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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.

BACKGROUND High-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 METHODS IgG 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. RESULTS The 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. CONCLUSION Excluding 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.

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 CD56

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.

Background and Objectives A human plasma-derived butyrylcholinesterase preparation manufactured on the industrial scale is described. Material and Methods The human butyrylcholinesterase (hBChE) product was extensively investigated for its purity using immunological and electrophoretic methods and characterized by thorough glycoproteomic approaches. A comprehensive preclinical testing programme addressing safety and pharmacokinetic parameters supplemented the biochemical characterization. Results The high-purity hBChE preparation is tetrameric and has high specific activity and molecular integrity of the protein backbone. Acute toxicity studies and in vivo thrombogenicity studies provided evidence of a sufficient safety margin for use in humans. Conclusion Extensive preclinical safety and pharmacokinetic testing confirmed that this hBChE preparation can be used for further efficacy testing as a bioscavenger for toxic organophosphate compounds in appropriate animal models and ultimately in humans.

Human butyrylcholinesterase (hBChE) is a highly glycosylated protein present in human plasma. The enzyme hydrolyses choline esters, for example benzoylcholine, butyrylthiocholine and acetylthiocholine as well as noncholine esters like heroin and aspirin. hBChE is primarily involved in neuronal transmission and is a potential bioscavenger of toxic organophosphates to protect acetylcholinesterase. A prerequisite for the therapeutic use of hBChE is a detailed characterization of this glycoprotein purified from human plasma. In this study, MS/MS could confirm most of the protein backbone, including the N- and the C-terminus. Site-specific analysis of all nine potential N-glycosylation sites revealed mainly mono- and disialylated N-glycans to be present on this glycoprotein. Sialic acids (Neu5Ac) are mainly alpha2,6-linked, however a fraction of the N-glycans contained Neu5Ac also in alpha2,3 linkage. On monosialylated N-glycans, sialic acid is exclusively located on the 3-arm and in alpha2,6 linkage, as verified by 2D-HPLC and exoglycosidase digests of 2-aminopyridine (PA)-labelled N-glycans. This first comprehensive glycoproteomic analysis of the important human plasma glycoprotein BChE did not give any indication of O-glycosylation or any other kind of PTMs as previously postulated.

Background and Objectives The ultimate goal was to generate an industrial-scale process suitable to produce a high-yield, safe and stable immunoglobulin G (IgG) preparation for intravenous administration, which is ready to use for customer convenience. This new liquid 10% IgG preparation (IGIV 10%) was compared to Gammagard SD, a licenced lyophilized immunoglobulin in biochemical and preclinical testing. Materials and Methods The new process, which includes three dedicated virus clearance steps, is a streamlined combination of the currently applied and well-established manufacturing procedures. The biochemical characterization is done by standard methods focusing on purity, integrity and functionality of the preparation. Efficacy is demonstrated in vivo by mouse protection testing and in vitro by opsonization and protein A affinity chromatography. Pharmacokinetics in rats is evaluated after a single intravenous dose. The anaphylactoid potential is determined in rats and in guinea pigs, while thrombogenicity is assessed in a rabbit model. The influence of the products on vital functions is tested on dogs, while acute toxicity studies are carried out on mice and rats. Results The biochemical characterization data demonstrate the high purity of monomeric IgG in the product. The mouse protection test showed that the protective activity against systemic bacterial infections of IGIV 10% is at least as good as the reference Gammagard SD. This result is supported by the broad spectrum of antibodies in high titres against bacteria and viruses and the high functional integrity of the IgG molecule (≥ 90% functionally intact IgG) in IGIV 10%. The opsonic activity of all IGIV 10% lots is similar to the one of the reference Gammagard SD. In safety and thrombogenicity studies, no adverse effects of IGIV 10% were observed. Pharmacokinetic studies showed no statistically significant differences between the two products. In the acute toxicity animal studies, IGIV 10% compared favourably to the reference Gammagard SD. Conclusions The new manufacturing process enables the production of a highly purified IgG preparation for intravenous administration. The product has an IgG subclass distribution similar to plasma and contains a broad spectrum of functionally intact antibodies. Preclinical studies demonstrate that the liquid IGIV 10% combines excellent qualities of efficacy, safety and tolerability.

