The Technology Of Microbiological Environmental · PDF fileThe Technology Of Microbiological...

© Silliker Australia 2010

The Technology OfMicrobiologicalEnvironmental

Monitoring

Stephen JaySenior Consultant Microbiologist

ChairmanSilliker Australia

CAPSIG Wednesday 7th July 2010

2© Silliker Australia 2010

Presentation PlanMicrobiological Environmental Monitoring

Standards and guidanceWhen to sampleSampling plansSample site selectionSampling frequencyHow to sample:•Sampling surfaces•Sampling air

ValidationQuality of growth mediaCharacterisation of microfloraAnalysis of dataEmphasis on cleanrooms and aseptic areas


Standards And Guidance

General :• ISO 14644 series - Cleanrooms and associated controlled environments• ISO 14698 series - Cleanrooms and associated controlled environments -

Biocontamination controlTherapeutic :• TGA - PIC/S Guide to Good Manufacturing Practice for Medicinal Products

•Annex 1 for manufacture of sterile medicinal products + technical interpretationof Annex 1

•USP chapter 1116• EU GMP Guidelines :

•Annex 1 for sterile products•PDA documents :

•Environmental Monitoring: A Comprehensive Handbook, Volume I, II, III, IV•Technical Report 13 - Fundamentals of an Environmental Monitoring Program

(1990)•Technical Report 49–Points to Consider for Biotechnology Cleaning Validation

(2010)• Journal articles, especially J Pharm Sci Technol•Pharmaceutical Microbiology Forum (PMF)–www.microbiologyforum.org


When To Sample

When microbiological contamination is important to theprocesses and products

When the facility is used for manufacture of therapeuticproducts including medical devices that are to be suppliedmicrobiologically clean

When the facility is used for manufacture of therapeuticproducts including medical devices that are to be suppliedsterile

Cleanrooms used for electronic assembly and the like usuallydo not need microbiological monitoring as viable particles arejust particles. An exception might be the water supply


When To Sample

When built–initial investigations for commissioning

Periodic certification

After breaches of alert/action levels

After breaches of hygiene security

In operation - routine monitoring for performancemaintenance


Sampling Plan

Logical to perform a large and intense sampling of areas (qualification orbaseline study) prior to deciding on the routine and usually reducedsampling plan. Qualification air sampling: Number of sites =√area in m2

Logical to sample more frequently as the criticality of the area increasesLogical to allow for investigative sampling within a formal plan whereparticular designated sites are always sampledShould address risk of sampling introduced contaminationSampling plans should be dynamic with monitoring frequencies andsample locations adjusted based on trending performance. It isappropriate to increase or decrease sampling based on this performance–R. Tirumalai USP, 2009, www.usp.orgSampling should occur at the same sites and at the same time of day.This will make analysis and trending of data more meaningful by reducingthe variables. It is recommended to sample during peak activity torepresent worse case - Anne Booth, www.gsfcc.orgUse worst-case situation?•Statistical analysis of environmental monitoring data: does a worst case time

for monitoring clean rooms exist? Cundell et al, J Pharm Sci Technol, Vol. 52, No.6, 1998


Sampling Plan

Frequency of sampling increases as the importance(criticality) of the zone increases

Zone 4Remote areas: hallways, doors, coolers, cafeteria, etc.

Zone 3Non-product contact surfaces:

Floors, walls, drains, leg supports, wheeled items, forklifts, etc.

Zone 2Non-product contact surfaces:

Exterior of equipment, control panel / buttons, tables, etc.

Zone 1Product contact surfaces:

Conveyors, fillers, pipes, storage vessels, utensils,scrapers


Sample Site Selection

Conduct a risk analysis and develop a rationale for thesampling locations and frequency:•Where microbial contamination is likely to occur•Where microbial proliferation is likely to occur•Where microbial accumulation is likely to occur•Where most activity occurs•Where it is difficult to clean•Where personnel are working

EM Qualification sample site selection, PharmaceuticalMicrobiology Forum Newsletter–Vol. 14 (8),www.microbiologyforum.orgEstablishment of critical contamination risk locations (“Hot Spots”) in environmental monitoring by means of three-dimensional airflow analysis and particulate evaluation,Katayama et al, J Pharm Sci Technol, Vol. 63, No. 3, 2009


Sample Site Selection

Far surfaces•Walls•Floor•Ceiling•Doors, especially areas that are

contacted by peopleNear surfaces–nearproduct contactsurfaces•Exterior of equipment•Control panels•Control buttons•Tables

Product contact surfaces•Bench and table tops•Crates or boxes that hold product

or parts•Equipment product contact

surfacesPersonnel•Gloves•Garments

AirCompressed gases if they exitinto a cleanroom or contactproductWaterAqueous solutions oremulsions


Frequency Of Sampling

Medical device manufacture, terminally sterilised devices andclean pharmaceutical manufacture - weekly, fortnightly,monthly, quarterly - depending on the process. Less thanquarterly is probably not sufficient

Aseptic fill/assembly - hourly, per operator session, per shift,daily - depending on the process. Less than per shift isprobably not sufficient

