Biochemical Characterization of a New 10% IVIG Preparation [IgG Next Generation (BT595)/Yimmugo®] Obtained from a Manufacturing Process Preserving IgA/IgM Potential of Human Plasma.

BACKGROUND AND OBJECTIVE: Human plasma is used for the generation of several life-saving drugs and contains valuable antibodies from the immunoglobulin classes IgG, IgM and IgA. Purified intravenous IgG solutions (IVIGs) form the majority of plasma-derived medicine to treat patients with various forms of immunodeficiencies. In conventional IVIG manufacturing processes, immunoglobulin classes IgM and IgA are often discarded as contaminants, but these antibody classes have been proven to be effective for the treatment of acute bacterial infections. Considering the increase in demand for human plasma-derived products and the ethical value of the raw material, a more resource-saving usage of human plasma is needed. Intensive research over the last decades showed that adverse reactions to IVIGs depend on the presence of thrombogenic factors, partially unfolded proteins, non-specific activation of the complement system, and blood group specific antibodies. Therefore, new IVIG preparations with reduced risks of adverse reactions are desirable. METHOD: A new manufacturing process that yields two biologics was established and quality attributes of the new IVIG solution (Yimmugo®) obtained from this process are presented. RESULTS: Here, we provide a biochemical characterization of Yimmugo®, a new 10% IVIG preparation. It is derived from human blood plasma by a combined manufacturing process, where IgM and IgA are retained for the production of a new biologic (trimodulin, currently under investigation in phase III clinical trials). Several improvements have been implemented in the manufacturing of Yimmugo® to reduce the risk of adverse reactions. Gentle and efficient mixing by vibration (called "vibromixing") during a process step where proteins are at risk to aggregate was implemented to potentially minimize protein damage. In addition, a dedicated process step for the removal of the complement system activator properdin was implemented, which resulted in very low anticomplementary activity levels. The absence of measurable thrombogenic activity in combination with a very high degree of functional monomeric antibodies predict excellent efficacy and tolerability. CONCLUSION: Yimmugo® constitutes a new high quality IVIG preparation derived from a novel manufacturing process that takes advantage of the full therapeutic immunoglobulin potential of human plasma.

Caldariomyces fumago DSM1256 Contains Two Chloroperoxidase Genes, Both Encoding Secreted and Active Enzymes.

Inspection of transcriptome data from the chloroperoxidase (CPO)-producing fungus Caldariomyces fumago DSM1256 led to the discovery of two distinct CPO mRNA sequences. This strain could be shown to contain the newly identified isogene as well as produce and secrete both isoenzymes. The CPO2 enzyme bears high sequence similarity to the well-characterized CPO (87% identity for the mature proteins). It shows two insertions in the signal peptide and in the C-terminal propeptide, and one deletion in the mature polypeptide close to the C-terminus. Furthermore, it lacks one of the serine residues known to be O-glycosylated in the CPO sequence. The demonstration of a CPO isogene which is expressed as a secreted and active CPO clarifies the nature of this isoenzyme already identified in earlier reports. A structure model comparison shows a high conservation of the active site and the substrate channel, suggesting very similar catalytic properties.

Role of Surf1 in heme recruitment for bacterial COX biogenesis.

Biogenesis of the mitochondrial cytochrome c oxidase (COX) is a highly complex process involving subunits encoded both in the nuclear and the organellar genome; in addition, a large number of assembly factors participate in this process. The soil bacterium Paracoccus denitrificans is an interesting alternative model for the study of COX biogenesis events because the number of chaperones involved is restricted to an essential set acting in the metal centre formation of oxidase, and the high degree of sequence homology suggests the same basic mechanisms during early COX assembly. Over the last years, studies on the P. denitrificans Surf1 protein shed some light on this important assembly factor as a heme a binding protein associated with Leigh syndrome in humans. Here, we summarise our current knowledge about Surf1 and its role in heme a incorporation events during bacterial COX biogenesis. This article is part of a Special Issue entitled: Biogenesis/Assembly of Respiratory Enzyme Complexes.

