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.     

Cold ethanol fractionation and heat inactivation of hepatitis a virus during manufacture of albumin from human plasma

The purpose of the present study was to examine the efficacy and mechanism of fraction IV cold ethanol fractionation and pasteurization (60°C heat treatment for 10h), involved in the manufacture of albumin from human plasma, in the removal and/or inactivation of the hepatitis A virus (HAV). Samples from the relevant stages of the production process were spiked with HAV and the amount of virus in each fraction then quantified using a 50% tissue culture infectious dose (TCID₅₀). HAV was effectively partitioned from albumin during the fraction IV cold ethanol fractionation with a log reduction factor of 3.43. Pasteurization was also found to be a robust and effective step in inactivating HAV, where the titers were reduced from an initial titer of 7.60 log TCID₅₀ to undetectable levels within 5 h of treatment. The log reduction factor achieved during pasteurization was≽4.76. Therefore, the current results indicate that the production process for albumin has sufficient HAV 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.     

High-pressure inactivation of hepatitis A virus within oysters

Previous results demonstrated that hepatitis A virus (HAV) could be inactivated by high hydrostatic pressure (HHP) (D. H. Kingsley, D. Hoover, E. Papafragkou, and G. P. Richards, J. Food Prot. 65:1605-1609, 2002); however, direct evaluation of HAV inactivation within contaminated oysters was not performed. In this study, we report confirmation that HAV within contaminated shellfish is inactivated by HHP. Shellfish were initially contaminated with HAV by using a flowthrough system. PFU reductions of >1, >2, and >3 log(10) were observed for 1-min treatments at 350, 375, and 400 megapascals, respectively, within a temperature range of 8.7 to 10.3 degrees C. Bioconcentration of nearly 6 log(10) PFU of HAV per oyster was achieved under simulated natural conditions. These results suggest that HHP treatment of raw shellfish will be a viable strategy for the reduction of infectious HAV.     

Use of macroreticular resins for the adsorption of urokinase from human urine

Summary 22 commercial macroreticular resins were screened for their suitability to adsorb the enzyme urokinase directly from human urine at the outlet of a urinal. Both batch and fixed bed techniques were used for the screening, which identified a phenolic resin (Duolite S-762) as having the best capacity and adsorption efficiency. A capacity in excess of 1800 IU/ml. resin is attainable, and elution with 50% ethanolamine followed by immediate neutralisation with HCl gives satisfactory recovery of activity.     

Removal and inactivation of human immunodeficiency virus (HIV-1) by cold ethanol fractionation and pasteurization during the manufacturing of albumin and immunoglobulins from human plasma

Viral safety is a prerequisite for manufacturing clinical albumin and immunoglobulins from human plasma pools. This study was designed to evaluate the efficacy of cold ethanol fractionation and pasteurization (60°C heat treatment for 10 h) for the removal inactivation of human immunodeficiency virus type 1 (HIV-1) during the manufacturing of albumin and immunoglobulins. Samples from the relevant stages of the production process were spiked with HIV-1, and the amount of virus in each fraction was quantified by the 50% tissue culture infectious dose (TCID₅₀). Both fraction IV fractionation and pasteurization steps during albumin processing were robust and effective in inactivating HIV-1, titers of which were reduced from an initial 8.5 log₁₀ TCID₅₀ to undetectable levels. The log reduction factors achieved were ≥4.5 and ≥6.5, respectively. In addition, fraction III fractionation and pasteurization during immunoglobulins processing were robust and effective in eliminating HIV-1. HIV-1 titers were reduced from an initial 7.3 log₁₀ TCID₅₀ to undetectable levels. The log reduction factors achieved in this case were ≥4.9 and ≥5.3, respectively. These results indicate that the process investigated for the production of albumin and immunoglobulins have sufficient HIV-1 reducing capacity to achieve a high margin of safety.     

