Enhanced virus safety of a solvent/detergent-treated antihemophilic factor IX concentrate by dry-heat treatment

With particular regards to the hepatitis A virus (HAV), a terminal dry-heat treatment (100°C for 30 min) process, following lyophilization, was developed to improve the virus safety of a solvent/detergent-treated antihemophilic factor IX concentrate. The loss of factor IX activity during dry-heat treatment was of about 3%, as estimated by a clotting assay. No substantial changes were observed in the physical and biochemical characteristics of the dry-heat-treated factor IX compared with those of the factor IX before dry-heat treatment. The dry-heat-treated factor IX was stable for up to 24 months at 4°C. The dry-heat treatment after lyophilization was an effective process for inactivating viruses. The HAV and murine encephalomyocarditis virus (EMCV) were completely inactivated to below detectable levels within 10 min of the dry-heat treatment. Porcine parvovirus (PPV) and bovine herpes virus (BHV) were potentially sensitive to the treatment. The log reduction factors achieved during lyophilization and dry-heat treatment were ≥5.60 for HAV, ≥6.08 for EMCV, 2.64 for PPV, and 3.59 for BHV. These results indicate that dry-heat treatment improves the virus safety of factor IX concentrates, without destroying the activity. Moreover, the treatment represents an effective measure for the inactivation of nonlipid enveloped viruses, in particular HAV, which is resistant to solvent/detergent treatment.     

Improvement of virus safety of an antihemophilc factor IX by virus filtration process

Viral safety is an important prerequisite for clinical preparations of plasma-derived pharmaceuticals. One potential way to increase the safety of therapeutic biological products is the use of a virus-retentive filter. In order to increase the viral safety of human antihemophilic factor IX, particularly in regard to non-enveloped viruses, virus removal process using a polyvinylidene fluoride membrane filter (Viresolve NFP) has been optimized. The most critical factor affecting the filtration efficiency was operating pH and the optimum pH was 6 or 7. Flow rate increased with increasing operating pressure and temperature. Recovery yield in the optimized productionscale process was 96%. No substantial changes were observed in the physical and biochemical characteristics of the filtered factor IX in comparison with those before filtration. A 47-mm disk membrane filter was used to simulate the process performance of the production-scale cartridges and to test if it could remove several experimental model viruses for human pathogenic viruses, including human hepatitis A virus (HAV), porcine parvovirus (PPV), murine encephalomyocarditis virus (EMCV), human immunodeficiency virus type 1 (HIV), bovine viral diarrhea virus (BVDV), and bovine herpes virus (BHV). Nonenveloped viruses (HAV, PPV, and EMCV) as well as enveloped viruses (HIV, BVDV, and BHV) were completely removed during filtration. The log reduction factors achieved were (i)v.12 for HAV, (i)t.28 for PPV, (i)u.33 for EMCV, (i)u.51 for HIV, (i)u.17 for BVDV, and (i)u.75 for BHV. These results indicate that the virus filtration process successfully improved the viral safety of factor IX.     

Removal and inactivation of viruses during the manufacture of a high-purity antihemophilic factor IX from human plasma

The purpose of this study was to evaluate the efficacy and mechanisms of the solvent/detergent (S/D) treatment, DEAE-toyopearl 650M anion-exchange column chromatography, heparin-sepharose 6FF affinity column chromatography, and Viresolve NFP filtration steps employed in the manufacture of high-purity antihemophilic factor IX (Green-Nine VF) from human plasma, with regard to removal and/or inactivation of blood-borne viruses. A variety of experimental model viruses for human pathogenic viruses, including human immunodeficiency virus (HIV), bovine herpes virus (BHV), bovine viral diarrhoea virus (BVDV), hepatitis A virus (HAV), murine encephalomyocarditis virus (EMCV), and porcine parvovirus (PPV), were all selected for this study. Samples from relevant stages of the production process were spiked with each virus and subjected to scale-down processes mimicking the manufacture of high-purity factor IX. Samples were collected at each step, immediately titrated using a 50% tissue culture infectious dose (TCID₅₀), and virus reduction factors were evaluated. S/D treatment using the organic solvent, tri (n-butyl) phosphate (TNBP), and the detergent, Tween 80, was a robust and effective step in inactivation of enveloped viruses. Titers of HIV, BHV, and BVDV were reduced from the initial titer of 6.06, 7.72, and 6.92 log₁₀ TCID₅₀, respectively, reaching undetectable levels within 1 min of S/D treatment. DEAE-toyopearl 650M anion-exchange column chromatography was found to be a moderately effective step in the removal of HAV, EMCV, and PPV with log reduction factors of 1.12, 2.67, and 1.38, respectively. Heparin-sepharose 6FF affinity column chromatography was also moderately effective for partitioning BHV, BVDV, HAV, EMCV, and PPV with log reduction factors of 1.55, 1.35, 1.08, 1.19, and 1.61, respectively. The Viresolve NFP filtration step was a robust and effective step in removing all viruses tested, since HIV, BHV, BVDV, HAV, EMCV, and PPV were completely removed during the filtration step with log reduction factors of ≥ 5.51, ≥ 5.76, ≥ 5.18, ≥ 5.34, ≥ 6.13, and ≥ 4.28, respectively. Cumulative log reduction factors of HIV, BHV, BVDV, HAV, EMCV, and PPV were ≥ 10.52, ≥ 12.07, ≥ 10.49, ≥ 7.54, ≥ 9.99, and ≥ 7.24, respectively. These results indicate that the production process for GreenNine VF has a sufficient virus reduction capacity for achievement of a high margin of virus safety.     

