CHAPTER 27 The puri cation of plasma proteins for ... · PDF fileCHAPTER 27 The puriﬁcation...

CHAPTER 27

The purification of plasma proteins for therapeutic use

Joseph Bertolini

CSL Behring, Broadmeadows, Victoria, Australia

IntroductionIt has been noted that blood plasma is the largest human proteome.1

As it is part of the circulation system, plasma contains other tissueproteomes as subsets through its contact with all organs of the bodyand their diverse cellular components. In addition it containsspecific plasma cells involved in the immune response andhomeostasis. In one study 289 proteins were detected in plasma,while in another 3700 protein spots were identified by two-dimen-sional electrophoresis, and 325 distinct proteins identified.1,2 Pro-teins found in plasma can constitute proteins secreted by solidtissues that act in plasma; immunoglobulin (Ig); “long-distance”receptor ligands, such as hormones; “local” receptor ligands, such ascytokines and growth factors; and tissue leakage products. If there isan existing disease state, there may be aberrant secretions andforeign proteins as a result of cell damage, or the presence of atumor or infectious organism.1

The true plasma proteins are considered to be those that carry outtheir functions in circulation.3 These constitute a number ofproteins predominantly produced by the liver and, the immuno-globulins from the bone marrow.4,5 The hepatic derived plasmaproteins encompass key proteins that constitute:• The coagulation pathway and its regulation; these include theclotting factors (fibrinogen [factor I] and factors V, VII, VIII, IX,X, XI, XII, and XIII) and the regulatory factors or proteaseinhibitors (antithrombin III, heparin cofactor H, and proteinsC, S, and Z);

• Components of the immune response and reaction to foreignbodies via the classic and nonclassic pathway, consisting of thecomplement factors and mannose binding lectin;

• Acute phase reactants such as, α1-acid glycoprotein, C-reactiveprotein, serum amyloid protein, serum amyloid A, and α2-HSglycoprotein, constitute part of the body’s response to injury andinfections; increased production of several hundredfold canoccur;

• Plasma proteinase inhibitors, which regulate the action of keyproteolysis enzymes such as plasmin cathepsin G, elastase, andkallikrein and include α1-antichymotrypsin, α2-macroglobulin,inter-α-trypsin inhibitor, C1 inhibitor, and α1-antitrypsin;

• Carrier proteins, which are involved in the transport of iron,copper, hemoglobin, heme, bilirubin, and fatty acids; theseinclude albumin transferrin, ceruloplasmin, transthryretin, reti-nal binding protein, haptoglobin, hemopexin, and vitamin bind-ing protein.

• Biochemical regulators involved in immune cell regulation suchas interleukins, interferons, and growth factors; for exampletumor necrosis factor and transforming growth factor β.The immunoglobulins, produced in the bone marrow exist as a

number of subtypes—IgG, IgM, IgA, IgE, and IgD. IgG and IgM arein the highest concentration, and have the prime role in immunesurveillance, the sequestration of foreign bodies and the initiation ofsubsequent immune cellular and humoral responses.6

With increased understanding of the biochemical basis ofdisease, researchers see plasma proteins as possible therapeuticagents in a number of conditions either as a means of supplemen-tation in cases of deficiency, or to regulate biochemical pathways toachieve desired therapeutic outcomes. The biochemistry, clinicaluse, and production of these proteins have been reviewedrecently.7,8 Of the many identified plasma proteins, 20 therapeuticproducts have been developed and are in clinical use, with approx-imately another five in various stages of development.9–30 These aretabulated in Table 27.1.The production of plasma proteins for therapeutic use has led to

the development of a large global industry involving the complexactivities of collecting blood and plasma donations and applyingbioprocessing procedures to produce products with validated effi-cacy and pathogen safety.31

It is generally agreed that the start of plasma fractionation can betraced to the work of Cohn in his Harvard Laboratory in the 1940s,where he developed an ethanol-based precipitation process for thepurification of albumin to be used as a resuscitative fluid in theatresof war.32

This chapter will describe the manufacture of plasma proteins fortherapeutic use and make extensive use of divergent examples toillustrate the range of approaches that can be taken. In addition, itwill detail the expected specifications of the final product. Thisinformation will be of use to the reader involved in the assessment,purchase, or use of plasma products, who is often confronted withliterature on products containing unfamiliar concepts and termi-nology relating to plasma fractionation, viral removal procedures,and product specifications. An increased understanding of theproduction of plasma protein therapeutics will facilitate communi-cation with plasma products manufacturers and aid in the assess-ment of existing products and the exploration of possibilities forfuture development.

302

Rossi’s Principles of Transfusion Medicine, Fifth Edition. Edited by Toby L. Simon, Jeffrey McCullough, Edward L. Snyder, Bjarte G. Solheim, andRonald G. Strauss.© 2016 John Wiley & Sons, Ltd. Published 2016 by John Wiley & Sons, Ltd.

Key components of the manufacture ofplasma protein therapeutics

Plasma for fractionationThe manufacture of plasma protein therapeutics commences withthe collection of plasma. This is a logistically and technicallycomplex activity due to the need to ensure product quality andsafety.33 The viral safety of plasma collected for fractionation isensured by what has been referred to as a “Five Layer Safety.”34

These layers are donor screening, blood testing, donor referral,quarantine, and investigation. Details of these activities are pro-vided in Chapters 44, 55 and 56.Plasma used in the United States for manufacture is either

recovered plasma—plasma derived from whole blood collection,or source plasma collected by plasmapheresis. No specific mono-graph exists in the United States that details the handling conditionsto be applied to collected plasma. However, the pharmacopeia of theEuropean Union (European Pharmacopeia) specifically details thefreezing conditions applicable for labile and nonlabile proteins.35

Thus, it recommends that plasma to be used in the manufacture oflabile proteins needs to be frozen within 24 hours of collection, andthat the rate of freezing be such that�25 °C is attained in 12 hours of

being placed in the freezing apparatus. For the recovery of nonlabileproteins the plasma must be frozen to �20 °C or below within24 hours of collection. During storage and transportation, thetemperature of the plasma can exceed �20 °C for not more than atotal of 72 hours, it must not transiently exceed�15 °C onmore thanone occasion and at no time must the temperature exceed �5 °C.The Cohn processThe procedure that continues to have a pivotal role in plasmafractionation was developed by Cohn in the 1940s. It is based on thedifferential precipitation of plasma proteins by manipulation ofethanol concentration and pH of a low-ionic-strength solutionmaintained at a subzero temperature.32 The original process, knownas the Cohn method,6 is presented in Figure 27.1 and shows theconditions for the generation of specific fractions. Also shown is therecovery of cryoprecipitate from freeze-thawed plasma by centrifu-gation, typically incorporated in the processing of plasma. Inaddition the source of current therapeutic proteins or those underdevelopment, listed in Table 27.1, are detailed. An example will beconsidered to specifically illustrate the principle of the Cohnprocess: If one takes Supernatant I, adding ethanol to a finalconcentration of 25% (v/v) and adjusting the solution to pH 6.9,

Table 27.1 Plasma proteins with established clinical use or under active investigation

Protein or Therapeutic Product Clinical Use

Established Clinical UseAlbumin Fluid replacement in trauma, burns, sepsis, and cirrhosisImmunoglobulins:- Intravenous

Replacement therapy in primary and secondary immunodeficiency, immunomodulation incertain autoimmune diseases including immune thrombocytopenia purpura, Kawasakidisease, Guillain–Barré syndrome, autoimmune polyneuropathy, and myasthenia gravis

- Hyperimmune to viral and bacterial antigens Treatment of hepatitis A, hepatitis B, cytomegalovirus, varicella–zoster, and tetanusinfections

- Anti RH(D) immunoglobulin Prevention of Rh(D) isoimmunization due to fetus–maternal rhesus D incompatibility,treatment of immune thrombocytopenia purpura

Factor VIII Replacement in hemophilia A–factor VIII deficiency; induction of immune tolerance foranti–factor VIII antibodies

Von Willebrand factor (vWF) Treatment of vWF deficiencyFactor IX Replacement in hemophilia B–factor IX deficiencyActivated prothrombin complex concentrate (aPCC)Composed predominantly of prothrombin and factors VIII, VII, VIIa, IX, X, Xa;and protein C. Brand name: Factor VIII Inhibitor Bypassing Activity (FEIBA)

Treatment of patients with inhibitory antibodies to factor VIII or factor IX

Prothrombin complex concentrate (PCC); factors II, VII, IX, and X; andregulatory proteins: protein C, S, and Z

Treatment of the rare conditions of factor VII and factor X deficiencies, reversal of theanticoagulant activity of warfarin and prevention of massive bleeding, often in combinationwith fibrinogen

Fibrinogen Replacement in congenital fibrinogen deficiency and in acquired deficiency followingmassive bleeding

Factor XI Treatment of Factor XI deficiency; incidence: 1:106

Factor XIII Treatment of Factor VIII deficiency; incidence: 1:2× 106

Factor X Treatment of Factor X deficiency; incidence: 1:1×106

Fibrin glue (fibrinogen and thrombin components) Used in surgery as a sealant to achieve hemostasis, as a surgical glue and to promote woundhealing

Alpha1-proteinase inhibitorAntithrombin III

Treatment of alpha1-proteinase inhibitor deficiencyTreatment of acquired and hereditary deficiency of Antithrombin III

C1-esterase inhibitor Treatment of hereditary angioedema

Under InvestigationPlasmin Dissolution of clots by direct application in peripheral arterial occlusion, stroke, deep vein

thrombosis, myocardial infarction, and clotted intravascular devicesReconstituted plasma-derived high-density lipoprotein (derived fromApolipoprotein A)

Atherosclerotic plaque stabilization in acute coronary syndrome through reduction in lipidcontent and other anti-inflammatory and antithrombotic mechanisms

Transferrin Treatment of hypotransferrinemia, enhancing erythropoiesis, and reducing anemia inβ-thalassemia.Used in hematological stem cell transplantation to sequester free iron-binding of ironreduces free-iron-mediated ischemia/reperfusion injury in transplanted organs

Plasminogen Treatment of ligneous conjunctivitis related to plasminogen deficiencyHaptoglobin Removal of free hemoglobin arising from hemolysis in burns, trauma, and surgical

interventionCeruloplasmin Use in Ukraine and Russia has been as an adjunct therapy in cancer therapy, emergency

medicine indications, and infection; outcomes thought to be enhanced by the antioxidantand ferroxidase activity of ceruloplasmin

Chapter 27: The purification of plasma proteins for therapeutic use 303

while ensuring the ionic strength and temperature remain atapproximately I= 0.09 and �5 °C respectively, results in the gener-ation of the immunoglobulin-enriched Fraction II+ III and Super-natant II+III containing albumin.It is important to have an understanding of the Cohn process as

the terminology associated with the process is often used in litera-ture associated with various plasma-derived products. Each Cohnfraction is an enriched, albeit crude, source of various plasmaproteins that can be further purified to generate a therapeuticproduct. How this is achieved will be covered in the subsequentsections. Prior to this however, a description of viral removalprocedures commonly used in manufacturing processes will beundertaken. These need to be incorporated in any manufacturingprocess to ensure the viral safety of the product.

