Removal of small non-enveloped viruses by nanofiltration.

BACKGROUND AND OBJECTIVES: Nanofiltration is one of the most effective virus reduction methods in the manufacturing process of plasma products. However, it is difficult to remove small viruses from high molecular weight protein preparations like immunoglobulin G or factor VIII complex by nanofiltration, because the size of the protein is similar to that of viruses. In order to separate the viruses from these proteins by nanofiltration, it is necessary to change the size of either one. In this study, we report that such non-enveloped viruses as human parvovirus B19 (B19), human encephalomyocarditis virus (EMC) or porcine parvovirus (PPV) aggregate in the presence of certain kinds of amino acids and could be easily removed by nanofiltration. MATERIALS AND METHODS: 0.3 M Glycine (or other amino acid) solution spiked with viruses was subjected to dead-end single filtration with a 35-nm pore-size filter. Virus removal by nanofiltration was either evaluated by PCR or by infectivity assay. RESULTS: B19 in a 0.3 M glycine solution was reduced to 1:10(7.5) (7.5-log) by nanofiltration with a 35-nm pore-sized filter, whereas in PBS it was not reduced. Similarly, B19 was also reduced when suspended in other amino acids solutions. This effect was also confirmed with the other small non-enveloped viruses EMC or PPV. When 5% globulin or 5% albumin was added to a 0.3 M glycine solution, the removal rate was decreased. CONCLUSIONS: These data suggest that viruses in the presence of certain kinds of amino acids could be aggregated and effectively removed by a filter that has a pore size larger than the size of the viruses.

Status of NAT screening for HCV, HIV and HBV: experience in Japan.

The first nationwide nucleic acid amplification testing (NAT) for hepatitis B virus (HBV), hepatitis C virus (HCV), and human immunodeficiency virus type 1 (HIV-1) of voluntarily donated blood after serological pre-screening and before release of cellular components and plasma for fractionation was implemented by the Japanese Red Cross Blood Transfusion Services. The NAT screening assay using multiplex reagent is time-saving, cost effective, and labour-saving procedure for all blood and blood products including short-shelf life platelets. During the 50-mini-pool NAT screening of serologically negative donations (February 1, 2001-April 30, 2001), we were able to screen out 112 HBV-positive, 25 HCV-positive, and 4 HIV-1 positive units from blood and blood components.

The first large-scale nucleic acid amplification testing (NAT) of donated blood using multiplex reagent for simultaneous detection of HBV, HCV, and HIV-1 and significance of NAT for HBV.

The first nationwide nucleic acid amplification testing (NAT) for hepatitis B virus (HBV), hepatitis C virus (HCV), and human immunodeficiency virus type 1 (HIV-1) of voluntarily donated blood after serological pre-screening and before release of cellular components and plasma for fractionation was implemented by the Japanese Red Cross Blood Transfusion Services. From February 1, 2000 to April 30, 2001, specimens from 6,805,010 units of serologically negative donation were screened in minipools of 50 samples within 24 hr after blood donation by NAT using multiplex HBV/HCV/HIV-1 reagent for blood transfusion including short shelf-life platelets. Among them, 112 HBV DNA-positives, 25 HCV RNA positives and 4 HIV-1 RNA positives were screened out and we could prevent transfusion of these NAT positive units. Subtypes/genotypes of HBV DNA, adr/C, adw/A, adw/B, adw/C, ayr/C and ayw/D were found and adr/C was predominant. A total of 61.6 % of them (69/112) were negative by overnight EIA. Sixth three of HBV NAT-positive samples carried virus loads less than 10(4) copies/mL and 92.1 % of them (58/63) were negative by overnight EIA. The virus growth curves of HBV in 6 cases obtained by retrospective and prospective follow-up study showed exponential straight lines in the early stage of serological window periods and the log times of HBV growth (10 fold increase) in serological window period were between 4.6 and 7.6 days. NAT screening with highly sensitive reagents in pool of specimens is useful to exclude blood units with low level of HBV and HBV mutants from blood transfusion.

