Quantifying the thrombogenic potential of human plasma-derived immunoglobulin products

Polyvalent immunoglobulin G (IgG) products obtained by fractionation of human plasma are used to treat a broad range of conditions, including immunodeficiency syndromes and autoimmune, inflammatory, and infectious diseases. Recent incidences of increased thromboembolic events (TEEs) associated with intravenous (IV) IgG (IVIG) led to recalls of some products and increased regulatory oversight of manufacturing processes in order to ensure that products are essentially free of procoagulant/thrombogenic plasma protein contaminants. Laboratory investigations have now identified activated factor XI (FXIa) as the likely causative agent of IVIG-related TEEs. Quantification of the thrombogenic potential is becoming a requirement made to fractionators (a) to validate the capacity of IVIG and subcutaneous IgG manufacturing processes to remove procoagulant contaminants and (b) to establish the safety of the final products. However, in the absence of a recommended test by the main regulatory authorities, several analytical approaches have been evaluated by fractionators, regulators, and university groups. This review focuses on the scientific rationale, merits, and applications of several analytical methods of quantifying the thrombogenic potential of IgG products and intermediates to meet the latest regulatory requirements.     

Technology of immunoglobulin production. I. Technological aspects of purification

Plasma-derived intravenous immunoglobulin (IVIG) preparations have become the major plasma products on the world blood product market due to the successful application for the prophylactic prevention of infectious diseases and replacement therapy in autoimmune and inflammatory diseases. In this review classical manufacturing processes as well as new chromatographic, membrane, and mixed industrial technologies are discussed with respect to the cost and amount of the final product of high quality and virus- and prion-safety which is to be obtained.     

Purification of IgG and albumin from human plasma by aqueous two phase system fractionation

The current shortages in human plasma products at global levels justify the development of new, cost effective plasma fractionation methods. We have developed a fractionation process to obtain immunoglobulin G (IgG) and albumin‐enriched fractions based on polymer‐salt aqueous two phase system (ATPS). A small‐scale (0.02 L) ATPS composed of polyethyleneglycol 3350 (PEG), potassium phosphate and sodium chloride, at pH 6.1, was evaluated and subjected to 50‐fold scale‐up (1 L). Further purification of the fractions was performed using caprylic acid precipitation and ion exchange chromatography. Similar yield and purity were obtained at both small and large scales. IgG precipitated in the PEG rich upper phase at 83% recovery and 2.75‐fold purification factor. An 81% pure albumin fraction was obtained in the salt rich bottom phase with a 91% yield. After polishing, IgG was obtained at a recovery of 70% and a purity of 92%. Corresponding values for albumin were 91% and 90%. This IgG and albumin fractionation technology deserves further evaluation as it may represent a potential alternative to conventional plasma fractionation methods. © 2012 American Institute of Chemical Engineers Biotechnol. Prog., 28: 1005–1011, 2012     

Functional integrity of intravenous immunoglobulin following irradiation with a virucidal dose of gamma radiation.

Although intravenous immunoglobulins (IVIG) and other plasma therapeutics have had a relatively good safety record, improved methods for viral clearance are constantly being evaluated and incorporated into new manufacturing processes. Gamma irradiation has been used routinely to assure sterility of healthcare products and medical devices, but it has not been applied successfully as a viral inactivation method for biologics. We examine whether virucidal doses of gamma irradiation (50 kGy) can be delivered to a manufacturing intermediate form of IVIG, a fractionated plasma paste, with negligible effect on structural and functional integrity of purified IgG product. Immunoglobulins from paste were examined for radiation-induced damage by SDS-PAGE and ELISAs utilizing viral antigens specific for rubella, CMV and mumps. Fc domain integrity was assessed by immunoblotting, quantitatively comparing the binding of irradiated and non-irradiated materials to cell surface Fcgamma receptors, and by employing quantitative RT-PCR to study the kinetics of accumulation of mRNA for the immune modulatory cytokines IL-1alpha, IL-1beta, IL-4, IL-8, IFNgamma, and TNFalpha. The results demonstrate that Fab and Fc domains of IVIG remain essentially intact and functional after gamma irradiation to virucidal doses, suggesting that this method could be used to enhance the safety of IVIG products.     