Sterility testing - per operator session, per test - depending onthe testing logistics. Less than per operator session is notadequate


How To Sample

Surfaces :•Swabs•Contact agar plates, films or

slides•PetrifilmTM

•Liquid rinse

Air :•Settle agar plates, films or slides•Active air sampling :

• Impact to agar sampling• Filtration• Liquid entrapment• Optical spectroscopy

Compressed gases :•Filtration•Liquid entrapment

Water and other liquids :•Filtration•Surface agar plating•Agar pour plates•MPN methods•ATP

Personnel :•Swabs•Agar contact


Sampling Surfaces

The methods are inaccurate and may provide highly variabledataSwab or agar contact may recover ~20-80% of the flora present–very dependent on operator training and experience. It iseasy to get false low results–e.g. dry swab on dry surfaceSwab developments:•Flocked swabs - Giblerto Dalmaso et al, J Pharm Sci Technol Vol. 62,

No. 3, 2008•MSSS™ swab system - Sandy Rubio, et al, J Pharm Sci Technol Vol. 64,

No. 2, 2010The liquid rinse technique is difficult, time consuming andshould be confined to special situations that might demand ithowever it is likely that it is the best technique•Microbiological sampling of surfaces, Faverom et al, J App Bact, Vol.

31 p 336-343, 1968


Sampling Surfaces

Press withconsistentpressure for 10s•bioMérieux has a

device.

Swabs must berolled over thesurfacerepeatedly to beeffective.•It is easy to get false

low counts fromswabbing.


Other Considerations

Microbiological methods do not recover all organisms thatmay be presentMedia residues are left on surfaces after samplingMicrobial counts under 25 CFU per test aliquot are statisticallyinaccurateA microbiological rule of thumb is that differences in microbialcounts of <1 log to 0.5 log do not indicate significantdifferences in population. In cleanroom monitoring, adifference of 1 CFU, depending on the type of productionoccurring, might be importantMicrobial counts from surfaces are at best semi-quantitative.They give an impression of the microbiological condition ofthe facility


Sampling Air

Settle agar plates :•Estimate the number of viable particles

that settle on to a surface in a particulartime - the rate of contamination

•They can therefore be useful indetermining the possible contaminationof products. Refer to the work of Whyte, JPharm Sci Tech, 1981, 1983, 1984, 1996and Slater and Johnston, J Pharm SciTechnol, 1988

•Active vs passive air (settle plate)monitoring in routine environmentalmonitoring programs, Andon, J Pharm SciTechnol, Vol. 60, No. 6, 2006 Settle plate stand for

monitoring cleanrooms


Sampling Air

Active air sampling - air samplers :

•Estimate the number of viable particles in air•Sampling technology has not been optimised :

•Data is device dependent:• Comparative study of airborne viable particles assessment methods in microbiological

environmental monitoring, Temprano, J Pharm Sci, Technol Vol. 58, No. 4, 2004• Impact to agar velocity is a balance between capturing small particles and not

causing excessive damage to the organisms• In liquid entrapment the velocity of air intake is important as is the bubble size•Filtration may dehydrate organisms and cause under estimation

No absolute scientific agreement on a relationship between theconcentration of nonviable particulates and viable microorganisms:•Airborne viable particles and total number of airborne particles:

comparative studies of active air sampling, Bengt Ljungqvist and BeritReinmu¨ Ller, J Pharm Sci Technol, Vol. 54, No. 2, 2000

•Environmental monitoring: A correlation study between viable andnonviable particles in clean rooms, Catherine Simõ Es De Abreu, J Pharm SciTechnol, Vol. 58 No. 1, 2004


Sampling Air

Agar Impact SamplersLiquid Entrapment

Sampler

Gas Samplers


Other Considerations

Specific fungal and/or anaerobic monitoring?•Preliminary work can be done to show that strict

anaerobes and fungi are not a significant part of theproduction facility microflora and this can be used tojustify use of TSA for routine monitoring

•Most companies use TSA for routine monitoring, someuse SDA also

•Many companies internationally use dual incubation ofTSA at 30−35°C and 20−25°C to capture organisms that require lower temperatures like fungi•Comparative mold and yeast recovery analysis (the effect of differing

incubation temperature ranges and growth media), Marshall et al, JPharm Sci Technol, Vol. 52, No. 4 1998


Validation

Validation of neutralisation of antimicrobial substances is important–bacteriostasis/fungistasisValidation of methods for recovery of microflora from surfaces, airand gases would be quite difficult for most laboratories and is usuallynot undertakenValidation of recovery from environmental sampling is not particularlyuseful because :•There is little scope for routine laboratories to improve methods and there

are many potential variables to investigate•Validation would result in a recovery correction factor, that is, a constant

used to adjust the data. Environmental microbial data is used to attempt todiscern trends. Trends can be determined without using a recovery constant–assuming methods remain constant

•Alert and action levels can be successfully applied to uncorrected data–assuming methods remain constant

•Many results from microbiologically critical areas are at the limit ofdetection for routine methods (0 CFU) and therefore applying a correctionfactor will give ‘less than’ data


Quality Of Growth Media

Must be growth promotion tested

All agar plates, slides and films must be either pre-incubated to check for sterility or gamma irradiated

Diluents or broths used for MPN tests must belikewise treated

There must be no risk of false positive results fromcontamination of the media - unnecessary heartacheoccurs!


