many medical products and their processes of manufacture are inn:nelyparticle generating. For instance, wilh cellulosic mar:erials used for manufactureof sterile dressings it is impossible to avoid both paniculate and microbiologicalcontamination. With plastic medical devices the silUatian is different. The con­ditions of pressure and temperature at which plastics are molded or extruded aresufficienl to sterilize such components at the point of manufacture. On the otherhand. these processes are significantly panicle generating. There is lillie point inendeavoring to operate most medical device manufacturing processes in thesame type of clean room conditions used for aseptic manufacture; they would beimpossible to achieve.

The concentration of effon in contamination control for sterile medicaldevices is therefore toward conlrolling the major sources of both microbiologicaland particulate contamination but without specific targets. There is no regula­tory necessity to manufacture medical devices in classified clean rooms.although Class 100.000 may be achievable in many processes. The requirementis for a controlled manufacturing area. with intact floors. walls. and ceilings. forcontrolled aocess (personnel and raw materials) and egress (personnel. finishedproduct. and waste), and for high standards of personal hygiene.

The process of injection molding. for instance. produces a sterile productat point of manufacture as Slated above. Molding polymers are. however. panic­ulate in nature. The raw material may lead to higher than necessary particulatecontamination.

This is best controlled by operating a centralized polymer room separatedfrom lhe molding process and capable of delivering me polymer to the moldingmachine lhrough a sealed system. The amount of vibration arising from theoperation of molding machines is innately particle generating. The mechanism

238 Chapler 8

of removing molded comJXmenlS from lheir sprue or runner may also be paniclegenerolting (lhis is best done automatically but in some insUUlces may requiremanual intervention).

Collection of molded components should be in lcrile nonparticle sheddingplastics. Robotic devices can be used to remove easily defonnable componenlSfrom the mold face and transfer them gently to receptaCles. Wooden palletsshould be avoided. There is no fundamental reason why injection molding pro­cesses should not operate in !.he IOlal a~nce of personnel. 'The ideal is OOl oftenachieved.

Thereafter. contamination of medical devices assembled from moldedplastic components is very much a function of their exposure to personnel.Superb air quality is nol required. so some base level of contamination as a resultof its absence is inevitable. Personnel should be trained in hygiene and equippedwith nonshedding overalls (usually smocks with high collars and fitted wrists).headcovers. and shoe covers. Gloves are not usually a considerolion for medicaldevice manufacture: unless for some particularly critical operation or a "crytightly specified tcrilization process. In general terms, thr level of microbio­logical contamination found on medical devices prior to sterilization has a crudeinverse rclationship to the Client of automalion used in the device's assembly. IIis paradoxical howevc,r that the amount of particulate: conlaminalion tends toincrease wilh automation due to the vibration from ITl<Ichinery.

The manufacture of lenninally sterilized medical and phamlaceulicalproducts is controlled therefore to minimize microbiological and particulatecontamination. 11lere: is a major divide separating the conditions necessary forparenteral prodUClS from conditions salisfaclory for nonparenteral pnxlUClS.Irrespective of whether they are being aseptically manufactured or tcnninallysterilized. the manufacture of parenteral products requires superbly controlledand monitored manufacturing conditions. This is in the main because of theneed to control nonviable particulate comaminalion as well as microbiologicalcontamination. For medical devices. where panicul:11e contamination is un·avoidable. the need is only to control microbiological contamination. Therequirement at best is about the same as the requirement for controlling gr.tyaseptic manufacturing areas. but for the mos. part it is 3 good deal Ies..li stringenl.

REFERENCES

I. Whyte. W.. and Donaldson. N. (1989). Cleaning a c1eanroom. M~dica/ De\'ic~ alldDiagno$tic /ndU$try It (2): 31-35.

2. Food and Drug Administration (1981). Gu;d~/iJle on St~ril~ Drug Prod"ets Pro­ducrd by Mrptic Procrssing. Washinglon, D.C.

3. Her M:tjes1y's Stationery OffK'C (1983). Guid~ to Good Manufacturing Practice.London.

4. Commission or the Europc'an Communities (1992). Thr. Rulrs Gm'f'rni"g Mf'dicinalProtium ill thl! European Community, Volunu' 4. GuidI! 10 Good Manufacluri"g

Copyrighted m "'n I

9Maintenance of Sterility

L Considerations for Containment of Sterile ProductsA. Materials of ContainmentB. Systems of Conlainment

n. MicrobiologicJl1 Evaluation of Maintenance of S,erilityA. Microbiological Immersion Challenge TestsB. Microbiological Aerosol Challenge TeslS

m. Physical Evaluation of MaintenllJlce of SterilityA. Glass AmpoulesB. Glass Vials with Ela.stomeric ClosuresC. Flexible Containers

242242243245245246249250251253

The sterile condition can ooly exist within barriers that protect it from the non­sterile general environment. An essential prerequisite to the maintenance ofsterility i for !he product or pan of !he product that is requirtd '0 be slerile '0 beisolated by containment within a material impenneable to microbial penetmtion.If the design of the containment system or primary package does not allow thematerial of containment to be continuous. aU-enveloping. and complete. then thesealing surfaces of !he material of containmen~ eilher 10 itself or to anolhermaterial of containment or to the product itself, must also be impermeable tomiaobial penetration. 1be barrier properties of the containment system 10

241

242 Chapter 9

microbial penetration must be sufficient to withstand the rigors of sterilizationand toleranr to extreme conditions of transponation and storage throughout thepnxlucCs shelf-life. Yet cont'ainmenl should not be so secure mat it becomesimpossible to use the product without compromising its sterility.