A Comparative Structural and Bioanalytical Study of IVIG Clinical Lots

Intravenous immunoglobulin are important bio-therapeutics used in the replacement therapy for primary and secondary immunodeficiencies, chronic inflammatory disorders and several autoimmune haematologic disorders. Currently, a number of immunoglobulin intravenous (IVIG) products have been approved by the Food and Drug Administration (FDA) and are available commercially. It is known that small differences in the manufacturing processes as well as in the formulations may affect their clinical efficacy and tolerability. Therefore, given the complexity of the multi-step process required for the isolation of IVIG from human plasma, it is necessary to ensure a rigorous quality control of final products. We show here that a set of different bioanalytical techniques can be conveniently used to comparatively characterize, at a quantitative and qualitative level, different lots of IVIG preparations and to unveil randomly occurring impurities which can also affect the overall product stability. We have used circular dichroism, surface plasmon resonance and two-dimensional electrophoresis (2DE), and have demonstrated that this combination of bioanalytical approaches is very useful to improve the quality control of antibodies and to monitor the reliability of the IVIG manufacturing process.     

Human intravenous immunoglobulin preparation and virus inactivation by pasteurization and solvent detergent treatment

Human intravenous immunoglobulin (IVIG) solutions were prepared by two different methods and compared to each other. The crude immunoglobulin fraction obtained from Cohn-Oncley fractionation of plasma was further purified and subjected to virus inactivation, either by polyethylene glycol precipitation and pasteurization at 60 degrees C for 10 hours, or by ion exchange chromatography and solvent/detergent treatment. The final preparations, formulated in 5% immunoglobulin solutions were characterized by in vitro analyses of biochemical and biological properties and compared with the samples of other manufacturer's IVIG solution products. The critical properties evaluated in this study were purity, molecular intactness, and the biological functions such as Fc function and anticomplementary activity. Virus inactivation and removal by processing steps and by deliberate virucidal steps, as described above, were tested on various human pathogenic viruses, such as human immunodeficiency and experimental model viruses. The tested viruses were successfully inactivated and removed. We conclude that the intravenous immunoglobulins prepared by two different methods, as described above, provide an equivalent viral safety and quality.     

IL-2 and GM-CSF are regulated by DNA demethylation during activation of T cells, B cells and macrophages

High-dose intravenous immunoglobulin (IVIG) preparations are currently used for the treatment of autoimmune diseases such as immune thrombocytopenic purpura (ITP). Although the mechanisms of IVIG efficacy remain enigmatic, some clinical and laboratory studies suggest that interaction of the Fc domain of IgG, especially the Fc domain of dimeric IgG, with its receptors (Fc gamma receptors; FcγRs) plays an essential role. In this study, IVIG was dimerized with chemical crosslinkers to augment its therapeutic efficacy. Dimerized IVIG was found to have a much higher affinity for FcγRs than monomeric IVIG. In a mouse ITP model, chemically dimerized IVIG abrogated the decrease in platelet numbers in the blood that was caused by an anti-platelet antibody at a dose that was one tenth of the required dose of IVIG. These results suggest that chemical dimerization of IVIG should greatly improve the efficacy of IVIG therapy of ITP.     

Analysis and functional consequences of increased Fab-sialylation of intravenous immunoglobulin (IVIG) after lectin fractionation

It has been proposed that the anti-inflammatory effects of intravenous immunoglobulin (IVIG) might be due to the small fraction of Fc-sialylated IgG. In this study we biochemically and functionally characterized sialic acid-enriched IgG obtained by Sambucus nigra agglutinin (SNA) lectin fractionation. Two main IgG fractions isolated by elution with lactose (E1) or acidified lactose (E2) were analyzed for total IgG, F(ab')(2) and Fc-specific sialic acid content, their pattern of specific antibodies and anti-inflammatory potential in a human in vitro inflammation system based on LPS- or PHA-stimulated whole blood. HPLC and LC-MS testing revealed an increase of sialylated IgG in E1 and more substantially in the E2 fraction. Significantly, the increased amount of sialic acid residues was primarily found in the Fab region whereas only a minor increase was observed in the Fc region. This indicates preferential binding of the Fab sialic acid to SNA. ELISA analyses of a representative range of pathogen and auto-antigens indicated a skewed antibody pattern of the sialylated IVIG fractions. Finally, the E2 fraction exerted a more profound anti-inflammatory effect compared to E1 or IVIG, evidenced by reduced CD54 expression on monocytes and reduced secretion of MCP-1 (CCL2); again these effects were Fab- but not Fc-dependent. Our results show that SNA fractionation of IVIG yields a minor fraction (approx. 10%) of highly sialylated IgG, wherein the sialic acid is mainly found in the Fab region. The tested anti-inflammatory activity was associated with Fab not Fc sialylation.     