Viral clearance during the manufacture of urokinase from human urine

The purpose of the present study was to evaluate the efficacies and mechanisms of the PAB (para-amino benzamidine) affinity column chromatography, virus filtration, pasteurization (60°C heat treatment for 10 h), and lyophilization steps employed in the manufacture of urokinase from human urine with regard to the removal and/or inactivation of human immunodeficiency virus (HIV), bovine viral diarrhoea virus (BVDV), bovine herpes virus (BHV), and murine encephalomyocarditis virus (EMCV). Samples from relevant stages of the production process were spiked with each virus and subjected to scale-down processes mimicking the manufacture of urokinase. Samples were collected at each step, immediately titrated using a 50% tissue culture infectious dose (TCID₅₀), and the virus reduction factors evaluated. PAB chromatography was found to be an effective step for removing BVDV, BHV, and EMCV with log reduction factors of 2.79, 6.50, and 5.96, respectively. HIV, BVDV, BHV, and EMCV were completely removed during the Viresolve NFP filtration step with log reduction factors of ≥6.06, ≥4.60, ≥5.44, and ≥6.87, respectively. Pasteurization was also found to be a robust and effective step in inactivating all the viruses tested, since there were no residual viruses detected after the pasteurization process. The log reduction factors achieved by pasteurization were ≥5.73 for HIV, ≥3.86 for BVDV, ≥6.75 for BHV, and ≥5.92 for EMCV. Lyophilization showed significant efficacy for inactivating BVDV, BHV, and EMCV with log reduction factors of 2.69, 1.37, and 4.70, respectively. These results indicate that the production process for urokinase exhibited a sufficient viral reducing capacity to achieve a high margin of virus safety.     

Virus removal capacity at varying ionic strength during nanofiltration of AlphaNine® SD

Nanofiltration is incorporated into the manufacturing processes of many protein biopharmaceuticals to enhance safety by providing the capacity to retain pathogens while allowing protein drugs to pass through the filter. Retention is mainly a function of size; however, the shape of the pathogen may also influence retention. The ability of the Viresolve^® Pro nanofilter to remove different sized viruses during the manufacture of a Coagulation Factor IX (Alphanine^® SD) was studied at varying ionic strength, a process condition with the potential to affect virus shape and, hence, virus retention. Eight viruses were tested in a scale-down of the nanofiltration process. Five of the viruses (EMCV, Reo, BVDV, HIV, PRV) were nanofiltered at normal sodium processing conditions and three (PPV, HAV and WNV) were nanofiltered at higher and lower sodium. Representative Reduction Factors for all viruses were ≥4.50 logs and removal was consistent over a wide range of ionic strength.     

Inactivation and clearance of viruses during the manufacture of high purity factor IX.

