Virus inactivation by solvent/detergent treatment using Triton X-100 in a high purity factor VIII

Virus inactivation by solvent/detergent treatment using 0.3% tri-n-butyl phosphate and 1% Triton X-100 in the high purity factor VIII concentrate Replenate((R)) has been investigated. A wide range of model enveloped viruses were confirmed to be inactivated by >4 to >6log after 30min at 22 degrees C under standard conditions. Using Sindbis as a representative enveloped virus, the effect of various parameters on the inactivation process was tested. Virus inactivation was confirmed to be effective in different batches of product and was not influenced by changing the process conditions with regard to protein and salt concentration or pH. Virus inactivation was effective even at a temperature as low as 4-5 degrees C. Although solvent/detergent concentration was the most critical parameter, a concentration as low as 0.15% TnBP/0.5% Triton X-100 was still completely effective. At a lower concentration an extended incubation period was required. These studies demonstrate the robustness of this solvent/detergent procedure based on Triton X-100 and allow suitable process limits to be set for this manufacturing step.     

Virus reduction in an intravenous immunoglobulin by solvent/detergent treatment, ion-exchange chromatography and terminal low pH incubation

Virus reduction by several steps in the manufacturing process for the intravenous immunoglobulin Vigam^®, has been investigated. The solvent/detergent step based on treatment with 0.3% tri-n-butyl phosphate and 1% polysorbate 80 at 37 °C, was confirmed to be effective for a range of enveloped viruses. Virus infectivity was undetectable i.e. >6 log inactivation within 30 min of the standard 6 h process. This was consistent over the range of conditions tested i.e. solvent/detergent and protein concentration, temperature and pH. The ion-exchange chromatography step in the process was also able to remove some viruses. Virus spiked followed by blank column runs confirmed the effectiveness of the sanitisation step for ensuring there was no virus cross contamination between column runs. The terminal low pH incubation step was also able to inactivate enveloped viruses, as well as some non-enveloped viruses. The combination of these three steps ensures a high margin of virus safety for this product.     

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.     

Virus inactivation in a factor VIII/VWF concentrate treated using a solvent/detergent procedure based on polysorbate 20

Treatment with solvent/detergent is a widely used method for ensuring the virus safety of plasma products. In the present study, virus inactivation by a novel solvent/detergent combination, i.e. TnBP (tri-n-butyl phosphate) and polysorbate 20 during the manufacture of the factor VIII/VWF concentrate Optivate^® has been investigated. The inactivation of most enveloped viruses was rapid, i.e. >5 log in 2 min, although the inactivation of vaccinia virus was slower, i.e. 4 log in 1 h. Virus inactivation was effective over a wide range of conditions, i.e. solvent/detergent concentration, protein concentration and temperature, irrespective of whether tested individually or in combination. This confirms the effectiveness and robustness of this alternative version of the solvent/detergent procedure, and allows appropriate control limits to be set for this manufacturing step. Polysorbate 20 provides an alternative to the non-ionic detergents currently in use with the solvent/detergent procedure.     

Purification of factor VIII and von Willebrand factor from human plasma by anion-exchange chromatography

Factor VIII (anti-hemophilia A factor) is isolated from human plasma. Purification is carried out by a combination of precipitation and chromatographic procedures. After precipitation, the first step in virus inactivation is achieved through the effect of a non-ionic detergent such as Tween 80, and a solvent, e.g. tri-n-butylphosphate (TnBP). By subsequent anion-exchange chromatography, a highly enriched product is isolated, consisting of a complex formed by factor VIII and von Willebrand factor (FVIII-vWF). This treatment also removes the virus-inactivating reagents to quantities in the low ppm range. The second step in virus inactivation is aimed specifically at the non-enveloped viruses and consists of pasteurization at temperatures higher than 60°C for 10 h. Through the addition of stabilizers, between 80% and 90% of the initial activity of FVIII is preserved during the modified pasteurisation. Along with the possibly denatured proteins the stabilizers, such as sugars, amino acids and bivalent cations, are subsequently removed by ion-exchange chromatography. The two-fold virus inactivation, by solvent/detergent treatment and subsequent pasteurisation, allows the destruction of both lipid-enveloped and non-enveloped viruses. During the procedure FVIII is stabilized through the high content of vWF. The complex consisting of FVIII and vWF can be dissociated by adding calcium ions. Subsequently both glycoproteins from this complex are separated from one another by further anion-exchange chromatography.     