Some systems of containment for sterile medical and pharmaceutical prod·UCIS have become so widely used that expectation has become conditioned totheir being the systems of first choice. Sterile pharmaceuticals arc expected (0

be contained in glass. mainly in the fonn of bollies. ampoules. or vials. bUI alsoin other presentations such as prefilled syringes. Glass has achieved its pre­dominance through an extensive body of knowledge confirming irs inertness andcompatibility with pharmaceutically active subSlances. It is also impenneable tomicroorganisms and slable at thermal sterilization temperatures. Other materialssuch as plastics arc also used for sterile pharmaceuticals, but not so extensively.Medical devices are expected to be contained within flexible packaging, mostoften nowadays consisting of polymeric film material, but originating from thetolerance of particular types of microbiologically impermeable paper to steam,gas, and radiation sterilization.

I. CONSIDERATIONS FOR CONTAINMENT OF STERILEPRODUCTS

The choice of appropriate containment systems for maintaining sterility is par­ticular to specific products. It may often be nllher complex. Factors relating (0

the materials of containment and 10 the overalJ system of conlainment should beconsidered separately and then in combination to achieve the best end result.

A. Malerials of Containment

Some of (he major factors affecting the choice of materials of containmentinclude

(a) Microbiological impermeability. Materials thai are impenncable tomolecular migration such as glass are guaranteed to be impermeable tomicrobiological penetration. Some forms of nexible packaging are delib­erately chosen to allow the passage of steam or ethylene oxide gas. Thesematerials must be evaluated specifically for their impemleability to micro­bial penetration. In this respect their properties can be compared direcLlyto the propenies of filter media.

(b) Biological propenies of the material. Materials should not leach bio­logically active substances into or onlO the product. In some inslances thismay be a simple decision; in others il may require knowledge of thebehavior of the material in relation to the specific product. In general

244 Chilpl~r 9

faclors affecting materials. their microbiological impermeability. Tht extradimension i the microbiological integrity of the overall system and panicularlyin relation to sealing one material surface to another.

11le choice of a system of containmenl must address the following majorfactors relating 10 container<losure integrity:

(a) Microbiological impermeability of the seal. How are surfaces sealedone to another? Molecular bonding is the method of choice, for installl'c inhe:u-sealed glass ampoules. and in heat-sealing paper to paper. paper topolymeric film. or polymeric film (0 polymeric film. Dimensional inter­ference fit for instance between the outside neck: diameter of a closure andthe inside ned: diameter of a vial. is an acceptable alternative. Adhesivesmay also produce microbiologically impenneable seals.

(b) Suitability for sterilization. Will the seal break down when sterilized?Will internal headspace pressure in 3.utoclaving vials blow the closuresout? WiU the pressure differentials in ethylene oxide slerilization burstflexible packaging seals open?

(c) Seal strength. This is a curiously ambiguous property. Seals should bestrong enough to maintain microbiological integrity Ihrough sterilization(if considering a terminal process), transportation, and storage. On theolher hand Ihey usually constitute the roule Whereby the pnxluct isremoved for use, so they should not be too difficult to open.

Other aspects of the overall syslems of containment for sterile productsinclude the shape and sile of the system. These factors may affect its fragility.the ways in which the produci may be $ilerilized, and the ways in which (heproduct itself may be removed from its contllinment system. The potential formovemenl of the product within its containment system should be restricted ifdamage and poten1ialloss of sterility are to be avoided.

Clearly, containment systems must be specified in some detail with regardto those propenies that may affect their resistance to microbiological penetr.ltion.For simple systems such as rubber stoppered glass vials, dimensional specifica­tions should addre s the internal diameter of the neck opening and its depth, theinternal and external diameter.;; of the flange. and the concentricity of the flange.the neck, and the body of the vial. Any angularity of the flange ver.;;us the verti·ca) center line of the vial must be specified; so must the physical finish of thesurfaces of flange and internal neck bore 10 ensure satisfactory maling with theclosure. Closures should be specified in leons of dillmelCr'S, depth, thickness.and elasticity.

Other containment systems must be specified in similar levels of detail ifthere is to be consistent assurance of maintenance of microbiological integrity.

Maintenance of Sterility

II. MICROBIOLOGICAL EVALUATION Of MAINTENANCE OfSTERILITY

245

A microbiologically contaminated product is nonslerile regardless of l.he mannerin which the contamination arose. Nonsterility rna)' have arisen through an inad­equate sterilization process, through contamination during aseptic processing. orlhrough failure of the system of containment. Systems of conlainment shouldtherefore. like all other systems contributing 10 the assurance of sierility. beshown 10 be capable of meeling their intended purpose. i.e., to maintain sterililY.

1.1 may be obvious thai a conlainment system has failed to mainlain steril­ity. The conlents of a broken ampoule have no assurance of sterility. A syringereceived in an open package cannot be supposed to be sterile. Other deficienciesin containment systems may pose grealer IhrealS to the user of the medical orpharm3CeUlical product because Ihey go unnoticed. It is Ihese mort' sublle prob·lems that demand serious consideralion.

