TR-05246; No of Pages 6 Thrombosis Research xxx (2014) xxx–xxx
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Review Article ®
Pathogen safety of Beriate Albrecht Gröner ⁎ CSL Behring GmbH, Marburg, Germany
a r t i c l e
i n f o
Available online xxxx Keywords: Haemophilia A Beriate® Plasma-derived product Pasteurization Virus filtration Pathogen safety
a b s t r a c t Plasma-derived factor VIII (FVIII) concentrates have been used successfully to treat haemophilia A since the late 1960s. To ensure the pathogen safety of the plasma-derived FVIII concentrate, Beriate® (formerly Beriate® P), donors of blood/plasma are carefully selected and all donations are screened for hepatitis B virus surface antigen (HBsAg), antibodies against HIV types 1 and 2 (HIV-1/HIV-2) and hepatitis C virus (HCV), and genomic material of hepatitis A virus (HAV), hepatitis B virus (HBV), HCV, and for high titres of parvovirus B19 (B19V). As additional quality control, plasma pools for fractionation are only released for further processing when nonreactivity has been demonstrated in serological and genome amplification assays. The manufacturing process for Beriate® comprises dedicated virus reduction steps such as pasteurization and the recently introduced virus filtration step, resulting in effective inactivation of various enveloped and non-enveloped viruses and effective removal of viruses and prion material larger than the mean pore size of the virus filter (19 nm). The effectiveness of these production steps has been demonstrated in virus and prion validation studies using a range of different viruses and prion preparations. The multiple precautionary measures inherent to the overall production process for Beriate® (and its predecessor Beriate® P) are reflected in an excellent safety record documented during 20 years of clinical use with no proven record of virus transmission, even before the introduction of the virus filtration step. Continued improvement of safety measures according to scientific knowledge and regulatory guidance maintains and even enhances the excellent safety profile of Beriate®. © 2013 Published by Elsevier Ltd.
Contents Introduction . . . . . . . . . . . . . . . . . . . . . . . . Pathogen safety: plasma collection . . . . . . . . Virus reduction during the manufacture of Beriate® . Prion safety of Beriate® . . . . . . . . . . . . Summary and conclusions . . . . . . . . . . . . . . . . Conflict of interest statement . . . . . . . . . . . . . . . Acknowledgements . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . .
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Abbreviations: Al(OH)3, aluminium hydroxide; B19V, parvovirus B19; BSE, bovine spongiform encephalopathy; BVDV, bovine viral diarrhoea virus; CJD, Creutzfeldt–Jakob disease; CMV, cytomegalovirus; CPV, canine parvovirus; DEAE, diethylamino ethanol; DNA, deoxyribonucleic acid; FDA, U.S. Food and Drug Administration; FVIII, factor VIII; HAV, hepatitis A virus; HBsAg, hepatitis B virus surface antigen; HBV, hepatitis B virus; HCV, hepatitis C virus; HIV, human immunodeficiency virus; NAT, nucleic acid amplification technology; PCR, polymerase chain reaction; PPV, porcine parvovirus; PrPc, cellular prion protein; PrPSc, pathogenic prion protein; PRV, pseudorabies virus; QAE, quaternary aminoethyl; RNA, ribonucleic acid; TSE, transmissible spongiform encephalopathy; vCJD, variant Creutzfeldt–Jakob disease; VWF, von Willebrand factor; YFV, yellow fever virus. ⁎ CSL Behring GmbH, P.O. Box 1230, 35002 Marburg, Germany. Tel.: +49 6421 392 725; fax: +49 6421 394 689. E-mail address: [email protected].
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Introduction When the high-purity, plasma-derived factor VIII (FVIII) concentrate, Beriate® P, was marketed in the early 1990s, measures implemented to control potential virus contamination in the final product covered donor selection and testing of donations according to the state-of-theart at that time. Virus reduction procedures in the original manufacturing process included pasteurization (heat treatment in aqueous stabilized solution at 60 °C for 10 hours), which was the proprietary virus inactivation method of Behringwerke (a predecessor company of CSL Behring), and other production processes such as chromatography, which also added towards the reduction of viruses. With the introduction
0049-3848/$ – see front matter © 2013 Published by Elsevier Ltd. http://dx.doi.org/10.1016/j.thromres.2013.10.013 ®
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of a new virus filtration step, Beriate® P was renamed Beriate® – a high purity FVIII concentrate containing sufficient von Willebrand factor (VWF) to stabilize FVIII. Multiple improvements in manufacturing procedures in terms of pathogen safety have been introduced since Beriate® P was initially developed, starting from plasma collection to fractionation pool testing up to the recent introduction of a virus filtration step. Refinements of serological assays have enabled the detection of donations from infected donors, with confirmatory assays resulting in the permanent exclusion of such donors. Serological assays for human immunodeficiency virus (HIV) were implemented initially in 1985 after the virus was detected and could be propagated in cell culture [1]. The first serological assays for hepatitis C virus (HCV) were implemented in 1990/1991 after the virus was detected by molecular biological methods [2,3]. The introduction of nucleic acid amplification technology (NAT) for the testing of virus deoxyribonucleic acid (DNA) and ribonucleic acid (RNA) in sample pools of donations (mini-pool testing) further reduced the residual risk [4]. The release of plasma pools for fractionation for further processing only if they were non-reactive for serological markers of hepatitis B virus (HBV), HCV, and HIV, was implemented in 1994. Further release testing was introduced at CSL Behring (and its predecessor companies) from 1997 for HCV RNA according to European guidance [5, referenced in 6]. On a voluntary basis, since 1997, all plasma pools for the production of Beriate® P and Beriate® are released only when nonreactive for HBV DNA and HIV-1 RNA (as well as for HCV RNA). From 2000, also on a voluntary basis, plasma pools are released only when non-reactive for hepatitis A virus (HAV) RNA and high titres of parvovirus B19 (B19V).1 Pathogen safety: plasma collection Plasma for the production of Beriate® (and previously Beriate® P) complies with the European Pharmacopoeia Monograph, Human Plasma for Fractionation (0853) [8] and other international requirements. Quality standards for the starting material of plasma-derived products consist of a continuum of interrelated steps to adequately select and control the source material to prevent or limit the occurrence of infectious agents in the fractionation pool. The principal complementary measures to ensure the virus safety of the starting material are: • 1. Rigorous selection of plasma centres according to the epidemiology of blood-borne viruses in the donor population. This selection of centres applies to centres collecting recovered plasma (from whole blood donations) and source plasma (from apheresis donations) according to European guidelines [9]. • 2. Rigorous selection of donors at each donor session with regard to health status and behavioural risk, based on physical examination and a predefined questionnaire. Donor deferral due to geographic risk (e.g. variant Creutzfeldt–Jakob disease [vCJD]) is implemented according to the guidance documents applicable where donations are collected and the product is marketed [10,11]. Donors who are not healthy or are at risk of being infected with blood-borne viruses are deferred, according to regulatory guidance, depending on the risk of developing a transmissible disease either temporarily or permanently. All donors confirmed to be reactive for an infection by HBV, HCV, or HIV, as well as donors at risk of developing vCJD, are permanently excluded from donating.