Viral removal proceduresTo ensure the viral safety of plasma protein therapeutics, as has beendiscussed in this chapter, a safe plasma supply is required, achieved

through careful donor selection and screening and testing ofdonations. A third important determinant is the presence ofinactivation or removal procedures in the manufacturing process.This is covered extensively in Chapter 56.The purification of plasma proteins typically results, at various

process steps, in the partitioning of virus away from the protein ofinterest. Thus, for example in the Cohn process, virus removal isachieved with the precipitation of Fractions III, I/III, I/II/III, andFraction IV-I/IV-4 in the manufacture of immunoglobulin andalbumin, respectively.36 Regulatory requirements, however, requirethe inclusion of at least two dedicated orthogonal viral removalprocedures that are effective against both enveloped and non-enveloped viruses.37–41

There is an ongoing interest in identifying effective yet conve-nient means of achieving viral removal. Viral filtration is a preferredoption as it can, in many cases, be added to an existing process andis usually benign with respect to its effect on product integrity.42–44

The preferred pore size for viral filters is 15 or 20 nm, thereby

Cryoprecipitate Cryosupernatant

Frozen plasma

Centrifugation

Ethanol 8%pH = 7.2T = –3 °C

Fraction I Supernatant I

Ethanol 25%pH = 6.9T = –5 °C

Fraction II + III Supernatant II + III

Ethanol 18%pH = 5.2T = –5 °C

Fraction IV-I Supernatant VI-I

Ethanol 40%pH = 5.8T = –5 °C

FractionIV-4 Supernatant IV-4

Ethanol 40%pH = 4.8T = –5 °C

Fraction V Supernatant V

Prothrombin complexFactor IXAntithrombin IIIC1-Esterase inhibitorActivated prothrombincomplex concentrateFactor XIFactor XFactor II (thrombin)

Antithrombin IIIProthrombin complexFactor IX

Factor VIIIvWFFibrinogen

Fibrinogen Factor XIII

ImmunoglobulinPlasminogen(plasmin)

Alpha1-Proteinase InhibitorApolipoprotein A

Alpha1-Proteinase InhibitorApolipoprotein ATransferrinCeruloplasminHaptoglobin

AlbuminHaptoglobin

Albumin

Figure 27.1 The Cohn plasma fractionation process with the fractions indicated that are the source of various therapeutic proteins.

304 Section II: Part IV: Plasma

ensuring the removal of small non-enveloped viruses such asparvovirus B19 and hepatitis A.45 This can limit the size of theprotein that can be filtered. Immunoglobulins with a molecularweight of 150,000 d, can readily be filtered.46,47 For larger proteins,viral filters with 35 nm pore size need to be used, although thiswould only be effective in removing large enveloped viruses such ashepatitis B and C and HIV. Examples of this include von Wille-brand factor (vWF) which has a molecular weight of 500,000 d butexists as a series of multimers to a molecular weight of 20,000 kd.48

Factor VIII–vWF has been filtered through a 20 nm filter by usingspecific conditions to dissociate the complex prior to filtration andsubsequent inducing reassembly.49 Viral filtration of fibrinogen,with a molecular weight of 340,000 d, through a 20 nm filter has alsobeen achieved.50

Chromatographic proceduresThe Cohn fractionation process can generate highly purified albu-min and immunoglobulin products.32,51 Today, albumin is stillpredominately purified by the Cohn procedure, and this was thecase until the late 1990s for the production of immunoglobulinproducts. The purification of other therapeutic proteins fromenriched Cohn fractions has only been able to be achieved bythe application of chromatographic techniques.52

Details of the purification of the major plasma-derived thera-peutic proteins will be provided in this chapter. However, anappreciation and understanding of these processes requires knowl-edge of the principles of chromatography and the orthogonalapproaches that need to be applied to obtain highly purifiedpreparations.Chromatographic techniques allow the purification of proteins

because of their differences in molecular weight, charge, hydropho-bicity, and specific affinity for ligands. This involves the use of sizeexclusion, ion exchange, hydrophobic interaction (HIC), and affin-ity chromatography, respectively.53–56 Chromatography is typicallyperformed with columns packed with resin beads of about 80 μm indiameter and derivatized with particular functional groups to allowthe particular chromatographic separation to be performed.Size exclusion separation occurs as proteins percolate through the

beads. Smaller proteins enter the pores of the beads, and hence theirpassage through the column is retarded, leading to the separation ofproteins on the basis of molecular weight, with higher molecularweight proteins eluting first followed by the smaller species.Ion exchange chromatographic resins exist in either the anion

(positively charged) or cation (negatively charged) forms. Typicalion exchange ligands include dietlylaminoethyl (DEAE), quaternaryamino ethyl (QAE), quaternary ammonium (Q), carboxymethyl(CM), sulfopropyl (SP), and methyl sulfonate (S) for anion andcation exchange chromatography, respectively. As proteins arezwitterion molecules, the net charge of the molecule can be modu-lated by changing the pH of the protein environment. Proteins havedifferent pI values-the pH at which the net charge of the protein iszero.56 Therefore, the net charge of a protein depends on pH, andthis determines the degree of interaction with an ion exchange resin.Interaction can be further modulated by changes in ionic strength.Thus, by utilizing conditions that promote differential binding toion exchange resins, coupled with defined elution conditions—again involving specific conditions of pH and conductivity—frac-tionation of a mixture of proteins can be achieved.Proteins exhibit differences in their hydrophobic profile, reflect-

ing differences in the composition of amino acids with hydrophobic(nonpolar) or hydrophilic (polar) properties.56 This difference isexploited in hydrophobic interaction chromatography (HIC).

Solution conditions can be manipulated so that the more hydro-phobic proteins are retained on a column while less hydrophobicproteins flow through.Proteins exhibit unique surface epitopes and functional

domains.56 Immobilized antibodies and specific ligands on chro-matographic resins, which can specifically interact with these partsof proteins, can be the basis of affinity chromatography for thepurification of proteins from complex mixtures. Immobilizedmonoclonal antibodies, heparin, and metal affinity ligands areused in the purification of several plasma proteins.52

Manufacturing processes for plasmaprotein therapeuticsIn this section, specific processes used in the recovery of all themajor plasma proteins for therapeutic use will be described. Thefocus will be on illustrating the range of techniques that can be usedin the manufacture of these products, and exposing the reader to awide range of concepts underpinning the fractionation of plasmaproteins. In addition, the expected pharmacopeial specifications ofthe final product, that ensure desired product safety and efficacy,will be detailed.

AlbuminThe Cohn process remains the dominant process for the manufac-ture of albumin (Figure 27.1).32 Details on the production ofalbumin by the Cohn process in a modern facility and the requiredquality attributes expected of the product have been reviewedelsewhere.19

As previously described, the Cohn process involves manipulationof ethanol concentration and pH of solutions at low ionic strength,while maintaining subzero temperatures, to achieve differentialprecipitation of proteins. The recovery of albumin involves thegeneration of Supernatant I, II+III, IV-I, IV-4, and finally theprecipitation of pure albumin as Fraction V at 40% (v/v) ethanol,pH 5.8, with temperature maintained at �5 °C. A variation to theCohn process was reported by Kistler and Nitschmann in 1962.57

The aim was to maximize albumin yield and decrease the use ofethanol. Fraction I is generated as by the Cohn process. PrecipitateA, the equivalent of Fraction II+III in the Cohn process, is producedwith 19% (v/v) ethanol, pH 5.85, and allows the recovery of thealbumin containing Supernatant A. This then progresses throughthe generation of Supernatant IV (40% v/v) ethanol, pH 5.85,�8 °C)and Precipitate C (40% v/v) ethanol, pH 4.8, �8 °C). Resuspensionof Precipitate C and recovery of Supernatant D (10% v/v) ethanol,pH 4.6, �3 °C) provide the final purified albumin. This process wasinitially used by the Swiss manufacturer ZLB, and continues to beused following its incorporation into CSL Behring.31 The albuminrecovered by ethanol fractionation is highly purified, although traceamounts of residual plasma proteins—typically, haptoglobin, ceru-loplasmin, α2-macroglobulin, acid2-glycoprotein, hemopexin, andtransferrin—can be detected58,59 (CSL data on file).An alternative chromatographic process, based on the method of

Curling et al., was developed by CSL Behring (Australia) in the1990s.58,60,61 The process was initially developed for the purificationof albumin from Supernatant II+ III but was later adapted for theprocessing of Supernatant I.62 The process is shown in Figure 27.2.The process incorporates three chromatographic steps—anion andcation exchange chromatography with DEAE and CM Sepharose,respectively, and size exclusion chromatography using Sephacryl

S200 resin. This last step markedly contributes to the purity of therecovered albumin, removing residual proteins and aggregates.