Microbiological evaluation oughl to be done as part of Ihe validation pro­gram for new or changed conlainment systems or new or changed malerials ofcontainment. Physical or chemical testing is best used for rourine purposes if itcan be assumed that these characteristics relate to the microbiological barrierpropenies of the containment system. There are two broad systems in use formicrobiologically evalualing barrier propenies of materials. These are microbi­ological immersion challenge methods and microbiological aerosol challengemelhods [I]. The microorganisms differ wil.bin each type of test. and belweentests; there is no accepl.ed sl3I1dard method. and delails differ from one· labora·tory to another.

A. Microbiological Immersion Challenge Tests

The microbiological immersion challenge test is probably the most severe test ofconlainmtnt system integrity. The lest accepts that microbiological penetrationinlo a container is most likely to occur in the event of a continuous film or bridgeof liquid forming belween the general contaminated environment and Ihe con­rained protected environment To a large extent it mimics Ihe possibility of thecontainment system becoming weued in lranspon. stonlge. or use. Wening mayarise from sources within the conlainment system. e.g.. a liquid fonnulationleaking out of a viaJ. or from outside the conlainmenl system, e.g.• splashing.minwiucr. condensation. Immersion is probably the most appropriate lestmethod for rigid containment systems. for containment systems containing liq­uids. and for any type of product where wening might arise bUI not be immedi­ately or obviously apparent

Frieben et at [2] filled vials with Soybean Casein Digt'~jl Met/illtn. sealedthem. and then immersed them invened in a suspension of 108 Eschuichia coli

248 Chapler 9

Microbiological aerosol challenges are panicularly appropriate for thetypes of flexible packaging used for medical devices. There is rarely any liquidcontent 10 leak out. External water damage is often obvious and should stimulaterejection of the product. On the other hand. there is a real risk of unnoticeableair-borne microorganisms passing through pinholes in the material or throughthe seals of flex.ible containment systems. The risk of failure to provide salis­factory containment may be increased if temperature or pressure differentialsbetween the air within the package and the generdJ environment give rise toforces that may drive microorganisms inward. Some flexible packaging materi­als, for instance those used in connection with radiation sterilization. can betotally impermeable to molecular movement; others. for instance those used inconnection with ethylene oxide and steam sterilization. must be penneable (orhave permeable insens) to molecular movement. The risk of encountering pin­holes is greatest in these permeable materials.

Powell 14J recommended the use of an aerosol suspension of Serraliamarc:escens in a respirator to lest product-filled sealed nexible pacbges. Thepackages were subjected to alternative positive and negative pressures at a fre­quency of 20 cycles per min over 2 h duration to simulate the changes in envi­ronmental temperature and pressure that might be encountered in transpon andstorage. On complelion of the challenge the contents of the package were testedfor sterility. This is a relatively unsophisticated system.

Experimental technologies become more elaborate as they auempl toaddress somc of the uncontrolled variables present in the type of tcst describedby Powell [4J. lypically

(a) Maintenance of viability. Some microorganisms may have only lim­ited viability in aerosol suspension and thus could provide a falsely highimpression of the severity of the challenge. Beuer experimental technolo­gies allow periodic withdrawal of samples from the aerosol to check via­bility.

(b) Humidity. The two major reasons for wishing to control humidity arefirst because of the effects of humidity on microbiological viability (mostmicroorganisms naturally encountered in atmospheric air are resistant todesiccation but many others are highly sensitive 10 dry conditions) andsecond to simulate the effects of moist atmospheric conditions on the bar·rier propenies of the containment system.

(c) Homogeneity of the suspension. It is sensible to prevent the microor·ganisms from settling out of suspension during the challenge period. Thisis normally done by maintaining air movement by fans or by recirculationduring the challenge.

a

Maintenance of Sterility 249

(d) Uniformity of panicle size. Uniform particle sizes are most usuallyobmined by nebulizing lhe microbial suspension into the challenge cham­ber.

(e) Qualitative or quanlilative penetration. The amounl of informationobtainable from quanml response experiments (penelration or no penetra­tion) is limited. For experimental purposes that attempl to relate physicalcharacteristics of the containmenl material or containment syslem it is nec­essary 10 obtain quantitative data. This may be done by arranging to col­lect and cultivate those microorganisms that penetrate the barrier systems.

One such system recommended for packaging films by Reich {5} describeda challenge of Bacillus sub,iUs atomized via a Devilbiss nebulizer into a testchamber to a concentration of J()4 spores per m3. The spores were mai.ntained inhomogeneous suspension by means of a fan, and the chamber was equipped witha pump to allow pressure differentials to be crealed across the samples of rnale­rial under lesl. The tesl films are silUated over baclcria-retenlive membranes toallow quantitative comparison of various pressure differentials and flow rates.Other elaborate experimental technologies include Ihose of Tillenlire and Sin~

clair [6]; using spores of Bacillus sub/iUs var niger these workers showed sig­nificant differences in microbiological penetration through paper and spun­bonded polyolefin webs over now rales from I to 100 cm3 per min per cm2.