Identification and characterization of a Triton X-100 replacement for virus inactivation

Triton X-100 detergent treatment is a robust enveloped virus inactivation unit operation included in biopharmaceutical manufacturing processes. However, the European Commission officially placed Triton X-100 on the Annex XIV authorization list in 2017 because a degradation product of Triton X-100, 4-(1,1,3,3-tetramethylbutyl) phenol (also known as 4-tert-octylphenol), is considered to have harmful endocrine disrupting activities. As a result, the use of Triton X-100 in the European Economic Area (EEA) would not be allowed unless an ECHA issued authorization was granted after the sunset date of January 4, 2021. This has prompted biopharmaceutical manufacturers to search for novel, environment-friendly alternative detergents for enveloped virus inactivation. In this study, we report the identification of such a novel detergent, Simulsol SL 11W. Simulsol SL 11W is an undecyl glycoside surfactant produced from glucose and C11 fatty alcohol. We report here that Simulsol SL 11W was able to effectively inactive enveloped viruses, such as xenotropic murine leukemia virus (XMuLV) and pseudorabies virus (PRV). By using XMuLV as a representative enveloped virus, the influence of various parameters on the effectiveness of virus inactivation was evaluated. Virus inactivation by Simulsol SL 11W was effective across different clarified bioreactor harvests at broad concentrations, pH, and temperature ranges. Simulsol SL 11W concentration, temperature of inactivation, and treatment time were identified as critical process parameters for virus inactivation. Removal of Simulsol SL 11W was readily achieved by Protein A chromatography and product quality was not affected by detergent treatment. Taken together, these results have shown the potential of Simulsol SL 11W as a desirable alternative to Triton X-100 for enveloped virus inactivation that could be readily implemented into biopharmaceutical manufacturing processes.     

Stability of ectromelia virus strain NIH-79 under various laboratory conditions

Ectromelia virus strain NIH-79 was suspended in fetal bovine serum (FBS), minimum essential medium, Hanks' base plus 10% FBS (MEMH + FBS), phosphate-buffered saline (PBS) or PBS plus 50% glycerol (PBS + G). Suspensions were held as liquids or as dry spots at various temperatures. Virus was most stable in FBS and least stable in PBS + G at 4 degrees C, room temperature (23-25 degrees C) or 37 degrees C. Virus held at 4 degrees C was more stable than virus held at higher temperatures, irrespective of supporting medium. Dried spots of blood or serum from ectromelia virus-infected mice remained infectious at room temperature for 11 days and 4 days, respectively. Dried spots of FBS that contained virus were infectious for 5 days, whereas virus retained infectivity for 1 day after drying in other media. Virus was inactivated completely in 10% serum in PBS exposed to 60 degrees C for 30 minutes. Virus was inactivated completely in slices of infected liver and spleen immersed in 10% neutral buffered formalin for 20 hours. These results show that the stability of ectromelia virus strain NIH-79 is medium and temperature dependent and that rapid inactivation occurs after treatments routinely used in diagnostic and research procedures.     

Human intravenous immunoglobulin preparation and virus inactivation by pasteurization and solvent detergent treatment

Human intravenous immunoglobulin (IVIG) solutions were prepared by two different methods and compared to each other. The crude immunoglobulin fraction obtained from Cohn-Oncley fractionation of plasma was further purified and subjected to virus inactivation, either by polyethylene glycol precipitation and pasteurization at 60 degrees C for 10 hours, or by ion exchange chromatography and solvent/detergent treatment. The final preparations, formulated in 5% immunoglobulin solutions were characterized by in vitro analyses of biochemical and biological properties and compared with the samples of other manufacturer's IVIG solution products. The critical properties evaluated in this study were purity, molecular intactness, and the biological functions such as Fc function and anticomplementary activity. Virus inactivation and removal by processing steps and by deliberate virucidal steps, as described above, were tested on various human pathogenic viruses, such as human immunodeficiency and experimental model viruses. The tested viruses were successfully inactivated and removed. We conclude that the intravenous immunoglobulins prepared by two different methods, as described above, provide an equivalent viral safety and quality.     