• 3. Donation testing: Each donation is tested for the absence of viral markers (hepatitis B surface antigen [HBsAg] and antibodies against HCV and HIV-1/HIV-2), according to the current European Pharmacopoeia [8]. Additionally, sample pools (mini-pools) of plasma donations, either source plasma or recovered plasma, are tested by NAT for genomic material of HAV, HBV, HCV, HIV-1/-2, and high titres of B19V and released only when non-reactive (i.e. below the limit of detection of a sensitive, validated NAT assay). The strategy of creating mini-pools for NAT/PCR (polymerase chain reaction) testing was implemented to avoid unnecessary plasma loss should NAT/PCR reactivity be detected in large plasma pools for fractionation. Furthermore, mini-pool vs. plasma-pool testing increases the sensitivity of the NAT assay. Results of NAT/PCR testing of donations used in the production of the former product Beriate® P in terms of positivity for HBV, HCV and HIV-1 are shown in Table 1 for qualified donors of plasmapheresis plasma (source plasma) – defined as those who have passed separate medical screens and had negative virus tests on two different occasions, with no more than a 6-month interval between each test – and all donors of recovered plasma derived from whole blood donations (i.e. first-time and repeattested donors). In addition, more than 35 million source plasma donations have been tested since the introduction of NAT testing for genomic material of HAV and B19V, demonstrating a low incidence of HAV (b 0.3 HAV NAT positive donations per 100,000 donations) and B19V (b170 B19V NAT positive donations per 100,000 donations) in the donor population. As HAV and B19V infections cause selflimiting diseases due to the development of specific antibodies, donors whose samples are reactive in NAT assays are not deferred, but all positive donations are destroyed so they do not enter a plasma pool for fractionation. Furthermore, antibodies against HAV and B19V are essential for high quality immunoglobulin products prepared from such plasma pools. The virus reduction capacity of the NAT/ PCR testing performed by CSL Behring is significant, as shown in Table 2 [12–16]. • 4. Manufacturing pool testing: In order to ensure that the testing and pooling of donations is performed without errors, the plasma pool for fractionation (the first homogeneous material, i.e. the cryo-depleted pool) is further tested for the absence of viral markers (HBsAg and antibodies against HIV-1/HIV-2) and genomic material of HAV, HBV, HCV, HIV-1/-2, and high titres of B19V as an in-process control measure. All plasma pools are only released for further processing if non-reactive at a sensitivity of the NAT test as listed in Table 3. The plasma pools for fractionation therefore contain a very low load, if any, of the viruses HAV, HBV, HCV, HIV-1 (below the limit of detection of a sensitive NAT assay) and the B19V load does not exceed 104 IU B19V DNA/mL. • 5. Post-donation information and inventory hold: Any source plasma donation stored in inventory hold for at least 60 days and retrospectively suspected to contain viruses (below the limit of the NAT assay applied to that stored donation) based on post-donation information (e.g. a subsequent donation that was found to have virus markers and/or genomic material) will be intercepted and destroyed. Due to the long inter-donation interval of whole blood donations, use of post-donation information is less helpful to ensure the removal and Table 1 Results of NAT/PCR testing of blood/plasma donations at CSL Behring for the production of Beriate® P (Test period 01.01.2003 to 31.12.2011 shown as an example for qualified [source plasma] donors and for whole blood [recovered plasma] donors). Number of donors
1
According to the current European Pharmacopoeia, the limit of B19V DNA in a plasma pool is 10 IU B19V DNA/μL (i.e. not exceeding 104 IU B19V DNA/mL) when used for the production of anti-D plasma [7]; the same limit, but for all pools, was requested by the U.S. Food and Drug Administration (FDA) in 2009 for products marketed in the USA from 2009 onwards. Between 2000 and 2003, the B19V plasma load in plasma pools used to produce Beriate® P did not exceed 105 IU B19V DNA/mL; after that time period, plasma pools were released only when the B19V load did not exceed 104 IU B19V DNA/mL. ®
a
Tested Positive Positive per 105 donors tested
HBV
HCV
HIV-1
22,261,389 698 3.1
22,261,389 1,986 8.9
22,261,389 298 1.3
HBV, hepatitis B virus; HCV, hepatitis C virus; HIV, human immunodeficiency virus; NAT, nucleic acid amplification technology; PCR, polymerase chain reaction. a Donations non-reactive for serological markers of HBV, HCV, and HIV-1/HIV-2.
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A. Gröner / Thrombosis Research xxx (2014) xxx–xxx Table 2 Efficacy of NAT/PCR at reducing the virus load in plasma pools for fractionation by discarding one virus-positive window donation (source plasma). Virus
Virus removal due to NAT/PCR screening(GE log10) a
Reference
HBV HCV HIV-1 HAV B19V
6.3 12.6 9.4 9.9 16.9
12 13 14 15 16
3
Plasma pool for fractionation Cryoprecipitation Al(OH)3 adsorption
B19V, parvovirus B19; HAV, hepatitis A virus; HBV, hepatitis B virus; HCV, hepatitis C virus; HIV, human immunodeficiency virus; NAT, nucleic acid amplification technology; PCR, polymerase chain reaction. a One donation (approximately 800 mL) can harbour up to the following amount of virus particles (GE, genome equivalent): only window donations (donations that are not reactive in serological marker testing for HBV, HCV, and HIV) are tested by NAT for the presence of genomic material of HAV, HBV, HCV, HIV-1 and high-titre B19V.
destruction of recovered plasma donations from a donor subsequently shown to be reactive, as a large proportion of these donations will already have been processed. The residual risk of pooling a donation from an infected donor with a virus load below the limit of detection is very low for recovered plasma and even lower for source plasma due to several factors including the short inter-donation interval (i.e. the health of the donor is continuously monitored) and the inventory hold of at least 60 days, which allows, due to the post-donation information, the interception and destruction of donations from a donor potentially testing positive at a subsequent donation. These measures of carefully selecting blood/plasma collection centres and donors, of testing each donation for the absence of virus markers (by serological assays) and virus genomes (by sensitive NAT assays) of transfusion-relevant viruses, and of intercepting reactive donations as a result of post-donation information ensure that the plasma used for Beriate® (and previously for Beriate® P) contains minimal, if any, blood-borne viruses. Virus reduction during the manufacture of Beriate® The manufacturing process for plasma derivatives is critical for product quality, safety, and efficacy. Virus inactivation/removal steps must maintain the integrity of the plasma-derived therapeutic protein, thus ensuring both its clinical efficacy and safety [17]. The high-purity FVIII concentrate, Beriate® (and previously Beriate® P) is produced from cryoprecipitate derived from plasma pools of high quality, purified by adsorption to different matrices, pasteurized (heat treated at 60 °C for 10 hours in aqueous stabilized solution), and finally purified by ion exchange chromatography and filtered through a virus-retentive filter (Fig. 1). After dialysing and formulation with glycine, sucrose, sodium chloride, and calcium chloride dihydrate for stability, sterile filtration is performed and the final bulk solution is filled into suitable vials and lyophilized. This manufacturing process has been studied in virus validation studies for its capacity to inactivate and/or remove viruses. Virus validation studies assess the safety of a biological product with regard Table 3 Criteria of NAT tests for release of plasma pools for fractionation. Virus
Release criterion for NAT test sensitivity
HBV HCV HIV-1 HAV B19V
b10 IU/mL ≤30 IU/mL b100 IU/mL b20 IU/mL ≤4 log10 IU/mL
B19V, parvovirus B19; HAV, hepatitis A virus; HBV, hepatitis B virus; HCV, hepatitis C virus; HIV-1, human immunodeficiency virus 1; NAT, nucleic acid amplification technology. ®
Al(OH)3/QAE Sephadex adsorption
Pasteurization (heat treatment at 60°C for 10 hours in aqueous stabilized solution) Ion-exchange chromatography (DEAE–Sepharose)
20N virus filtration
Dialysis/formulation (with sucrose, glycine and salts) Sterile filtration Filling/lyophilization Fig. 1. Manufacturing process for Beriate®. Al (OH)3, aluminium hydroxide; DEAE, diethylamino ethanol; QAE, quaternary aminoethyl.