The same principles of controlled aerosol challenge can be applied 10sealed packages. excepl that flow inlo sealed packs is far more difficult toachieve Ihan now across samples of films or webs. II is very doubtful. also,whether this type of aerosol lesling can be meaningfully applied 10 rigid con­tainment syslems. It is unlikely thai aerosols can pose a severe challenge to sayglass vials with oversealed rubber closures. Toward the end of the nineleenlhcentury Pasleur demonstrated me intrinsic unlikelihood of air-borne microor­ganisms Iraversing a coovoluled pathway in the absence of some specific drivingforce 10 contaminate nulrienl infusions. II is similarly unlikely for microorgan­isms 1'0 traverse a tonuous channel between an elastomeric closure and a glassnange.

III. PHYSICAL EVALUATION OF MAINTENANCE OF STERILITY

Physical evaluation of the containment syslem used in the mainltnance ofsterility is an acceptab.le alternative 10 microbiological evaluation. because it isthe inlegrily of physical syst.ems thai creales baniers 10 microbiological penetra­tion of conlainmenl materials and systems. For instance. a pinhole in a flexiblepackage or a crack in a glass vialls a loss of integrity of the containment system;the fact thai microorganisms can Iheoretically traverse ilial crack or pinhole issufficienl 10 compromise slerility without hav~ng to set OUI specifically to

Maintenance of Sierilily 251

If it is to be considered thai microorganisms can penetrate capillaries withdiameters as small as 0.4 ~m (and this is a reasonable assumption, becausePseudomonas diminuta was first encountered from having passed through filtermembranes with 0.45 IlID pore size ralings), PoiseuHlc's law can be used to cal­culate the dimensions of a satisfactory vacuum dye penetration test.

Given ampoules with wall thicknesses of 4 x 10-2 em, methylene blue witha viscosity of 10-2 poise in I% solution, and a pressure differential of 2 x 106

dynlcm. the minimum time required for 10-5 mL (a reasonable guess al theminimum perceptible volume) Co penetrate through a 0.4 11m pinhole would be 9h. Practical testing times in the vacuum dye penetration lest are in the region of15 min.

A funher complication in relation to lhe dye penetration test is the possi­bility of microbiologically contaminated dye penetrating some of the ampoulesand going unooticed during inspection, Sterile dye solutions are usually speci­fied for this test

An alternative to the dye test is high-voltage electronic pinhole deteelion.The subjectivily of visual inspection is eliminated, the time taken to complete thetest is shaner, and il is usually automated and can be ruD as a conlinuous inspec­tion system rather than as a Ixttchwise inspection system. Ampoules are individ­ually exposed to a high-frequency voltage. Ohm's law dictates mat the currentflowing in the system is proportional to the voltage applied and inversely pr0­

portional to the resistance of the system. Glass has insulating properties lhalwhen intact confer high resistance 10 the now of current. Pinholes or cracks inthe glass, and even regions of thinner than normal glass, atlow lhe discharge cur­rent to entcr the ampoule; the resistance of lhe system decreases measurably. andthe resultant signal can be channell~d into automatic rejection of the corre­sponding ampoule. Sensitivity to 0.5 Ilm is claimed.

High-voltage methods arc not restricted to glass ampoules: indeed. theymay be used for other materials with high resistances such as plastics, and forOlher types of containers such as vials.

B. Glass Vials with Elastomeric Closures

There is a United States Federal Specification dated 1976 that describes a physi­cal tesl for vial integrity consisting of suspending the vials upside down at roomtemperature for 2 h and then at 49"C for 4 h_ Evidence of leakage may be byweight or by visual inspection. This test is of somewhat doubtful relevance tomicrobiological penelration,

Vials are a little less prone to pinhole cracks than ampoules. They are usu­any more robust and are not deliberately weakened in particular areas to allowopening. 1be main CODcern with vials is the integrily of the seal between the vialir:self and its elastomeric closure.

c ,

252 Chapter 9

Some degree of evaluation of the microbiological integrity of the viaVclosure system can be made from the specified characteristics of the twO compo­nents. In the first instance it would be reasonable to have a clear picture of thequality characteristics of the vial flange and the vial neck that affect con­tainer/closure integrity; concurrently to have a clear picture of the quality char­acteristics of !he closure.

Closures may be flat discs or wads thai rest on the vial. making contactwith the nange, and being held in place by an overseal. This type of presentationis nOI used commonly nowadays. having been replaced in the main by plugs thatfil in'o the neck of lhe vial (Fig. 2). These plugs also make conlac' with theflange of the vial and are held in place by an overseaI.

The factors thai ensure microbiological integrity are

(a) An interference fit between the outside diameter of (he plug and theinside diameter of [he viaJ neck. Any ovality within the vial neck mayhave an adverse effect on interference. but this may be compensated bydistonion of the plug which is in tum a function of the composition of (heelastomer. The finish of the two mating surfaces may also bear someinfluence.

(b) The completeness of the seal belween lhe lOp surface of the flange ofthe vial and the bouom surface of the flange of the closure. This isaffected by the compression forces e;\ened by (he. overseal and the elastic·ity of the closure. The finish of the (wo mating surfaces is of majorimponance.

It is generally understood that the principal means of ensuring the microbi­ological integrity of vial container/closure systems is via the two mating naoges.Monon et al. (7) studied container/closure characteristics by measuring the rateof pressure decay from sealed vials. Although relaliog directly to molecular

FlAT DISC OR WAD

CLOSURE

CLOSURE WITH SH.t.NK

..­...

