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

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.

Local tolerance and stability up to 24 months of a new 20% proline-stabilized polyclonal immunoglobulin for subcutaneous administration.

Subcutaneous administration of human IgG is an alternative to intravenous replacement therapy that is associated with more stable serum IgG levels and fewer systemic adverse events. Highly concentrated IgG solutions are most convenient to minimize infusion volume, but their preparation and stability presents substantial technical difficulties. We report on the stability and local tolerance of IgPro20, an l-proline-stabilized, 20% polyvalent human IgG developed for subcutaneous administration. Stability was tested according to ICH guidelines. Local tolerance and vasoactivity were examined in rabbit and rat models, respectively. The presence of l-proline in IgPro20 reduced viscosity and addition of Polysorbate 80 and inert gassing improved the appearance of the solution. After storage at 25 °C for 24 months, monomer + dimer content, aggregates, and fragments were within specification (≥ 90.0%, ≤ 4.0%, and ≤ 10.0%, respectively), and Fc function and antibody activities were maintained. In rats, intravenous injection of IgPro20 produced mild and transient hypotension comparable to that seen with intravenous IgG products. Local tolerance of IgPro20 in rabbits was comparable to that of a marketed subcutaneous IgG, Beriglobin P. Functionality and quality of IgPro20 are maintained during storage at 25 °C for at least 24 months. The product is well tolerated as assessed in animal models.

L-Proline reduces IgG dimer content and enhances the stability of intravenous immunoglobulin (IVIG) solutions.

Liquid intravenous immunoglobulin (IVIG) products offer improved convenience in preparation but often lack sufficient stability to allow room temperature storage. Furthermore, clinical tolerability may be affected due to formation of idiotype/anti-idiotype IgG dimers and/or aggregates. Here we report on the development of a 10% IVIG formulation with optimized stability achieved by the use of l-proline. The stability of concentrated liquid IVIG was strongly pH dependent. Aggregate formation, yellowish discoloration of the solution and loss of anti-hepatitis B surface antigen (HBs) antibody activity was minimal at intermediate pH (pH 4.8-5.3). Fragmentation of IgG was highest at low pH (pH 4.1). Idiotype/anti-idiotype IgG dimer formation was highest at neutral pH and was reduced with decreasing pH. The presence of L-proline further improved stability by inhibiting protein aggregation, reducing loss of anti-HBs antibody activity and decreasing coloring, particularly compared with glycine formulations. The IgG dimer content was up to 30% lower in solutions containing L-proline compared with those containing glycine or other stabilizers. In conclusion, a weakly acidic pH of approximately 5 and L-proline as stabilizer are optimal conditions for long-term stability of a liquid IVIG. L-proline, an amphiphilic, naturally occurring amino acid, is superior to glycine in restricting IgG dimer formation.

Monomerization of dimeric IgG of intravenous immunoglobulin (IVIg) increases the antibody reactivity against intracellular antigens.

Intravenous immunoglobulin (IVIg) preparations are derived from pooled plasma from up to 60,000 healthy human donors and reflect the immunologic experience of the donor population. IVIg contains monomeric and dimeric IgG populations which are in a dynamic equilibrium depending on concentration, pH, temperature, donor pool size, time and stabilizers added in order to keep the portion of dimeric IgG below a certain level. In the present study, monomeric and dimeric fractions were isolated by size exclusion chromatography. The dimeric fractions, however, showed a dynamic instability and tended to dissociate. Both dimeric and monomeric IgG fractions were acid treated (pH 4) in order to dissociate the dimeric IgG. Western-blot analysis identified a sub-population of SDS resistant IgG dimers. Furthermore, the reactivities of the fractions were tested against a panel of self- and exo-antigens. There was a marked increase in activity of the dimeric compared to the monomeric IgG fraction against various intracellular self-antigens. Our data indicates that the increased reactivities of pH 4-treated fractions can mainly be attributed to dimer dissociation, as pH 4-treated monomers do not show significantly increased activities against a range of antigens.

Self-reactivity in the dimeric intravenous immunoglobulin fraction.

Therapeutic intravenous immunoglobulin (IVIg) preparations contain antibodies reflecting the cumulative antigen experience of the donor population. IVIg contains variable amounts of monomeric and dimeric IgG, but there is little information available on their comparative antibody specificities. We have isolated highly purified fractions of monomeric and dimeric IgG by size-exclusion chromatography. Following treatment of all fractions at pH4, analyses by immunodot and immunocytology on human cell lines showed a preferential recognition of autoantigens in the dimeric IgG fraction. Investigation of the HEp-2 cytoplasmic proteome by 2D-PAGE, Western blot, and subsequent identification of IVIg reactive spots by mass spectrometry (LC-MS/MS) showed that IVIg recognized only a restricted set of the total proteins. Similar experiments showed that more antigens were recognized by the dimeric IgG fraction, especially when the dissociated dimer fraction was used, as compared to its monomeric counterpart. These observations are consistent with idiotype-anti-idiotype masking of auto-specific Abs in the dimeric fraction of IVIg.

Effect of IVIG Formulation on IgG Binding to Self- and Exo- Antigens In Vitro and In Vivo.