冻干静脉注射人血丙种球蛋白的研制

作者参照瑞典ＫａｂｉＶｉｔｒｕｂ药厂的“Ｇａｍｍｏｎａｔｉｖ”的制备方法,以低温乙醇法的ＦⅡ沉淀为原料,经ＤＥＡＥ－ＳｅｐｈａｄｅｘＡ－５０吸附,加入人血白蛋白作为稳定剂制备静脉注射人血丙种球蛋白（ＩＶＩＧ）。该制剂的γ－球蛋白纯度＞９４％,ＩｇＧ单体二聚体含量＞９７％,ＩｇＧ各亚类均存在且构成比与正常人血浆相似。抗补体活性（ＡＣＡ）和前激肽释放酶激活剂活性（ＰＫＡ）符合规程要求,安全性和稳定性好。该制剂为冻干剂型,便于运输和保存。     

Both the Fab and Fc domains of IgG are essential for ROS emission from TNF-α-primed neutrophils by IVIG

Intravenous immunoglobulin (IVIG) is currently a very important therapeutic used for not only infectious diseases, but also for autoimmune diseases such as idiopathic thrombocytopenic purpura (ITP). Untoward reactions of IVIG have been thought to result from complement activation by aggregated IgG in IVIG. In addition, the aggregates have been known to activate neutrophils, which may result in the untoward reactions. However, the effect and mechanism of IVIG on neutrophils remain unclear. In this study, we investigated the activation of neutrophils by IVIG in terms of their reactive oxygen species (ROS) emission to elucidate the mechanisms. IVIG-induced ROS emission from purified neutrophils was remarkably augmented by TNF-α priming of the cells. The ROS emission from TNF-α-primed neutrophils occurred by activation with whole gammaglobulin (GG) molecules, but not F(ab')(2), Fc, or a mixture of F(ab')(2) and Fc. ROS emission by GG was inhibited by the F(ab')(2) fragment and an inhibitory antibody against FcγRIII. These results suggest that binding of IVIG to not only surface antigen(s), but also FcγRIII on neutrophils, is involved in IVIG-induced ROS emission from TNF-α-primed neutrophils, and contribute to the untoward reactions of IVIG.     

A microassay for measurement of Fc function of human immunoglobulin preparations by using tetanus toxoid as antigen.

In order to minimize possible adverse reactions, the functional integrity of proteins in products derived from human plasma has to be unaffected by methods of preparation and storage conditions. Numerous biologically relevant functions of IgG, a major component of immunoglobulin for intravenous use preparations (IVIG), rely on the integrity of Fc fragments. Manufacturers are obliged to prove that Fc-mediated functions are maintained in IVIG preparations. The European Pharmacopoeia's monograph proposes a Rubella antigen-based test for Fc function of immunoglobulins. We present a modification of the proposed method achieved by using more convenient and readily available tetanus toxoid as an alternative antigen target and by adapting the procedure for the use on microtitre plates, thus greatly enhancing its feasibility and sample throughput. The test conditions were optimized so that batch-to-batch variability in tetanus antibody content did not influence the result. The precision of the test was within +/- 5%. By using this test, we compared Fc functionality of 9 commercial IVIG-7S preparations, which were prepared by using different virus inactivation/removal approaches. No significant differences between them have been found.     

Investigations of prion and virus safety of a new liquid IVIG product.