Fig. 2 Types of vial closure.

M.lintenance of Sterility 253

leakage from vials, lhe conclusions drawn by these workers are by inferenceapplicable also to microbiological integrity. The most imponant faclor con~

triOOting to conUliner/dosure integrity was the compression force applied by theoverscal. Various di{[erences in elastomeric propenies of closures and defects inthe surface finish of flanges could be compensated by closure compression.

Evaluation of the compression forces applied by overseals lO sealed vials istherefore crilical to the evaluation of microbiological integrily of glass vials withelastomeric closures. This oughl to be determined at validation and moniloredroutinely in production. Equipmenl has been developed [8) to allow in-processmeasuremenl of the forces required to crimp overseals onto vial closures, lhusrevealing and automatically rejecting broken glass flanges. defective glass fin­ishes. damaged or incorrecl closures. elc.

II is DOt necessarily correct to assume that the neck fit is of no importanceto microbiological integrity of vials. It may ralher be of secondary imJ,X)rtance tomating flanges. Interference is clearly of importance. for instance, to themicrobiological integrily of prefiUed syringes. where there are no mating flangesurfaces. The primary microbiologkal seal in the prefilled syringe is effected byinlerference between lhe outside diameters of two or lhree ribs molded into theelastomeric plunge-nip and the inside diameter of the syringe barrel.

Other physical tests may allow routine in-place evaluation of the microbi­ological integrity of vials. Headspace gas analysis is one such method. Manysterile products are held under nitrogen or some other gas in vials. The gas con­tent of the headspace should. with a perfect seal, remain conslanl over limeraLher than becoming equilibrated with the atmosphere under a less lhan perfectseal. This lype of analysis is amenable to chemical methodology and is likelydone routinely in pharmaceutical production for reasons other than evalualion ofmicrobiological integrity.

1be evaluation of viaJ seal integrity may also be done routinely by similardifferential pressure measurement methodology to thai used experimentally byMorton et aI. [7]. The principle of the melhod is to detect leakage through thecontainer/closure interface by reducing the pressure in an inspection head thatsurrounds the suspect region. Any change in pressure (leakage outward) fromthe item under test is detected by comparison with a blank for which no pressurechange is po ible. A fully autornaled 100% inspection system allOWing auto­matic rejection has been described [9].

C. Flexible Containers"the solid conlenlS of a flexible package containing a sterilized medical deviceare very unlikely to be small enough to leak out through a pinhole in the systemof containment and be immediately noticeable. Yel pinholes are aU thai isneeded to compromise the sterility of the product. 1be Iwo issues surroundingl1exible conUline" are the quality of the material and the quali.y of the seals.

, I

Copyrighted m "'n I

10Parametric Release and Other

Regulatory Issues

l. The PharmacopoeiasU. Good Manufacluring Pmctices

m. 1be FDA's Prefen:nce for Terminal SterilizationIV. Parametric Release

A. Barriers to Parametric ReleaseB. The Price to Pay for Parametric Release

V. FDA PreapprovallnspectionA. Preapprovallnspection for PhannaceutiC1ll ProductsB. Preapprovallnspection for MediC1ll Devices

260261264266267269271273273

Sterility knows no frontiers. Sterile products are manufactured in all countries;they are u,;.,d within national boundaries. they are imported. and they areex-poRed. However. most governments will not pennia the marketing of sterilep/larmaceutical produClS or mediC1ll devices unless the products have been noti·fied 10 them. scrutinized for safety and efficacy. and approved. [t is nol theintention of this chap"er to describe in detail the activities of governmentalauthorities throughout the workt in relation to sterile products bul (0 dmw atten­lion to some of the issues that are peculiar to sterile products and sterilization.

159

260 Chapter 10

The greatest amounts of governmental activit>, in the regulation of medical prod­ucts are in the U.S.A. and in Europe. More often than not, the regulation of ster­ile products in olher continents and countries tends 10 follow U.S. or westernEuropean models.

I. THE PHARMACOPOEIAS

Historically. the control of pharmaceutically active products has rested with thepharmac.opoeias. Phanllacopoeias in one guise or other have been publishedsince antiquity (I]. Wilh the beginnings of greater mobility of commerce in theseventeenth and eighteenth centuries. local treatises on the purily of medicinalsubslances began (0 take on national stature, for instance (he London Phanna­copoeia of 1618. Ihe Phannacopoeia Danica of J772. elc. The first edition of theUnilcd Siaies Phannacopeia (USP) of t 820 predates the first British Phanna­copoeia (BP) of J864.

Interest in international pharmacopoeial standards was taken up by (heWorld Health Organization's International Pharmacopoeia of 1952 and its subse­quent revisions. This differs from national pharmacoJX>eias in that it has no legalauthority. Within the beginnings of the development of whal is now the Euro­pean Community, a European Phannac0JX>eial Convention was signed in 1964,by whose terms the signatories undenook to make the standards of the EuropeanPhannacopoeia (EP) the official slandards of lheir countries. The majorily ofcountries in western Europe are now signalories.