to potential virus transmission, and they identify the production steps that are effective in reducing the level of infectious viruses. Consequently, an estimate of the overall ability of a manufacturing process to inactivate and/or to remove any contaminating infectious viruses can be obtained. A prerequisite of virus validation studies is a validated scaling down of the manufacturing process to a laboratory scale, as these studies have to be performed in dedicated laboratories suitable for handling viruses according to their respective biosafety levels. In these laboratories, cell culture-derived stocks of either blood-borne viruses or appropriately selected model viruses are added (“spiked”) to intermediates of production steps derived from Beriate® (and previously Beriate® P) production lots. The manufacturing process is performed according to the validated downscale model taking into consideration all relevant process parameters: concentration of proteins and other components, temperature, pH, reaction time, and step efficacy. During the development of the former product Beriate® P, a wide range of different viruses have been studied to demonstrate the capacity of the pasteurization step, in particular, to inactivate viruses
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of different physicochemical properties. During this development period, when HCV was still called non-A, non-B hepatitis virus, studies demonstrating the reduction of HBV and non-A, non-B hepatitis virus by the manufacturing process for Beriate® P were performed in chimpanzees. In addition, a range of other viruses was employed in pasteurization studies using cell cultures (Table 4). Subsequently, in line with regulatory guidance, viruses chosen for validation studies using cell culture infectivity assays closely resemble the viruses that may potentially contaminate human plasma. Cell culture infectivity assays employing HIV and HAV are used; as currently, no cell culture infectivity assay is available for HBV or HCV, model viruses are used in virus validation studies: the specific model virus used for HCV is bovine viral diarrhea virus (BVDV; genus pestivirus), which is also a member of the same Flaviviridae family as HCV (genus hepacivirus) and is more closely related to HCV than members of the genus flavivirus [18]. For HBV, no practical test system is available [6,17], thus, unspecific model viruses such as herpesviruses (e.g. pseudorabies virus [PRV], an enveloped DNA virus, as is HBV) are used. In order to employ the relevant human parvovirus in virus validation studies, a complex cell culture infectivity assay for B19V has been developed at CSL Behring, utilizing B19V high-titre donations for spiking. As B19V has only an abortive replication cycle in cell culture, the detection of B19V-encoded proteins in the inoculated cell cultures by immunofluorescence is used to assess the titre of infectious B19V. Since animal parvoviruses as canine parvovirus (CPV) or porcine parvovirus (PPV) are considerably more resistant to physiochemical methods for virus inactivation than B19V [19–21], the inactivation capacity of pasteurization for B19V was studied. In order to assess the ability of the production process to reduce viruses in general, a range of viruses with different physicochemical properties that replicate to high titres in cell culture can be assayed in an effective, sensitive, and reliable in vitro infectivity assay were chosen by CSL Behring for their virus validation studies (Table 5) [22]. The continued improvement of safety measures, in line with current scientific knowledge and regulatory guidance, has recently led to the incorporation of an additional virus reduction step into the manufacturing process for Beriate®: virus filtration utilizing a virus filter (Planova 20 N) with a mean pore size of approximately 19 nm (Fig. 1). The virus reduction capacity of the manufacturing process for Beriate® is shown in Table 6. Virus filtration of Beriate®, a FVIII concentrate with a low VWF:FVIII ratio, has no impact on the stability of FVIII and only a minimal impact on the amount and multimeric structure of VWF (Fig. 2). Prion safety of Beriate® Transmissible spongiform encephalopathies (TSEs) are a group of fatal neurodegenerative diseases of animals (scrapie in sheep, bovine spongiform encephalopathy [BSE] in cattle) and man (CJD) that are characterized by the accumulation of the disease-associated prion protein (PrPSc) – an abnormal isoform of the cellular prion protein Table 4 Inactivation of viruses by pasteurization – studies performed during the development of Beriate® P. Virus
Virus reduction factor (log10)
HBV a Non-A, non-B hepatitis virus (HCV) a HIV CMV Poliovirus YFV BVDV Rubella virus Measles virus
≥5.9 ≥5.4 ≥5.7 ≥6.0 ≥5.6 ≥5.5 ≥5.1 ≥4.1 ≥4.6
Table 5 Panel of viruses used in validation studies at CSL Behring for Beriate® P (in accordance with CPMP/BWP/268/95) [22]. Transfusion Test virus relevant virus (model virus)
Genome Enveloped Size(nm) Resistance to treatment
HIV HCV HBV HAV B19V
RNA RNA DNA RNA DNA DNA DNA
–
HIV-1 BVDV None available a HAV B19Vb CPVc Herpes virus (nonspecific model)
Low Low Medium High Very high Very high Medium
(PrPC) – in the central nervous system. PrPSc may also accumulate in other tissues, depending on the host and type of TSE involved. In 1996, a new variant of CJD, vCJD was identified [23]. Epidemiological and scientific data indicate a strong association between vCJD and consumption of meat products derived from BSEinfected cattle [24]. In humans with vCJD, abnormal prions are present in both the central nervous system and the lymphoreticular system, which has given rise to the possibility of blood-borne transmissions. Transmission of vCJD by transfusion of non-leuco-depleted red blood cell concentrates in the UK [25] and experimental studies in sheep and rodents in either the preclinical or clinical stage of disease have confirmed transmission of TSE through blood [26]. Plasma used for the production of Beriate® P previously and now Beriate®, is always leuco-depleted due to the collection method (plasmapheresis plasma) or processing (leuco-depleted, recovered plasma from a whole blood collection). In addition to leuco-depletion, precautionary measures have been instigated to minimize the theoretical risk of contaminating plasma pools with human TSEs by exclusion of donors at risk of developing CJD and vCJD (geographic deferral). CSL Behring and its predecessor companies have never used plasma for the production of Beriate® P collected in the UK, and did not use plasma collected in France from the time the first case of vCJD was reported in that country. Despite the very low risk of vCJD contamination of plasma pools for fractionation, prion removal studies have been performed to evaluate the ability of the overall manufacturing process to remove prion proteins. As the physicochemical properties of potential prion contamination in plasma is not known, two different prion spike preparations have been used in spiking studies. These were prepared from the brains of hamsters inoculated with the hamster-adapted scrapie prion strain 263 K: microsomes (membrane-associated infectious prions) and purified PrPSc (a detergent-extracted, non-membrane-associated infectious prion) [27]. The prion material in the spiked starting material Table 6 Virus reduction capacity of the manufacturing process of Beriate®.
Pasteurization Ion exchange chromatography 20 N Virus filtration Overall virus reduction factor
®
80–100 50–70 45 25–30 18–24 18–24 120–200
B19V, parvovirus B19; BVDV, bovine viral diarrhoea virus; CPV, canine parvovirus; DNA, deoxyribonucleic acid; HAV, hepatitis A virus; HBV, hepatitis B virus; HCV, hepatitis C virus; HIV-1, human immunodeficiency virus type 1; RNA, ribonucleic acid. a According to the European Medicines Agency, 1996. b Blood-borne virus B19V studied for inactivation capacity of pasteurization step. c Model virus CPV studied for removal capacity of the manufacturing process.
Manufacturing process
BVDV, bovine viral diarrhoea virus; CMV, cytomegalovirus; HBV, hepatitis B virus; HCV, hepatitis C virus; HIV, human immunodeficiency virus; YFV, yellow fever virus. a Manufacturing process of Beriate® P studied in chimpanzees.
Yes Yes Yes No No No Yes
Virus reduction factors (log10) HIV
BVDV
PRV
HAV
Parvovirus a
≥6.8 3.3 ≥6.0 ≥16.1
≥9.3 3.0 ≥5.8 ≥18.1
4.7 2.1 ≥7.2 ≥14.0
3.9 1.3 ≥5.5 ≥10.7
≥3.8 b 3.4 c 3.4 c ≥10.6
B19V, parvovirus B19; BVDV, bovine viral diarrhoea virus; CPV, canine parvovirus; HAV, hepatitis A virus; HIV, human immunodeficiency virus; PRV, pseudorabies virus. a Virus reduction factor demonstrated for: b B19V (relevant human parvovirus); c CPV (model virus for B19V).
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A. Gröner / Thrombosis Research xxx (2014) xxx–xxx
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2
3
4
Summary and conclusions
Fig. 2. Multimeric analysis of von Willebrand factor in Beriate® after virus filtration. Line 1, starting material for virus filtration; line 2, pool after pre-filtration (0.1 μm); line 3, pool after virus filtration (20 N); line 4, standard human plasma.
and the final sample was quantified using a conformation-dependent immunoassay [28,29]. These prion evaluation studies were performed in a similar manner to the virus validation studies using a validated downscale of the manufacturing process in a dedicated laboratory [30] and spiking prion preparations to product intermediates derived from production lots. The prion reduction capacities of individual steps are added together to produce an overall prion reduction factor, as in virus validation studies. However, the results generated using this approach must be interpreted with caution as the spike preparation may be heterogeneous, with one fraction preferentially removed by one manufacturing step and the same sub-fraction removed by another step [30]. As the manufacturing process of Beriate® may alter the physicochemical features of the spiked preparation and modify its removability, the overall manufacturing process of Beriate® was studied in one experiment covering nearly the whole manufacturing process by spiking dissolved cryoprecipitate and studying the filtrate of the virus filtration for residual PrPSc signal. The overall prion reduction capacity of the Beriate® manufacturing process is shown in Table 7.