Chromatographically purified albumin is purer than that derived byethanol fractionation and also exhibits a lower aggregate content.58

This increased purity has been associated with a decreased rate ofadverse reactions with chromatographically purified albumin.62

The Cohn process for the manufacture of albumin contributes tothe viral safety of the product through partitioning virus away fromthe product stream.63–66 With the alternative chromatographicprocess, similar partitioning is achieved at the chromatographicsteps.67,68 In addition, a specific viral inactivation step is included inthemanufacturing process—pasteurization at 60 °C for 10hours.63–66

This has been part of the production of albumin from the verybeginning and undoubtedly accounts for the fact that there has neverbeen a viral transmission from an albumin product.69 The pasteur-ization of albumin was possible due to the finding that N-acetyltryptophanate and sodium octanoate (caprylate), or sodium octa-noate alone, stabilized the molecule during heating.70,71

The European Pharmacopoeia (EP) and US regulations (CFR21)stipulate that pasteurization be performed in the final containerfollowing dispensing in order to completely remove the risk ofrecontamination.72,73 Reflecting advances in bioprocessing execu-tion and control, CSL Behring (Australia), in addition to developinga novel chromatographic manufacturing process, obtained approvalfrom the Australian regulatory authority to implement bulk pas-teurization of its albumin product prior to dispensing.58 Thisproduct (Albumex) is registered and used in Australia, NewZealand, Hong Kong, Singapore, Taiwan, Malaysia, and Indonesia(CSL data on file). Albumex has an additional viral inactivationstep consisting of incubation at low pH in the presence of caprylicacid at 30 °C for 10 hours.74 This step was introduced to add furtherviral safety to the product and reflected the regulatory expectationsthat plasma protein products have at least two dedicated viralinactivation or removal steps.37

The specifications of the product are governed by EP and USPharmacopeia and National Formulary (USP) or CFR pharmaco-peial requirements, which provide limits for physical properties,biological safety, purity, excipients, and contaminants.72,73,75 Purif-ied albumin solutions are supplied by various manufacturers as 4,

4.5, 5, 20, and 25% (w/v) solutions and typically exhibit a shelf-lifeof up to five years at room temperature (25–30 °C).19 It is arequirement that solutionmust be clear and can be almost colorless,yellow, amber, or green (EP). The variation in color reflectsdifferences in the presence of residual heme as well as coloredprotein impurities such as haptoglobin, ceruloplasmin, transferrin,and hemopexin.59,76 Chromatographically purified albumin is typ-ically a greenish color, reflecting its higher purity. It contains only asmall amount or absence of colored proteins and heme, butincreased levels of albumin bound bilirubin and the green oxidizedderivative—biliverdin.76 The product should not change in appear-ance with heating at 57 °C for 50 hours (CFR21).The product should have a pH (at 20 °C) of 6.7–7.3 (EP) or

6.4–7.4 (USP). Osmolality should be equivalent to that of plasma(USP). With respect to excipients, sodium should not exceed160mmol/L (EP) or 130–160mmol/L (USP). Sodium caprylate(sodium octanoate) should be 0.16mmol/g albumin if it is thesingle stabilizer (CFR21). In preparation where dual stabilizers,sodium caprylate and N-acetyl tryptophanate, are used, the con-centration of each should be 0.08mmol/g albumin (CFR21).Albumin purity should be at least 95% (EP) or 96% (USP), with

aggregate content not more than 10% as determined by the area ofthe chromatogram determined by adsorption at 280 nm. Hemelevels as determined by adsorption at 403 nm should not exceed0.15 AU (EP). Chromatographically purified albumin contains nodetectable heme-related absorbance.76

Prekallikrein activator (PKA) activity should not be more than35 IU/mL (EP). This is an important specification as PKA (alsoknown as factor XIIa or Hageman factor fragment), if present, canlead to a clinical hypotensive reaction.77

Contaminant levels, of which potassium and aluminum arespecified, should be 0.05mmol/g albumin (EP) or 2meq/L (USP)and 200 μg/L (EP), respectively. The limit for aluminum wasintroduced into the British Pharmacopoeia in 1993 to ensurealbumin safety for use in renal dialysis and with prematurebabies.78 Another concern was the accumulation of aluminumwith the treatment of burn patients.79 The source of aluminum isfrom the glass containers used for the product. Leaching ofaluminum is abetted by even very low concentrations of citrate(<0.1mmol/L), which originates from the anticoagulant usedduring plasma collection.80 Through optimization of diafiltrationprocesses used in manufacturing, to minimize citrate content,current albumin products meet the required aluminum pharma-copeial limit.19,81

The solution should be sterile, nonpyrogenic, and with endotoxinlevels not more than 0.5 EU/mL (EP/USP). The pharmacopeias alsospecify that the final product be incubated for a period prior to finalinspection to provide additional assurance of product sterility.CFR21 requires incubation for at least 14 days at 20–35 °C, andthe EP states not less than 14 days at 30–32 °C, or not less than fourweeks at 20–25 °C.

ImmunoglobulinsThe Cohn process also served as the starting point for the purifica-tion of immunoglobulins.32 Oncley developed a process for therecovery of purified immunoglobulins from Cohn Fraction II+III.51 In the process, the fraction is resuspended and reprecipitatedwith 20% v/v ethanol, at pH 7.6 (modifications exist with pH 6.7),and temperature at �5 °C. The recovered precipitate is then re-suspended, and the solution adjusted to 17% v/v ethanol, at pH 5.2,with temperature maintained at �6 °C. This results in precipitationof IgM and other impurities (Fraction III), whereas IgG remains in

Supernatant I

Delipidation

DEAE anion exchange chromatography

CM cation exchange chromatography

Gel filtration chromatography

Final product

Viral inactivation (x2)

• Low pH/caprylic acid• Bulk pasteurization

Figure 27.2 Chromatographic process for the purification of albumin—Albumex (CSL Behring, Australia).


the supernatant (Supernatant III). IgG is then recovered as FractionII by precipitation with 25% v/v ethanol, pH 7.4, with the tempera-ture at �5 °C.A modification of the Oncley process was developed by Kistler

and Nitschmann in 1962.57 The aim was to increase the yield ofIgG and reduce ethanol use. In this process, Precipitate A (corre-sponding to Cohn Fraction I+ II+ III) is obtained at 19% v/vethanol, pH 8.5, temperature �5 °C. Following resuspension of theprecipitate in water, the solution is adjusted to pH 5.1 and ethanolis added to 12% v/v while maintaining the temperature at 5 °C. Thisresults in the precipitation of impurities, including IgM (PrecipitateB), with IgG remaining in the supernatant (Supernatant b) (equiv-alent of Cohn Supernatant III). The IgG is recovered by precipita-tion at 25% v/v ethanol, pH 7.0, at �7 °C. This precipitate Gcorresponds to Cohn Fraction II. These processes, with variousmodifications, for many years, have accounted for the bulk ofcommercially produced immunoglobulins.82,83 The Kistler–Nitschmann process was particularly identified with the ZLB (laterCSL Behring) facility in Bern, where the process was used for theproduction of Sandoglobulin.84

The development of new immunoglobulin manufacturing pro-cesses from the mid-1990s has reflected the desire of manufacturersto improve product characteristics and safety. This has led to theincrease in final formulation protein concentration in order toincrease the convenience of use, the introduction of viralinactivation steps to improve safety; and the introduction ofprocedures to improve yield and manufacturing efficiency.82,83,85

A number of companies have developed hybrid processes where thecrude IgG precipitate generated by ethanol precipitation (FractionII+ III or Precipitate A) is subjected to further chromatographicprocessing to recover the purified immunoglobulin.20,82,83,85,86 CSLBehring (Australia) developed a unique completely chromato-graphic procedure, and in 2000, its use commenced for the pro-duction of immunoglobulin for the Australian market and other tollcustomers, which included New Zealand, Hong Kong, Singapore,and Taiwan.87–89 A key feature of this process was the increasedyield in IgG that was achieved when compared to the Cohn-basedprocess.90

In the following, a brief description is presented of the manu-facturing processes of all the major immunoglobulin productscurrently produced and the expected pharmacopeial specificationsof immunoglobulin products. This area has also been reviewedextensively elsewhere.20

A number of products are purified essentially by the completeapplication of the Oncley ethanol fractionation process. In themanufacture of Octogam (Octopharma), purified IgG is recoveredas Fraction II (Figure 27.3). In addition to the viral partitioningthat occurs during the fractionation process, there is solvent–detergent (S/D) treatment and low-pH incubation, as describedabove, for viral inactivation. The oil/solid phase extraction step is ameans of removing the S/D used during the viral inactivationprocedure.20,91,92

In the BIVIGAM (Biotest, AG) process, pure IgG as SupernatantIII is produced by the Oncley process.93 This is then subjected to thefirst viral inactivation procedure involving S/D treatment. It is thenfurther purified by Q anion exchange chromatography before under-going viral filtration through a 35 nm pore filter (Figure 27.4). Thespecific viral inactivation procedures in this process targetinactivation and removal of enveloped viruses (HIV, hepatitis B,and hepatitis C). Removal of non-enveloped viruses (parvovirus B19and hepatitis A) relies on partitioning into the discarded Fraction IIIduring the recovery of Supernatant III.93

Octagam®

Fraction I + II + III

Supernatant I + III

Fraction II

S/D treatment

pH 4 treatment (24 hours at 37ºC)

Formulation

Filling

Oil/solid phase extraction

Cryoprecipitate-depleted plasma

Figure 27.3 Process steps for Octagam (Octapharma).

BIVIGAM®

Fraction I + III

Supernatant III

C18 Adsorption

S/D treatment

Viral filtration, 35 nm

Ultrafiltration/diafiltration

Formulation

Q anion exchange chromatography


Filling

Figure 27.4 Process steps for BIVIGAM (Biotest).