National pharmacopoeias, and EP, have. legal slalUs. They are legally con­stituted compendia of standards. some of which are mandatory and some ofwhich are not Taking the USP as an example.lhe pharmacopoeia is divided intomonographs and General Chapters. The first purpose of the pharmacopoeias isto publish monographs. These are official specifications for drugs or devices. Itis illegal for an item that is alleged to meet the requirements of a USP mono­graph to fail to do so. A product specified as being sterile in a phannacopoeialmonograph is required to be capable of passing the Test for Sterility. The T~st

for Sterility is not described in a monograph; it is described in a Geneidl Chapler.The SP and abe EP are structured in the same way.

The General Chapters of the USP may be mandatory or nonmandatory.The mandatory chapters are themselves standards that define official "referee"tesl methods intended to be used in the event of litigation or arbitration con·cerning the status of items alleged to comply with the requirements of panicularmonographs. The Tut for Sterility is a mandatory test. This does not in Jawmean that there is any obligation on a manufacturer to use the Tist for Sterility asa release test or as part of ;] quality conuoUassurance program. although manydo.

262 Chapter 10

(lhe USP, for instance, is published in revision every five years). In theseinstances. official permission to market a product may predate its recognition bythe pharmacopoeias.

Furthermore. in addition to the compendial restrictions on end·produclspecifications and referee tests. a need has been found for legal we:lponsextending into Lhe ways and means by which prodUCIS are manufactured. Regu­latory agencies are justifiably concerned mat particular drug substances may. ifmanufactured by a different method from that used for the original product onwhich the phannacopoeial monograph was based. contain undesirable impuritiesthat are not defined in the monograph. The U.S. Pood. Drug and Cosmetic Actof 1938 gave the FDA the authority to inspect manufacturing facilities. The1962 Kefauver-Hanis amendments to the Act provided aulhority for publicationofGo<xl Manufacturing Practice Regulations in the U.S.A.

This movement to governmental regulation has led to publication of arange of governmental rules and guidelines affecting the manufacture of medicalproducts. The main sources of regulations are. in the U.S.A.. the Code of Fed·eral Regulatiolls. which publishes. in title 21. the basic requirements for regis·tration of new drugs (the NDA or New Drug Application) and the Good Manu­facturing Practice (GMP) Regulations for medical devices (Section 820) andpharmaceuticals (Section 821); in Europe, EEC Guide 10 Good ManufacturingPractice for Pharmaceuticals 12] and Ihe U.K. "Blue Guide" [3J for sterilemedical devices.

The U.S. Code of Federal Regulations has Ihe force of law. As such, theU.S. GMPs are rather stark in their slyle and content. Two U.K. documents thathave been of significant influence on the development of Slandards in Europe.the "Blue Guide" [3J and 'he "Orunge Guide" [4J. differ from the Code of Fed­eral R(!gulatiolls in Ihat they have no statutory force. As such, their content(within three revisions of the Orange Guide, in 1971, 1977. and 1983, and tworevisions of the Blue Guide. in 1981 and 1990) has always been more user­friendly. The EEC Guide to Good Manufacturillg Practice (2] has arisen froman EEC Directive. The puJ1>OSC of an EEC Directive is to set out principles andcriteria that must be embodied in the national laws of the member states, and sothe EEC guide (2} will have, in due coune, the force of law for pharmaceuticalGMPs throughout Europe. The EEC guide, however. has retained the user­friendly style of its origins in the Orange Guide. Ie has yet to be seen how legalauthority will affect this in the future.

The Blue Guide is the only one of the pivotal regulatory documents that isparticular to sterile products ("Sterile Medical Devices and Surgical Products").In its 1990 revision, it has adopted the principles, slyle, and contenl of Interna­tional (ISO 9000 to 90(4). European (EN 29000 '0 290(4). and Bri'ish 5'ondardsOrganizations (BS 5750) requirements for integrated quality assurance systems.Additional requirements specific to sterile products are included.

264 Chpter 10

In Ute area of sterile medical devices. the principal U.S. voluntary stan­dards bodies have been the Health Industry Manufacturers' Association (HIMA)and the Association for the Advancement of Medical Instrumentation (AAMI).Other bOOies working in the area of standards for sterile products and steriliza­lion technologies include the American National Standards Institute (ANSI) andthe International Standards Organization (ISO). The interactions of the variousbodies involved in preparing standards is complicated. nuid. and not uncompli­c~ned by political undel1Ones. Some hannonizalion of lhese diverse activitiesoccurred in 1989 with the AAMI. under the auspices of the ISO. being endorsedby the FDA. lhe HlMA, and the ANSI as the organization that should lead stan­dards development in several medically related areas including sterili7.3tiontechnologies. It is likely that sterilization standards already published or to bepublished by the ISO TechnicaJ Commiuee 198 will be considered state-of-lhe­an by the FDA (51.

In Europe. sterile medical device legislation is to be published in 1993 10

1994 to allow the free passage of goods across national borders. The key to Ihislegislation is "harmonized" standards. A list of harmonized standards is to bt:published in the "Official Journal of the European Communities:' and manufac­turers who comply with these hannonized standards will be presumed to complywith the statutory requirements. Harmonized slandards for sterile products willhave to be endorsed by the European Standards Organization (eEN) under theauspices of Technical Committee 204. CEN technical subcommittees areactively working in the area of medical device sterilization standards: in otherareas such as GMP they have agreed 10 adopt the voluntary standards of theEuropean Confederation of Medical Suppliers Associations (EUCOMED). Thiswill certainly improve the confusing mish-mash of standards that have in thepast existed in Europe.

