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.     

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.     

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.     

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.     

Inactivation kinetics of model and relevant blood-borne viruses by treatment with sodium hydroxide and heat.

To determine the efficacy of a clean-in-place system for the inactivation of viruses present in human plasma, the effect of 0.1 M sodium hydroxide at 60 degrees C on viral infectivity was investigated. Inactivation of the following model and relevant viruses were followed as a function of time: human hepatitis A virus (HAV), canine parvovirus (CPV; a model for human parvovirus B-19) pseudorabies virus (PRV, a model for hepatitis B virus), and bovine viral diarrhoea virus (BVDV, a model for hepatitis C virus and human immunodeficiency virus). Infectivity of CPV was determined by a novel in situ EIA method which will prove useful for studies to validate parvovirus inactivation or removal. Infectivity of BVDV, PRV and CPV were shown to be reproducibly inactivated below the limit of detection by 0.1 M NaOH at 60 degrees C within 30 s. HAV was inactivated to below the limit of detection within 2 min. Treatment with heat alone also resulted in some log reduction for all viruses tested except for CPV which remained unaffected after heating at 60 degrees C for 16 min. Treatment of HAV with hydroxide alone (up to 1.0 m) at 15 degrees C did not lead to rapid inactivation. Collectively, these data suggest that 0.1 M NaOH at 60 degrees C for two min should be sufficient to inactivate viruses present in process residues.     

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.     

Evaluation of viral clearance in the production of HPV-16 L1 virus-like particles purified from insect cell cultures

Biopharmaceutical products produced from cell cultures have a potential for viral contamination from cell sources or from adventitious introduction during production. The objective of this study was to assess viral clearance in the production of insect cell-derived recombinant human papillomavirus (HPV)-16 type L1 virus-like particles (VLPs). We selected Japanese encephalitis virus (JEV), bovine viral diarrhea virus (BVDV), and minute virus of mice (MVM) as relevant viruses to achieve the aim of this study. A downstream process for the production of purified HPV-16 L1 VLPs consisted of detergent lysis of harvested cells, sonication, sucrose cushion centrifugation, and cesium chloride (CsCl) equilibrium density centrifugation. The capacity of each purification/treatment step to clear viruses was expressed as reduction factor by measuring the difference in log virus infectivity of sample pools before and after each process. As a result, detergent treatment (0.5% v/v, Nonidet P-40/phosphate-buffered saline) was effective for inactivating enveloped viruses such as JEV and BVDV, but no significant reduction (< 1.0 log(10)) was observed in the non-enveloped MVM. The CsCl equilibrium density centrifugation was fairly effective for separating all three relevant adventitious viruses with different CsCl buoyant density from that of HPV-16 L1 VLPs (JEV, BVDV, and MVM = 4.30, 3.10, > or = 4.40 log(10) reductions). Given the study conditions we used, overall cumulative reduction factors for clearance of JEV, BVDV, and MVM were > or = 10.50, > or = 9.20, and > or = 6.40 log(10) in 150 ml of starting cell cultures, respectively.     

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.     

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.     

Enveloped virus inactivation by caprylate: a robust alternative to solvent-detergent treatment in plasma derived intermediates.

Solvent-detergent treatment, although used routinely in plasma product processing to inactivate enveloped viruses, substantially reduces product yield from the human plasma resource. To improve yields in plasma product manufacturing, a new viral reduction process has been developed using the fatty acid caprylate. As licensure of plasma products warrants thorough evaluation of pathogen reduction capabilities, the present study examined susceptibility of enveloped viruses to inactivation by caprylate in protein solutions with varied pH and temperature. In the immunoglobin-rich solutions from Cohn Fraction II+III, human immunodeficiency virus, Type-1, bovine viral diarrhea virus (BVDV), and pseudorabies virus were inactivated by caprylate concentrations of >/=9 mM, >/=12 mM, and >/=9 mM, respectively. Compared to solvent-detergent treatment, BVDV inactivation in Fraction II+III solution was significantly faster (20-60 fold) using 16 mM caprylate. Caprylate-mediated inactivation of BVDV was not noticeably affected by temperature within the range chosen manufacturing the immunoglobulin product. In Fraction II+III solutions, IgG solubility was unaffected by 相似文献   

Virus safety of a porcine-derived medical device: evaluation of a viral inactivation method