Table 7 Prion reduction capacity of the improved Beriate® process. Overall manufacturing process
Al(OH)3 adsorption, Al(OH)3/QAE Sephadex adsorption, pasteurization, ion exchange chromatography, virus filtration (Planova 20 N)
5
Prion reduction factors [log10] Microsomes
Purified PrPSc
≥3.6
≥4.0
Microsomes, membrane-associated prion preparation; purified PrPSc, non-membrane associated prion preparation; Al(OH)3, aluminium hydroxide; QAE, quaternary aminoethyl. ®
The virus safety of Beriate® (and previously Beriate® P) is ensured through careful selection of donor centres and donors, screening of each donation for transfusion-relevant viruses by serological and NAT assays, the release of the plasma pool for fractionation based on nonreactivity for HBsAg and antibodies against HIV-1/HIV-2 and genomic material of HAV, HBV, HCV, HIV-1/-2, and high titres of B19V by sensitive NAT/PCR assays, and the virus reduction capacity of the manufacturing process. This virus reduction capacity is especially enhanced through the dedicated virus reduction steps of pasteurization and virus filtration (also called nanofiltration) employing a virusretentive filter with a pore size of approximately 19 nm. Further manufacturing steps for protein purification such as chromatography reliably contribute to virus removal. The manufacturing process for Beriate® (and previously Beriate® P) removes prions efficiently. An assessment by the U.S. Food and Drug Administration (FDA) has concluded that the risk of transmission of prions by FVIII products with a prion reduction factor of 4 log10 or more is very remote [31]. In conclusion, the measures taken in the production of Beriate® (and previously Beriate® P) ensure a high margin of pathogen safety for this product and are effective for enveloped viruses such as HIV, HBV and HCV and the non-enveloped viruses HAV and B19V. No proven cases of transmission of blood-borne viruses have been reported during clinical trials and through post-marketing surveillance over 20 years of clinical use, even before the virus filtration step was introduced. The incorporation of a virus filtration step into the manufacturing process is part of our continuous effort to improve the quality and safety of Beriate®. Conflict of interest statement The author is an employee of CSL Behring. Acknowledgements The contribution of the entire team of the Department of Virus Safety Development at CSL Behring GmbH, most notably to Dr T Nowak, Dr W Schäfer and Dr B Popp, to the execution of the virus validation studies is acknowledged. References [1] Busch MP, Young MJ, Samson SM, Mosley JW, Ward JW, Perkins HA, et al. Risk of human immunodeficiency virus (HIV) transmission by blood transfusions before the implementation of HIV-1 antibody screening. Transfusion 1991;31:4–11. [2] Choo QL, Kuo G, Weiner AJ, Overby LR, Bradley DW, Houghton M. Isolation of a cDNA clone derived from a blood-borne non-A, non-B viral hepatitis genome. Science 1989;244:359–62. [3] Kuo G, Choo QL, Alter HJ, Gitnick GL, Redeker AG, Purcell RH, et al. An assay for circulating antibodies to a major etiologic virus of human non-A, non-B hepatitis. Science 1989;244:362–4. [4] Schreiber GB, Busch MP, Kleinman SH, Korelitz JJ. The risk of transfusion-transmitted viral infections. The Retrovirus Epidemiology Donor Study. N Engl J Med 1996;334:1685–90. [5] The European Agency for the Evaluation of Medicinal Products. The introduction of nucleic acid amplification technology (NAT) for the detection of hepatitis C virus RNA in plasma pools. CPMP/BWP/390/97. 24 March 1998. (Annex V of CPMP/BWP/ 269/95, rev. 3), 24 March 1998. Available at: http://www.ema.europa.eu/docs/ en_GB/document_library/Scientific_guideline/2009/09/WC500003613.pdf [last accessed November 3, 2013]. [6] The European Agency for the Evaluation of Medicinal Products. Note for guidance on plasma-derived medicinal products. CPMP/BWP/269/95, rev. 3, 25 January 2001. Available at http://www.ema.europa.eu/docs/en_GB/document_library/Scientific_ guideline/2009/09/WC500003613.pdf [last accessed November 3, 2013]. [7] Council of Europe. European Pharmacopoeia. Nucleic acid amplification techniques European Pharmacopoeia; 2011 20621 [7.0–07/2010]. [8] Council of Europe. European Pharmacopoeia. Human plasma for fractionation European Pharmacopoeia; 2011 0853 [7.0–07/2008]. [9] European Medicines Agency. Guideline on epidemiological data on blood transmissible infections. EMEA/CPMP/BWP/125/04, 20 January 2005. Available at: http://www.ema.europa.eu/docs/en_GB/document_library/Scientific_guideline/2009/ 09/WC500003718.pdf [last accessed November 3, 2013].
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[10] European Medicines Agency. CHMP position statement on Creutzfeldt-Jakob disease and plasma-derived and urine-derived medicinal products. EMA/CHMP/BWP/ 303353/2010, 23 June 2011. Available at http://www.ema.europa.eu/docs/en_GB/ document_library/Position_statement/2011/06/WC500108071.pdf [last accessed November 3, 2013]. [11] U.S. Department of Health and Human Services, Food and Drug Administration, Center for Biologics Evaluation and Research (CBER). Guidance for Industry: Revised Preventive Measures to Reduce the Risk of Transmission of Creutzfeldt-Jakob Disease (CJD) and Variant Creutzfeldt-Jakob Disease (vCJD) by Blood and Blood Products, May 2010. Available at: http://www.fda.gov/downloads/BiologicsBlood Vaccines/GuidanceComplianceRegulatoryInformation/Guidances/UCM213415.pdf [last accessed November 3, 2013]. [12] Biswas R, Tabor E, Hsia CC, Wright DJ, Laycock ME, Fiebig EW, et al. Comparative sensitivity of HBV NATs and HBsAg assays for detection of acute HBV infection. Transfusion 2003;43:788–98. [13] Glynn SA, Wright DJ, Kleinman SH, Hirschkorn D, Tu Y, Heldebrant C, et al. Dynamics of viremia in early hepatitis C virus infection. Transfusion 2005;45:994–1002. [14] Fiebig EW, Wright DJ, Rawal BD, Garrett PE, Schumacher RT, Peddada L, et al. Dynamics of HIV viremia and antibody seroconversion in plasma donors: implications for diagnosis and staging of primary HIV infection. AIDS 2003;17:1871–9. [15] Weimer T, Streichert S, Watson C, Gröner A. Hepatitis A virus prevalence in plasma donations. J Med Virol 2002;67:469–71. [16] Brown KE, Young NS. Parvovirus B19 infection and hematopoiesis. Blood Rev 1995;9:176–82. [17] European Medicines Agency. Guideline on plasma-derived medicinal products. EMA/CHMP/BWP/706271/2010, 21 July 2011. Available at: http://www.ema. europa.eu/docs/en_GB/document_library/Scientific_guideline/2011/07/ WC500109627.pdf [last accessed November 3, 2013]. [18] Choo QL, Richman KH, Han JH, Berger K, Lee C, Dong C, Gallegos C, et al. Coit D, Medina-Selby A, Barr J, Weiner AJ, Bradley DW, Kuo G, Houghton M. Genetic organization and diversity of the hepatitis C virus. Proc Natl Acad Sci U S A 1991;88:2451–5. [19] Schwarz TF, Serke S, von Brunn A, Hottenträger B, Huhn D, Deinhardt F, Roggendorf M. Heat stability of parvovirus B19: kinetics of inactivation. Zentralbl Bakteriol 1992;277:219-2.