Haemophilia is a bleeding disorder characterised by a deficiency in Factor IX. Replacement therapy in the form of a Factor IX concentrate is a widely accepted practice. In this paper we describe a double virus inactivated chromatographic process for producing a high purity Factor IX product, MonoFIX((R))-VF. The process involves separation of the prothrombin complex by cryoprecipitation, fraction I precipitation and DEAE-cellulose adsorption, further ion-exchange chromatography of crude Factor IX, followed by solvent/detergent treatment. Heparin affinity chromatography is then used to further purify Factor IX. Final nanofiltration is sequential through 35 nm then 15 nm membrane filters. The principal virus inactivation/removal steps are solvent/detergent treatment and nanofiltration and the partitioning of relevant and model viruses provides further reduction in virus load through the production process.Solvent/detergent treatment was shown to achieve log reduction factors of 4.5 for HIV-1, 5.1 for Sindbis virus, 6.1 for vesicular stomatitis virus (VSV), 5.1 for bovine viral diarrhoea virus (BVDV) and 5.3 for pseudorabies virus (PRV). BVDV is a model for hepatitis C virus (HCV), and pseudorabies virus (PRV), like hepatitis B virus (HBV) is an enveloped DNA virus. Using scaled down models of the production process, we have also demonstrated the neutralization/partitioning of at least 6 logs of hepatitis A virus (HAV) during cryoprecipitation, Fraction I precipitation, and the DEAE adsorption and elution step, and a further 1.6 log reduction in HAV load as a result of heparin affinity chromatography. The log reduction factors for HAV as a result of the second ion-exchange chromatography step and as a result of enhanced neutralisation associated with solvent/detergent treatment were not significant. Nanofiltration was shown to contribute a further log reduction factor of 6.7 for HAV and 5.8 for BVDV indicating that log reduction factors of this order would be obtained with other viruses of a similar or larger size, such as HIV, HBV and HCV.Overall, these studies indicate that MonoFIX-VF is a product with an extremely high level of viral safety.     

Evaluation of wet pasteurization of a factor VIII concentrate produced by controlled-pore silica adsorption.

In the routine production of a factor VIII concentrate (produced by adsorption of contaminating proteins in cryoprecipitate to controlled-pore silica and concentration of the factor VIII effluent by ultrafiltration) the terminal dry-heat treatment has been replaced by pasteurization in the liquid state. High effectivity of this procedure with respect to virus inactivation was demonstrated using a variety of both lipid- and protein-enveloped model viruses, including HIV. Pair-wise quality control of dry-heated and pasteurized product revealed no significant differences, except in the composition of the formulation buffer. In a clinical study in which 17 patients with haemophilia A participated the pasteurized product was well tolerated and in vivo recovery and half-life of factor VIII were in the same (normal) range as found for the dry-heated counterpart.     

Use of lipid solvents for viral inactivation in factor VIII concentrates

Model virus inactivation studies with lipid solvents were carried our in antihemophilic factor concentrates. The procoagulant activity obtained was >/=80% recovery with 20% amyl acetate-0.1% deoxycholate. A concurrent reduction of four logs of virus titer was obtained for model viruses provided the viral mass contained significant amounts (>/=20%) of lipid. From this preliminary study it appears that further investigations in animal models may be warranted to demonstrate the inactivation of hepatitis B virus, non-A-non-B virus, and AIDS virus with 20% amyl acetate-0.1% deoxycholate in antihemophilic factor concentrates.     

Virus safety of avital bone tissue transplants: evaluation of sterilization steps of spongiosa cuboids using a peracetic acid-methanol mixture.

The aim of this study was to validate the virus-inactivating/eliminating capacity of the manufacturing process of spongiosa cuboids. Both the sterilization step with peracetic acid (PAA)/ethanol and the defatting step of bones with chloroform/methanol (2:1, v/v) were investigated. Relevant enveloped, non-enveloped, and model viruses belonging to different virus families were included in the investigation: human immunodeficiency virus type 2 (HIV-2), hepatitis A virus (HAV), poliovirus (PV-1), pseudorabies virus (PRV), porcine parvovirus (PPV), and bovine virus diarrhoea virus (BVDV). Treatment of virus-spiked spongiosa cuboids for 4 hours at room temperature (RT) with 1% PAA/24% ethanol (PES) efficiently inactivated most viruses. Titres were reduced by more than 4 log(10)with the exception of HAV. The defatting step with chloroform/methanol reduced HAV titres by a factor of >/=7.0 log(10). From these results it can be concluded that the treatment of spongiosa cuboids with (i) chloroform/methanol and (ii) 1% PAA/24% ethanol solution leads to a virus-safe medicinal product.     

Effect of gamma irradiation on human cortical bone transplants contaminated with enveloped and non-enveloped viruses.