A highly purified, liquid, 10% immunoglobulin product stabilized with proline, referred to as IgPro10 has recently been developed. IgG was purified from human plasma by cold ethanol fractionation, octanoic acid precipitation and anion-exchange chromatography. The manufacturing process includes two distinctly different partitioning steps and virus filtration, which were also assessed for the removal of prions. Prion removal studies used different spike preparations (brain homogenate, microsomes, purified PrP(sc)) and three different detection methods (bioassay, Western blot, conformation-dependent immunoassay). All of the investigated production steps were shown to reduce significantly all different spike preparations, resulting in an overall reduction of >10log(10). Moreover, the biochemical assays proved equally effective to the bioassay for the demonstration of prion elimination. Four of the manufacturing steps cover three different mechanisms of virus clearance. These are: i) virus inactivation; ii) virus filtration; and iii) partitioning. These mechanisms were assessed for their virus reduction capacity. Virus validation studies demonstrated overall reduction factors of >18log(10) for enveloped and >7log(10) for non-enveloped model viruses. In conclusion, the IgPro10 manufacturing process has a very high reduction potential for prions and for a wide variety of viruses resulting in a state-of-the-art product concerning safety towards known and emerging pathogens.     

Intravenous immunoglobulin treatment of four patients with juvenile polyarticular arthritis associated with persistent parvovirus B19 infection and antiphospholipid antibodies

Children with rheumatic oligoarthritis and polyarthritis frequently establish persistent parvovirus B19 infections that may be associated with the production of antiphospholipid antibodies (anti-PL IgG). In this study we analysed the influence of high-dose intravenous immunoglobulin (IVIG) therapy on virus load, on the level of anti-PL IgG and its potential capacity to improve the patients' clinical status. Four juvenile patients with long-lasting polyarticular rheumatic diseases and persistent parvovirus B19 infection, associated in three cases with the presence of antibodies against beta2-glycoprotein I (anti-beta2GPI IgG), were treated with two cycles of IVIG on five successive days (0.4 g/kg per day). Clinical parameters including scores of disease activity, virus load and anti-PL IgG levels were determined before, during and after treatment. Two patients showed a complete remission that has lasted 15 months. During that period they showed neither clinical nor laboratory signs of inflammation. Viral DNA was not detectable in serum, and a decrease in anti-beta2GPI IgG was observed. As assessed by the Childhood Health Assessment Questionnaire and the Health-related Quality of Life Questionnaire for Children, both patients were no longer restricted in their activities of daily living and no impact on the health-related quality of life was observed. In one patient the therapy failed: there was no improvement of symptoms and no decrease in virus load or inflammatory parameters. In the fourth patient, clinical and laboratory parameters did not improve despite a decrease in both viral load and anti-PL IgG. Our results show that the use of IVIG to treat parvovirus B19-triggered polyarticular rheumatic disease of childhood might offer an opportunity to improve this disabling condition.     

Different IVIG Glycoforms Affect In Vitro Inhibition of Anti-Ganglioside Antibody-Mediated Complement Deposition

Intravenous immunoglobulin (IVIG) is the first line treatment for Guillain–Barré syndrome and multifocal motor neuropathy, which are caused by anti-ganglioside antibody-mediated complement-dependent cytotoxicity. IVIG has many potential mechanisms of action, and sialylation of the IgG Fc portion reportedly has an anti-inflammatory effect in antibody-dependent cell-mediated cytotoxicity models. We investigated the effects of different IVIG glycoforms on the inhibition of antibody-mediated complement-dependent cytotoxicity. Deglycosylated, degalactosylated, galactosylated and sialylated IgG were prepared from IVIG following treatment with glycosidases and glycosyltransferases. Sera from patients with Guillain–Barré syndrome, Miller Fisher syndrome and multifocal motor neuropathy associated with anti-ganglioside antibodies were used. Inhibition of complement deposition subsequent to IgG or IgM autoantibody binding to ganglioside, GM1 or GQ1b was assessed on microtiter plates. Sialylated and galactosylated IVIGs more effectively inhibited C3 deposition than original IVIG or enzyme-treated IVIGs (agalactosylated and deglycosylated IVIGs). Therefore, sialylated and galactosylated IVIGs may be more effective than conventional IVIG in the treatment of complement-dependent autoimmune diseases.     