KIOVIG or Gammagard produced by Baxalta (European andUS trade names, respectively) is also produced by the Oncleyprocess resulting in the generation of purified IgG as FractionII94 (Figure 27.5). The purified immunoglobulin is then subjectedto three viral inactivation or removal procedures—S/D treatment,viral filtration with a 35nm filter, and low-pH formulation andincubation of the dispensed product. The cation exchange chroma-tography step is used to bind the immunoglobulin and thereby allowthe removal of the S/D used in the viral inactivation procedure.Passage through the anion exchange column retains impurities andresults in further purification of the immunoglobulins.The manufacture of Gammunex (Grifols) is one of a number of

hybrid manufacturing processes that involves the generation of acrude IgG fraction by ethanol precipitation followed by furtherpurification by chromatography108,109 (Figure 27.6). Cohn FractionII+ III is suspended and caprylic acid added to precipitateimpurities. The caprylic acid concentration is adjusted in therecovered filtrate and incubated at 25 °C for one hour. This con-stitutes the first viral inactivation procedure in the manufacturingprocess. The generation of pure IgG is achieved by sequentialpassage through a strong (Q) and a weak (DEAE) anion exchangechromatographic columns. Following buffer exchange, concentra-tion, and formulation, where pH is adjusted approximately 4.25, theproduct is dispensed and then incubated at 28 °C for 21 days. Thisconstitutes the second viral inactivation step. The viral inactivationprocedures of Gammunex are effective for enveloped viruses

(HIV, hepatitis B, and hepatitis C) but are not recognized foruse against non-enveloped viruses (hepatitis A and parvovirusB19). The virus clearance for non-enveloped virus in theGammunex process is accounted for by caprylic acid precipitation,depth filtration following the caprylate incubation, and partitioningduring anion exchange chromatography.85

In the manufacture of Flebogamma DIF, Cohn Fraction II+IIIis resuspended, partially purified by precipitation of impurities bythe addition of polyethylene glycol (PEG), and then further purifiedby anion exchange chromatography under conditions wherebyimpurities are retained. The purified immunoglobulin recovery issubjected to three validated viral removal or inactivation proce-dures: pasteurization, S/D treatment, and viral filtration through20 nm filters. The PEG precipitation of the resuspended CohnFraction II+ III is also associated with significant viral clearance.There is a second PEG precipitation after the low-pH treatment,pasteurization, and S/D treatment, which serves to remove immu-noglobulin aggregates and facilities passage of the solution in thesubsequent viral filtration step60,95 (Figure 27.7).The purification of Clair Y g (LFB) involves the use of Cohn

Fraction I+ II+ III. Following resolubilization, impurities areremoved by caprylic acid precipitation. The filtrate is ultrafilteredto remove caprylic acid and the retenate is subjected to S/Dtreatment. The immunoglobulin is then captured on an anionexchange column allowing the solvent and detergent of the viralinactivation procedure to be removed. The eluted immunoglobulin

KIOVIG®

Fraction II + III

Fraction II

CM cation exchange chromatography (IgG binding)

S/D treatment

ANX anion exchange chromatography (IgG flow-through)


Formulation

pH adjustment


Filling

Low pH treatment

Figure 27.5 Process steps for KIOVIG/Gammagard (Baxalta).

Gamunex®

Fraction II + III

Caprylic acid precipitation

Adjustment of caprylic acid, pH, and incubation of filtrate

Filtration

Q/DEAE anion exchange chromatography (IgG flow-through)

Ultrafiltration/diafiltration

Formulation

Filtration


Filling

Low pH treatment

Figure 27.6 Process steps for Gamunex (Grifols).


is then passed through an affinity resin to reduce the anti-A andanti-B isoagglutinin titer. The immunoglobulin then undergoesviral filtration, the second viral removal step, prior to formulationand filling96 (Figure 27.8).The manufacture of Privigen (CSL Behring) is also a hybrid

manufacturing process that involves the generation of a crude IgGfraction by ethanol precipitation followed by further purification bychromatography.97 Kistler–Nitschmann precipitate A or CohnFraction II+ III is resuspended, and caprylic acid addition isused to precipitate impurities. The recovered partially purifiedimmunoglobulin solution is subjected to low-pH incubation inthe presence of a low concentration of detergent as part of thefirst viral inactivation procedure incorporated in the process. Finalpurification of the immunoglobulins is achieved by passage througha Q anion exchange chromatographic column under conditionswhere impurities (predominantly IgM and IgA) are bound and IgGflows through. This in turn is passed over an affinity column todecrease the anti-A and anti-B hemagglutinin titers.98 The recov-ered immunoglobulin solution is then subjected to viral filtrationthe second viral removal procedure in the process, prior to formu-lation and dispensing (Figure 27.9).The final manufacturing process to be described is a completely

chromatographic process used by CSL Behring (Australia) tomanufacture Intragam P87,88 (Figure 27.10). As has been men-tioned in this chapter, a key feature of this process is the achievedincreased recovery of immunoglobulins. Supernatant I, which is

Flebogamma DIF®

Cohn Fraction II + III

PEG precipitation

pH 4 treatment, 4 hours at 37ºC


S/D treatment

PEG precipitation

Tangential flow filtration /resuspension

Pasteurization


Viral filtration, 35 and 20 nm

Filling

Figure 27.7 Process steps for Flebogamma DIF (Grifols).

ClairYg®

Fraction II + III


S/D treatment

Ultrafiltration

Hemagglutinin reduction: affinity chromatography


Formulation

DEAE anion exchange chromatography (lgG binding)


Filling

Figure 27.8 Process steps for ClairYg (LFB).

Privigen®

Kistler–Nitschmann Precipitate Aor Cohn Fraction II + III


HQ anion exchange chromatography(IgG dropthrough)

Low-pH incubation

Hemagglutinin reduction:affinity chromatography


Formulation


Filling

Figure 27.9 Process steps for Privigen (CSL Behring).


essentially fibrinogen-depleted plasma, is delipidated to minimizechromatographic resin fouling, prior to being passed through aDEAE anion exchange column. Albumin, a protein of interest,adheres to the resin while immunoglobulins drop through. As hasbeen discussed in the albumin section, albumin is eluted andundergoes further processing. The recovered immunoglobulinfraction undergoes further purification by passage through a Qanion exchange chromatographic column at a pH that promotesthe binding of the major protein impurities, IgA, IgM, and trans-ferrin, but results in the flow-through of the IgG immunoglobulins.The process includes pasteurization as the first viral inactivationprocedure. Following formulation at a low pH (pH 4.25) and filling,the dispensed product is incubated at 27 °C for 14 days as thesecond viral inactivation procedure of the process.Gamimune N (Miles Cutter/Bayer) in 1992 was the first

immunoglobulin for intravenous use to be available as a 10%(w/v) formulation.99 There has been a consistent trend over thelast 20 years of moving from a final protein concentration of 5 or 6%(w/v) to 10% (w/v). The increased protein concentration reducesthe volume that needs to be infused and hence, is more convenientfor the patient.100 Today, all the major immunoglobulin productson the market that have been described here are available at the 10%w/v formulation.101 CSL Behring has developed a 20% w/v formu-lation immunoglobulin product called Hizentra intended forsubcutaneous administration.102

A key challenge in the development of an immunoglobulinpreparation suitable for intravenous use was identifying a meansof preventing the development of anti-complementary activity inthe product arising from the formation of aggregates. These led tospontaneous activation of the complement system and causedsevere adverse reactions.103 Early success in inactivating anti-com-plementary aggregates was achieved in 1962 by Barandum andIsliker through mild proteolysis with pepsin at pH 4.0.103 Improve-ments in processing, such as the use of ultrafiltration and diafiltra-tion at pH 4.0 for the removal of ethanol from Supernatant III andfor the concentration of IgG, instead of precipitation and lyophili-zation, minimized aggregate formation and generated a clinically

tolerated product.104 It was then shown that formulation of theimmunoglobulin at approximately pH 4.25 generated a liquid stableproduct with low anti-complementary.105,106 The stability of immu-noglobulin at low pH is due to the fact that this is belowtheir isoelectric point, and results in non-interacting positivelycharged molecules. Consequently, many immunoglobulin prod-ucts are now formulated at less than pH 5.0.101,105,107 This is thecase for many of the products described above—Privigen,Hizentra, KOIVIG (Gammagard), BIVIGAM, IntragamP, and Gammunex. Formulation pH of Flebogamma DIF

and Octogam is still much lower than neutral at pH 5–6.0 andpH 5.1–6.0, respectively.Excipients are used in the formulation of immunoglobulins.

These serve to stabilize the immunoglobulin molecules in solutionby minimizing protein–protein interactions to ensure the clinicallyacceptable tonicity of the product.108,109 Most immunoglobulinproducts in fact can be stored at room temperature for up to36 months.101,110 Currently used excipients comprising of aminoacids, sugars and sugar alcohols have a long history of use andare known to be well tolerated.108 Glycine is most commonlyused, but as illustrated by the products described above, proline,maltose, sorbitol, and mannitol are also used. Low concentrationsof sodium chloride and polysorbate detergent may also be addedto adjust tonicity and enhance stability.111–115

As immunoglobulins for intravenous use are manufactured byvarying processes, the properties of the different products pro-duced have been examined.96,116 In addition, consideration hasbeen given to whether formulation differences could affect clinicaltolerability.111–115 Although differences have been noted, there isno evidence that they significantly correlate with differences inproduct efficacy and tolerability. As registered products, theseimmunoglobulin products meet the standards as prescribed byrelevant regulatory authorities.The European Pharmacopoeia has a comprehensive monograph

on immunoglobulins for intravenous use.117 The product isexpected to be sterile and free of pyrogens and endotoxins. A liquidformulation is expected to be clear or slightly opalescent, colorless,or pale yellow. The allowable pH is 4.0–7.4. Osmolality must begreater than 240mOsmol/kg. The minimum allowable proteinconcentration of a formulation should not be less than 30 g/L(3% w/v). As modern immunoglobulin products are typicallyformulated at 10% (w/v), this limit is not usually relevant. Purityis expected to be greater than 95%—with the currently usedmanufacturing processes, this is readily achieved. Aggregate contentmust not exceed 3% of total protein. This is an important qualityattribute as aggregates typically result in complement activation-mediated adverse reactions. Any propensity for this is monitoredthrough the measurement of anti-complementary activity, with thespecification set at<1 CH50/mg IgG. Specifically, this means that analiquot of immunoglobulin solution will not sequester more than50% of a given amount of complement.The allowable maximum limit of prekallikrein activator (PKA)

(factor XIIa), which can mediate the formation of the vasoactivepeptide bradykinin from kinnogen and lead to hypotensivereactions, is set at 35 IU/mL.118

Hemagglutinins, anti-A and anti-B antibodies to red cell anti-gens, can result in hemolytic reactions with the infusion ofimmunoglobulins. Therefore, the EP stipulates a maximum allow-able titer of 1:64. Given that high-volume administration ofimmunoglobulins is required in some indications, specificallythose associated with achieving immune modulation, manufac-turers are introducing specified hemagglutinin removal steps in

Supernatant I

Delipidation


Q anion exchange chromatography

Pasteurization

Formulation, filling

Low pH incubation

Final product

Figure 27.10 Process steps for Intragam P (CSL Behring, Australia).


manufacturing their process to lower the titer in their prod-ucts.96,116 Therefore, the titer of some products will be considera-bly lower than this limit.The EP does not define a maximum level of IgA allowed in

immunoglobulin products but the product must comply with thelevel stated on the label. The range for the products described hereis 5–200 μg/mL.101 Knowledge of the IgA content of an immuno-globulin product is important for a clinician when confrontedwith an IgA deficient patient who could exhibit an anaphylacticreaction if infused with a higher IgA concentration immuno-globulin product.119