West Nile virus (WNV)-neutralizing intravenous immune globulins (IVIG) were fractionated into IgG subclasses, and the contribution of each subclass to in vitro neutralization of and in vivo protection against WNV was evaluated. The results indicate that IgG1 (i) is the main subclass induced following WNV infection of humans, (ii) contained nearly all the in vitro WNV neutralization capacity, and (iii) mediates effector functions in vivo that render it superior to other subclasses in protection against WNV. The importance of human IgG1 indicates that a candidate WNV vaccine should induce an immune response that includes WNV-specific IgG1.

The physiological effects of human plasma-derived butyrylcholinesterase (huBuChE) administration and its modulation of the effects of percutaneous VX challenge are poorly understood. Percutaneously administered nerve agents are more slowly absorbed than inhaled agents; consequently, signs of poisoning occur later, with a longer duration. Telemetry was used to monitor heart rate, EEG, temperature and activity in guinea-pigs. Treatment with huBuChE at 30 or 120 min following percutaneous VX challenge ( approximately 2.5 x LD(50)) provided 100% protection from lethality. When huBuChE administration was delayed until the onset of observable signs of poisoning only 1 out of 6 animals survived to the end of the experiment at 7 days. This study adds to the body of evidence demonstrating the efficacy of huBuChE in animals by describing the successful therapeutic use of a protein bioscavenger as a post-exposure treatment against dermal exposure to VX up to 2h post-exposure. This study simultaneously used telemetric methods to show that the efficacy of huBuChE is linked to the prevention of detrimental physiological changes observed in control VX-treated animals. Post-exposure therapy is a promising additional indication for the concept of use of this material, and one that has particular relevance in a civilian exposure scenario.

rom arge poo s o uman p asma onat ons, as een shown to contain amyloid-β (Aβ) conformer-specific IgG autoantibodies [2] that could be involved in the beneficial effects observed in the phase II study. Here we asked the question whether GGL contains other antibodies and soluble proteins that can modulate the Aβ transport/ clearance apart from these anti-Aβ auto-antibodies. In N-terminal human RAGE peptide AQNITARIGEPLVLKCKGAPKKPP QRLEWKLN Y

Background and Objectives A new 10% liquid human intravenous immunoglobulin (US trade name: Gammagard Liquid; European trade name: KIOVIG) manufactured by a process with three dedicated pathogen inactivation/removal steps (solvent/detergent treatment, 35-nm nanofiltration and low pH/elevated temperature incubation) was developed. The ability of the manufacturing process to inactivate/remove viruses and prions was investigated. Materials and Methods Virus and prion removal capacities were assessed with down-scale spiking experiments, validated for equivalence to the large-scale process. Results Lipid-enveloped viruses were completely inactivated/removed by each of the three dedicated virus clearance steps, and for human immunodeficiency virus 1 (HIV-1) and pseudorabies virus (PRV), also by the upstream cold ethanol fractionation step. Relevant non-enveloped viruses [i.e. hepatitis A virus (HAV) and parvovirus B19 (B19V)] were effectively removed by nanofiltration and the cold ethanol fractionation step, and partial inactivation of non-enveloped viruses was achieved by low pH incubation. Overall log reduction factors were > 20·0 for HIV-1, > 18·1 for bovine viral diarrhoea virus, > 16·3 for West Nile virus, > 10·0 for influenza A virus subtype H5N1, > 21·8 for PRV, 12·0 for HAV, > 12·1 for encephalomyocarditis virus, 10·6 for B19V and 10·3 for mice minute virus. Prions (Western blot assay) were completely removed (≥ 3·2 mean log reduction) by a step of the cold ethanol fractionation process. Conclusions Introducing three dedicated virus-clearance steps in the manufacturing process of immunoglobulins from human plasma provides high margins of safety.