The safely of the patient is lhe issue at stake when deb.aling free trade andmovement of sterile products across national and international borders. A goodundentanding of the alternative ways of meeting the same end is the basis ofharmonization. and signjficant progress is being made in Europe and the U.S.A..separately and to a Jesser extent between the two continents. National govern­ments have 3 responsibility to prolecl the heahh of their populace. and differ­ences in legislation will always arise according 10 the balance of expecl.3tionsthat exist between governments and industry in differing cultures.

III. THE FDA'S PREFERENCE FOR TERMINAL STERILIZATION

In the Federal Register of Oclober 11. 199 I. the FDA formally proposed thai arule should be established whereby aseptic processing of allegedly sterile phar­maceuticals should only be justifiable on the basis of the product being proven 10

be unsuitable for tenninal sterilization. On ratification of the rule and after a

P.....metric Rf'leue and Other Regulatory Issues 265

phasing·in period of 18 months. manufacturers wishing to pursue aseptic pro­cessing of a new or existing sterile product must provide documentary evidenceto Ihe FDA of the product's unsuitability for terminal sterilization. Manufactur­ers requiring to change their existing aseptic processes to teoninal sterilizationwill have their regulatory documentation fast-tracked through the FDA's bureau­cracy. The rule was expected to be published in the Federal Register sometimein 1992.

The reaction of the pharmaceutical industry was so lukewarm to the pro·posal that 1992 and J993 passed without ratification. This is not to say that theFDA has reconciled itself to shelving the rule; indeed, the inspection branch isplacing greater and greater pressw-e on aseptic manufacturers toward stricter andstricter compliance witb FDA guidelines and policy statements mat in law haveno authority. It would nOl be uncommon to see seemingly minor technical con­cerns like the failure of a laboratory to use magnification and illumination aidsfor plale counts cited alongside the absence of adequate validation protocols inIhe same FDA inspection "'pons (fonn 483). The", is little doubt thaI the FDAis 50 convinced of the correctness of its view Ihal it will continue 10 pursue Iheelimination of aseptic manufacture unless there is a step change improvement inavailable technology.

In maJcing this proposal, the FDA recognizes thaI a dual standard of steril­ity assurance has been in operation. Tenninal sterilization processes for par­enteral pharmaceutical products are currently required to be validated 10 sterilityassurance levels of 10-6; aseptic processes can only be demonsLrated to achievesterility assurance levels of 10.3. This is clearly an example of dual standards.Fwthennore, to the FDA it appears to be fundamentally wrong for produclS thatare quite capable of tolerating terminal sterilization 10 be manufactured aseplj·cally.

In practical terms. terminal sierilization of liquid parenteral prodUCISmeans sterilization by saturated sleam. Production of free radicals in water pro­hibits the application of radiation sterilization to aqueous prodUCIS. but mdiationsteriliulion may be suitable for some solid dosage forms. Dry heat and ethyleneoxide are unlikely to be of any value. In the first instance, lherefore. saturatedsleam should be the process of first c,hoice for sterilization of thennally stabledrug substances; dosage forms should not be fonnulated in ways that compro­mise thermal stability.

Beyond Ihe ways in which the proposal affects drug subslances and dosagefomu. there are some uncertainties associated with various presentations. Forinstance. the FDA exempts pre-tilled syringes from their proposal, alleging thatthey are unsuitable for tenninal sterilization. This is not necessarily true with aUtechnologies. for instance sterilization by steam in ballasted autoclaves.

Moreover, some phannaceutical products may be contained in plastics orolber terminal sterilization labile malerials. It is not clear whether the choice of

266 Chapter 10

materials for containers is sufficient justification to retain aseptic processing fordosage forms capable of withstanding terminal sterilization. The assessment ofacceptability in such circumstances has been stated (6)10 be based on demon­strable evidence thai the panicular package or container is of positive benefit tothe patient Some tricky decisions are likely to arise in Ihis area.

The impact of the proposal may reopen a debale on !.he way in which SALsappear to be applied 10 aseptic processing. The origins of the SAL concept lie interminal sterilization and rest heavily on the eXlrapolated effects of uniform ster­ilization treatments to populations of contaminants defined in terms of resistanceand of numbers of contaminants (bioburden). The process of aseptic manufac­ture is a process of contamination control; the frequency of occurrence of a con­taminated item within a population of aseptically filled items is a measure ofbioburden. nm a measure of the SAL. The SAL is the probability of those con­taminants surviving, and this is a function of the types of contaminants and thefonnulation of the product Fonnulations can be made to be antimicrobial. Inprinciple this is no different from chemical sterilization.

If this principle is extended, then SALs of 10-6 may be attainable by asep­tie manufacture followed by mild heal treatment.

All manufacturers seeking approval of new sterile products will need astrategy to justify the method of sterilization. tenninal or aseptic or something inbetween. The technology chosen should minimally jeopardize the chemical,physical. and pharmacological propenies of the dosage forms. Most cases arenot going to be "open and shut," and the FDA and the industry have an interest­ing time ahead developing these strategies and decision-making processes.