Although not prescribed in the EP, immunoglobulin products areexpected to exhibit a subclass distribution comparable to thatnormally found in plasma.119 Partial depletion of IgG3 and IgG4

is, however, encountered in a number of manufacturing processes.20

There is no evidence that this affects the efficacy of the product.The EP requires that a developed immunoglobulin purificationprocess maintain the integrity of the Fc portion of the immuno-globulin molecule—key to its immunomodulatory role throughinteraction with the Fc receptor and its effector role in complementactivation.119 Monitoring of Fc function is not a product releaserequirement but is part of process validation. An immunoglobulinproduct should exhibit an Fc function of >60% of that of an EPstandard. However, current products typically have values of 100%.20

The EP also stipulates that antibody titer to hepatitis B be atleast 0.5 IU/g IgG. There is no limit for antibodies to hepatitis A,but regulatory authorities expect levels to be above 10 IU/ml. Thismay be increasingly difficult to maintain due to the consistentdecline in population titers from at least 2003 due to increasedhygiene and the availability of an effective hepatitis A vac-cine.20,120 Product specifications for the United States also includeminimum antibody levels for measles, diphtheria, and polio.121

With respect to measles, there are interesting trends suggestingthat as disease prevalence is decreasing as a result of increasedvaccination, antibody titer in plasma and hence immunoglobulinproducts is decreasing.122

In 2010, increased incidences of thromboembolic events werereported in patients using Octogam.123 Analysis showed this wasdue to increased levels of factor XIa caused by a process change. Theregulatory agencies requested that manufacturers confirm that theirprocesses have the capability of removing thrombogenic activityand generate a product low in procoagulant activity.117,124,125

Procoagulants have been shown to be partitioned away from theproduct during purification and inactivated by pasteurization andexposure to low pH.123–125 The most commonly used methods arenon-activated partial thromboplastin time (NaPTT) and the throm-bin generation assay (TGA). The latter is typically calibrated relativeto factor XIa concentration. NaPTT values with clotting time >150seconds are considered acceptable. With respect to TGA-derivedresults, existing products typically have very low to nondetectablelevels of factor XIa.126,127

Factor XIIIThe development of manufacturing processes for factor XIII hasbeen extensively reviewed.15 The focus here will be on describing theproduction of a major commercial factor XIII product, Fibrogam-min P (in Europe) and Corifact (in the United States), asundertaken by CSL Behring. The process utilizes Cohn FractionI precipitate derived from cryoprecipitate-depleted plasma. Follow-ing resolubilization and removal of fibrinogen by treatment withaluminum hydroxide and Vitacel (a cellulose fiber), the clarifiedsolution is subjected to DEAE anion exchange chromatography.

The purified factor XIII is then subjected to two viral inactivationand removal procedures—pasteurization (60 °C for 10 hours) andviral filtration with 20 nm filters. These dedicated viral removalprocedures, together with viral partitioning achieved during thechromatography procedure, result in excellent viral clearancethrough the manufacturing process. Following formulation to thetarget potency and with the addition of albumin, glucose, andsodium chloride, the solution is sterile filtered, dispensed, andlyophilized.128,129 There is no monograph for factor XIII product,but these products have been extensively characterized with respectto purity, function, and factor XIII integrity. This includes factorXIII activity, specific activity, factor XIII subunit A content, fibrin-ogen clotting time, PKA, and activated factor XIII.15

Factor XThe manufacture of a therapeutic factor X produced by BPL (UK) isshown in Figure 27.11. The key steps in the process are adsorptionof factor X from cryoprecipitate-depleted plasma by DEAE anionexchange chromatography. Further purification is achieved by


Elution of prothrombim complex factors

Copper – metal affinity chromatography

S/D treatment

Wash out S/D chemicals

Factor X elution



Factor X elution


Formulation

Filling

Lyophilization

Heat treatment, 72 hours at 80ºC

Figure 27.11 Manufacturing process for factor X (BPL).


metal affinity chromatography with a copper chelate resin. Viralremoval of enveloped and non-enveloped viruses is achieved by S/Dtreatment and viral filtration with 15 nm filters. In addition, thelyophilized product is subject to heat treatment at 80 °C for 72hours.15 The product has been shown to comply with requiredtoxicity, thrombogenicity, and immunogenicity requirements. Inaddition, in vitro tests confirm the product is potent, highly purified,and free of potentially hazardous residues. The testing includesdetermination of factor X potency, specific activity, the presence offactor II, factor IX, proteins C and S, and NaPTT.15

Activated prothrombin complex concentrate(aPCC)aPCC, known as factor VIII inhibitor bypassing activity (FEIBA),is manufactured by Baxalta. Details on the development, produc-tion, mechanism of action, and clinical use of the product haverecently been reviewed.11 The manufacturing process is shown inFigure 27.12. It involves purification of prothrombin complexfactors (II, VII, IX, and X) from cryoprecipitate-depleted plasmaby DEAE anion exchange chromatography. The recovered materialundergoes viral filtration with a 35 nm filter. The factors are thencontact activated, generating activated Factor VII and some acti-vated factor X. These activated factors, together with prothrombin,are thought to be important for the clinical efficacy of the prod-uct.11,130 The product undergoes a second viral inactivation treat-ment involving vapor heat treatment at 60 °C and 80 °C for at least8.5 hours and 1 hour, respectively, on the lyophilized product. Thisstep is effective against both enveloped and non-enveloped viruses.The product is then formulated with sodium citrate and sodiumchloride, and dispensed in 500, 1000, and 2500 arbitrary FEIBAunits. One unit of activity is defined as that amount of product thatshortens the activated partial thromboplastin time (aPTT) of ahigh-titer factor VIII inhibitor reference plasma to 50% of the blankvalue. The final product is a lyophilized preparation.131

Alpha1-proteinase inhibitorThe manufacturing processes for the four major alpha1-proteinaseinhibitor (API) products on the market, Aralast NP (Baxalta),Zemaira (CSL Behring), Prolastin C (Grifols), and Glassia

(Kamada), are presented in Figure 27.13.132–136 A comprehensiveexamination of these manufacturing processes is availableelsewhere.23

Cohn Fraction IV-I is the source material. The Aralast NP

process utilizes a unique step involving, an extended thaw of thepaste and dissolution in pH 6.0 buffer that allows the recovery of anAPI-enriched precipitate. The generated initial crude API solutionsundergo an initial “activation hold” step at approximately pH 9 andat an elevated temperature of approximately 45 °C. This results inincreased recovery of functional protein, which could be related torefolding of denatured protein. The commencement of the purifi-cation process typically involves polyethylene glycol (PEG) precipi-tation of impurities, destabilization of unwanted proteins byreduction with dithiothrietol (DTT), and adsorption with fumedsilica (Aerosil). This treatment takes advantage of the fact thatthere are no disulfide bonds in the API molecule.137 All processesutilize anion exchange chromatography for further purification.Further polishing is achieved by adsorption with bentonite (alumi-num phyllosilicate clay), hydrophobic interaction chromatography,or cation exchange chromatography. The Aralot NP, Glassia, andProlastin-C processes all incorporate S/D treatment as the first viralinactivation procedure. Pasteurization is used in the Zemaira

process. All processes include viral filtration as the second viralremoval step, and this is effective for both enveloped and non-enveloped viruses. Aralast NP, Zemaira, and Prolastin C arepresented as lyophilized final products, whereas Glassia is a liquidproduct.23

The EP prescribes certain product qualities for API prepara-tions.138 The specific activity should not be less than 0.35mg ofactive API per mg of total protein. The ratio of API activity toantigen should not be less than 0.7. With respect to appearance, theliquid preparation should be clear or slightly opalescent, colorless,or pale yellow, pale green, or pale brown; the freeze-dried prepara-tion should be a powder or solid friable mass, hygroscopic, andwhite, pale yellow, or pale brown.It is required that the pH be in the range of 6.5–7.8, that the

preparation be completely soluble, and that osmolality be greaterthan 240mOsmol/kg. In addition, the water content of the freeze-dried preparation must be within the limits approved for theproduct by the competent authority. The product must be sterileand free of pyrogens and bacterial endotoxins.

Factor XIThe manufacture of factor XI is undertaken by LFB Biotechnol-ogies (France).14 Cryoprecipitate-depleted plasma (cryosuperna-tant) is passed through a negatively charged filter to capture thefactor XI. It is then eluted with 1M NaCI containing antithrom-bin III (ATIII), which inhibits any proteolytic activity that coulddegrade the product. Following buffer exchange by ultrafiltration,the solution is subjected to viral inactivation treatment with S/D.The solution is passed through a sulfate(S) cation exchangechromatographic resin to capture the protein and allow thewashing away of the virus-inactivating chemicals. The factor XI isrecovered with eluate buffer containing lysine and arginine byincreasing the salt content and pH to 170mM and 7.5, respectively.Heparin and ATIII were added to the eluate, and then it was sterile


Activation



Formulation

Filling

Final product

Viral inactivation, 2-step vapor heat, 60°C/80°C

Figure 27.12 Manufacturing process for factor VIII bypassing activity(FEIBA) (Baxalta).


filtered, dispensed, and lyophilized. The product is highly purified,containing only trace amounts of fibrinogen, albumin, C1-esteraseinhibitor, fibronectin, alpha2-macroglobulin, and IgG. The levels ofother coagulation factors, kinin system components, and proteasesare also low or nondetectable.139

The EP required that the freeze-dried final product be white oralmost white powder or friable solid. The water content must bewithin the limits approved by the competent authority. It shouldcompletely dissolve within 10 minutes. The potency must be within

80–120% of that stated on the label. The reconstituted productshould be of pH 6.8–7.4 and have a minimum osmolality of240mOsmol/kg. Heparin and ATIII added as stabilizers mustcomply with the level stated on the label. Ensuring the absenceof activated coagulation factors, the NaPTT coagulation time shouldnot be less than 150 seconds. There should not be any significantpresence of anti-A and anti-B hemagglutinins, with no agglutina-tion occurring at a 1:64 dilution of the product. The product must besterile and free of bacterial endotoxins.140

Aralast NP® Zemaira® Glassia® Prolastin-C®

IV-1 paste IV-1 paste IV-1 paste IV-1 paste

Extended thaw

Dissolution andrecovery of precipitate

Dissolution Dissolution Dissolution

Aerosil®

addition

Suspension of crudeAPI precipitate

Activation API Activation API Activation API Activation API

PEG precipitation DTT andAerosil®

treatment

PEGprecipitation

PEGprecipitation

S/D treatment insucrose

S/D treatment

Anion exchangechromatography




Bentonitetreatment

Hydrophobicinteraction

chromatography

Cation exchangechromatography

Cation exchangechromatography

Pasteurizationin sucrose

S/D treatment

Anion exchange chromatography

Viral filtration Viral filtration Viral filtration Viral filtration

Formulatation,Filling

Lyophilization


Lyophilization


Liquid


Lyophilization

ZnCl2 precipitation

Figure 27.13 Comparison of alpha1-proteinase inhibitor production methods.