Novel ligands for the affinity-chromatographic purification of antibodies.

Affinity chromatography represents one of the most powerful fractionation techniques for the large-scale purification of biotechnological products. Despite its potential, the use of this methodology is limited by the availability of specific ligands for each target. Combinatorial chemistry and molecular modeling, often combined, have become interesting and innovative methods for generating novel ligands, tailored to specific biotechnological needs. One of the greatest area of application has been the discovery of novel ligands for the purification of antibodies, which represent an emerging but very important class of innovative therapeutic agents for the treatment of a vast array of diseases. Naturally available affinity ligands, such as Protein A or G for IgG purification or lectins for IgA and IgM purification, which are obtained from microorganisms or genetically modified bacteria through complex and expensive procedures, are not well suited for large-scale purification and require moreover time-consuming analytical controls to check for the presence of contaminants which may affect the safety of the purified antibody for clinical purposes. Recent results suggest that the application of combinatorial technologies and molecular modeling for the discovery of synthetic ligands may open new avenues for the development of more efficient, less expensive and--more importantly--safer procedures for antibody purification at the industrial level.     

IgG subclass distribution among antibodies to varicella-zoster virus in human varicella/zoster immunoglobulin preparations and the corresponding donor plasma

IgG subclasses to varicella-zoster virus (VZV) were detected in plasma from different sources used for the production of varicella/zoster immunoglobulin (VZIG). IgG1 and IgG3 were the principal virus antibodies in plasma from healthy donors as well as from convalescents after primary and reactivated disease. Anti-VZV IgG3 antibodies were predominant among varicella convalescents while IgG1 antibodies dominated among zoster convalescents. IgG4 antibodies were present in zoster convalescents and healthy donors but were rarely detected in varicella convalescents. Antiviral IgG2 antibodies were found only in a few cases. Studies of plasma samples collected from one varicella convalescent during a period of seven months following an outbreak of disease, demonstrated a rapid fall in antiviral IgG1 and IgG3, while IgG4 increased to reach a maximum six months after the onset of symptoms. The relative distribution of VZV-specific subclasses in a plasma pool was conserved during a fractionation procedure combining polyethyleneglycol 6000 precipitation with ion exchange chromatography, thus suggesting that the protective efficacy is maintained in the resulting immunoglobin preparations.     

Analyzing titers of antibodies against bacterial and viral antigens,and bacterial toxoids in the intravenous immunoglobulins utilized in Taiwan

Intravenous immunoglobulin (IVIG) manufactured from human plasma contains IgG as the primary ingredient, and is used for indications such as immunodeficiency syndrome. Available IVIGs in Taiwan are either manufactured from Taiwanese or North American plasma. The effectiveness of the national immunization program of Taiwan can be evaluated by analyzing and comparing IVIG antibody titers that are induced through the corresponding vaccines (tetanus, diphtheria, and pertussis, measles, rubella, hepatitis A, hepatitis B and varicella). Both enzyme-linked immunosorbent assay (ELISA) and the in vitro neutralization test demonstrated that all IVIGs provide adequate clinical protection against diphtheria and tetanus toxins. ELISA results further revealed that plasma of Taiwanese subjects contains higher levels of pertussis toxin and filamentous hemagglutinin antibodies, when compared to foreign IVIGs. This may be related to the later adoption of acellular pertussis vaccine in Taiwan. Antibodies titers against measles, rubella, hepatitis A, and varicella-zoster virus were otherwise low. Low titers of hepatitis B surface antigen antibodies are present in Taiwanese plasma IVIG, indicating immune memory decline or loss. In conclusion, our results show that Taiwanese IVIG contains varying titers of vaccine-induced antibodies, and serves as a guide for future amendments to Taiwan's immunization program.     