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[20] Blümel J, Schmidt I, Willkommen H, Löwer J. Inactivation of parvovirus B19 during pasteurization of human serum albumin. Transfusion 2002;42:1011–8. [21] Boschetti N, Niederhauser I, Kempf C, Stühler A, Löwer J, Blümel J. Different susceptibility of B19 virus and mice minute virus to low pH treatment. Transfusion 2004;44:1079–86. [22] The European Agency for the Evaluation of Medicinal Products. Note for guidance on virus validation studies: the design, contribution and interpretation of studies validating the inactivation and removal of viruses. CPMP/BWP/268/95, 14 February 1996. Available at: http://www.ema.europa.eu/docs/en_GB/document_library/ Scientific_guideline/2009/09/WC500003684.pdf [last accessed November 3, 2013]. [23] Will RG, Ironside JW, Zeidler M, Cousens SN, Estibeiro K, Alperovitch A, et al. A new variant of Creutzfeldt-Jakob disease in the UK. Lancet 1996;347:921–5. [24] Bruce ME, Will RG, Ironside JW, McConnell I, Drummond D, Suttie A, et al. Transmissions to mice indicate that ‘new variant’ CJD is caused by the BSE agent. Nature 1997;389:498–501. [25] Hewitt PE, Llewelyn CA, Mackenzie J, Will RG. Creutzfeldt-Jakob disease and blood transfusion: results of the UK Transfusion Medicine Epidemiological Review Study. Vox Sang 2006;91:221–30. [26] Houston F, McCutcheon S, Goldmann W, Chong A, Foster J, Sisó S, et al. Prion diseases are efficiently transmitted by blood transfusion in sheep. Blood 2008;112:4739–45. [27] Vey M, Baron H, Weimer T, Gröner A. Purity of spiking agent affects partitioning of prions in plasma protein purification. Biologicals 2002;30:187–96. [28] Safar J, Wille H, Itri V, Groth D, Serban H, Torchia M, et al. Eight prion strains have PrP(Sc) molecules with different conformations. Nat Med 1998;4:1157–65. [29] Bellon A, Seyfert-Brandt W, Lang W, Baron H, Gröner A, Vey M. Improved conformation-dependent immunoassay: suitability for human prion detection with enhanced sensitivity. J Gen Virol 2003;84:1921–5. [30] European Medicines Agency. Guideline on the investigation of manufacturing processes for plasma-derived medicinal products with regard to vCJD risk. CPMP/ BWP/CPMP/5136/03, 21 October 2004. Available at http://www.ema.europa.eu/ docs/en_GB/document_library/Scientific_guideline/2009/09/WC500003741.pdf [last accessed November 3, 2013]. [31] Transmissible Spongiform Encephalopathies Advisory Committee meeting June 12. Available at: http://www.fda.gov/downloads/AdvisoryCommittees/ Calendar/UCM164322.pdf; 2009 [last accessed November 3, 2013].
Please cite this article as: Gröner A, Pathogen safety of Beriate , Thromb Res (2014), http://dx.doi.org/10.1016/j.thromres.2013.10.013
Contents lists available at ScienceDirect
Thrombosis Research journal homepage: www.elsevier.com/locate/thromres
Review Article ®
Pathogen safety of Beriate Albrecht Gröner ⁎ CSL Behring GmbH, Marburg, Germany
a r t i c l e
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Available online xxxx Keywords: Haemophilia A Beriate® Plasma-derived product Pasteurization Virus filtration Pathogen safety
a b s t r a c t Plasma-derived factor VIII (FVIII) concentrates have been used successfully to treat haemophilia A since the late 1960s. To ensure the pathogen safety of the plasma-derived FVIII concentrate, Beriate® (formerly Beriate® P), donors of blood/plasma are carefully selected and all donations are screened for hepatitis B virus surface antigen (HBsAg), antibodies against HIV types 1 and 2 (HIV-1/HIV-2) and hepatitis C virus (HCV), and genomic material of hepatitis A virus (HAV), hepatitis B virus (HBV), HCV, and for high titres of parvovirus B19 (B19V). As additional quality control, plasma pools for fractionation are only released for further processing when nonreactivity has been demonstrated in serological and genome amplification assays. The manufacturing process for Beriate® comprises dedicated virus reduction steps such as pasteurization and the recently introduced virus filtration step, resulting in effective inactivation of various enveloped and non-enveloped viruses and effective removal of viruses and prion material larger than the mean pore size of the virus filter (19 nm). The effectiveness of these production steps has been demonstrated in virus and prion validation studies using a range of different viruses and prion preparations. The multiple precautionary measures inherent to the overall production process for Beriate® (and its predecessor Beriate® P) are reflected in an excellent safety record documented during 20 years of clinical use with no proven record of virus transmission, even before the introduction of the virus filtration step. Continued improvement of safety measures according to scientific knowledge and regulatory guidance maintains and even enhances the excellent safety profile of Beriate®. © 2013 Published by Elsevier Ltd.
Contents Introduction . . . . . . . . . . . . . . . . . . . . . . . . Pathogen safety: plasma collection . . . . . . . . Virus reduction during the manufacture of Beriate® . Prion safety of Beriate® . . . . . . . . . . . . Summary and conclusions . . . . . . . . . . . . . . . . Conflict of interest statement . . . . . . . . . . . . . . . Acknowledgements . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . .
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Abbreviations: Al(OH)3, aluminium hydroxide; B19V, parvovirus B19; BSE, bovine spongiform encephalopathy; BVDV, bovine viral diarrhoea virus; CJD, Creutzfeldt–Jakob disease; CMV, cytomegalovirus; CPV, canine parvovirus; DEAE, diethylamino ethanol; DNA, deoxyribonucleic acid; FDA, U.S. Food and Drug Administration; FVIII, factor VIII; HAV, hepatitis A virus; HBsAg, hepatitis B virus surface antigen; HBV, hepatitis B virus; HCV, hepatitis C virus; HIV, human immunodeficiency virus; NAT, nucleic acid amplification technology; PCR, polymerase chain reaction; PPV, porcine parvovirus; PrPc, cellular prion protein; PrPSc, pathogenic prion protein; PRV, pseudorabies virus; QAE, quaternary aminoethyl; RNA, ribonucleic acid; TSE, transmissible spongiform encephalopathy; vCJD, variant Creutzfeldt–Jakob disease; VWF, von Willebrand factor; YFV, yellow fever virus. ⁎ CSL Behring GmbH, P.O. Box 1230, 35002 Marburg, Germany. Tel.: +49 6421 392 725; fax: +49 6421 394 689. E-mail address: [email protected].