In the production of bone grafts intended for transplantation, basic safety measures to avoid the transmission of pathogens are selection and serological screening of donors for markers of virus infections. As an additional safety tool we investigated the effect of gamma irradiation on the sterility of human bone diaphysis transplants and evaluated its impact on the virus safety of transplants. Model viruses were included in the study to determine the dose necessary to achieve a reduction factor for the infectivity titres of at least 4 log(10) at a temperature of -30+/-5 degrees C. The following viruses were used: human immunodeficiency virus type 2 (HIV-2), hepatitis A virus (HAV), and poliovirus (PV-1), and the following model viruses: pseudorabies virus (PRV) as a model for human herpesviruses, bovine viral diarrhoea virus (BVDV) for HCV, and bovine parvovirus (BPV) for parvovirus B19. A first approach was to determine the D(10) values (kGy) for the different viruses (virus inactivation kinetics: BPV 7.3; PV-1 7.1; HIV-2 7.1; HAV 5.3; PRV 5.3; BVDV <3.0 kGy). Based on these results, inactivation of these viruses was studied in experimentally contaminated human bone transplants (femoral diaphyses). For BPV, the most resistant one of the viruses studied, a dose of approximately 34 kGy was necessary to achieve a reduction of infectivity titres of 4 log(10). We therefore recommend a dose of 34 kGy for the sterilisation of frozen bone transplants.     

Virus Validation of PH 4-treated Human Immunoglobulin Produced by the Cohn Fractionation Process

To assess the virus reducing capacity of Cohn's cold ethanol fractionation process for the production of intravenous (IVIg) and intramuscular (IMIg) immunoglobulin products, and treatment of these products at pH 4, a validation study of virus removal and/or inactivation was performed using both lipid-enveloped viruses [human immunodeficiency virus (HIV), bovine viral diarrhoea virus (BVDV) and pseudorabies virus (PSR)], and non-lipid-enveloped viruses [(simian virus 40 (SV40) and encephalomyocarditis virus (EMC)]. For the cold ethanol fractionation process, overall reduction factors of 3.0 logs, > or = 2.6 (< 5.5) logs, 4.6 logs, 5.8 logs and > or = 2.6 (< 6.2) logs were found for HIV, BVDV, PSR, SV40 and EMC, respectively. For all tested viruses the precipitation of fraction III from fraction II + III was the most effective step. From the overall reduction factors it appears that cold ethanol fractionation, although capable of reducing viral infectivity to a significant extent, is not sufficient to meet the requirements of regulatory bodies for viral safety of immunoglobulin products. However, pH 4 treatment contributes effectively to the viral safety of the final products. Treatment at pH 4.05 and 37 degrees C for 16 h, as is applied to IVIg, yields reduction factors of > or = 8.4 logs, > or = 4.0 logs, > or = 7.1 logs, 4.8 logs and 1.4 logs for HIV, BVDV, PSR, SV40 and EMC, respectively. The effectiveness of this process step could be enhanced by extending incubation to 40 h at pH 4.25 compared to 16 h at pH 4.05. The extended incubation, as applied in the production of IMIg, yields a reduction of infectivity of SV40 by > or = 5.5 (< 8.0) logs and of EMC by > or = 4.1 (< 7.1) logs. Storage of IMIg, which is formulated as a solution, at 2-8 degrees C also contributes to virus safety. For storage periods of 8 weeks or longer, reduction factors of 2 to 6 logs were found for all viruses, except for BVDV which remained unaffected. These data indicate that the production processes for IVIg and IMIg as described here have sufficient virus reducing capacity to achieve a high margin of virus safety.     

Removal of Viruses from Human Intravenous Immune Globulin by 35 nm Nanofiltration