C1-esterase inhibitorA recent review comprehensively deals with the biology, purification,and clinical use of C1-esterase inhibitor.24 This section will focus ondescribing the manufacturing processes of two of the major C1-esterase inhibitor products on the market: Berinert (CSL Behring),andCinryze (US) orCeton (Europe) (Sanguine).24,141,142As canbeseen in Figure 27.14, the processes are quite distinct in the use ofchromatographic processes. In the production of Berinert, cryo-precipitate-depleted plasma is initialed; passed through a DEAEanion exchange column, under conditions in which the C1-esteraseinhibitor flows through; and subsequently captured on a QAE anionexchange resin. Furtherpurification is achievedbyammoniumsulfateprecipitation steps and hydrophobic interaction chromatography(HIC). In the Cinryze process, the C1-esterase inhibitor is capturedfrom the starting material by anion exchange chromatography, andfurther purification is achieved by PEG precipitation and two proce-dures involving cation exchange chromatography. In both processes,viral removal involves pasteurization and virus filtration requiringtwo filters in series.141,143 The recovered virally filtered product isformulated, dispensed, and lyophilized. In the case of Berinert,excipients used in its formulation are glycine, sodium chloride, andsodium citrate.144

There are no pharmacopoeial requirements for C1-esterase inhib-itor concentrates. However, as with other lyophilized therapeutic

protein products, it must have an acceptable appearance in thelyophilized state and comply with registered residual moisture con-tent. The product must readily dissolve and present as a colorless andclear solution with clinically acceptable osmolality and insolubleparticle count. It must be sterile and free of bacterial endotoxins.Manufacturers determine and report the potency of the product.144

The integrity of the product is determined by monomer content,which is a quality control release specification. Thus, for example,Berinert must have a monomer content of >89% to meet releasespecifications (CSL data on file). Recently, a biochemical and puritycomparison of four commercially available C1-esterase inhibitorproducts (Berinert, Cinryze, Cetor, and Ruconest [transgenicproduct]) was published consisting of characterization of molecularweight, determination of specific activity, and quantitation andidentification of residual proteins. It was the hope of the authorsthat the work would contribute to the establishment of regulatoryrequirements for determining purity and setting allowable thresholdlevels.145

Antithrombin IIIAs examples of manufacturing processes for Antithrombin III(ATIII), theprocesses forKyberninP (CSLBehring), Thrombotrol

(CSL Behring [Australia]), and Thrombate III (Grifols) are

Berinert® Cinryze®

Cryoprecipitate-depleted plasma Cryoprecipitate-depleted plasma

DEAE anion exchange chromatography DEAE anion exchange chromatography

QAE anion exchange chromatography PEG precipitation

Ammonium sulfate precipitation CM cation exchange chromatography

Pasteurization Pasteurization

Ammonium sulfate precipitation

Phenyl hydrophobic interaction chromatography CM cation exchange chromatography

Virus filtration (20 nm and 15 nm in series) Virus filtration (two 15 nm filters in series)

Ultrafiltration Ultrafiltration

Formulation Formulation

Filling, lyophilization Filling, lyophilization

Figure 27.14 Processes for the manufacture of C1-esterase inhibitor.


described and shown in Figure 27.15.146,147 Further information onthe biology, purification, and clinical indications for ATIII is availablein a recent review.18

From either Supernatant I, cryoprecipitate-depleted plasma, or aFraction IV-I suspension, ATIII is captured by heparin affinitychromatography. The Kybernin P process then uses sequentialammonium sulfate steps to remove impurities and to concentratethe ATIII by precipitation. All processes incorporate pasteurizationas a viral inactivation step. In the Kybernin P process, a subsequentammonium precipitation of ATIII allows the removal of pasteur-ization stabilizers and the production of a concentrated solution,which is formulated, dispensed, and lyophilized. In theThrombotrol and Thrombate III processes, further purificationis achieved by size exclusion and heparin affinity chromatography,respectively. Both processes incorporate viral filtration. The recov-ered filtrate is then concentrated and diafiltered prior to formula-tion, dispensing, and lyophilization. Thrombotrol and ThrombateIII processes incorporate two dedicated viral inactivation andremoval steps that are effective for both enveloped and non-enveloped viruses. The Kybernin P process has only one dedicatedviral inactivation step—pasteurization, which is effective againstboth enveloped and non-enveloped viruses. In addition, viralclearance is achieved by precipitation at the first ammonium sulfateprecipitation step.146

The EP prescribes a number of product specifications.148 Atleast a 60% fraction of the product must bind heparin. The specificactivity of the product should not be less than 3 IU ATIII per mgof protein (excluding albumin excipient). The lyophilized productshould be a white or almost white, hygroscopic, friable solid orpowder. The water content must be within limits approved by thecompetent authority. The product should completely dissolve

within 10 minutes with gentle swirling, giving a clear or slightlyturbid, colorless or almost colorless solution. The limits for pHand osmolality are 6.0–7.5 and >240mOsmol/kg, respectively.The maximum allowed heparin content is 0.1 IU per IU ATIII.The product must be sterile and free of bacterial endotoxins.

FibrinogenRiastap (US) or Haemocomplettan (Europe) produced by CSLBehring is the leading fibrinogen concentrate available on themarket. The product is derived from cryoprecipitate residue afterextraction of factor VIII. The manufacturing process is based on aseries of glycine-based precipitations—a procedure initiallydescribed by Bromback and Bromich in 1956.149,150 The cryopre-cipitate extract is initially adsorbed with aluminum hydroxide, andthen the fibrinogen is precipitated with high-concentration glycine.The reconstituted fibrinogen is then pasteurized. Following theaddition of glycine to promote precipitation of residual proteins, apurified fibrinogen is recovered by precipitation by increasing theconcentration of glycine. The pure fibrinogen precipitate is redis-solved, concentrated and diafiltered, formulated, dispensed, andlyophilized. The pasteurization step is effective against envelopedand non-enveloped virus. Further viral clearance is obtained at thecryoprecipitation and glycine precipitation steps.151,152

The EP requires that the lyophilized product be white or paleyellow, in a hygroscopic powder or friable solid; that the watercontent is within approval limits; and that it dissolves within 30minutes at room temperature, forming an almost colorless,slightly opalescent solution. The solution should contain not lessthan 10 g/l fibrinogen with pH of 6.5 to 7.5 and a minimumosmolality of 240mOsmol/kg. The product should be sterile andfree of bacterial endotoxins.153

Kybernin P® Thrombotrol® Thrombate III®

Cryoprecipitate-depletedplasma

Cryoprecipitate-depletedplasma

Fraction IV-I

Supernatant I Suspension

Heparin affinity

chromatography

Heparin affinity

chromatography

Heparin affinity

chromatography

Ammonium sulfateprecipitation of impurities

Ultrafiltration/Diafiltration

Pasteurization Pasteurization Pasteurization

Ammonium sulfateprecipitation of ATIII

Size exclusionchromatography

Heparin affinitychromatography

Resuspension Viral filtration, 20 nm Viral filtration 15 nm

Formulation Ultrafiltration/Diafiltration Ultrafiltration/Diafiltration

Filling, lyophilization Filling, lyophilization Filling, lyophilization

Figure 27.15 Comparison of Antithrombin III manufacturing processes.


The development of another fibrinogen product by Octa-pharma, called Octofibrin, which is highly purified and incor-porates two dedicated viral removal procedures (S/D treatmentand viral filtration), has been reported. It is currently undergoingclinical trial.154

Prothrombin complex concentrateThe manufacture of commercially available prothrombin complexconcentrates (PCC) is predominantly achieved by the use of anionexchange chromatography. Incorporated viral removal proceduresinclude S/D treatment, heat pasteurization, vapor heat or dry heat,and viral filtration. The final formulation may involve the additionof heparin and antithrombin (ATIII) as specific clotting factorstabilizers (for the prevention of activation), and albumin as anexcipient.12,155

A manufacturing process for PCC should ensure the generationof a product with a balanced content of vitamin K–dependentfactors (II, VII, IX, and X) and the presence of physiologicallyrelevant amounts of the regulatory inhibitory proteins, proteins C,S, and Z.12 The properties of various PCCs have been compared. Allhad similar levels of coagulation factors, but there were differencesin purity and content of the inhibitory proteins.156

To specifically illustrate the principles of the manufacture of PCC,the process used for the production of Kcentra (CSL Behring)(Beriplex in Europe) and Octaplex (Octapharma)—two commer-cial products in wide use—will be examined (Figure 27.16). Themanufacture of Kcentra is initiated by the adsorption of the PCC

factors from cryoprecipitate-depleted plasma with DEAE anionexchange resin. The factors are eluted and stabilized by the additionof ATIII, heparin, and CaCl2. The eluate is pasteurized to ensure viralsafety. Further purification involves ammonium precipitation ofimpurities, binding to calcium phosphate, followed by elution andAerosil (fumed silica) adsorption of the eluate. The recoveredsolution is formulated by the addition of albumin, ATIII, and heparinto stabilize the active components, and then subjected to viralfiltration through two 20 nm filters. Following dilution to a targetedfactor IX concentration and the adjustment of the final albuminconcentration, the product is dispensed and lyophilized157 (CSLinformation on file). In the Octoplex process, PCC factors areadsorbed from cryoprecipitate-depleted plasma with a Q anionexchange chromatographic resin following pH addition and heparinaddition to prevent factor activation. The recovered eluate undergoesS/D treatment for viral inactivation. Subsequent binding to a DEAEanion exchange resin allows thewashing away of the viral inactivationchemicals, and provides further purification. The product is thensubjected to viral filtration with a 20 nm filter. Following ultra-filtration, the product is formulated with the addition of heparin(but no albumin), then dispensed and lyophilized.12,158