Intravenous immunoglobulin treatment of four patients with juvenile polyarticular arthritis associated with persistent parvovirus B19 infection and antiphospholipid antibodies

Children with rheumatic oligoarthritis and polyarthritis frequently establish persistent parvovirus B19 infections that may be associated with the production of antiphospholipid antibodies (anti-PL IgG). In this study we analysed the influence of high-dose intravenous immunoglobulin (IVIG) therapy on virus load, on the level of anti-PL IgG and its potential capacity to improve the patients' clinical status. Four juvenile patients with long-lasting polyarticular rheumatic diseases and persistent parvovirus B19 infection, associated in three cases with the presence of antibodies against β₂-glycoprotein I (anti-β₂GPI IgG), were treated with two cycles of IVIG on five successive days (0.4 g/kg per day). Clinical parameters including scores of disease activity, virus load and anti-PL IgG levels were determined before, during and after treatment. Two patients showed a complete remission that has lasted 15 months. During that period they showed neither clinical nor laboratory signs of inflammation. Viral DNA was not detectable in serum, and a decrease in anti-β₂GPI IgG was observed. As assessed by the Childhood Health Assessment Questionnaire and the Health-related Quality of Life Questionnaire for Children, both patients were no longer restricted in their activities of daily living and no impact on the health-related quality of life was observed. In one patient the therapy failed: there was no improvement of symptoms and no decrease in virus load or inflammatory parameters. In the fourth patient, clinical and laboratory parameters did not improve despite a decrease in both viral load and anti-PL IgG. Our results show that the use of IVIG to treat parvovirus B19-triggered polyarticular rheumatic disease of childhood might offer an opportunity to improve this disabling condition.     

Inactivation and removal of influenza A virus H1N1 during the manufacture of plasma derivatives

Although transmission of pandemic influenza A virus H1N1 2009 is still occurring globally, little has been reported about how this outbreak has affected the safety of plasma derivatives. To evaluate the safety of plasma derivatives, dedicated virus clearance processes used during their production were investigated for their effectiveness in eliminating this virus of recent concern. In this study, influenza A virus H1N1 strain A/NWS/33 (H1N1) was chosen as a surrogate. H1N1 was completely inactivated by fraction IV fractionation as well as pasteurization during the manufacture of albumin. H1N1 was also effectively removed into the precipitate by fraction III fractionation and completely inactivated by low pH incubation as well as pasteurization during the manufacture of intravenous immunoglobulin. H1N1 was completely inactivated within 1 min of solvent/detergent treatment using 0.3% tri (n-butyl) phosphate and 1.0% Triton X-100 and also completely inactivated within 10 min of dry-heat treatment at 98 °C during the manufacture of factor VIII. H1N1 was completely removed by virus filtration process using Viresolve NFP filter and also completely inactivated by pasteurization during the manufacture of anti-thrombin III. These results indicate that all the virus clearance processes commonly used have sufficient H1N1 reducing capacity to achieve a high margin of safety.     

Flebogamma® DIF (intravenous immunoglobulin) purification process effectively eliminates procoagulant activities

BackgroundStudies have demonstrated that traces of activated factor XI (FXIa) present in specific brands of intravenous immunoglobulin (IVIG) concentrates may pose a thrombogenic risk.AimTo characterize procoagulant activity during fractionation and the elimination capacity of the Flebogamma^® DIF (Grifols' IVIG) manufacturing process.MethodsFlebogamma^® DIF fractionation steps included cryoprecipitate supernatant (Cryo/S), Fraction (Fr) I supernatant, and Fr II + III suspension. Purification steps included ultrafiltrate I, acid treatment, and pasteurization. Samples were assessed for total protein, IgG, and procoagulant activation markers.ResultsCryo/S showed no procoagulant activity for prekallikrein activator (PKA), kallikrein-like, and non-activated partial thromboplastin time (NaPTT) with normal (-PPP) or FXI-deficient (-FXI) platelet poor plasma. Thrombin generation test (TGT)-PPP and TGT-FXI were <83–148 and <53–197 nM thrombin, respectively. Shortened NaPTTs (100–296 s), high PKA (51–119 IU/mL), kallikrein-like activities (0.043–0.075 ΔAU/min), positive TGTs (98–298 nM), and FXIa (9.5–14.0 ng/mL) were detected in Fr II + III. After pasteurization, no residual evidence of any procoagulant activity marker was observed, including the final IVIG concentrate at 5% or 10% protein. Results were similar in Fr II + III from different IVIG manufacturing facilities.ConclusionsThe Flebogamma^® DIF production process is capable of eliminating procoagulant activity because of its purification steps.