Reduction of Isoagglutinin in Intravenous Immunoglobulin (IVIG) Using Blood Group A- and B-Specific Immunoaffinity Chromatography: Industry-Scale Assessment

Abstract

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 <1, respectively, when measured by the Ph. Eur. indirect method. The isoagglutinin flow cytometry assay showed an 87-90 % reduction in isoagglutinins in post-IAC versus pre-IAC fractions. IAC alone reduced anti-A and anti-B of the IgMs isotype by 92.5-97.8 % and 95.4-99.2 %, respectively. Other product quality characteristics were similar with and without IAC.

Conclusions: IAC is an effective method for reducing isoagglutinin levels in IVIG, and it is feasible on an industrial scale.

Fig. 1

Original and modified processes for the manufacture of the intravenous immunoglobulin product, Privigen. ^aWhen assessing the clearance of immunoglobulin M isoagglutinins by the anion-exchange chromatography and immunoaffinity chromatography steps, the process steps outlined in orange (ethanol- and octanoic acid fractionation) were omitted to minimize the loss of immunoglobulin M, and therefore the loss of the immunoglobulin M isoagglutinins. This was done because the octanoic acid fractionation step is the main elimination step of total immunoglobulin M. The intention was to retain as much immunoglobulin M as possible for the subsequent isoagglutinin removal steps. AIC immunoaffinity chromatography, AIEX anion exchange chromatography, DF diafiltration, UF ultrafitration. Adapted with permission from Hoefferer et al. [19]© 2015 AABB. Hoefferer L, Glauser I, Gaida A, Willimann K, Marques Antunes A, Siani B, et al. Isoagglutinin reduction by a dedicated immunoaffinity chromatography step in the manufacturing process of human immunoglobulin products. Transfusion. 2015;55(Suppl 2):S117–21. doi:10.1111/trf.13088

Fig. 2

Anti-A (a) and anti-B (b) titers in final intravenous immunoglobulin product lots, measured by the European Pharmacopoeia direct method. IAC immunoaffinity chromatography, IgG immunoglobulin G

Fig. 3

Immunoglobulin G isoagglutinin levels with and without the immunoaffinity chromatography step in the production process, measured using an isoagglutinin flow cytometry assay. Anti-A and anti-B relative fluorescence intensity, measured relative to the internal standard. RFI relative fluorescence intensity, IAC immunoaffinity chromatography

Fig. 4

Anti-A and anti-B titers in intravenous immunoglobulin lots produced with the immunoaffinity chromatography step (Privigen) and intravenous immunoglobulin lots produced using Cohn-like fractionation (Sandoglobulin/Carimune), measured by the European Pharmacopoeia direct method. IAC immunoaffinity chromatography, IgG immunoglobulin G, IVIG intravenous immunoglobulin