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Background Hemolysis, a rare but potentially serious complication of intravenous immunoglobulin (IVIG) therapy, is associated with the presence of antibodies to blood groups A and B (isoagglutinins) in the IVIG product. An immunoaffinity chromatography (IAC) step in the production process could decrease isoagglutinin levels in IVIG. Objectives Our objectives were to compare isoagglutinin levels in a large number of IVIG (Privigen®) batches produced with or without IAC and to assess the feasibility of the production process with an IAC step on an industrial scale. Methods The IAC column comprised a blend of anti-A and anti-B resins formed by coupling synthetic blood group antigens (A/B-trisaccharides) to a base bead matrix, and was introduced towards the end of the industrial-scale IVIG manufacturing process. Isoagglutinin levels in IVIG were determined by anti-A and anti-B hemagglutinin direct and indirect methods according to the European Pharmacopoeia (Ph. Eur.) and an isoagglutinin flow cytometry assay. IVIG product quality was assessed with respect to the retention of immunoglobulin G (IgG) subclasses, specific antibodies, and removal of IgM using standardized procedures. Results The IAC step reduced isoagglutinins in IVIG by two to three titer steps compared with lots produced without IAC. The median anti-A and anti-B titers with IAC were 1:8 and 1:4, respectively, when measured by the Ph. Eur. direct method, and 1:2 and

BACKGROUND The passive transfer of antibodies specific to blood groups A and B (also called isoagglutinins) contained in immunoglobulin (Ig)G products for intravenous administration (IVIG) is believed to be largely responsible for rare but sometimes serious IVIG-related hemolytic events. We present in this work a modification of the manufacturing process of Privigen—a 10% l-proline–stabilized IVIG product—that allows extensive reduction of isoagglutinin concentrations in the final product. STUDY DESIGN AND METHODS An additional immunoaffinity chromatography (IAC) step was introduced toward the end of the manufacturing process of Privigen. Isoagglutinin titers were measured using the indirect agglutination method and a published flow cytometry–based binding assay. Quality attributes, such as microorganism counts and concentration of endotoxins, IgG, IgA, IgM, aggregates, and so forth were measured using standardized procedures. RESULTS The introduction of an IAC step in the manufacturing process of Privigen resulted in an 88% to 90% reduction in isoagglutinins between the feed of the chromatography column and the flow-through fraction. All other product quality attributes measured were nearly identical before and after IAC. This process modification resulted in a three-titer-step reduction in isoagglutinin levels in the final IgG product compared to Privigen lots produced by the unmodified process. CONCLUSION Introducing an isoagglutinin-specific IAC step in the manufacturing process of Privigen is an efficient strategy for reduction of anti-A and anti-B titers. Such reductions might help minimize the risk of hemolytic events in patients receiving IVIG therapy.

BACKGROUND Hemolysis reaction is a rare class effect of therapy with intravenously administered human normal immunoglobulin (IVIG). Anti-A/B isoagglutinins (isohemagglutinins) originating from donor plasma are considered a probable risk factor for hemolysis. We hypothesized that screening and exclusion of plasma obtained from donors with high isoagglutinin titers from the manufacturing process would produce a meaningful reduction of anti-A/B isoagglutinin titers of the final IVIG product. STUDY DESIGN AND METHODS A donor screening method for anti-A isoagglutinins using an automated indirect agglutination test (IAT) in gel cards was developed. Industry-scale donor plasma pools and final IVIG product were prepared according to the manufacturing process of Privigen (human 10% liquid IVIG). Anti-A/B isoagglutinin levels were measured by IAT, direct agglutination test, and a flow cytometry–based assay. RESULTS Screening of plasma from 705 randomly selected donors identified 6.8% donors with high anti-A isoagglutinin titers in plasma. Exclusion of plasma from these donors resulted in a one-titer-step reduction of anti-A isoagglutinin in laboratory-scale pooled plasma. The same donor screening method applied to industry-scale production resulted in exclusion of 5.1% of donors and produced a one-titer-step reduction of both anti-A and anti-B isoagglutinin titers in the final IVIG product. CONCLUSION Anti-A/B isoagglutinin titers in IVIG products can be reduced on an industrial scale through implementation of anti-A donor screening, which may lower the risk of hemolysis after IVIG therapy.

The passive transfer of antibodies specific to blood groups A and B (also called isoagglutinins) contained in immunoglobulin (Ig)G products for intravenous administration (IVIG) is believed to be largely responsible for rare but sometimes serious IVIG-related hemolytic events. We present in this work a modification of the manufacturing process of Privigen-a 10% l-proline-stabilized IVIG product-that allows extensive reduction of isoagglutinin concentrations in the final product.An additional immunoaffinity chromatography (IAC) step was introduced toward the end of the manufacturing process of Privigen. Isoagglutinin titers were measured using the indirect agglutination method and a published flow cytometry-based binding assay. Quality attributes, such as microorganism counts and concentration of endotoxins, IgG, IgA, IgM, aggregates, and so forth were measured using standardized procedures.The introduction of an IAC step in the manufacturing process of Privigen resulted in an 88% to 90% reduction in isoagglutinins between the feed of the chromatography column and the flow-through fraction. All other product quality attributes measured were nearly identical before and after IAC. This process modification resulted in a three-titer-step reduction in isoagglutinin levels in the final IgG product compared to Privigen lots produced by the unmodified process.Introducing an isoagglutinin-specific IAC step in the manufacturing process of Privigen is an efficient strategy for reduction of anti-A and anti-B titers. Such reductions might help minimize the risk of hemolytic events in patients receiving IVIG therapy.

Hemolysis reaction is a rare class effect of therapy with intravenously administered human normal immunoglobulin (IVIG). Anti-A/B isoagglutinins (isohemagglutinins) originating from donor plasma are considered a probable risk factor for hemolysis. We hypothesized that screening and exclusion of plasma obtained from donors with high isoagglutinin titers from the manufacturing process would produce a meaningful reduction of anti-A/B isoagglutinin titers of the final IVIG product.A donor screening method for anti-A isoagglutinins using an automated indirect agglutination test (IAT) in gel cards was developed. Industry-scale donor plasma pools and final IVIG product were prepared according to the manufacturing process of Privigen (human 10% liquid IVIG). Anti-A/B isoagglutinin levels were measured by IAT, direct agglutination test, and a flow cytometry-based assay.Screening of plasma from 705 randomly selected donors identified 6.8% donors with high anti-A isoagglutinin titers in plasma. Exclusion of plasma from these donors resulted in a one-titer-step reduction of anti-A isoagglutinin in laboratory-scale pooled plasma. The same donor screening method applied to industry-scale production resulted in exclusion of 5.1% of donors and produced a one-titer-step reduction of both anti-A and anti-B isoagglutinin titers in the final IVIG product.Anti-A/B isoagglutinin titers in IVIG products can be reduced on an industrial scale through implementation of anti-A donor screening, which may lower the risk of hemolysis after IVIG therapy.

We determined the complete genome sequences of both biotypes of a virus pair of bovine viral diarrhea virus (BVDV) subgenotype 1k. The viruses were isolated from a persistently infected calf suffering from mucosal disease. Compared to the noncytopathic biotype, the cytopathic biotype contains an insertion of 84 nucleotides and 22 nucleotide changes.