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Introduction When the high-purity, plasma-derived factor VIII (FVIII) concentrate, Beriate® P, was marketed in the early 1990s, measures implemented to control potential virus contamination in the final product covered donor selection and testing of donations according to the state-of-theart at that time. Virus reduction procedures in the original manufacturing process included pasteurization (heat treatment in aqueous stabilized solution at 60 °C for 10 hours), which was the proprietary virus inactivation method of Behringwerke (a predecessor company of CSL Behring), and other production processes such as chromatography, which also added towards the reduction of viruses. With the introduction
0049-3848/$ – see front matter © 2013 Published by Elsevier Ltd. http://dx.doi.org/10.1016/j.thromres.2013.10.013 ®
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of a new virus filtration step, Beriate® P was renamed Beriate® – a high purity FVIII concentrate containing sufficient von Willebrand factor (VWF) to stabilize FVIII. Multiple improvements in manufacturing procedures in terms of pathogen safety have been introduced since Beriate® P was initially developed, starting from plasma collection to fractionation pool testing up to the recent introduction of a virus filtration step. Refinements of serological assays have enabled the detection of donations from infected donors, with confirmatory assays resulting in the permanent exclusion of such donors. Serological assays for human immunodeficiency virus (HIV) were implemented initially in 1985 after the virus was detected and could be propagated in cell culture [1]. The first serological assays for hepatitis C virus (HCV) were implemented in 1990/1991 after the virus was detected by molecular biological methods [2,3]. The introduction of nucleic acid amplification technology (NAT) for the testing of virus deoxyribonucleic acid (DNA) and ribonucleic acid (RNA) in sample pools of donations (mini-pool testing) further reduced the residual risk [4]. The release of plasma pools for fractionation for further processing only if they were non-reactive for serological markers of hepatitis B virus (HBV), HCV, and HIV, was implemented in 1994. Further release testing was introduced at CSL Behring (and its predecessor companies) from 1997 for HCV RNA according to European guidance [5, referenced in 6]. On a voluntary basis, since 1997, all plasma pools for the production of Beriate® P and Beriate® are released only when nonreactive for HBV DNA and HIV-1 RNA (as well as for HCV RNA). From 2000, also on a voluntary basis, plasma pools are released only when non-reactive for hepatitis A virus (HAV) RNA and high titres of parvovirus B19 (B19V).1 Pathogen safety: plasma collection Plasma for the production of Beriate® (and previously Beriate® P) complies with the European Pharmacopoeia Monograph, Human Plasma for Fractionation (0853) [8] and other international requirements. Quality standards for the starting material of plasma-derived products consist of a continuum of interrelated steps to adequately select and control the source material to prevent or limit the occurrence of infectious agents in the fractionation pool. The principal complementary measures to ensure the virus safety of the starting material are: • 1. Rigorous selection of plasma centres according to the epidemiology of blood-borne viruses in the donor population. This selection of centres applies to centres collecting recovered plasma (from whole blood donations) and source plasma (from apheresis donations) according to European guidelines [9]. • 2. Rigorous selection of donors at each donor session with regard to health status and behavioural risk, based on physical examination and a predefined questionnaire. Donor deferral due to geographic risk (e.g. variant Creutzfeldt–Jakob disease [vCJD]) is implemented according to the guidance documents applicable where donations are collected and the product is marketed [10,11]. Donors who are not healthy or are at risk of being infected with blood-borne viruses are deferred, according to regulatory guidance, depending on the risk of developing a transmissible disease either temporarily or permanently. All donors confirmed to be reactive for an infection by HBV, HCV, or HIV, as well as donors at risk of developing vCJD, are permanently excluded from donating.
• 3. Donation testing: Each donation is tested for the absence of viral markers (hepatitis B surface antigen [HBsAg] and antibodies against HCV and HIV-1/HIV-2), according to the current European Pharmacopoeia [8]. Additionally, sample pools (mini-pools) of plasma donations, either source plasma or recovered plasma, are tested by NAT for genomic material of HAV, HBV, HCV, HIV-1/-2, and high titres of B19V and released only when non-reactive (i.e. below the limit of detection of a sensitive, validated NAT assay). The strategy of creating mini-pools for NAT/PCR (polymerase chain reaction) testing was implemented to avoid unnecessary plasma loss should NAT/PCR reactivity be detected in large plasma pools for fractionation. Furthermore, mini-pool vs. plasma-pool testing increases the sensitivity of the NAT assay. Results of NAT/PCR testing of donations used in the production of the former product Beriate® P in terms of positivity for HBV, HCV and HIV-1 are shown in Table 1 for qualified donors of plasmapheresis plasma (source plasma) – defined as those who have passed separate medical screens and had negative virus tests on two different occasions, with no more than a 6-month interval between each test – and all donors of recovered plasma derived from whole blood donations (i.e. first-time and repeattested donors). In addition, more than 35 million source plasma donations have been tested since the introduction of NAT testing for genomic material of HAV and B19V, demonstrating a low incidence of HAV (b 0.3 HAV NAT positive donations per 100,000 donations) and B19V (b170 B19V NAT positive donations per 100,000 donations) in the donor population. As HAV and B19V infections cause selflimiting diseases due to the development of specific antibodies, donors whose samples are reactive in NAT assays are not deferred, but all positive donations are destroyed so they do not enter a plasma pool for fractionation. Furthermore, antibodies against HAV and B19V are essential for high quality immunoglobulin products prepared from such plasma pools. The virus reduction capacity of the NAT/ PCR testing performed by CSL Behring is significant, as shown in Table 2 [12–16]. • 4. Manufacturing pool testing: In order to ensure that the testing and pooling of donations is performed without errors, the plasma pool for fractionation (the first homogeneous material, i.e. the cryo-depleted pool) is further tested for the absence of viral markers (HBsAg and antibodies against HIV-1/HIV-2) and genomic material of HAV, HBV, HCV, HIV-1/-2, and high titres of B19V as an in-process control measure. All plasma pools are only released for further processing if non-reactive at a sensitivity of the NAT test as listed in Table 3. The plasma pools for fractionation therefore contain a very low load, if any, of the viruses HAV, HBV, HCV, HIV-1 (below the limit of detection of a sensitive NAT assay) and the B19V load does not exceed 104 IU B19V DNA/mL. • 5. Post-donation information and inventory hold: Any source plasma donation stored in inventory hold for at least 60 days and retrospectively suspected to contain viruses (below the limit of the NAT assay applied to that stored donation) based on post-donation information (e.g. a subsequent donation that was found to have virus markers and/or genomic material) will be intercepted and destroyed. Due to the long inter-donation interval of whole blood donations, use of post-donation information is less helpful to ensure the removal and Table 1 Results of NAT/PCR testing of blood/plasma donations at CSL Behring for the production of Beriate® P (Test period 01.01.2003 to 31.12.2011 shown as an example for qualified [source plasma] donors and for whole blood [recovered plasma] donors). Number of donors
1
According to the current European Pharmacopoeia, the limit of B19V DNA in a plasma pool is 10 IU B19V DNA/μL (i.e. not exceeding 104 IU B19V DNA/mL) when used for the production of anti-D plasma [7]; the same limit, but for all pools, was requested by the U.S. Food and Drug Administration (FDA) in 2009 for products marketed in the USA from 2009 onwards. Between 2000 and 2003, the B19V plasma load in plasma pools used to produce Beriate® P did not exceed 105 IU B19V DNA/mL; after that time period, plasma pools were released only when the B19V load did not exceed 104 IU B19V DNA/mL. ®
a
Tested Positive Positive per 105 donors tested
HBV
HCV
HIV-1
22,261,389 698 3.1
22,261,389 1,986 8.9
22,261,389 298 1.3
HBV, hepatitis B virus; HCV, hepatitis C virus; HIV, human immunodeficiency virus; NAT, nucleic acid amplification technology; PCR, polymerase chain reaction. a Donations non-reactive for serological markers of HBV, HCV, and HIV-1/HIV-2.
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A. Gröner / Thrombosis Research xxx (2014) xxx–xxx Table 2 Efficacy of NAT/PCR at reducing the virus load in plasma pools for fractionation by discarding one virus-positive window donation (source plasma). Virus
Virus removal due to NAT/PCR screening(GE log10) a
Reference
HBV HCV HIV-1 HAV B19V
6.3 12.6 9.4 9.9 16.9
12 13 14 15 16
3
Plasma pool for fractionation Cryoprecipitation Al(OH)3 adsorption
B19V, parvovirus B19; HAV, hepatitis A virus; HBV, hepatitis B virus; HCV, hepatitis C virus; HIV, human immunodeficiency virus; NAT, nucleic acid amplification technology; PCR, polymerase chain reaction. a One donation (approximately 800 mL) can harbour up to the following amount of virus particles (GE, genome equivalent): only window donations (donations that are not reactive in serological marker testing for HBV, HCV, and HIV) are tested by NAT for the presence of genomic material of HAV, HBV, HCV, HIV-1 and high-titre B19V.