Background and Objectives Baxter AG has developed a new liquid intravenous immunoglobulin product [Immune Globulin Intravenous (IGIV) 10%] using a new manufacturing procedure. A modified Cohn fractionation and ion exchange chromatography is used to produce an IgG solution with no alterations to the Fc region. Three dedicated virus reduction steps are included: solvent-detergent treatment, nanofiltration, and incubation at low pH and elevated temperature in final formulation. We applied the reference method of the European Pharmacopoeia (EP) together with a flow-cytometric binding assay for the evaluation of the Fc function of the new product. Materials and Methods The EP reference method was done as described in the EP. The flow-cytometric method measured binding of IgG to Fc receptors of human monocytic THP-1 cells after exclusion of apoptotic cells. Results Sixteen lots of the new product expressed Fc functions between 84% and 110% when analysed with the EP reference method and Fc-binding activities between 82% and 121% when determined by the flow-cytometric method. Conclusion All tested lots of the new product demonstrated a high level of Fc activity and met the requirements of the EP for Fc function.

SummaryOur study investigated the immunomodulatory activities of human plasma-derived serum immunoglobulin (Ig)A. Previous findings seem contradictory indicating either pro- or anti-inflammatory activities. We used serum IgA purified from large plasma pools and studied the modulation of the release of cytokines and chemokines from resting and lipopolysaccharide (LPS, endotoxin)-stimulated human adherent monocytes and human peripheral blood mononuclear cells (PBMC). Our results indicate that IgA down-modulates the release of the pro-inflammatory chemokines monocyte chemoattractant protein (MCP) 1, macrophage inflammatory protein (MIP) 1α and MIP1β from LPS-stimulated PBMC and the release of MCP1, MIP1α and MIP1β from LPS-stimulated monocytes. Furthermore, we confirmed previous reports that plasma-derived serum IgA down-modulates the release of the pro-inflammatory cytokines, interleukin (IL)-6 and tumour necrosis factor (TNF)-α, from LPS-stimulated monocytes and PBMC, and up-regulates the release of IL-1 receptor antagonist (IL-1RA) from resting and LPS-stimulated monocytes and resting PBMC. This IgA-mediated up-regulation of IL-1RA is independent of the simultaneous up-regulation of IL-1β release, as shown by blocking the biological activity of IL-1β with a neutralizing antibody. On the other hand, we also found an IgA-induced pro-inflammatory activity, namely IgA-mediated up-regutation of the release of pro-inflammatory IL-1β as well as down-regulation of the anti-inflammatory cytokines IL-10 and IL-12p40 from LPS-stimulated monocytes and PBMC and a down-regulation of transforming growth factor (TGF)-β from resting and LPS-stimulated PBMC. We conclude that human serum IgA has both an anti-inflammatory and a pro-inflammatory capacity and this dual capacity might contribute to the feedback mechanisms maintaining a balance between pro-inflammatory and anti-inflammatory activities.

Phosphofructokinase from Escherichia coli binds fructose-6-phosphate with the sugar moiety of the substrate interacting with one subunit and the phosphate group with another one, so that bound fructose-6-phosphate lies across the interface between the subunits [(1988) J. Mol. Biol. 204, 973-994]. When this interface is ‘cross-linked’ by fructose-6-phosphate. it becomes more stable because of the extra interactions between subunits: inactivation upon dissociation occurs only above 5 M urea, instead of 1 M urea for the free protein. At saturation in fructose-6-phosphate. this interface is no longer the first to dissociate as in the free protein [(1989) Biochemistry 28, 6836-6841]: instead, the addition of urea to phosphofructo-kinase in the presence of fructose-6-phosphate induces a conformational change within the tetramer which alters the environment of Trp-311 and distorts the regulatory site.

Rationale In preclinical testing the efficacy, safety, pharmacokinetics, and toxicity of a new liquid 10% intravenous immunoglobulin preparation (IGIV, 10%TVR) was compared with Gammagard® S/D, a licensed lyophilized immunoglobulin for intravenous infusion. Methods Efficacy is proved in vivo by mouse protection testing and in vitro by opsonization and affinity chromatography using protein A Sepharose. The blood-pressure-lowering effect in spontaneously hypertensive rats and the bronchospastic effect in guinea pigs are used as indicators of the anaphylactoid potential. Pharmacokinetics in rats is evaluated by the variables "in vivo recovery" and "half-life" after a single intravenous dose of 1000 mg/kg. Thrombogenicity is assessed in a rabbit model and the influence of the products on vital functions like cardiovascular, respiratory and blood coagulation variables is tested in dogs. Acute toxicity studies are carried out in mice and rats. Results The mouse protection test showed that the protective activity against systemic bacterial infections of IGIV, 10%TVR is at least as good as the reference Gammagard S/D. This result is supported by the broad spectrum of antibodies in high titers against bacteria and viruses and the high functional integrity of the IgG molecule (≥90% functional intact IgG) in IGIV, 10%TVR. In safety and thrombogenicity studies, no adverse effects of IGIV, 10%TVR were observed. Pharmacokinetic studies showed no statistically significant differences between the two products. In the acute toxicity animal studies, IGIV, 10%TVR was better tolerated than the reference Gammagard S/D. Conclusions Liquid IGIV, 10%TVR combines excellent qualities of efficacy, safety and tolerability.