The goal of this study was to evaluate the efficacy of a virus-inactivating process for use during the preparation of porcine-derived extracellular matrix biomaterials for human clinical implantation. Porcine small intestine, the source material for the tissue-engineered, small intestinal submucosa (SIS) biomaterial, was evaluated. Relevant enveloped, non-enveloped, and model viruses representative of different virus families were included in the investigation: porcine parvovirus (PPV), porcine reovirus, murine leukemia retrovirus (LRV), and porcine pseudorabies (herpes) virus (PRV). Samples of small intestine were deliberately inoculated with approximately 1 x 10(7) plaque-forming units (PFU) of virus which were thereafter exposed to a 0.18% peracetic acid/4.8% aqueous ethanol mixture for time periods ranging from 5 minutes to 2 hours. Enveloped viruses were more easily inactivated than non-enveloped viruses, but material processed for 30 minutes or longer inactivated all of the viruses. D(10) values were calculated and used to extrapolate the extent of inactivation after 2 hours. Viral titers were reduced by more than 14.0 log(10) PPV, 21.0 log(10) reovirus, 40.0 log(10) PRV, and 27.0 log(10) LRV, meeting international standards for viral sterility. These results demonstrate that treatment of porcine small intestine with a peracetic acid/ethanol solution leads to a virus-free, non-crosslinked biomaterial safe for xenotransplantation into humans.     

Dry-heat treatment process for enhancing viral safety of an antihemophilic factor VIII concentrate prepared from human plasma

Viral safety is a prerequisite for manufacturing clinical antihemophilic factor VIII concentrates from human plasma. With particular regard to the hepatitis A virus (HAV), a terminal dry-heat treatment (100 degrees for 30 min) process, following lyophilization, was developed to improve the virus safety of a solvent/detergent-treated antihemophilic factor VIII concentrate. The loss of factor VIII activity during dry-heat treatment was of about 5%. No substantial changes were observed in the physical and biochemical characteristics of the dry-heat-treated factor VIII compared with those of the factor VIII before dry-heat treatment. The dry-heat-treated factor VIII was stable for up to 24 months at 4oC. The dry-heat treatment after lyophilization was an effective process for inactivating viruses. The HAV, murine encephalomyocarditis virus (EMCV), and human immunodeficiency virus (HIV) were completely inactivated to below detectable levels within 10 min of the dry-heat treatment. Bovine herpes virus (BHV) and bovine viral diarrhea virus (BVDV) were potentially sensitive to the treatment. However porcine parvovirus (PPV) was slightly resistant to the treatment. The log reduction factors achieved during lyophilization and dry-heat treatment were > or =5.55 for HAV, > or =5.87 for EMCV, > or =5.15 for HIV, 6.13 for BHV, 4.46 for BVDV, and 1.90 for PPV. These results indicate that dry-heat treatment improves the virus safety of factor VIII concentrates, without destroying the activity. Moreover, the treatment represents an effective measure for the inactivation of non-lipid-enveloped viruses, in particular HAV, which is resistant to solvent/detergent treatment.     

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.     

Resistance of vaccinia virus to inactivation by solvent/detergent treatment of blood products.

The inactivation of enveloped viruses by two different solvent/detergent combinations, i.e. tri-n-butyl phosphate (TNBP)/Triton X-100 or TNBP/Tween 80, has been investigated using a high purity factor VIII (Replenate) and factor IX (Replenine) respectively. Treatment with TNBP/Triton X-100 rapidly inactivated all the typical enveloped viruses tested, i.e. Sindbis, semliki forest virus (SFV), herpes simplex virus type-1 (HSV-1) and vesicular stomatitis virus (VSV), by 3.7-5.8 log within 15 seconds. While virus inactivation with TNBP/Tween 80 was slower, effective inactivation of Sindbis, HSV-1, VSV and human immunodeficiency virus type-1, i.e. 4.1-->6.3 log, occurred within 30 minutes. In contrast, vaccinia virus was relatively resistant to inactivation in either of these solvent/detergent combinations. Incubation times of 10 minutes for TNBP/Triton X-100 or 6-24 hours for TNBP/Tween 80, were required to reach inactivation levels of about 4 log.     

