IMMUNE GLOBULIN MANUFACTURING AND RISK MITIGATION Isoagglutinin reduction by a dedicated immunoaffinity chromatography step in the manufacturing process of human immunoglobulin products Liane Hoefferer,1 Isabelle Glauser,2 Annette Gaida,3 Katharina Willimann,4 Adriano Marques Antunes,4 Brigitte Siani,4 Sandra Wymann,3 Eleonora Widmer,4 Ibrahim El Menyawi,2 Reinhard Bolli,5 Martin Spycher,3 and Martin Imboden6

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.

A

ntibody-mediated hemolysis is a documented rare adverse event of intravenous immunoglobulin (IVIG) therapy. Because IVIG-related hemolytic events have been observed almost exclusively in patients with non-O blood groups, one important causative factor is believed to be the passive transfer of antibodies specific to blood group A and B antigens (also referred to as isoagglutinins).1,2 The isoagglutinins contained in IVIG products originate from the plasma donated by healthy individuals, from which IgG products are manufactured. In the past, Cohn-like fractionation methods were used to separate the IgG fraction from other components of human plasma.3 To meet the increasing global demand for IgG products, new chromatography-based manufacturing processes with improved IgG recovery were developed.4 Chromatography-based methods also allow improved preservation of the functional diversity of antibodies present in donor plasma; however, this leads to increased isoagglutinin concentrations in the resulting IgG preparations.5 To counteract this effect, we developed

ABBREVIATION: IAC 5 immunoaffinity chromatography. From 1Research and Development, 2Process Development, 3

Product Development, 4Biochemical Quality Control,

5

Biochemistry Research and Development, and Pharmaceutical Development, CSL Behring AG, Berne,

6

Switzerland. Address reprint requests to: Liane Hoefferer, CSL Behring AG, Wankdorfstrasse 10, 3014 Berne, Switzerland; e-mail: [email protected]. doi:10.1111/trf.13088 C 2015 AABB V

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Fig. 1. Introduction of an IAC step in the manufacturing process of Privigen. A new IAC step was introduced in the manufacturing process of Privigen and Hizentra between the AIEX chromatography and nanofiltration steps. Blood group A and B trisaccharides were coupled to a bead matrix to create anti-A/B–specific resins for IAC. AIEX 5 anion exchange; UF/DF 5 ultrafiltration/diafiltration.

measures to reduce anti-A/B isoagglutinin levels in IgG products. In this work we describe a modification of the manufacturing process of IgPro10 (Privigen, CSL Behring, Berne, Switzerland)—a 10% L-proline–stabilized IVIG— that allows efficient and selective depletion of isoagglutinins in the final product. The modification consists of the addition of a specific immunoaffinity chromatography (IAC) step toward the end of the manufacturing process and was validated in a large-scale production facility.

anion-exchange chromatography. Subsequently, virus filtration (nanofiltration) is applied as another dedicated virus elimination step. Manufacture of the IgG product is completed by concentration via ultrafiltration-diafiltration and final formulation. To selectively remove anti-A/B isoagglutinins, we introduced a dedicated IAC step in the manufacturing process downstream of the anion-exchange

MATERIALS AND METHODS Privigen manufacturing process The Privigen manufacturing process has been described elsewhere.6 Briefly, pooled donor plasma undergoes cold ethanol precipitation, yielding an IgG-enriched precipitate. The resuspended precipitate is subjected to octanoic acid fractionation, the principal purification step in the process. The resulting precipitate, which contains contaminants such as lipids, high-molecular-weight proteins, and proteases, is removed by depth filtration. The filtrate is diafiltered and subjected to a pH 4 incubation, the first dedicated virus inactivation step. After adjusting pH and a second depth filtration step, residual protein impurities (mainly IgA and IgM) and aggregates are removed by S118 TRANSFUSION Volume 55, July 2015

Fig. 2. Isoagglutinin concentration before and after IAC. Isoagglutinin binding to blood group A ( ) and B ( ) RBCs was measured by the fluorescence-activated cell sorting antiA/B assay. The values measured before IAC were set to 100%.

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TABLE 1. Specific antibody concentrations before and after IAC Mean antibody concentration (IU/mg) Specific antibody

Feed of IAC column (n 5 10)*

Flow-through (n 5 10)*

3.6 6 0.6 0.07 6 0.02

3.4 6 0.6 0.07 6 0.02

16.2 6 2.3 0.41 6 0.05 0.31 6 0.14 0.19 6 0.01 0.23 6 0.07 0.56 6 0.06 0.07 6 0.003

16.3 6 2.0 0.38 6 0.03 0.30 6 0.15 0.19 6 0.01 0.29 6 0.06 0.51 6 0.04 0.07 6 0.01

Anti-parvovirus B19 Anti-hepatitis B surface antigen Anti-streptolysin Anti-tetanus toxin Anti-poliomyelitis Anti-varicella Anti-measles Anti-cytomegalovirus Anti-diphtheria toxin * Privigen and Hizentra lots.

chromatography, while leaving all other steps unchanged (Fig. 1). Customized anti-A– and anti-B–specific affinity resins with blood group A and B trisaccharides coupled to the bead matrix were employed to reduce the levels of both anti-A and anti-B in one chromatographic step.