destruction of recovered plasma donations from a donor subsequently shown to be reactive, as a large proportion of these donations will already have been processed. The residual risk of pooling a donation from an infected donor with a virus load below the limit of detection is very low for recovered plasma and even lower for source plasma due to several factors including the short inter-donation interval (i.e. the health of the donor is continuously monitored) and the inventory hold of at least 60 days, which allows, due to the post-donation information, the interception and destruction of donations from a donor potentially testing positive at a subsequent donation. These measures of carefully selecting blood/plasma collection centres and donors, of testing each donation for the absence of virus markers (by serological assays) and virus genomes (by sensitive NAT assays) of transfusion-relevant viruses, and of intercepting reactive donations as a result of post-donation information ensure that the plasma used for Beriate® (and previously for Beriate® P) contains minimal, if any, blood-borne viruses. Virus reduction during the manufacture of Beriate® The manufacturing process for plasma derivatives is critical for product quality, safety, and efficacy. Virus inactivation/removal steps must maintain the integrity of the plasma-derived therapeutic protein, thus ensuring both its clinical efficacy and safety [17]. The high-purity FVIII concentrate, Beriate® (and previously Beriate® P) is produced from cryoprecipitate derived from plasma pools of high quality, purified by adsorption to different matrices, pasteurized (heat treated at 60 °C for 10 hours in aqueous stabilized solution), and finally purified by ion exchange chromatography and filtered through a virus-retentive filter (Fig. 1). After dialysing and formulation with glycine, sucrose, sodium chloride, and calcium chloride dihydrate for stability, sterile filtration is performed and the final bulk solution is filled into suitable vials and lyophilized. This manufacturing process has been studied in virus validation studies for its capacity to inactivate and/or remove viruses. Virus validation studies assess the safety of a biological product with regard Table 3 Criteria of NAT tests for release of plasma pools for fractionation. Virus
Release criterion for NAT test sensitivity
HBV HCV HIV-1 HAV B19V
b10 IU/mL ≤30 IU/mL b100 IU/mL b20 IU/mL ≤4 log10 IU/mL
B19V, parvovirus B19; HAV, hepatitis A virus; HBV, hepatitis B virus; HCV, hepatitis C virus; HIV-1, human immunodeficiency virus 1; NAT, nucleic acid amplification technology. ®
Al(OH)3/QAE Sephadex adsorption
Pasteurization (heat treatment at 60°C for 10 hours in aqueous stabilized solution) Ion-exchange chromatography (DEAE–Sepharose)
20N virus filtration
Dialysis/formulation (with sucrose, glycine and salts) Sterile filtration Filling/lyophilization Fig. 1. Manufacturing process for Beriate®. Al (OH)3, aluminium hydroxide; DEAE, diethylamino ethanol; QAE, quaternary aminoethyl.
to potential virus transmission, and they identify the production steps that are effective in reducing the level of infectious viruses. Consequently, an estimate of the overall ability of a manufacturing process to inactivate and/or to remove any contaminating infectious viruses can be obtained. A prerequisite of virus validation studies is a validated scaling down of the manufacturing process to a laboratory scale, as these studies have to be performed in dedicated laboratories suitable for handling viruses according to their respective biosafety levels. In these laboratories, cell culture-derived stocks of either blood-borne viruses or appropriately selected model viruses are added (“spiked”) to intermediates of production steps derived from Beriate® (and previously Beriate® P) production lots. The manufacturing process is performed according to the validated downscale model taking into consideration all relevant process parameters: concentration of proteins and other components, temperature, pH, reaction time, and step efficacy. During the development of the former product Beriate® P, a wide range of different viruses have been studied to demonstrate the capacity of the pasteurization step, in particular, to inactivate viruses
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of different physicochemical properties. During this development period, when HCV was still called non-A, non-B hepatitis virus, studies demonstrating the reduction of HBV and non-A, non-B hepatitis virus by the manufacturing process for Beriate® P were performed in chimpanzees. In addition, a range of other viruses was employed in pasteurization studies using cell cultures (Table 4). Subsequently, in line with regulatory guidance, viruses chosen for validation studies using cell culture infectivity assays closely resemble the viruses that may potentially contaminate human plasma. Cell culture infectivity assays employing HIV and HAV are used; as currently, no cell culture infectivity assay is available for HBV or HCV, model viruses are used in virus validation studies: the specific model virus used for HCV is bovine viral diarrhea virus (BVDV; genus pestivirus), which is also a member of the same Flaviviridae family as HCV (genus hepacivirus) and is more closely related to HCV than members of the genus flavivirus [18]. For HBV, no practical test system is available [6,17], thus, unspecific model viruses such as herpesviruses (e.g. pseudorabies virus [PRV], an enveloped DNA virus, as is HBV) are used. In order to employ the relevant human parvovirus in virus validation studies, a complex cell culture infectivity assay for B19V has been developed at CSL Behring, utilizing B19V high-titre donations for spiking. As B19V has only an abortive replication cycle in cell culture, the detection of B19V-encoded proteins in the inoculated cell cultures by immunofluorescence is used to assess the titre of infectious B19V. Since animal parvoviruses as canine parvovirus (CPV) or porcine parvovirus (PPV) are considerably more resistant to physiochemical methods for virus inactivation than B19V [19–21], the inactivation capacity of pasteurization for B19V was studied. In order to assess the ability of the production process to reduce viruses in general, a range of viruses with different physicochemical properties that replicate to high titres in cell culture can be assayed in an effective, sensitive, and reliable in vitro infectivity assay were chosen by CSL Behring for their virus validation studies (Table 5) [22]. The continued improvement of safety measures, in line with current scientific knowledge and regulatory guidance, has recently led to the incorporation of an additional virus reduction step into the manufacturing process for Beriate®: virus filtration utilizing a virus filter (Planova 20 N) with a mean pore size of approximately 19 nm (Fig. 1). The virus reduction capacity of the manufacturing process for Beriate® is shown in Table 6. Virus filtration of Beriate®, a FVIII concentrate with a low VWF:FVIII ratio, has no impact on the stability of FVIII and only a minimal impact on the amount and multimeric structure of VWF (Fig. 2). Prion safety of Beriate® Transmissible spongiform encephalopathies (TSEs) are a group of fatal neurodegenerative diseases of animals (scrapie in sheep, bovine spongiform encephalopathy [BSE] in cattle) and man (CJD) that are characterized by the accumulation of the disease-associated prion protein (PrPSc) – an abnormal isoform of the cellular prion protein Table 4 Inactivation of viruses by pasteurization – studies performed during the development of Beriate® P. Virus
Virus reduction factor (log10)
HBV a Non-A, non-B hepatitis virus (HCV) a HIV CMV Poliovirus YFV BVDV Rubella virus Measles virus
≥5.9 ≥5.4 ≥5.7 ≥6.0 ≥5.6 ≥5.5 ≥5.1 ≥4.1 ≥4.6
Table 5 Panel of viruses used in validation studies at CSL Behring for Beriate® P (in accordance with CPMP/BWP/268/95) [22]. Transfusion Test virus relevant virus (model virus)
Genome Enveloped Size(nm) Resistance to treatment
HIV HCV HBV HAV B19V
RNA RNA DNA RNA DNA DNA DNA
–
HIV-1 BVDV None available a HAV B19Vb CPVc Herpes virus (nonspecific model)
Low Low Medium High Very high Very high Medium
(PrPC) – in the central nervous system. PrPSc may also accumulate in other tissues, depending on the host and type of TSE involved. In 1996, a new variant of CJD, vCJD was identified [23]. Epidemiological and scientific data indicate a strong association between vCJD and consumption of meat products derived from BSEinfected cattle [24]. In humans with vCJD, abnormal prions are present in both the central nervous system and the lymphoreticular system, which has given rise to the possibility of blood-borne transmissions. Transmission of vCJD by transfusion of non-leuco-depleted red blood cell concentrates in the UK [25] and experimental studies in sheep and rodents in either the preclinical or clinical stage of disease have confirmed transmission of TSE through blood [26]. Plasma used for the production of Beriate® P previously and now Beriate®, is always leuco-depleted due to the collection method (plasmapheresis plasma) or processing (leuco-depleted, recovered plasma from a whole blood collection). In addition to leuco-depletion, precautionary measures have been instigated to minimize the theoretical risk of contaminating plasma pools with human TSEs by exclusion of donors at risk of developing CJD and vCJD (geographic deferral). CSL Behring and its predecessor companies have never used plasma for the production of Beriate® P collected in the UK, and did not use plasma collected in France from the time the first case of vCJD was reported in that country. Despite the very low risk of vCJD contamination of plasma pools for fractionation, prion removal studies have been performed to evaluate the ability of the overall manufacturing process to remove prion proteins. As the physicochemical properties of potential prion contamination in plasma is not known, two different prion spike preparations have been used in spiking studies. These were prepared from the brains of hamsters inoculated with the hamster-adapted scrapie prion strain 263 K: microsomes (membrane-associated infectious prions) and purified PrPSc (a detergent-extracted, non-membrane-associated infectious prion) [27]. The prion material in the spiked starting material Table 6 Virus reduction capacity of the manufacturing process of Beriate®.