Characterisation Of Flora

The effort expended on characterisation should be related to thepossible significance of the data

Mostly the flora of dry areas, e.g. cleanrooms, does not vary greatlywith respect to the main flora groupings. Microflora from cleanroomenvironments involved in aseptic production should be characterisedroutinely

Tracking variation in strain or genetic make up is a challenge foraseptic operations

Microbial identification strategies in the pharmaceutical industry,Cundell, J Pharm Sci Technol, Vol. 60, No. 2, 2006


Cleanroom Flora

Common microorganisms :•Gram positive cocci•Gram positive sporing rods - Bacillus spp. - probably present as

sporesLess common microorganisms :•Moulds - probably present as spores and hyphael fragments•Yeast•Gram negative bacteria - usually associated with water,

wetted surfaces, solutions and emulsions. Common in watersystems

•CoryneformsThe bacterial diversity of pharmaceutical clean rooms analyzedby the fatty acid methyl ester technique, Pacheco and Pinto, JPharm Sci Technol, Vol. 64, No. 2, 2010


Alert And Action Levels

The Alert Level is a level above which a drift from normalconditions is shown - prompt investigation and follow uprequired with possible remedial actionThe Action Level is a level above which results are notacceptable - immediate investigation and remedial actionrequiredAlert Levels often set at the 95 percentile of historical data, orat 2 x SD from the mean valueAction Levels often set at the 99 percentile of historical data, orat 3 x SD from the mean valueAction Levels also might be based on industry/compendialrecommendationsJudgement is needed to determine if the baseline obtained istoo highAseptic and sterility testing areas in operation set by–Annex 1and USP <1116>


Trending Microbiological Data

The objective of a microbial monitoring program is to obtainrepresentative estimates of environmental bioburden

When data are compiled and analysed, any trends should beevaluated by trained personnel

While it is important to review environmental results on thebasis of recommended and specified frequency, it is alsocritical to review results over extended periods to determinewhether trends are present

The microbial control of controlled environments can beassessed, in part, on the basis of these trend dataR. Tirumalai USP, 2009 www.usp.org



Bar graphsLine graphsScatter chartsControl charts e.g.individuals chart•Pharmaceutical Microbiology

Forum Newsletter –Vol. 15(2),www.microbiologyforum.org

0

0.2

0.4

0.6

0.8

0 100 200 300 400 500 600 700 800

Observations

Mean UCL

I Chart

CF

U



CuSum analysis :•Is sensitive to small shifts in the

performance indicator•Is more sensitive to small changes

than a moving average chart•Not useful for cleanroom data that

are very low with many zero results•Computer assisted evaluation of

microbiological environmental controldata, Russell et al, J Pharma SciTechnol, Vol. 38, No. 3, 1984

-1.5-1

-0.50

0.51

1.52

CuS

UM

0 100 200 300 400 500 600 700Observations

CuSUM Graph


For aseptic areas statistical analysis is often difficultbecause the results are too low –mostly zero

Mostly such facilities only yield 1 or 2 CFU fromhundreds of determinations

Use frequency between non-zero results as an event

Frequency of non-zero results is monitored

Environmental monitoring: data trending using afrequency model, Caputo and Huffman, J Pharm SciTechnol, Vol. 58, No. 5, 2004

Analysis of environmental microbiology data fromcleanroom samples, Hussong & Madson, PharmaTechnol January 1, 2004

0.00%

20.00%

40.00%

60.00%

80.00%

100.00%

0 1 2 3

CFU per Plate

Location A Location B

~1,200 Results fromSterility Testing Facility


% Standard Deviation

+ 100/√Count

SD of 1 CFU is + 1 CFU


Other References

Environmental monitoring: Myths and misapplications, Akers &Agallaco, J. Pharm. Sci Technol, Vol. 55, No. 3, 2001Environmental monitoring: Misconceptions and misapplications,Wilson, J. Pharm. Sci Technol, Vol. 55, No. 3, 2001Pharmaceutical Microbiology Forum (PMF)www.microbiologyforum.orgThe Microbiology Network - www.microbiol.orgControlled Environments Magazine–www.cemag.usMedical Device and Diagnostic Industry - www.mddionline.comPharmaceutical Technology - pharmtech.findpharma.comGlobal Society for Contamination Control - www.gsfcc.orgEvaluation of the Biovigilant IMD-A™, A novel optical spectroscopy technology for the continuous and real-time environmental monitoringof viable and nonviable particles, Parts 1 and 2, Miller et al, J PharmSci Technol, Vol. 63, No. 3, 2009


Microbiological EnvironmentalMonitoring = Front Line Action

The Technology Of Microbiological Environmental · PDF fileThe Technology Of Microbiological...

Documents

Transcript of The Technology Of Microbiological Environmental · PDF fileThe Technology Of Microbiological...