Isoagglutinin titer determination A flow cytometry–based anti-A and anti-B isoagglutininbinding assay (fluorescence-activated cell sorting anti-A/ B) was used to determine anti-A and anti-B isoagglutinin binding to blood group A and B red blood cells (RBCs), respectively, as described previously.7 A standard RBC preparation obtained from a single donor was used in all tests. The level of RBC binding before entry into the IAC column was compared to that in the flow-through fraction

in 10 industry-scale IgG preparations using a cell analyzer (FACSCanto II, Becton-Dickinson AG, Allschwil, Switzerland). Anti-A and anti-B isoagglutinin titers in final IgG product lots were determined with the indirect agglutination test as described previously.7

IgG product quality assessments To ensure that the new IAC step did not jeopardize the overall quality of the IgG product, a complete impurity profile including more than 25 variables was recorded using in-house assays before and after the IAC step. These included quality attributes such as total viable counts of microorganisms and concentration of endotoxins, IgG, IgA, IgM, aggregates, proteolytic activity, and anticomplementary activity. To verify the specificity of the IAC step toward anti-A/B isoagglutinins, the levels of antibodies specific to parvovirus B19, hepatitis B surface antigen, streptolysin, tetanus toxin, poliomyelitis, varicella, measles, cytomegalovirus, and diphtheria were measured before and after IAC using standard immunoassays, nephelometry, and neutralization assays. No new human or animal studies were performed for this study.

RESULTS Isoagglutinin depletion by the new IAC step The anti-A and anti-B isoagglutinin depletion ability of the IAC step was evaluated by measuring the blood group A and B RBC-binding capacities of the IgG product upand downstream of the IAC column. The IAC step reduced anti-A and anti-B isoagglutinin levels (RBC-binding capacity) in the IgG product by 88 and 90%, respectively

Fig. 3. Anti-A/B titer in IgG products produced with and without the IAC step. Anti-A (left) and anti-B (right) titers of final IgG , n 5 651) and modified manufacturing processes (with IAC; , product lots prepared by the unmodified (no IAC step; n 5 10), measured by the indirect agglutination test. neg 5 negative (no agglutination in undiluted sample). Volume 55, July 2015 TRANSFUSION S119

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Fig. 4. Anti-A/B titers in Privigen/Hizentra and Carimune/Sandoglobulin. Anti-A (left) and anti-B (right) titers measured in Privigen and Hizentra produced with the additional IAC step ( Cohn-like fractionation (Carimune/Sandoglobulin,

, n 5 10) and in product lots of an intravenous IgG preparation produced by , n 5 855). neg 5 negative (no agglutination in undiluted sample).

(Fig. 2). This decrease in isoagglutinin content is equivalent to a reduction of approximately three titer steps (a 50% reduction corresponds to one titer step; 75%, to two titer steps; 87.5%, to three titer steps).

Product quality after IAC Assessment of quality attributes and impurity profiles of intermediate and final IgG products demonstrated that introduction of the IAC step in the manufacturing process of Privigen did not affect the quality of the final IgG product (data not shown). Moreover, we found that the concentrations of antibodies specific to several selected infectious agents were nearly identical in the feed of the IAC column and in its flow-through fraction (Table 1).

Isoagglutinin concentrations in final IgG product lots The concentrations of anti-A and anti-B isoagglutinins in final IgG product lots were reduced compared to historical lots. In 651 Privigen historical lots produced by the unmodified manufacturing process without IAC, the most frequent anti-A and anti-B titers were 1:16 and 1:8, respectively. Isoagglutinin levels were measured in 10 final IgG product lots produced by the modified process: six lots of Privigen and four lots of Hizentra (CSL Behring, Berne, Switzerland; a 20% subcutaneous IgG product manufactured by the same process as Privigen). The most frequent anti-A and anti-B titers were three titer steps lower (1:2 and 1:1, respectively; Fig. 3). Carimune/Sandoglobulin (CSL Behring AG, Berne Switzerland) is an IV IgG preparation produced by CohnS120 TRANSFUSION Volume 55, July 2015

like fractionation. The highest anti-A and anti-B titers recorded in 855 lots of this product were both equal to 1:8; most frequent anti-A and anti-B titers were 1:4 and 1:2, respectively. Upon introduction of an IAC step in the manufacturing process of Privigen and Hizentra, the distribution of isoagglutinin titers in final product lots became comparable to the one of Carimune/Sandoglobulin (Fig. 4).