IV. PARAMETRIC RELEASE

There are various definitions of parametric release. for instance

(a) ... a sterility release procedure based upon effective control. moni­toring. and documentation of a validated sterilization process cycle in lieuof release based upon end product sterility testing (FDA Compliance Pol­iey Guide. 1987. Chapler 32a Parametric Release-Tenninally Heat Ster­ilized Drug Products).

(b) ... use of a specified set of engineering and microbiological dala ...10 determine that a desired sterility assurance level is provided and main­lained without Ihe need of finished product sterility (esting (ParenteralDrug Association Technical Repon No.8, 1987. Parametric Release ofParenteral Solutions Sterilized by Moisl Heat Sterilization).

Whatever definilions may have been proposed by regulalory bodies and othercommiuees for whalever specific purposes or processes, parametric release con­cerns eliminating end-product compendial sterility lesting as the final criterion

Parametric Releue and Other Regulatory Issues 267

required to release sterile products. AU of the major compendia recognize Lbesevere statistical and technical limitations of the test for sterility. The test isfonnally acknowledged in the US? and the EP as a "referee test" in cases wherethere may be a dispute over sterility (or more likely nonsterility). So where arethe barriers to parametric release?

A. Barriers to Parametric Release

The barriers 10 parametric "'lease are complex, partly legal. partly historical ortraditional. and muddled by issues of consistency.

The consistency issues are those of consistency among the various meth­ods of achieving sterility (should some be eljgible for parametric release whileothers should Rot be eligible?) and consistency among lhe sterility test and lhevarious end-product chemical and physical tests required to release phannaceuti­cal products to market

Among the four principal methods of achieving sterility (radiation. S3lU­

£Bted steam, elhylene oxide, and aseptic fiUing), only one has been whol~heart­

edly accepted for parametric release: sterilization by gamma irradiation, End­product steriJity testing has not been done on irradiated products for twodecades. The justification for this lies in reliable knowledge of how microor­ganisms are killed by radiation. and in the reliability of the methods of pn:x.~ess

control and measurement of absorbed dose.Sterilization by s:uurated steam has a longer history of praclical application

than irradiation. and the factors affecting microbial inactivation (temperature andtime) are equally well known and thoroughly researched. yet parametric releasefor terminal sterilization by saluraled steam has been addressed on paper farmore regularly than it has been applied in practice. One barner 10 parametricrelease for steam sterilization processes has been its technology. Steam steril­ization has been around for a long Lime. and its technology is improving all thetime; modern microprocessor<ontroUed autoclaves are pieces of very fine preci­sion equipment. but there are many older. less well-conlfolled, autoclaves sulI inregular usc. Patients have suffered and died from administration of nonsterileauloclaved products. Any mechanism therefore for allowing parametric releaseof steam sterilized products should emphasize the need for sound and reliabletechnology. but in doing this there is the danger of appearing to endorse the end·product sterility test as a justification for using unreliable technology that oughtnot to be used for sterilizing medical products in the fU'St place, This is a para­dox. that can only be resolved through wider usage of parametric release forsleam sterilized products, and indeed regulatory agencies might find parametricrelease a useful lever 10 force replacement or upgrading of obsolescent aUlO­claves.

Whereas radiation sterilization and steam sterilization technologies areclearly suitable for parametric release. ethylene ox.ide sterilization and aseptic

about tha book . • •

DeuiJing the scientific priDciples uDderlying the ochievement ofsterlJity, this uniqueref= exlmioes both I broad spectrum of pradkaJ, commooly used sterilizatioDprocedures and the methods .vailable to CODfirm .lerility-......ing the .trengthsaad limitatioDl of each tecboology.

Deli",,'iDg OIITOIIt regulatory requin:meIl15 for sterlJity, AdWIIIJIg SI"UJIy IJJMtdk:4J tutd Ph4rrruJautlalJ Protluch dis<:uIseo steriJizaliOD approach.. thai utilizeI8IUr8l<ld steam ... dry heal ... ethyl..... oxide ... gamma radialiaD ... sterile filtratioD•.• aDd more.

ContaiDing more thaa 200 up-to-<!ale IilUalUre citatioDl, usefUl equatiODl, aDdiDlightful tsbles aDd drawiDg., Ath/,lIillg SI'rlJIJ] /11 Mtdk:4J tutd P1uJnruu:IutiaIJProtIUdJ i. aa ....Dtial resource for pbarmacis15, pbarmacologisl5, cIiDk:al micro­biologisl5, virologisl5, quality ...u18..... aDd productioD maoagen in the pbarm_u.tical iDdustry, sterilization scientists and eGli.DCCil, biochemists, and upper·levelundergraduate aDd graduate ItudeD15 iD these discipli.....

about tho author • • .

NIGEL A. HAu..s is Head of Biolop:aJ Quality at Olexo MaaufacturiDg Servioea,COUDty Durham, United Kingdom. Dr. Hal1J is • FeUow of both the lnstilllte ofBiology (U.K.) aDd the l..tilllte of Quality Assurance (U.K.) as well as • memberof the Society for Applied Bacleriology (U.K.). He received the B.Tech. degree(1968) in biological science from the Univ....ity of Bradford and the Ph.D. degree(1972) in applied microbiology from the Univenity of Bath, both in the UnitedKiDgdom.

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