The complete amino acid sequence of low molecular mass urokinase from human urine

The sequence of all 253 amino acids of the heavy (B-) chain of human urinary urokinase was determined. The fragmentation strategy employed included cyanogen bromide cleavage of S-carboxymethylated B-chain at Met and/or Trp residues, cleavage of acid-labile Asp-Pro bonds, and the use of the specific endoproteinases Lys-C and Arg-C for generation of overlapping fragments. For sequence determination automated solid- or liquid-phase techniques of Edman degradation were used. The amino acid sequence obtained substantiates the serine protease character of the B-chain of urokinase: a considerable homology with other serine proteinases, especially with the B-chain of human plasmin, was proved. The pertinent active site amino acids were localized: His-46, Asp-97, and Ser-198. A carbohydrate side chain, containing at least 4 glucosamine and 2 galactosamine residues, was demonstrated to be fixed at asparagine in position 144. The sequence data presented, together with the sequence of the second (A1-) chain of low molecular mass urokinase which was reported by us in an earlier communication, complete the knowledge of the whole primary structure of an active form of human urinary urokinase.     

亚甲兰光化学法对血浆乙肝病毒的灭活

目的:研究灭活病毒对乙肝病毒和血浆成分的影响。方法:在已知HBV-DNA阳性血浆中加入MB,调整MB终浓度分别为1.0μmol/l、5.0μmol/l、10.0μmol/l,光照度为40000LUX,照射时间分别为0min、20min、40min、60min,在各点分别取样抽提基因组DNA用于荧光定量分析,测定HBV-DNA的浓度,推断在不同时间和浓度下的灭活效果。在灭活效果最好的点取样检测血浆正常成分有无显著变化,其中包括生化指标,酶学指标,凝血因子FⅧ:C活性,SDS聚丙烯酰胺凝胶电泳观察蛋白亚基的变化,蛋白免疫印迹观察血浆的抗体蛋白活性变化。结果:当MB为10.0μmol/L,光照60min时,灭活乙肝病毒的效果最好,对于高拷贝数的病毒浓度,在本实验的条件下,尚无法完全灭活病毒,但可以使HBV-DNA浓度明显下降;从病毒灭活的表格看,灭活效果与时间和MB浓度呈正相关,作用60min时血浆中主要成分的生化指标无显著变化及凝血因子FⅧ:C活性无明显改变,部分血浆酶活性有明显下降(P<0.05)。血浆蛋白的亚基数目和迁移速度未见明显变化,血浆的抗体蛋白也具有正常的生物学活性。结论:MB10μmol/L辅助荧光...     

Effect of dithiothreitol on activity and protein structure of human urine urokinase

Human urine urokinase [EC 3.4.21.31] was found to be inactivated by dithiothreitol (DTT) much more severely than by 2-mercaptoethanol at the same concentration on the basis of -SH groups. Removal of DTT by dialysis restored the activities of esterase toward acetyl-glycyl-L-lysine methyl ester, plasminogen activation, and amidase toward 7-(glutaryl-glycyl-L-arginine-amido)-4-methyl coumarin. But the restoration of amidase activity was much less than that of esterase activity. The addition of DTT mediated the conversion of high molecular weight urokinase to low molecular weight urokinase, releasing several peptides. This suggests that the urokinase consists of several polypeptides linked by disulfide bonds. The molecular weight of urokinase produced with DTT was smaller than that of low molecular weight urokinase obtained by autodigestion of high molecular weight urokinase. The autodigestion was also accompanied by liberation of some peptides. But, those peptides released on autodigestion of high molecular weight urokinase were different from those appearing in the presence of DTT.     

A technology of preparing hepatitis A virus (HAV) from human stools

Two-step differential centrifugation through 40% and 20% sucrose of 20% water extracts of HAV-positive stools yielded specific defined antigens for hepatitis A serological diagnosis by 3rd-generation methods. The antigens would be suitable also for other purposes. Products of comparable quality were obtained from HAV-positive stools yielding different amounts of virus. Optimal were incubation-period stools, where the loss in HAV yield was minimal; these preparations displayed the highest antigenic capacity and could be diluted up to 150 fold for serological reactions. Results obtained in extensive preliminary experiments, outlined in the Discussion, corroborated the advantages and efficiency of the proposed technology.     

Survival curve during virus inactivation]

The survival curves for bacteriophage lambda and vaccinia virus were shown theoretically and in experiments to have a plateau at prolonged inactivation by UV-irradiation or 8-methoxypsoralen. The level of the plateau is dependent of the accuracy of the repair process. The method for extrapolation of the survival curves is proposed.