The mechanism of the efficacy of Intravenous immunoglobulins (IVIG) in autoimmune and inflammatory diseases is not well understood. This study aimed at understanding mechanisms of IVIG-mediated suppression of effector cell activities of peripheral blood mononuclear cells (PBMC) in antibody-dependent cellular cytotoxicity (ADCC). We were particularly interested in CD56dim NK cells, the main ADCC effector cells in PBMC. Exposure of PBMC to IVIG for at least 48 h induced a caspase-3-dependent apoptotic cell death of CD56dim NK cells without affecting CD56bright NK cells. Induction of apoptosis in CD56dim NK cells and concomitant suppression of ADCC effector activities of PBMC was associated with the monomer fraction of IVIG. Moreover, it was independent of IgG sialyation, did not depend on engagement of FcγRIII and could not be mimicked by IVIG (Fab’)2 or IVIG Fc preparations. The described effect could contribute to the reduction of peripheral NK cells observed during IVIG therapy in patients.

BACKGROUNDHigh-dose intravenous immunoglobulin (IVIG) treatments are implicated in hemolytic events in some patients receiving treatment. The passive transfer of IgG anti-A and anti-B agglutinin is thought to play a role in the development of these events. The purpose of this study was to determine the prevalence of high-titer IgG anti-A and anti-B in plasma donors and investigate if there is any advantage of excluding these donors from the donor pool to limit anti-A and anti-B content in IVIG product.STUDY DESIGN AND METHODSIgG anti-A and anti-B levels were assessed from group O donor plasma, manufacturing IgG plasma pools, and finished IVIG product (Gammagard Liquid). Antibody level in group O donors was also assessed by sex and age for their relative contribution of antibody to the plasma pool.RESULTSThe majority of group O donors (80%) had antibody titers of less than 1000. Of those with titers of at least 1000, theoretical estimates provide further evidence that the effects of high-titer donors are minimal. Antibody levels in plasma pools both during the manufacturing process and from the final IVIG product also support that anti-A and anti-B levels are low. In general, there were more females than males with higher antibody titer levels, with significantly more females than males with anti-A.CONCLUSIONExcluding donors with high anti-A and anti-B titers has minimal impact on the finished IVIG product titers due to ABO antibody neutralization and the dilution factor in the manufacturing pool.

Various types of excipients are added to immunoglobulin preparations to stabilize the product and prevent aggregation and dimer formation. These excipients, which are also called stabilizers or additives, are not inert chemicals and may have clinical implications. This is one reason why immunoglobulin products are not interchangeable. Herein, immunoglobulin preparation, excipient types and the differences among sugar stabilizers and the amino acids, glycine and proline as excipients, are presented. Preclinical studies that unravel the complexities of dimer reduction are summarized. Details of patient considerations with respect to excipient content are outlined focusing on patients with renal insufficiency, diabetes, corn allergy, hereditary fructose intolerance, inborn errors of proline metabolism, DiGeorge Syndrome and neuropsychiatric disorders associated with hyperprolinemia. Excipients are essential components of immunoglobulin preparations and their presence should be a consideration when matching patient needs to product characteristics.

A thromboembolic event is a rare, yet serious side effect of IGIV administration. Recently higher than expected levels of these events traced to the increased presence of Factor XIa (FXIa) after a manufacturing process change led to a product recall. The removal capacity of pro-coagulant impurities of Baxter's KIOVIG (trade name in Europe)/Gammagard Liquid (trade name in the US) process and its robustness is demonstrated through process intermediate analysis and by FXIa spiking experiments.FXI zymogen/FXIa ELISA, non-activated partial thromboplastin time, thrombin generation assay and a specific FXIa assay were used to show that the KIOVIG process has robust pro-coagulant impurities removal capacity in alcohol fractionation and chromatographic purification.

This abstract provides follow-up data of previously presented 6 months' data of stability of IGIV in glycine and L-proline. Immunoglobulin intravenous 10% solutions formulated in either 0.25M glycine or 0.25M L-proline at a target pH of 4.8 were now investigated for up to 18 months. Three lots of IGIV process intermediates were divided into two parts and further processed to the final IGIV 10% formulated either in 0.25M glycine or 0.25M L-proline. Products were stored at 25oC in 60% relative humidity for 18 months. Immunoglobulin monomers, dimers, aggregates and fragments were measured by high-performance size-exclusion chromatography. Anti-hepatitis B surface (anti-HBs) antigen antibodies were measured by an enzyme immunoassay. The data were analyzed using the “paired t-test”. There were no statistically significant differences in total “monomers+dimers” (97.5% vs. 97.1%, p=0.540), aggregates and fragment content (0.19% vs. 0.18%, p=0.921, 2.27% vs. 2.71%, p=0.481) or the protective anti-HBs antigen antibody level (4.52 IU/mL vs. 4.44 IU/mL, p=0.636) between the formulations in glycine and L-proline after 18 months' storage. Dimer level was found to be well below 10% (6.3% in glycine, 4.6% in L-proline), considered to be the upper limit for good tolerability, in both formulations. The results obtained from IGIVs formulated in 0.25M glycine or 0.25M L-proline after 18 months' storage at 25°C indicate that these amino acids provide similar stabilization of the IgG molecule in liquid formulations at low pH.

Chromatography for protein purification on an industrial scale was developed supplementary to the use of different precipitating agents (e.g.polyethylene glycol (PEG) (Polson et al., 1964)) or batch-adsorption on DEAE Sephadex (Hoppe et al., 1967), starting from plasma itself or intermediates derived from Cohn's, Oncley's or Kistler & Nitschmann's processes (Falksveden & Lundblad, 1980, Hoppe et al., 1967, Kistler & www.intechopen.com

RATIONALE: Fc-receptor (FcR)-mediated activities of IGIV are of major importance for their therapeutic efficacy. Previously, we reported the development of a FACS-based binding assay for the assessment of IGIV binding to Fc-receptors (FcR) expressed on human THP-1 cells. We now present a further development of this assay for the assessment of FcR-mediated effector functions of IGIV products. METHODS: Human monocytic THP-1 cells were incubated for 20h with IGIV and the upregulation of the pro-inflammatory surface marker CD54 (ICAM-1) was analyzed by FACS and the release of the anti-inflammatory cytokine IL-1RA was measured by ELISA. Blocking anti-CD64 and anti-CD32 antibodies were included to prove that the effector functions analyzed were dependent on the binding of IGIV to FcR expressed on THP-1 cells. RESULTS: Incubation of THP-1 cells with IGIV resulted in a dose-dependent upregulation of CD54 and a substantial release of IL1-RA into cell culture supernatants which could be further amplified by the addition of a FcR-crosslinking antibody, mimicking the binding of immune complexes to FcR. Both the upregulation of CD54 and the release of IL1-RA were completely dependent on the binding of IGIV to FcRII expressed on THP-1 cells. Comparison of the FcR-dependent functional activities of six different IGIV lots revealed a variation between the lots of 11% and 14% for the induction of IL1-RA release and the upregulation of CD54, respectively. CONCLUSIONS: We developed novel assays to assess Fc-dependent functional activities of IGIV. We believe that these assays will be suitable for monitoring important functional activities of new IgG products during product development.