Viral safety is an important prerequisite for clinical immunoglobulin preparations. A common manufacturing practice is to utilize several virus removal/inactivation process steps to ensure the safety of human intravenous immunoglobulin (IVIg). In this regard, we examined the use of Planova 35 nm filters to reduce potential loads of both non-enveloped and enveloped viruses prior to end-stage solvent detergent treatment. The nanofiltration process was validated for removal of a variety of enveloped and non-enveloped viruses ranging in size from 70 nm to 18 nm including: Sindbis virus, Simian Virus 40 (SV40), Bovine Viral Diarrhoea virus (BVDV), Feline Calicivirus, Encephalomyocarditis virus (EMC), Hepatitis A virus (HAV), Bovine Parvovirus (BPV) and Porcine Parvovirus (PPV). The filtration procedure was carried out by first spiking a 7% solution of IVIg with < 10(8) virus. The spiked IVIg solution was then filtered through a 75 nm Planova filter followed by two Planova 35 nm filters in series (75/35/35). The 75 nm prefilter is incorporated into this process to increase the capacity of the 35 nm viral removal filters. As a result of the inclusion of the 75 nm pre-filtration step it was possible to assess the removal of virus by the 35 nm filters independent of possible aggregation of the initial viral spiking material. Samples were collected at each step and immediately titred by viral plaque assay. A process control sample of the spiked load solution was held at the same conditions for the duration of the filtration process and then titred to determine the extent to which antibody neutralization may have contributed to overall viral reduction. Control assays of spiked IVIg were performed to establish the degree of toxicity of the IVIg solution to the indicator cell lines and the extent to which the IVIg interfered with plaque formation in the assay system. This combined data was used to establish assay sensitivity for the calculation of log removal by the filtration process. It was noted that toxicity/interference effects could have a significant effect upon apparent log reductions, and these effects could vary greatly, even within viruses of the same family. The results of these studies indicate that 35 nm filtration is very effective for removing substantial quantities of both non-enveloped and enveloped viruses from IVIg. Complete clearance (to the limits of detection of the assay) was obtained for all viruses larger than 35 nm. Interestingly, viruses reported to have mean diameters of less than 35 nm (EMC and HAV) were at least partially removed by the filtration (4.3 and > 4.7 logs removal, respectively). Even small viruses such as PPV were to some extent removed from the IVIg solution by the filters (2.6 logs removal). Reduction of BPV would not be assessed due to extensive neutralization and interference with plaque formation by the IVIg. Sindbis and SV40 also were subject to neutralization and assay interference due to the IVIg, though to a lesser extent. We conclude from these studies that the 35 nm mean pore size is functionally efficient in removal of smaller size viruses from spiked IVIg concentrates.     

Partitioning and inactivation of viruses by the caprylic acid precipitation followed by a terminal pasteurization in the manufacturing process of horse immunoglobulins

Caprylic acid (octanoic acid), has been used for over 50 years as a stabilizer of human albumin during pasteurization. In addition caprylic acid is of great interest, by providing the advantage of purifying mammalian immunoglobulins and clearing viruses infectivity in a single step. Exploiting these two properties, we sequentially used the caprylic acid precipitation and the pasteurization to purify horse hyperimmune globulins used in the manufacturing of Sérocytol. To evaluate the effectiveness of the process for the removal/inactivation of viruses, spiking studies were carried out for each dedicated step. Bovine viral diarrhoea virus (BVDV), pseudorabies virus (PRV), encephalomyocarditis virus (EMCV) and minute virus of mice (MVM) were used for the virological validation. Our data show that the treatment with caprylic acid 5% (v/v) can effectively be used as well to purify or to ensure viral safety of immunoglobulins. Caprylic acid precipitation was very efficient in removing and/or inactivating enveloped viruses (PRV, BVDV) and moderately efficient against non-enveloped viruses (MVM, ECMV). However the combination with the pasteurization ensured an efficient protection against both enveloped and non-enveloped viruses. So that viruses surviving to the caprylic acid precipitation will be neutralized by pasteurization. Significant log reduction were achieved > or =9 log(10) for enveloped viruses and 4 log(10) for non-enveloped viruses, providing the evidence of a margin of viral safety achieved by our manufacturing process. Its a simple and non-expensive manufacturing process of immunoglobulins easily validated that we have adapted to a large production scale with a programmable operating system.     

Evaluation of virus decontamination techniques for porcine embryos produced in vitro