In relation to the recent trials of Intravenous Immunoglobulin (IVIG) in Alzheimer's Disease (AD) it was demonstrated that different IgG preparations contain varying amounts of natural anti-amyloid ß (Aß) antibodies as measured by ELISA. We therefore investigated the relevance of ELISA data for measuring low-affinity antibodies, such as anti-Aß. We analysed the binding of different commercial Immunoglobulin G (IgG) preparations to Aß, actin and tetanus toxoid in different binding assays to further investigate the possible cause for observed differences in binding to Aß and actin between different IgG preparations. We show that the differences of commercial IgG preparations in binding to Aß and actin in ELISA assays are artefactual and only evident in in vitro binding assays. In functional assays and in vivo animal studies the different IVIG preparations exhibited very similar potency. ELISA data alone are not appropriate to analyse and rank the binding capacity of low-affinity antibodies to Aß or other endogenous self-antigens contained in IgG preparations. Additional analytical methods should be adopted to complement ELISA data.

Comparative analysis of antigen specificities in the monomeric and dimeric fractions of intravenous immunoglobulin.

Intravenous immunoglobulin (IVIG) preparations are derived from the pooled plasma of thousands of healthy donors and contain a complex mix of antibodies. Depending on the formulation, IVIG preparations contain variable amounts of monomeric and dimeric IgG. The biological and therapeutic significance of these IVIG fractions is still ill defined. Kinetic analysis of monomeric and dimeric IgG isolated by size-exclusion chromatography revealed a stable monomeric versus an unstable dimeric IgG fraction tending to dissociation. Biochemical analysis by 2D gel electrophoresis and isotype analysis showed no significant differences between the fractions. In contrast, comparative analysis by immunodot, ELISA, FACS, and immunohistology of monomeric and dimeric IgG fractions showed a preferential reactivity of the dimeric IgG on a variety of both self-antigens and exoantigens.

Intravenous immunglobulin binds beta amyloid and modifies its aggregation, neurotoxicity and microglial phagocytosis in vitro.

Intravenous Immunoglobulin (IVIG) has been proposed as a potential therapeutic for Alzheimer's disease (AD) and its efficacy is currently being tested in mild-to-moderate AD. Earlier studies reported the presence of anti-amyloid beta (Aß) antibodies in IVIG. These observations led to clinical studies investigating the potential role of IVIG as a therapeutic agent in AD. Also, IVIG is known to mediate beneficial effects in chronic inflammatory and autoimmune conditions by interfering with various pathological processes. Therefore, we investigated the effects of IVIG and purified polyclonal Aß-specific antibodies (pAbs-Aß) on aggregation, toxicity and phagocytosis of Aß in vitro, thus elucidating some of the potential mechanisms of action of IVIG in AD patients. We report that both IVIG and pAbs-Aß specifically bound to Aß and inhibited its aggregation in a dose-dependent manner as measured by Thioflavin T assay. Additionally, IVIG and the purified pAbs-Aß inhibited Aß-induced neurotoxicity in the SH-SY5Y human neuroblastoma cell line and prevented Aß binding to rat primary cortical neurons. Interestingly, IVIG and pAbs-Aß also increased the number of phagocytosing cells as well as the amount of phagocytosed fibrillar Aß by BV-2 microglia. Phagocytosis of Aß depended on receptor-mediated endocytosis and was accompanied by upregulation of CD11b expression. Importantly, we could also show that Privigen dose-dependently reversed Aß-mediated LTP inhibition in mouse hippocampal slices. Therefore, our in vitro results suggest that IVIG may have an impact on different processes involved in AD pathogenesis, thereby promoting further understanding of the effects of IVIG observed in clinical studies.

An adult passive transfer mouse model to study desmoglein 3 signaling in pemphigus vulgaris.

Evidence has accumulated that changes in intracellular signaling downstream of desmoglein 3 (Dsg3) may have a significant role in epithelial blistering in the autoimmune disease pemphigus vulgaris (PV). Currently, most studies on PV involve passive transfer of pathogenic antibodies into neonatal mice that have not finalized epidermal morphogenesis, and do not permit analysis of mature hair follicles (HFs) and stem cell niches. To investigate Dsg3 antibody-induced signaling in the adult epidermis at defined stages of the HF cycle, we developed a model with passive transfer of AK23 (a mouse monoclonal pathogenic anti-Dsg3 antibody) into adult 8-week-old C57Bl/6J mice. Validated using histopathological and molecular methods, we found that this model faithfully recapitulates major features described in PV patients and PV models. Two hours after AK23 transfer, we observed widening of intercellular spaces between desmosomes and EGFR activation, followed by increased Myc expression and epidermal hyperproliferation, desmosomal Dsg3 depletion, and predominant blistering in HFs and oral mucosa. These data confirm that the adult passive transfer mouse model is ideally suited for detailed studies of Dsg3 antibody-mediated signaling in adult skin, providing the basis for investigations on novel keratinocyte-specific therapeutic strategies.

Pemphigus vulgaris identifies plakoglobin as key suppressor of c-Myc in the skin.

The autoimmune disease pemphigus vulgaris (PV) manifests as loss of keratinocyte cohesion triggered by autoantibody binding to desmoglein (Dsg)3, an intercellular adhesion molecule of mucous membranes, epidermis, and epidermal stem cells. Here we describe a so far unknown signaling cascade activated by PV antibodies. It extends from a transient enhanced turn over of cell surface-exposed, nonkeratin-anchored Dsg3 and associated plakoglobin (PG), through to depletion of nuclear PG, and as one of the consequences, abrogation of PG-mediated c-Myc suppression. In PV patients (6/6), this results in pathogenic c-Myc overexpression in all targeted tissues, including the stem cell compartments. In summary, these results show that PV antibodies act via PG to abolish the c-Myc suppression required for both maintenance of epidermal stem cells in their niche and controlled differentiation along the epidermal lineage. Besides a completely novel insight into PV pathogenesis, these data identify PG as a potent modulator of epithelial homeostasis via its role as a key suppressor of c-Myc.