Rationale Intravenous Immunoglobulin products (IVIG) are indicated as the treatment of choice in a number of immune-mediated disorders, including inflammatory diseases and autoimmune diseases. Interactions with Fc-receptors have been described as an important mechanism of action of IVIG. Therefore, we have evaluated a recently described flow-cytometric method for the quantification of the Fc-function of IVIG products. Methods Human monocytic THP-1 cells were incubated with IVIG. The IgG bound to the cells was detected using an anti-human IgG antibody conjugated with FITC. Cells were analyzed by flow cytometry. The specificity of IVIG binding via the Fc-part was evaluated by competition experiments using F(ab)2 fragments of IVIG. Furthermore, the release of cytokines into the supernatants of THP-1 cells was analyzed using a Bio-Plex array system and intracellular cytokines were analyzed by flow-cytometry. Results A dose-dependent binding of IVIG to THP-1 cells was found. Maximum binding was observed at 10 μg/ml IVIG. The results obtained were highly reproducible and showed little variation between different passages of THP-1 cells. Competition experiments using F(ab)2 fragments and the analysis of cytokine production by THP-1 cells are currently ongoing. Conclusions The flow-cytometric method that was recently described for the quantification of the Fc-function of IVIG preparations offers a fast and highly reproducible assay. Results of competition and validation experiments will show whether this new methodology is suitable for the quality control of IVIG products.

Rationale While immune globulin products after introduction of dedicated virus inactivation steps have an excellent safety record, patient concerns around transmission of particularly viruses still exist, and are fueled by newly emerging viruses. Methods To address these concerns, the manufacturing process of this new liquid IGIV contains three dedicated virus reduction steps: (1) a three component solvent-detergent (SD) treatment, (2) a 35 N nanofiltration step, and (3) a prolonged incubation period at low pH and elevated temperature. Results (1) SD treatment resulted in complete inactivation of all the lipid-enveloped viruses tested, already after only 1 minute of the 60 minutes treatment, and also at drastically reduced concentrations of the SD reagents. (2) Nanofiltration effectively removed lipid-enveloped viruses. Also, HAV and parvovirus B19, viruses which are smaller than the nominal pore size of the filter used, were reproducibly and robustly removed by antibody-enhanced nanofiltration which occurred at a wide range of HAV and B19 antibody concentrations. (3) Incubation at low pH and elevated temperature effectively inactivated lipid-enveloped viruses, and resulted in significant levels of inactivation for non-lipid enveloped viruses, including parvoviruses. Inactivation was shown to depend on a combination of low pH and elevated temperature. Conclusions The incorporation of three dedicated and mechanistically different virus reduction steps for this new liquid immunoglobulin product results in unprecedented safety margins.

A conversational program for the acquisition of experimental data in a multi-user, multi-instrument computer system is described. It assists the researcher when recording on-time data. Due to the simple structure of the dialogue, no special knowledge of computer handling is required by the experimenter. Whereas the experimental methods are versatile, a uniform concept of the dialogue and the file structure is realized.