The objective of this study was to explore approaches to decontaminate embryos either contaminated naturally or under experimental conditions with different viruses. Embryos were obtained from in vitro maturation and fertilisation of porcine oocytes. After 7 days of development, morula and blastocyst stages were exposed for 1 h to the following viruses: encephalomyocarditis virus (EMCV), porcine circovirus type 2 (PCV2), porcine parvovirus (PPV), porcine reproductive and respiratory syndrome virus (PRRSV), and bovine viral diarrhea virus (BVDV) at an infectivity of 100 TCID50/mL. Embryos samples were treated with different washing procedures, which all included the following standard washing solutions: PBS+0.4% BSA (five times for 10 s), Hank's+0.25% trypsin (two times for 60-90 or 120-150 s, or one time of 5 min), Hank's+0.1 mg/mL DNase 1+20 U/mL RNase One (one time of 30 min) and PBS+0.4% BSA again (five times for 10s). Two new approaches were used to improve trypsin treatment, 0.1% hyaluronidase (one time for 5 min) instead of trypsin and a pre-incubation with oviductal cells. Therefore, in the first experiment, oocytes received standard maturation treatments and in the second, they were also co-incubated with oviductal cells for the last 3 h of maturation. The effectiveness of the different washing techniques in removing viruses was evaluated by polymerase chain reaction (PCR) analysis. In the first experiment, trypsin treatment did not eliminate PRRSV, PPV, PCV, and EMCV from contaminated embryos. Surprisingly, treatment with hyaluronidase eliminated all tested viruses. In the second experiment, all viruses tested were removed from the oocytes following the different enzymatic treatments. In conclusion, in vitro embryo decontamination was more effective following exposure to oviductal secretions and hyaluronidase eliminated more virions than trypsin in washing techniques.     

Comparison of the inactivation of canine and bovine parvovirus by freeze-drying and dry-heat treatment in two high purity factor VIII concentrates.

The inactivation of bovine parvovirus (BPV) and canine parvovirus (CPV) by freeze-drying and terminal dry-heat treatment at 80 degrees C for 72 h has been investigated in two high purity factor VIII concentrates. In one product, CPV was slightly more resistant to freeze-drying compared to BPV, i.e. 0.7 vs. 1.4 log. However, BPV was substantially more resistant to heat-treatment compared to CPV, i.e. 1.3 vs. > 3.1 log inactivation after 72 h at 80 degrees C. In a second product, CPV was also slightly more resistant to freeze-drying than BPV, i.e. 0.2 vs. 1.3 log inactivation. However, heat-treatment gave essentially similar inactivation for both viruses, i.e. 2.8-3.4 log after 72 h at 80 degrees C. In conclusion, the resistance of these parvovirus models is dependent both on the type of virus and on the specific product involved.     

Replication of hepatitis A viruses with chimeric 5' nontranslated regions.

The role of the 5' nontranslated region in the replication of hepatitis A virus (HAV) was studied by analyzing the translation and replication of chimeric RNAs containing the encephalomyocarditis virus (EMCV) internal ribosome entry segment (IRES) and various lengths (237, 151, or 98 nucleotides [nt]) of the 5'-terminal HAV sequence. Translation of all chimeric RNAs, truncated to encode only capsid protein sequences, occurred with equal efficiency in rabbit reticulocyte lysates and was much enhanced over that exhibited by the HAV IRES. Transfection of FRhK-4 cells with the parental HAV RNA and with chimeric RNA generated a viable virus which was stable over continuous passage; however, more than 151 nt from the 5' terminus of HAV were required to support virus replication. Single-step growth curves of the recovered viruses from the parental RNA transfection and from transfection of RNA containing the EMCV IRES downstream of the first 237 nt of HAV demonstrated replication with similar kinetics and similar yields. When FRhK-4 cells infected with recombinant vaccinia virus producing SP6 RNA polymerase to amplify HAV RNA were transfected with plasmids coding for these viral RNAs or with subclones containing only HAV capsid coding sequences downstream of the parental or chimeric 5' nontranslated region, viral capsid antigens were synthesized from the HAV IRES with an efficiency equal to or greater than that achieved with the EMCV IRES. These data suggest that the inherent translation efficiency of the HAV IRES may not be the major limiting determinant of the slow-growth phenotype of HAV.     

Viral safety of C1-inhibitor NF

We studied the efficacy of virus reduction by three process steps (polyethylene glycol 4000 (PEG) precipitation, pasteurization, and 15 nm virus filtration) in the manufacturing of C1-inhibitor NF. The potential prion removing capacity in this process was estimated based on data from the literature. Virus studies were performed using hepatitis A virus (HAV) and human immunodeficiency virus (HIV) as relevant viruses and bovine viral diarrhea virus (BVDV), canine parvovirus (CPV) and pseudorabies virus (PRV) as model viruses, respectively. In the PEG precipitation step, an average reduction in infectious titer of 4.5 log₁₀ was obtained for all five viruses tested. Pasteurization resulted in reduction of infectious virus of >6 log₁₀ for BVDV, HIV, and PRV; for HAV the reduction factor was limited to 2.8 log₁₀ and for CPV it was zero. Virus filtration (15 nm) reduced the infectious titer of all viruses by more than 4.5 log₁₀. The overall virus reducing capacity was >16 log₁₀ for the LE viruses. For the NLE viruses CPV and HAV, the overall virus reducing capacities were >8.7 and >10.5 log₁₀, respectively. Based on literature and theoretical assumptions, the prion reducing capacity of the C1-inhibitor NF process was estimated to be >9 log₁₀.     