Removal and inactivation of hepatitis B virus from contaminated pooled plasma in a large-scale manufacturing process for factor VIII and human serum albumin.

BACKGROUND AND OBJECTIVES: The Japanese Red Cross Society recalled one lot of monoclonal-antibody-purified factor VIII (F VIII) and two lots of human serum albumin (HSA) 5 months after preparation of the final products, because of a procedural error that led to contamination by a unit of plasma positive for hepatitis B surface antigen (HBsAg). We evaluated the effectiveness of virus inactivation/removal in a large-scale process for manufacturing F VIII and HSA. MATERIALS AND METHODS: HBV DNA in the retained samples in process was measured by the polymerase chain reaction (PCR). The kinetics of virus inactivation by solvent-detergent (S/D) treatment was examined using model viruses. We also did a look-back survey of the patients who received corresponding products. RESULTS: Contaminated hepatitis B virus (HBV) DNA became undetectable beyond fraction S IV-I in the albumin process and immunoaffinity chromatography in the F VIII process, respectively. The model viruses were inactivated within 5 s by S/D treatment. There is no evidence that patients were infected by HBV after transfusion of these products. CONCLUSION: We conclude that virus inactivation/removal was effectively achieved in a large-scale manufacturing process for F VIII and HSA.

Bovine albumin-like protein in commercial human albumin for clinical use.

We have demonstrated that several lots of commercial human serum albumin (HSA), prepared for clinical use by two manufacturers, reacted with anti-bovine whole serum in the double-diffusion test. The antigenic proteins were purified by affinity chromatography from these HSA lots and their chemical and immunological properties characterized. The purified proteins showed the same molecular weight as HSA and bovine serum albumin (BSA). By CNBr treatment and trypsin digestion, the purified protein produced the same fragments as BSA. Moreover, the purified protein had an amino acid composition very similar to BSA and also 30 residues identical to those of the N-terminal amino acid sequence of BSA. In addition, it was shown that heterophilic Hanganutziu-Deicher antigenicity was present in the purified protein. The protein reactive against-bovine whole serum, contained in commercial HSA, was called BSA-like protein.

Immunochemical comparison of histidine-rich protein in keratohyalin granules and cornified cells.

(1) Combination of techniques for extraction and purification of histidine rich protein established by several investigators were employed for comparison of histidine-rich protein in granular cells and cornified cells of newborn rats. (2) Histidine-rich protein extracted from the same cell fraction by two different techniques either in 1 M potassium phosphate buffer (Ugel) or in 4 M urea (Dale) showed identical elution profiles on CM 52 cellulose ion exchange chromatography and the same SDS polyacrylamide gel electrophoretic patterns. (3) Histidine-rich protein from granular cells contained polypeptides of larger molecular sizes than those in histidine-rich protein from cornfield cells, although amino acid composition of the two histidine-rich protein was non-distinguishable (histidine residue was more than 7%). (4) Antibodies raised in rabbits by injection of histidine rich protein from granular cells and that from cornfield cells immunologically cross-reacted. Furthermore, the antisera were found to be reactive over both keratohyalin granules and cornified cells, but not epidermal cells of the lower strata.

Divalent cation stimulation of in vitro fibre assembly from epidermal keratin protein.

Keratin was extracted from purified cornified cells of newborn rats in Tris-HCl-buffered 8 M urea containing beta-mercaptoethanol. Microfilaments were assembled in vitro by reducing the ionic strength of buffer and the urea concentration. One millimolar concentration of KCl and NaCl did not affect filament formation, but the same concentration of divalent cations greatly altered this process. CaCl2 and MgCl2 induced gelation of keratin by formation of bundles of birefringent macrofilaments. ZnCl2, CuSO4 and HgCl2 formed greater numbers of macrofilaments and the protein aggregated.