Both processes generate highly a purified PCC with balancedfactor content and regulatory proteins.181–183 The dedicated viralremoval steps and partitioning of virus, which occur at specificprocess steps, ensure the viral safety of these products.159,160

There is an EP monograph for PCC products.161 It stipulatesthat a product must contain factor IX together with variable

Kcentra® Octaplex®

Cryoprecipitate-depleted plasma Cryoprecipitate-depleted plasma

DEAE anion exchange resin adsorption Heparin addition, pH adjustment

Elution and ATIII, heparin, CaCl2 added as

stabilizers

QAE anion exchange resin adsorption

Pasteurization Elution

Ammonium sulfate precipitation Solvent/detergent treatment

Calcium phosphate adsorption DEAE Anion exchange resin adsorption

Elution from calcium phosphate Viral filtration, 20 nm

Aerosil® adsorption Ultrafiltration/diafiltration

Albumin, ATIII, heparin addition Adsorption and pH adjustment

Viral filtration, 75 nm, 35 nm Formulation (albumin not used)

Formulation Filling, lyophilization

Filling, lyophilization

Figure 27.16 Comparison of the manufacture of two prothrombin complex concentrate products.


amounts of factor II, VII, and X. The preparation may containstabilizers (e.g., albumin, heparin, and ATIII). The lyophilizedproduct should be a white or slightly colored, very hygroscopicpowder or friable solid. Water content should be within approvedlimits. The preparation must dissolve completely within 10 min-utes with gentle swirling. The potency of the reconstituted prepa-ration should be not less than 20 IU of factor IX per milliliter. ThepH of the solution should be between 6.5 and 7.5, and osmolalitynot less than 240mOsmol/kg. If the content of any of the otherfactors is stated, it should not be less than 80% or more than 120%of the stated range. The specific activity of factor IX should not beless than 0.6 IU/mg of total protein (before addition of anystabilizer). The presence of any activated coagulation factorsshould not result in the coagulation time in a NaPTT assay tobe less than 150 seconds. If heparin has been used in theformulation, the amount should comply with that on the labeland not be more than 0.5 IU per IU of factor IX. The preparationmust be sterile and free of bacterial endotoxins.

Factor IXThe approaches taken for the purification of high-purity factor IXhas recently been extensively documented, and the key processstages have been collated for all the registered factor IX prod-ucts.13,155 Predominantly, the process commences with the initialcapture of prothrombin complex factors (factors II, VII, IX, and X).The isolation of factor IX can then involve the use of ion exchange,affinity, hydrophobic, or metal-chelating chromatography, oradsorption with insoluble metal salts. The manufacturing processesincorporate specific viral removal procedures, usually S/D treat-ment and viral filtration.13,155

Details of the manufacture of four products—Alphanine (Gri-fols), Betafact (LFB), Berinin P (CSL Behring), and Mononine

(CSL Behring)—are shown in Table 27.2. It can be seen that allproducts involve initial adsorption of factor IX (and the other PCCfactors II, IX, and VIII) from cryoprecipitate-depleted plasma byDEAE anion exchange chromatography. In the manufacture ofAlphanine, final purification involves adsorption to a barium citrateprecipitate, followed by two sequential dextran sulfate affinitychromatography steps. Viral removal is achieved by S/D treatmentand viral filtration.13,162 The recovery of purified factor IX in theBetafact process involves further purification by DEAE anionexchange chromatography and heparin affinity chromatography.Viral removal is achieved by S/D treatment and viral filtration.13,163

In the Berinin P process, the crude preparation initiallyobtained by DEAE anion exchange chromatography, undergoesa viral inactivation procedure—pasteurization—and then is

partially purified by ammonium sulfate precipitation, prior torecovery of factor IX by calcium phosphate adsorption and finalpurification by heparin affinity chromatography (CSL data on file).The purification of Mononine involves passage of a DEAE anionexchange chromatography eluate, which contains PCC factorsadsorbed from cryosupernatant, through a specific anti–factor IXmonoclonal antibody affinity resin. The pure factor IX is eluatedwith sodium thiocyanate and is then diafiltered. Thematerial is thenpassed through a viral removal filter. Subsequent passage throughan amino-hexanoic affinity column removes any leached monoclo-nal antibody ligand from the previous column.164–167 The productsare then formulated dispensed and lyophilized.The required properties of a purified factor IX product are

detailed by an EP monograph.168 The lyophilized product shouldbe a white or pale yellow hygroscopic powder or friable solid. Thewater content should be within limits approved by the competentauthority. The preparation should dissolve completely with gentleswishing within 10 minutes. The reconstituted product should havea pH between 6.5 and 7.5, and a minimum osmolality of 240mOs-mol/kg. Potency should be not less than 20 IU/mL, and specificactivity not less than 50 IU/mg of total protein. Any presence ofactivated factors should not cause the coagulation time in theNaPTT test to exceed 150 seconds. If heparin has been used inthe formulation, it should be stated on the label but in all casesshould not exceed 0.5 IU of heparin per IU of factor IX. The productshould be sterile and free of bacterial endotoxins.

von Willebrand factorHaemate P/Humate P (CSL Behring) was one of the firstdeveloped intermediate-purity factor VIII–vWF products. Sinceits licensing in Germany in 1981 for the treatment of hemophiliaA, it has also been used to this day for the treatment of vonWillebrand disease.169 The manufacture of the product is fromcryoprecipitate and involves initial adsorption of impurities fromthe reconstituted solution with aluminum hydroxide and thenpurification of the factor VIII–vWF complex by a series of glycine-and sodium chloride–mediated precipitation steps. Pasteurization isincorporated as a viral inactivation step155 (CSL data on file).In the late 1980s, new purification processes were developed

incorporating chromatographic procedures to improve the effi-ciency of manufacture, improve product purity, and facilitate theintroduction of viral inactivation procedures. A detailed examina-tion of the chromatographic approaches that can be used topurify vWF, and an overview of the manufacturing processesand the viral removal procedures utilized for registered vWFproducts has been published elsewhere.10,155 They show that a

Table 27.2 Examples of manufacturing processes for Factor IX

Brand Name (Manufacturer) Purification Process Specific Viral Removal Steps

Alphanine

(Grifols)DEAE anion exchange chromatographyBarium citrate adsorptionDextran sulfate affinity chromatography (x2)

Solvent/detergent treatmentViral filtration (15nm)

Betafact

(LFB)DEAE anion exchange chromatography(x2)Heparin affinity chromatography

Solvent/detergentViral filtration (15nm)

Berinin P

(CSL Behring)DEAE anion exchange chromatographyAmmonium sulphate precipitation of residuesCa2PO4 adsorption/elutionHeparin affinity chromatography

Pasteurisation (60 °C, I0 h)

Mononine

(CSL Behring)DEAE anion exchange chromatographyMonoclonal affinity chromatography,Amino-hexanoic affinity chromatography

Viral filtration (15 nm)


range of chromatographic procedures are used consisting of anionexchange, size exclusion, and affinity chromatography. Viralremoval procedures include S/D treatment, pasteurization, dryheat treatment, and viral filtration.The purification processes and viral removal procedures for

some specific vWF products are shown in Table 27.3. The startingmaterial in all cases is cryoprecipitate. The manufacture ofAlphonate involves an initial purification involving PEG precip-itation to remove impurities, followed by heparin affinity chro-matography to recover the purified vWF.10,170 The manufacture ofWilate and Wilfactin involves the use of size exclusion chro-matography and gelatin affinity chromatography respectively, tofurther purify the DEAE anion exchange chromatographic fractionobtained through the processing of a cryoprecipitate solu-tion.10,171,172 The manufacturing processes incorporate S/D treat-ment and dry heat as viral inactivation steps. In the Wilfactin

process, viral filtration is included.The manufacturing process for a vWF product must deliver a

consistent product with respect vWF multimer composition. TheEP requires that during process development, quantitative analysisof the product must be undertaken using electrophoretic anddensitometric techniques to confirm that the product closelyapproximates the multimer distribution of a plasma referencepreparation.173–175 The dispensed product must contain not lessthan 20 IU/ml of vWF, and the measured value must be within 20%of that stated on the label. Factor VIII must be tested if there isgreater than 10 IU of factor VIII per 100 IU of vWF. The measuredvalue must be within 40% of the stated value. vWF potency mustonly be measured by the ristocetin cofactor assay. vWF products arecharacterized on the basis of specific activity and a vWF activity–FVIII ratio. For the former, it must be greater than 1U vWF/mg oftotal protein excluding any protein stabilizer. A value greater than80U vWF/mg protein is usual, although products with lowerspecific activity exist.155 The vWF activity–FVIII ratio is notprescribed but must comply with the approved limit for the productwith the competent authority. Values for the various products arefrom 0.75 to 2.4. The exception is Wilfactin, where the ratio isapproximately 60.155 This reflects the design of the manufacturingprocess which reduces the co-purification of factor VIII, therebyproducing a predominantly vWF product.171

With respect to other product characteristics, the EP requiresthat the lyophilized final product be a white or pale yellow,hygroscopic powder or friable solid with a water content thatis within the approved limits of the competent authority and thatdissolves completely with gentle swirling within 10 minutes,giving a clear or slightly opalescent, colorless yellow solution.