DISCUSSION We report a modification of the manufacturing process of Privigen and Hizentra designed for anti-A and anti-B reduction. We showed in a large-scale production facility that the introduction of a dedicated IAC step toward the end of the production process allowed reduction of isoagglutinin levels in the final IgG product by up to three titer steps. Very recently, we demonstrated the feasibility of reducing anti-A/B titers by screening donors and excluding plasma with high anti-A levels from the plasma pools used to produce Privigen.7 On average, this measure resulted in a one-titer-step reduction of isoagglutinin titers in final IgG product lots. The strategy presented here results in a more efficient reduction of anti-A/B levels without the need to discard a highly valuable and limited resource: donor plasma. This strategy can be implemented in all industry-scale production facilities, independently of the plasma sources. The isoagglutinin content in Privigen and Hizentra lots produced with the additional IAC step was similar to that recorded in Carimune/Sandoglobulin, an IgG product manufactured by Cohn-like fractionation. With this IgG

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product, lower rates of hemolytic events were recorded: zero of 71 patients receiving Carimune/Sandoglobulin versus 13 of 154 patients on IVIG from chromatographybased manufacturing processes (Gamunex, Gammagard Liquid, or Privigen).1 Furthermore, the isoagglutinin levels in IAC-purified Privigen and Hizentra lots are among the lowest reported for the IVIG products currently available, similar to those reported for another IgG preparation, ClairYg (LFB Biotechnologies, Courtaboeuf, France), that also relies on IAC for isoagglutinin reduction.8 The IAC column was shown to deplete isoagglutinins selectively. In fact, the additional purification step did not alter the concentration of any of the pathogen-specific antibodies tested in this work. In addition, no effect was observed on any other quality attributes. It seems that selective isoagglutinin reduction in Privigen and Hizentra is feasible without a negative impact on product quality. Introducing this IAC step in the manufacturing process was successfully validated in a large-scale production facility. Whether the achieved reduction of isoagglutinin levels in final IgG products would have a clinically relevant impact on the incidence of hemolysis has not yet been evaluated. An observational, noninterventional clinical study in patients receiving IgG replacement therapy is planned in the years after implementation of IAC in all production facilities. ACKNOWLEDGMENTS The authors thank Alphonse Hubsch from CSL Behring AG, Berne, Switzerland, for the critical review of the manuscript. We also acknowledge the editorial support of Nathalie Preiswerk, PhD, from Fishawack Communications GmbH, member of the Fishawack Group of Companies, supported by CSL Behring.

CONFLICT OF INTEREST All authors are employees of and have financial interest in CSL Behring.

REFERENCES 1. Kahwaji J, Barker E, Pepkowitz S, et al. Acute hemolysis after high-dose intravenous immunoglobulin therapy in highly HLA sensitized patients. Clin J Am Soc Nephrol 2009;4: 1993-7. 2. MacLennan S. High titer anti-A/B testing of donors within the National Blood Service (NBS). Information document INF/ MED/MA/004/02. London: NBS; 2002. 3. Cohn EJ, Strong LE, Hughes WI, et al. Preparation and properties of serum and plasma proteins. IV. A system for the separation into fractions of the protein and lipoprotein components of tissues and fluids. J Am Chem Soc 1946;68:459-75. 4. Radosevich M, Burnouf T. Intravenous immunoglobulin G: trends in production methods, quality control and quality assurance. Vox Sang 2010;98:12-28. 5. Bellac CL, Polatti D, Hottiger T, et al. Anti-A and anti-B haemagglutinin levels in intravenous immunoglobulins: are they on the rise? A comparison of four different analysis methods and six products. Biologicals 2014;42:57-64. 6. Stucki M, Boschetti N, Schaefer W, et al. Investigations of prion and virus safety of a new liquid IVIG product. Biologicals 2008;36:239-47. 7. Siani B, Willimann K, Wymann S, et al. Isoagglutinin reduction in human immunoglobulin products by donor screening. Biol Ther 2014;4:15-26. 8. Dhainaut F, Guillaumat PO, Dib H, et al. In vitro and in vivo properties differ among liquid intravenous immunoglobulin preparations. Vox Sang 2013;104:115-26.

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Isoagglutinin reduction by a dedicated immunoaffinity chromatography step in the manufacturing process of human immunoglobulin products.

The passive transfer of antibodies specific to blood groups A and B (also called isoagglutinins) contained in immunoglobulin (Ig)G products for intrav...
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