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.     

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.     

Does an increase in membrane unsaturated fatty acids account for Tween 80 stimulation of glucosyltransferase secretion by Streptococcus salivarius?

When Streptococcus salivarius was grown in batch culture in the presence of various Tween detergents, the fatty acid moiety of the detergent was incorporated into the lipids of its membrane. Tween 80 (containing primarily oleic acid) markedly stimulated the production of extracellular glucosyltransferase and also increased the degree of unsaturation of the membrane lipid fatty acids. The possibility that an increase in membrane unsaturated fatty acids promoted extracellular glucosyltransferase production was examined by growing cells at different temperatures in the presence or absence of Tween 80. The membrane lipids of cells grown at 30 degrees C, 37 degrees C and 40 degrees C without Tween 80 exhibited unsaturated/saturated fatty acid ratios of 2.06, 1.01 and 0.87 respectively. A significant increase in the production of extracellular glucosyltransferase was observed at 30 degrees C compared to cells grown at 40 degrees C. However, cells produced much more exoenzyme at all temperatures when grown with Tween 80. The results indicated that an increase in the unsaturated fatty acid content of the membrane lipids was not by itself sufficient to account for the stimulation of extracellular glucosyltransferase production by Tween 80, but that the surfactant also had to be present.     

S/D处理人纤维蛋白原的病毒灭活验证

在人纤维蛋白原制备工艺中增加Ｓ／Ｄ处理灭活病毒步骤,ＴＮＢＰ和Ｔｗｅｅｎ８０终浓度分别为０．３％和１％,在２５℃处理６小时能有效灭活指示病毒ＶＳＶ（〉３．７５Ｌｏｇ）、Ｓｉｎｄｂｉｓ（〉４．４６Ｌｏｇ）、ＨＩＶ（〉３．６７Ｌｏｇ）,盲传三代未检出病毒     

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.     

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.     

Laboratory-scale inactivation of African swine fever virus and swine vesicular disease virus in pig slurry

Two methods were evaluated for the inactivation of African swine fever (ASV) and swine vesicular disease (SVD) viruses in pig slurry: chemical treatment and heat treatment. The addition of NaOH or Ca(OH)2 at different concentration/time combinations at 4 degrees C and 22 degrees C was examined, as was virus stability at different temperature/time combinations. ASF virus (ASFV) was less resistant to both methods than SVD virus (SVDV). In slurry from one source, ASFV was inactivated at 65 degrees C within 1 min, whereas SVDV required at least 2 min at 65 degrees C. However, it was found that thermal inactivation depended on the characteristics of the slurry used. Addition of 1% (w/v) of NaOH or Ca(OH)2 caused the inactivation of ASFV within 150 s at 4 degrees C; 0.5% (w/v) NaOH or Ca(OH)2 required 30 min for inactivation. NaOH or Ca(OH)2 (1% (w/v)) was not effective against SVDV at 22 degrees C after 30 min, and 1.5% (w/v) NaOH or Ca(OH)2 caused inactivation of SVDV at both 4 degrees C and 22 degrees C. At higher chemical concentrations or temperatures, ASFV and SVDV inactivation was faster in slurry than in buffered medium.     

Issues in the development of medical products based on human plasma

Product development and process validation are shown in the case of several products obtained from human plasma. These are virus-inactivated plasma, intravenous immunoglobulins and the clotting factors VIII and IX. Different analytical methods are presented, which are used for product control and in-process control. For the production of virus-inactivated human plasma a down-scale protocol is presented, allowing a simulation of the production on a laboratory scale. Virus validation has shown that the reduction of transfusion-relevant viruses in the process was higher than six log steps. Determination of leachables from the RP-column, which was used in this production, proved that they appear in the final product in quantities below the detection limits only. It was also shown that the chemicals used for virus inactivation could be quantitatively removed from the product. For the isolation of other products, here intravenous gamma globulins and the clotting factors VIII and IX, similar validation steps had to be taken. In the case of clotting factor VIII the following data were determined, the reduction of viruses, the amount of leachables from the column, the residues of chemicals from the solvent/detergent treatment for virus inactivation. Virus reduction was successfully performed as well as the removal of chemicals used for virus inactivation. The amount of leachables from the columns used for chromatographic purification was found to be far below the permissible levels.     