Some products allow for filtration to remove flakes or particlespresent after reconstitution. If this is the case, it must be shown invalidation studies that there is no impact on product potency.The reconstituted product must have a pH between 6.5 and 7.5,an osmolality greater than 240mmol/kg, and no anti-A- or anti-B-mediated agglutination at a 1:64 dilution at a defined dilution ofthe reconstituted preparation. The product must be sterile andendotoxin free.175

Factor VIIIThe production of cryoprecipitate for clinical use by Judith Pool in1965 was a pivotal event in the treatment of hemophilia as itprovided a high-potency product and an alternative to what hith-erto had been used-fresh frozen plasma.176 In the following years,purification processes were developed that further improved thepurity and viral safety of factor VIII products. Haemate P/HumateP, in Germany in 1981, was the first registered intermediate-purityproduct containing a dedicated viral inactivation procedure. Theproduct contained both factor VIII and vWF, and it became arecognized therapeutic for both hemophilia A and von Willebranddisease.169 The purification process increased the specific activity ofvWF from 1 IU/mg of protein for cryoprecipitate to approximately38 IU/mg of protein in Haemate P/Humate P.155

In subsequent years, the application of chromatographic stepsfurther increased the purity of factor VIII products.9 Commonlyreferred to as high-purity products, the specific activity was typicallygreater than 100 IU/mg and for some products as high as 180 IU/mg.9,155 The different manufacturing processes resulted in a varia-tion in the vWF content, which in some cases limited their use to thetreatment of hemophilia A and the exclusion of von Willebranddisease. With the development of these new processes, the oppor-tunity was also taken to introduce two specific viral removalprocedures. Typically, these were S/D treatment and dry-heattreatment of the lyophilized final product. Vapor heat and viralfiltration have also been applied.9,155 The use of monoclonal affinitychromatography allowed the generation of factor VIII concentrateswith higher specific activity: >3000 IU/mg. These products, ofcourse, do not have vWF.9,155

Table 27.4 presents the manufacturing process of a number ofcommercially available factor VIII products. The aim is to specifi-cally present details to illustrate the range of processes that havebeen used to purify Factor VIII and ensure viral safety. In themanufacture of the high-purity products BeriateP (CSL Behring),Factane (LFB), and Immunate (Baxalta), there is typically aninitial Al(OH)3 adsorption step. This serves to remove vitamin K–dependent prothrombin complex factors. Further purification then

Table 27.3 Examples of manufacturing processes for von Willebrand factor

Brand Name (Manufacturer) Purification Process Specific Viral Removal Steps

Haemate P/Humate P

(CSL Behring)Aluminum hydroxide treatmentGlycine precipitationSodium chloride precipitationNaCl/glycine precipitation

Pasteurization (60 °C for 10 hours)

Alphanate

(Grifols)Polyethylene glycol precipitationHeparin affinity chromatography

Solvent/detergent treatment.Dry heat (80 °C for 72 hours)

Wilate

(Octapharma)DEAE anion exchange chromatographySize exclusion chromatography

Solvent/detergent treatment.Dry heat (10 °C for 2 hours)

Wilfactin

(LFB)Alumina gel treatmentDEAE anion exchange chromatography (X2)Gelatin affinity chromatography

Solvent/detergent treatmentViral filtration, 35nm.Dry heat (80 °C for 72 hours)


occurs by anion exchange chromatography.177–179 Factane has aunique viral filtration step involving passing the product through a15 nm filter. It had been thought that the factor VIII–vWF complexwas too large to be able to be successfully filtered. However, this wasachieved by dissociating the complex prior to filtration by exposureto calcium chloride and then reassociating the complex by itsremoval from the filtrate. The manufacturing process forAlphanate differs in that there is an initial PEG precipitationstep that is effective in removing fibrinogen and fibronectin. Finalpurification utilizes heparin affinity chromatography.180–182

The examples of the manufacture of very-high-purity factor VIIIproducts offer some interesting and distinct differences. In the caseof Hemofil M, following a cold precipitation clarification step ofthe cryoprecipitate suspension, the solution undergoes S/D treat-ment to effect viral inactivation. Factor VIII purification is achievedby anti–factor VIII immunoaffinity chromatography. This step isvery effective in achieving the removal of fibrinogen, fibronectin,and vWF. There is then a viral filtration step. A subsequent Q anionexchange chromatographic step binds factor VIII eluted from theaffinity column and allows any leached monoclonal antibodyaffinity ligand to flow through.183,184 In the Monoclate process,the cryoprecipitate suspension is treated with aluminum hydroxidefor clarification and removal of vitamin K–dependent clottingfactors. The recovered solution is then pasteurized to provide aviral inactivation step. The factor VIII–vWFr complex is capturedby an immobilized anti-vWF antibody. Factor VIII is dissociatedwith the used elution conditions, recovered, and passed over anamino hexanoic affinity column to remove any possible leachedmonoclonal antibody affinity ligand185,186 (CSL data on file).All factor VIII products are lyophilized. Various excipients are

used and can include amino acids, sugar alcohols, sugars, and salts.In some cases, albumin is also included.155

The required properties of factor VIII are detailed in the EP.187

The specific activity should not be less than 1 IU of factor VIII permilligram of total protein before the addition of any proteinstabilizer. But, as has been noted, with modern purified products

this is readily achieved. The potency of the preparation as stated onthe label should result in not less than 20 IU factor VIII permilliliter. With respect to the appearance of the lyophilized prepa-ration, water content, solubility, pH, osmolality of the reconstitutedmaterial, anti-A and anti-B hemagglutinin titer, sterility and endo-toxin content, the requirements previously described for the vWFproduct apply.

ConclusionThe production of plasma protein-based therapeutic products is acomplex process commencing with the collection of plasma by asystem incorporating donor selection, viral testing and qualityassurance measures that ensure the viral safety of the plasma forfractionation.33 Starting with the work of Cohn, and through thesubsequent application of knowledge of protein chemistry, biopro-cessing technology, and virology, processes have been developed forthe production of therapeutic products for a range of clinicalindications, which meet the high standards of safety and efficacyprescribed by the regulatory authorities.7

Today, the production of plasma protein therapeutic products isa large global industry.31,188 Despite the development of manyrecombinant alternatives, the production of products by fractiona-tion of plasma remains cost-effective and continues to grow.189

There is particular growth in the provision of albumin and immu-noglobulins.190 Albumin is required in large amounts, and immu-noglobulins have unique biological properties. These factors make itcommercially unattractive or scientifically unjustifiable to producethese products by recombinant means, and therefore they will mostlikely continue to be produced from plasma for the foreseeablefuture.19,189,191

Plasma fractionators are continuing to explore the clinicalutility of additional plasma proteins and improve existing prod-ucts. Proteins with therapeutic potential that are being investi-gated are shown in Table 27.1. Plasmin and reconstituted HDL arethe most advanced in the clinical development pathway.19,25

Table 27.4 Representative manufacturing processes for factor VIII

Brand Name (Manufacturer) Purification Process Specific Viral Removal Steps Specific Activity (IU/mg)

Intermediate-Purity ProductHaemate P/Humate P

(CSL Behring)Aluminum hydroxide treatmentGlycine precipitationSodium chloride precipitationNaCl/glycine precipitation

Pasteurization (60 °C for 10 hours) 38

High-Purity ProductsBeriate P

(CSL Behring)Al(OH)3 adsorptionAl(OH)3/QAE anion exchange resin adsorptionDEAE anion exchange chromatography

PasteurizationViral filtration, 20 nm

170

Factane (LFB) Al(OH)3 adsorptionDEAE anion exchange chromatographyFVIII/vWF dissociation-0.3M CaCI2FVIII/vWF reassociation

Viral filtration, 35 nm, 15 nm >100

Immunate (Baxalta) Al(OH)3 adsorptionDEAE anion exchange chromatography

Solvent/detergent treatmentVapor heat (60 °C for 10 hours, 7–8% moisture, 190 bar)

75

Alphanate (Grifols) PEG precipitationHeparin affinity chromatography

Solvent/detergent treatmentDry heat (80 °C for 72 hours)

>100

Very-High-Purity ProductsHemofil M (Baxalta) Recovery of cryoprecipitate

Anti–factor VIII affinity chromatographyQAE anion exchange chromatography

Solvent/detergent treatmentViral filtration, 20 nm

>3000

Monoclate

(CSL Behring)Al(OH)3 adsorptionAnti-vWF affinity chromatographyAmino hexanoic affinity chromatography

Pasteurization ∼2000


With respect to existing products, the focus is on improvingformulations and addressing issues that can lead to adversereactions. The development of high-concentration immunoglo-bulins for subcutaneous use, ensuring that procoagulant activity isremoved during the manufacture of immunoglobulins, the intro-duction of a procedure to remove anti-A and anti-B hemagglu-tinins from immunoglobulin products and the development of aliquid formulation for alpha1-protease inhibitor are examples ofthese activities.Plasma manufacturers are confronted with a limited and expen-

sive starting raw material—plasma.192 An efficient means of pro-ducing plasma-derived products that is high yielding and cost-effective would be of commercial benefit to the manufacturer andwelcomed by the healthcare community, which is seeking anaffordable and adequate supply of products. Recent trends in theuse of disposable technologies and the developing concept ofcontinuous processing may help to decrease the costs of establishingand operating a manufacturing facility.193–195

The developed countries are well served for fractionation capac-ity. Many developing countries such as China and Brazil areextending and improving their capabilities.31 However, there aremany developing countries that do not have adequate access toplasma protein products and cannot afford to purchase the require-ments for their population.196 Arguments for and against theestablishment of a localized fractionation facility have been dis-cussed.192,197 The adoption of improvements in bioprocessingapproaches could make the design and establishment of a fraction-ation facility less complex, cheaper and hence feasibile for con-struction and operation in a developing country.

The challenge for the future is aptly captured by the followingreflection: “Nearly 2 billion people—or a third of the world’spopulation—lack access to essential medicines. We need to askourselves: what use is our scientific endeavour and innovation whenthey do not come to the aid of people who need it the most? Should adrug be described a ‘blockbuster’ by a billion-dollar label or abillion-patients label?”198

The plasma fractionation industry will continue to deliverunique therapeutics essential for human health. It is likely thatthe future will be as eventful as the period from Cohn to thepresent day.

Key referencesA full reference list for this chapter is available at: http://www.wiley.com/go/simon/transfusion

7 Bertolini J, Goss N, Curling J, Eds. Production of plasma proteins for therapeuticuse. Hoboken, NJ: John Wiley and Sons Inc., 2013.

8 Lundblad RL. Biotechnology of plasma proteins. Boca Raton, FL: CRC Press,2012.

117 European Directorate for the Quality of Medicines and Healthcare (EDQM).Normal immunoglobulin for intravenous use. In: European Pharmacopoeia. 8thed. Strasbourg, France: EDQM, 2013.

155 Brooker M. Registry of clotting factor concentrates. 9th ed. Montreal: WorldFederation of Hemophilia, 2012.

188 Burnouf T. Plasma fractionation in the world: current status. Transfus Clin Biol2007;14:41–50.

190 Robert P. The worldwide plasma proteins market 2012. Orange, CT: MarketingResearch Bureau Inc., 2013.


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