Pasteurization Ion exchange chromatography 20 N Virus filtration Overall virus reduction factor
®
80–100 50–70 45 25–30 18–24 18–24 120–200
B19V, parvovirus B19; BVDV, bovine viral diarrhoea virus; CPV, canine parvovirus; DNA, deoxyribonucleic acid; HAV, hepatitis A virus; HBV, hepatitis B virus; HCV, hepatitis C virus; HIV-1, human immunodeficiency virus type 1; RNA, ribonucleic acid. a According to the European Medicines Agency, 1996. b Blood-borne virus B19V studied for inactivation capacity of pasteurization step. c Model virus CPV studied for removal capacity of the manufacturing process.
Manufacturing process
BVDV, bovine viral diarrhoea virus; CMV, cytomegalovirus; HBV, hepatitis B virus; HCV, hepatitis C virus; HIV, human immunodeficiency virus; YFV, yellow fever virus. a Manufacturing process of Beriate® P studied in chimpanzees.
Yes Yes Yes No No No Yes
Virus reduction factors (log10) HIV
BVDV
PRV
HAV
Parvovirus a
≥6.8 3.3 ≥6.0 ≥16.1
≥9.3 3.0 ≥5.8 ≥18.1
4.7 2.1 ≥7.2 ≥14.0
3.9 1.3 ≥5.5 ≥10.7
≥3.8 b 3.4 c 3.4 c ≥10.6
B19V, parvovirus B19; BVDV, bovine viral diarrhoea virus; CPV, canine parvovirus; HAV, hepatitis A virus; HIV, human immunodeficiency virus; PRV, pseudorabies virus. a Virus reduction factor demonstrated for: b B19V (relevant human parvovirus); c CPV (model virus for B19V).
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Summary and conclusions
Fig. 2. Multimeric analysis of von Willebrand factor in Beriate® after virus filtration. Line 1, starting material for virus filtration; line 2, pool after pre-filtration (0.1 μm); line 3, pool after virus filtration (20 N); line 4, standard human plasma.
and the final sample was quantified using a conformation-dependent immunoassay [28,29]. These prion evaluation studies were performed in a similar manner to the virus validation studies using a validated downscale of the manufacturing process in a dedicated laboratory [30] and spiking prion preparations to product intermediates derived from production lots. The prion reduction capacities of individual steps are added together to produce an overall prion reduction factor, as in virus validation studies. However, the results generated using this approach must be interpreted with caution as the spike preparation may be heterogeneous, with one fraction preferentially removed by one manufacturing step and the same sub-fraction removed by another step [30]. As the manufacturing process of Beriate® may alter the physicochemical features of the spiked preparation and modify its removability, the overall manufacturing process of Beriate® was studied in one experiment covering nearly the whole manufacturing process by spiking dissolved cryoprecipitate and studying the filtrate of the virus filtration for residual PrPSc signal. The overall prion reduction capacity of the Beriate® manufacturing process is shown in Table 7.
Table 7 Prion reduction capacity of the improved Beriate® process. Overall manufacturing process
Al(OH)3 adsorption, Al(OH)3/QAE Sephadex adsorption, pasteurization, ion exchange chromatography, virus filtration (Planova 20 N)
5
Prion reduction factors [log10] Microsomes
Purified PrPSc
≥3.6
≥4.0
Microsomes, membrane-associated prion preparation; purified PrPSc, non-membrane associated prion preparation; Al(OH)3, aluminium hydroxide; QAE, quaternary aminoethyl. ®
The virus safety of Beriate® (and previously Beriate® P) is ensured through careful selection of donor centres and donors, screening of each donation for transfusion-relevant viruses by serological and NAT assays, the release of the plasma pool for fractionation based on nonreactivity for HBsAg and antibodies against HIV-1/HIV-2 and genomic material of HAV, HBV, HCV, HIV-1/-2, and high titres of B19V by sensitive NAT/PCR assays, and the virus reduction capacity of the manufacturing process. This virus reduction capacity is especially enhanced through the dedicated virus reduction steps of pasteurization and virus filtration (also called nanofiltration) employing a virusretentive filter with a pore size of approximately 19 nm. Further manufacturing steps for protein purification such as chromatography reliably contribute to virus removal. The manufacturing process for Beriate® (and previously Beriate® P) removes prions efficiently. An assessment by the U.S. Food and Drug Administration (FDA) has concluded that the risk of transmission of prions by FVIII products with a prion reduction factor of 4 log10 or more is very remote [31]. In conclusion, the measures taken in the production of Beriate® (and previously Beriate® P) ensure a high margin of pathogen safety for this product and are effective for enveloped viruses such as HIV, HBV and HCV and the non-enveloped viruses HAV and B19V. No proven cases of transmission of blood-borne viruses have been reported during clinical trials and through post-marketing surveillance over 20 years of clinical use, even before the virus filtration step was introduced. The incorporation of a virus filtration step into the manufacturing process is part of our continuous effort to improve the quality and safety of Beriate®. Conflict of interest statement The author is an employee of CSL Behring. Acknowledgements The contribution of the entire team of the Department of Virus Safety Development at CSL Behring GmbH, most notably to Dr T Nowak, Dr W Schäfer and Dr B Popp, to the execution of the virus validation studies is acknowledged. References [1] Busch MP, Young MJ, Samson SM, Mosley JW, Ward JW, Perkins HA, et al. Risk of human immunodeficiency virus (HIV) transmission by blood transfusions before the implementation of HIV-1 antibody screening. Transfusion 1991;31:4–11. [2] Choo QL, Kuo G, Weiner AJ, Overby LR, Bradley DW, Houghton M. Isolation of a cDNA clone derived from a blood-borne non-A, non-B viral hepatitis genome. Science 1989;244:359–62. [3] Kuo G, Choo QL, Alter HJ, Gitnick GL, Redeker AG, Purcell RH, et al. An assay for circulating antibodies to a major etiologic virus of human non-A, non-B hepatitis. Science 1989;244:362–4. [4] Schreiber GB, Busch MP, Kleinman SH, Korelitz JJ. The risk of transfusion-transmitted viral infections. The Retrovirus Epidemiology Donor Study. N Engl J Med 1996;334:1685–90. [5] The European Agency for the Evaluation of Medicinal Products. The introduction of nucleic acid amplification technology (NAT) for the detection of hepatitis C virus RNA in plasma pools. CPMP/BWP/390/97. 24 March 1998. (Annex V of CPMP/BWP/ 269/95, rev. 3), 24 March 1998. Available at: http://www.ema.europa.eu/docs/ en_GB/document_library/Scientific_guideline/2009/09/WC500003613.pdf [last accessed November 3, 2013]. [6] The European Agency for the Evaluation of Medicinal Products. Note for guidance on plasma-derived medicinal products. CPMP/BWP/269/95, rev. 3, 25 January 2001. Available at http://www.ema.europa.eu/docs/en_GB/document_library/Scientific_ guideline/2009/09/WC500003613.pdf [last accessed November 3, 2013]. [7] Council of Europe. European Pharmacopoeia. Nucleic acid amplification techniques European Pharmacopoeia; 2011 20621 [7.0–07/2010]. [8] Council of Europe. European Pharmacopoeia. Human plasma for fractionation European Pharmacopoeia; 2011 0853 [7.0–07/2008]. [9] European Medicines Agency. Guideline on epidemiological data on blood transmissible infections. EMEA/CPMP/BWP/125/04, 20 January 2005. Available at: http://www.ema.europa.eu/docs/en_GB/document_library/Scientific_guideline/2009/ 09/WC500003718.pdf [last accessed November 3, 2013].
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