Issues in the development of medical products based on human plasma

Product development and process validation are shown in the case of several products obtained from human plasma. These are virus-inactivated plasma, intravenous immunoglobulins and the clotting factors VIII and IX. Different analytical methods are presented, which are used for product control and in-process control. For the production of virus-inactivated human plasma a down-scale protocol is presented, allowing a simulation of the production on a laboratory scale. Virus validation has shown that the reduction of transfusion-relevant viruses in the process was higher than six log steps. Determination of leachables from the RP-column, which was used in this production, proved that they appear in the final product in quantities below the detection limits only. It was also shown that the chemicals used for virus inactivation could be quantitatively removed from the product. For the isolation of other products, here intravenous gamma globulins and the clotting factors VIII and IX, similar validation steps had to be taken. In the case of clotting factor VIII the following data were determined, the reduction of viruses, the amount of leachables from the column, the residues of chemicals from the solvent/detergent treatment for virus inactivation. Virus reduction was successfully performed as well as the removal of chemicals used for virus inactivation. The amount of leachables from the columns used for chromatographic purification was found to be far below the permissible levels.     

Inactivation mechanism of the membrane protein diacylglycerol kinase in detergent solution

We have examined the irreversible inactivation mechanism of the membrane protein diacylglycerol kinase in the detergents n-octyl-beta-D-glucopyranoside (OG) at 55 degrees C and n-decyl-maltopyranoside (DM) at 80 degrees C. Under no inactivation conditions did we find any direct evidence for the chemical modifications that are commonly found in soluble proteins. Moreover, protein inactivated at 55 degrees C in OG could be reactivated by an unfolding and refolding protocol, suggesting that the protein is inactivated by a stable conformational change, not a covalent modification. We also found that the inactivation rate decreased with both increasing protein concentration and increasing thermodynamic stability, consistent with an inactivation pathway involving transient dissociation and/or unfolding of the protein. Our results suggest that the primary cause of diacylglycerol kinase inactivation is not low solubility, but poor intrinsic stability in the detergent environment.     

A highly efficient second-step concentration technique for bacteriophages and enteric viruses using ammonium sulfate and Tween 80

Addition of Tween 80 to a 1.5% solution of beef extract was found to enhance the elution of bacteriophages adsorbed to electronegative filters. When reconcentration of the eluate was attempted by ammonium sulfate precipitation, a floating layer containing most of the viruses was formed. This floating layer can be obtained with several nonionic detergents including Tween 80 and under a salt saturation of 55% with ammonium sulfate, potassium tartrate, and sodium phosphate. Virus recovery ranged from 91 to 103% and was obtained with several bacteriophage strains. With poliovirus type 1, coxsackievirus B-4, and rotavirus SA-11 the recoveries were 100, 20, and 80%, respectively, but toxicity to cell culture was encountered: after removal of the detergent by a second floating layer method the recovery was 32% for poliovirus. Compared with organic flocculation, this method also had both improved recovery for bacteriophages and protective properties for samples frozen at -70 degrees C.     

Binding of an influenza A virus to a neomembrane measured by surface plasmon resonance

Neomembranes composed of either bovine brain lipid that contains sialoglycolipids or egg yolk lecithin that does not, were formed on an HPA sensor chip and used to study the binding of influenza A virus in real time by surface plasmon resonance. Virus bound only to the bovine brain lipid membrane. This was confirmed by an 84% reduction in virus binding after treatment of the neomembrane with neuraminidase. Binding was temperature dependent, being highest at 30-35 degrees C and lower at 10 degrees C. Surprisingly, the rate of complex formation was enhanced, rather than inhibited, by the presence of 1.34-25.2 x 10(6) molecules of free NANA per virus binding site and the rate of dissociation was lower suggesting that the complex was more stable. The free energy of association to form the transition complex was increased by 3 kJ mol(-1) and there was an almost 10-fold increase in the enthalpy of complex formation in the presence of free NANA. These results show the value of surface plasmon resonance for measuring complex molecular interactions in real time, and provide a model that can be used to study the effectiveness of inhibitors of attachment of influenza virus to its receptor molecules.