The influence of urea on the allosteric phosphofructokinase from Escherichia coli has been studied by measuring the changes in enzymatic activity, protein fluorescence, circular dichroism, and retention in size-exclusion chromatography. Tetrameric, dimeric, and monomeric forms of the protein can be discriminated by their elution from a high-performance liquid chromatography gel filtration column. Three successive steps can be detected during the urea-induced denaturation of phosphofructokinase: (i) the dissociation of the native tetramer into dimers which abolishes the activity; (ii) the dissociation of dimers into monomers which exposes the unique tryptophan, Trp-311, to the aqueous solvent; (iii) the unfolding of the monomers which disrupts most of the secondary structure. This pathway involves the ordered dissociation of the interfaces between subunits and supports a previous hypothesis (Deville-Bonne et al., 1989). Phosphofructokinase can be quantitatively renatured from urea solutions, provided that precautions are taken to avoid the aggregation of one insoluble monomeric state. The renaturation of phosphofructokinase from urea implies three steps: an initial folding reaction within the monomeric state is followed by two successive association steps. The faster association step restores the native fluorescence, and the slower regenerates the active enzyme. The renaturation and denaturation of phosphofructokinase correspond to the complex pathway: tetramer in equilibrium dimer in equilibrium folded monomer in equilibrium unfolded monomer. It is found that the subunit interface which forms the regulatory site is more stable and associates 40 times more rapidly than the subunit interface which forms the active site.

The folding and association pathway of the allosteric phosphofructokinase from Escherichia coli has been investigated after complete denaturation of the protein in guanidine hydrochloride by spec- troscopical methods, fluorescence and circular dichroism. Three successive processes can be observed during the renaturation of this protein. First, a fast reaction, detected by fluorescence, results in the formation of a (partially) structured monomer. Second, two monomers associate into a dimeric species. This step involves the shielding of the unique tryptophan residue, Trp 3 1 1, from the aqueous solvent, and it corresponds to the formation of the interface containing the effector binding site. The presence of ATP during renaturation increases the rate of formation of this dimeric species. The other ligands of the enzyme have no effect on this reaction as well as on the whole reactivation. Finally, the enzymatic activity is regained during the third slowest step. This last reaction is due to the association of two dimers into the native tetrameric structure. The presence of fructose 6-phosphate does not increase the rate of reactivation, even though this ligand strongly stabilizes the native enzyme against denaturation by bridging the interface corresponding to the active site. The self-assembly of phosphofructokinase from E. coli from its unfolded and separated chains follows a specific order in the formation of the interactions between subunits and involves a dimeric intermediate with a defined geometry.

Two types (isoenzymes) of octopine dehydrogenase (A and B) from Pecten jacobaeus adductor muscle were purified to homogeneity, applying affinity chromatography as an efficient final step of purification. Both forms of the enzyme differ in their electrophoretic mobility. All other physico-chemical and enzymatic properties, as well as the folding behaviour were found to be identical. Interconversion of one form into the other was not detectable. Sedimentation equilibrium, gel permeation chromatography, and NaDodSO4/polyacrylamide gel electrophoresis yield a relative molecular mass of 45000 +/- 1500 for both native and denatured enzyme. The unfolding transition at varying guanidine X HCl concentrations is characterized by a two-step profile: at 0.4-0.8 M, partial unfolding is parallelled by inactivation; at 2.0-2.4 M the residual structure is destroyed in a second unfolding step. Beyond 2.8 M no further changes in fluorescence emission and dichroic absorption are observed. At 0.4-1.8 M guanidine X HCl, partial unfolding is superimposed by aggregation. The emission maximum of the intrinsic protein fluorescence at 327 nm is shifted to 352 nm upon denaturation in 6 M guanidine X HCl. Changes in the far-ultraviolet circular dichroism indicate complete loss of the overall backbone structure in this denaturant, including the native helix content of about 33%. Denaturation in 6 M guanidine X HCl, as monitored by the decrease of protein fluorescence, is fast (less than 8s). Upon reactivation after short denaturation, about 25% of the activity is recovered in a fast initial phase (less than 20s). The product of this phase has a similar stability towards destabilizing additives or proteases as the native enzyme. The slow phase of reactivation, which predominates after long-term denaturation, is determined by a single first-order reaction characterized by tau = 29 +/- 3 min (20 degrees C). This reaction must be a relatively late event on the folding pathway, preceded by the fast formation of a structured intermediate, as indicated by the immediate recovery of the native fluorescence. The structural rearrangements, which are rate-limiting for reactivation after long-term denaturation, are characterized by a high energy of activation (112 +/- 8 kJ/mol). The slow reactivation step is compatible in rate with the first-order folding reactions involved in the reconstitution of several oligomeric dehydrogenases [c.f. R. Jaenicke and R. Rudolph (1983) Colloq. Ges. Biol. Chem. Mosbach 34, 62-90].