Comparison of the efficacy of virus inactivation methods in allogeneic avital bone tissue transplants

Severalprocedures for inactivating viruses are used presently in the context of bonetissue transplants. Common methods used are gamma irradiation (25kGy), treatment with moist heat (82.5°C/15min., lobator-sd2-system) as well as chemical sterilisation usingperacetic acid-ethanol treatment (PES, 2% peracetic acid, 96% ethanol, Aqua[2:1:1], 200 mbar, agitation, 4 hours). Based on national andinternational guidelines, we tested the antivirucidal effectiveness of thesemethods in human bone transplants. Three enveloped viruses: humanimmunodeficiency virus type 2 (HIV-2), pseudorabies virus (PRV), bovine virusdiarrhoea virus (BVDV), and three non–enveloped viruses were used:hepatitis A virus (HAV), poliovirus (PV-1), porcine/bovine parvovirus (PPV,BPV). Defatted spongiosa cuboids served as model in chemical treatmentexperiments, while cortical diaphyses were used in gamma irradiationexperiments, and the effects of thermal treatment were tested in preparedfemoral heads. The log₁₀ reduction was measured by cytopathogeniceffects after virus titration (TCID₅₀/mL). A dose of at least 33.9kGy (bone model) at –30 ± 5°C wasnecessary to achieve a sufficient reduction (4 log₁₀ steps) of BPV,the most resistant one of all viruses investigated. Thermal treatment as wellasPES treatment led to a reduction of virus titres by more than 4log₁₀. Only HAV showed a reduction below 4 log₁₀ (2.87)with PES. After validation of the defatting step included for HAV-infectedcells, a HAV-reduction of over 7 log₁₀ was found. All threesterilisation methods tested are recommended for bone transplant sterilisation,but only provided that additional safety measures (anamnestic informations,infectious serology, PCR in case of multiorgan donors) are taken.     

Assessment of Needs for Plasma for Fractionation in Europe

In the early 1990s, a series of outbreaks of hepatitis C (HCV) infections clustering among recipients of certain lots of plasma-derived medicinal products (PDMP) alarmed regulatory authorities, manufacturers and the public alike. Also, a few episodes of Hepatitis A (HAV) infections occurred in haemophiliacs receiving solvent-detergent-treated factor VIII concentrates. Thus, several measures were brought into effect to reestablish the safety of the incriminated products and to further increase the margin of safety of PDMP in general. Therefore, intramuscular immunoglobulins had to be free of HCV RNA as shown by nucleic acid amplification technology (NAT) in the final products. Furthermore, the manufacturing process of PDMP had to be validated for both viral inactivation and elimination. Finally, HCV-NAT was to be standardised and implemented as a validated test of plasma pool samples.In 1994, a joint meeting of EPFA, EAPPI and Regulatory Authorities was held in Brussels to outline the state of the art and to delineate the actions to be taken. Five years later, in 1999, the incidence rates of HIV, HBV and HCV in unpaid blood donors have been minimized, especially in European countries. With probabilities for window period donations as low as 0.6 in 1 million for both HIV and HCV and 2.1 in 1 million for HBV in Switzerland, labile blood products have reached extreme, but not absolute safety. The introduction of HCV-NAT roughly doubles this safety resulting in a 1 in 3 million probability of a window donation.Concomittantly, extensive viral validation studies document effective inactivation and removal of viruses in PDMP. The demonstrated margins of safety, expressed as logarithmical reduction factors (LRF), range from 4 to over 20 log(10), depending on product, virus, and inactivation procedure used. Further progress to even safer PDMP shall be acomplished by consolidating the GMP processes, abandoning of obsolete requirements and harmonising national regulations within Europe. Before introducing new measures for additional agents such as HAV or Parvovirus B 19, gains and risks and even potential new threats have to be carefully assessed. Alternative efforts for the safeguard of patients, e.g. vaccination for HAV, need to be balanced against the risks of changing established and validated manufacturing procedures of PDMP with long-lasting safety records.