辛酸盐灭活静注人免疫球蛋白中脂包膜病毒效果验证的研究

选用不同核酸类型的脂包膜病毒,其中RNA病毒为水疱性口炎病毒(VSV),DNA病毒为伪狂犬病毒(PRV),将两种指示病毒分别用于验证一定浓度的辛酸盐对某一厂家生产的人血静脉注射用丙种球蛋白(IVIG)的病毒灭活效果。结果表明,液体IVIG在辛酸钠(0.7±0.2mmol/g蛋白)、pH(5.1±0.1)、29.5~30.5℃,孵放90min可灭活VSV和PRV,两种指示病毒的灭活效果分别为≥4.00~4.12和≥5.25~5.75log TCID50/0.1ml。因此,辛酸盐是一种安全、有效、快速的灭活脂包膜病毒的灭活剂。     

S/D法灭活血液制剂中脂包膜病毒效果验证的研究

选用不同核酸的脂包膜病毒,其中RNA病毒为水疱性口炎病毒(VSV),DNA病毒为伪狂犬病毒(PRV),将两种指示病毒分别用于验证S/D法处理对纤维蛋白原、凝血酶原复合物、凝血因子Ⅷ、静注丙种球蛋白、免疫血浆等血液制剂的病毒灭活效果。结果该法对所有被处理的血液制剂中的PRV及VSV灭活能力分别为≥3.38~5.88和≥3.50~4.75logTCID50/0.1ml,表明S/D法对两种病毒核酸类型的脂包膜病毒有良好的灭活效果。     

S/D处理结合低pH放孵灭活静注人免疫球蛋白

在低ｐＨ静脉注射用人免疫球蛋白（ＩＶＩＧ）的制备中,采用有机溶剂／表面活性剂处理和低ｐＨ放孵（２３°Ｃ～２５°Ｃ,２１天）进行病毒灭活,以提高ＩＶＩＧ的安全性,两法累积灭活效果为：＞８ｌｏｇＨＩＶ－Ⅰ;＞１１．３ＬｏｇＶＳＶ和＞１０．８ＬｏｇＳｉｎｄｂｉｓ。     

Comparable virus inactivation by bovine or vegetable derived Tween 80 during solvent/detergent treatment.

A mixture of Tri-n-butyl phosphate (TNBP) and Polysorbate 80 (Tween 80) is often used for virus inactivation during the manufacture of medicinal products derived from human plasma. This procedure, known as solvent/detergent treatment, is of high effectiveness for inactivation of enveloped viruses. Tween 80 can be manufactured from bovine tallow or from vegetable material. As the bovine-derived Tween 80 is normally used for the solvent/detergent treatment, the question has been raised whether vegetable-derived Tween 80 can be applied as an alternative substance for the solvent/detergent treatment. Comparable inactivation studies were therefore performed using Vesicular Stomatitis Virus (VSV), Pseudorabiesvirus (PRV), Semliki Forest Virus (SFV) and Bovine Diarrhoea Virus (BVDV). In principle, no differences were observed in the effectiveness of the solvent/detergent treatment when bovine or vegetable-derived Tween 80 was used. The comparability in the efficiency of both detergents for virus inactivation was shown to be independent of solvent/detergent concentration, of temperature (16 degrees C and 6 degrees C vs. 27 degrees C and 25 degrees C) and protein concentration (10% and 5% human albumin). In summary, vegetable-derived Tween 80 is of the same effectiveness as bovine-derived Tween 80, when used for virus inactivation by the solvent/detergent treatment.     

Safety of snake antivenom immunoglobulins: Efficacy of viral inactivation in a complete downstream process

Viral safety remains a challenge when processing a plasma‐derived product. A variety of pathogens might be present in the starting material, which requires a downstream process capable of broad viral reduction. In this article, we used a wide panel of viruses to assess viral removal/inactivation of our downstream process for Snake Antivenom Immunoglobulin (SAI). First, we screened and excluded equine plasma that cross‐reacted with any model virus, a procedure not published before for antivenoms. In addition, we evaluated for the first time the virucidal capacity of phenol applied to SAI products. Among the steps analyzed in the process, phenol addition was the most effective one, followed by heat, caprylic acid, and pepsin. All viruses were fully inactivated only by phenol treatment; heat, the second most effective step, did not inactivate the rotavirus and the adenovirus used. We therefore present a SAI downstream method that is cost‐effective and eliminates viruses to the extent required by WHO for a safe product. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 29:972–979, 2013     

低pH孵放法灭活静注人免疫球蛋白中脂包膜病毒效果验证的研究

选用不同核酸类型的脂包膜病毒,其中RNA病毒为水疱性口炎病毒(VSV),DNA病毒为伪狂犬病毒(PRV),将两种指示病毒分别用于验证低pH孵放法对不同厂家生产的人血静脉注射用丙种球蛋白(IVIG)的病毒灭活效果。结果表明,液体IVIG的pH值为3.8～4.4,在23～25℃环境中,孵放21天可灭活VSV和PRV,两种指示病毒的灭活效果分别为≥5.50～6.62和≥5.38～6.62logTCID50/0.1ml。因此,低pH孵放法是一种安全、有效且简便实用的灭活脂包膜病毒的方法。