Characterization of heme-binding properties of Paracoccus denitrificans Surf1 proteins.

Biogenesis of cytochrome c oxidase (COX) is a highly complex process involving >30 chaperones in eukaryotes; those required for the incorporation of the copper and heme cofactors are also conserved in bacteria. Surf1, associated with heme a insertion and with Leigh syndrome if defective in humans, is present as two homologs in the soil bacterium Paracoccus denitrificans, Surf1c and Surf1q. In an in vitro interaction assay, the heme a transfer from purified heme a synthase, CtaA, to Surf1c was followed, and both Surf proteins were tested for their heme a binding properties. Mutation of four strictly conserved amino acid residues within the transmembrane part of each Surf1 protein confirmed their requirement for heme binding. Interestingly the mutation of a tryptophan residue in transmembrane helix II (W200 in Surf1c and W209 in Surf1q) led to a drastic switch in the heme composition, with Surf1 now being populated mostly by heme o, the intermediate in the heme a biosynthetic pathway. This tryptophan residue discriminates between the two heme moieties, apparently coordinates the formyl group of heme a, and most likely presents the cofactor in a spatial orientation suitable for optimal transfer to its target site within subunit I of cytochrome c oxidase.

Surf1, associated with Leigh syndrome in humans, is a heme-binding protein in bacterial oxidase biogenesis.

Biogenesis of mitochondrial cytochrome c oxidase (COX) relies on a large number of assembly factors, among them the transmembrane protein Surf1. The loss of human Surf1 function is associated with Leigh syndrome, a fatal neurodegenerative disorder caused by severe COX deficiency. In the bacterium Paracoccus denitrificans, two homologous proteins, Surf1c and Surf1q, were identified, which we characterize in the present study. When coexpressed in Escherichia coli together with enzymes for heme a synthesis, the bacterial Surf1 proteins bind heme a in vivo. Using redox difference spectroscopy and isothermal titration calorimetry, the binding of the heme cofactor to purified apo-Surf1c and apo-Surf1q is quantified: Each of the Paracoccus proteins binds heme a in a 1:1 stoichiometry and with Kd values in the submicromolar range. In addition, we identify a conserved histidine as a residue crucial for heme binding. Contrary to most earlier concepts, these data support a direct role of Surf1 in heme a cofactor insertion into COX subunit I by providing a protein-bound heme a pool.

Biogenesis of cytochrome c oxidase--in vitro approaches to study cofactor insertion into a bacterial subunit I.

Biogenesis of cytochrome c oxidase is a complex process involving more than 30 known accessory proteins in yeast for the regulation of transcription and translation, membrane insertion and protein processing, cofactor insertion, and subunit assembly. Here, we focus on the process of cofactor insertion into subunit I of cytochrome c oxidase using the soil bacterium Paracoccus denitrificans as a model organism. The use of bacterial systems facilitates biogenesis studies, as the number of required assembly factors is reduced to a minimum. Both, co- and posttranslational cofactor insertion scenarios are discussed, and several approaches to shed light on this aspect of biogenesis are presented. CtaG, the Paracoccus homolog of yeast Cox11 which is involved in copper delivery to the Cu(B) center, has been purified and characterized spectroscopically. A previously unreported signal at 358 nm allows monitoring copper transfer from copper-loaded CtaG to an acceptor. Both CtaG and apo-subunit I were purified after expression in Escherichia coli to develop an in vitro copper transfer system, probing the posttranslational insertion hypothesis. To mimic a potential cotranslational insertion process, cell-free expression systems using E. coli and P. denitrificans extracts have been established. Expression of subunit I in the presence of the detergent Brij-35 produces high amounts of "solubilized" subunit I which can be purified in good yield. With this system it may be feasible to trap and purify assembly intermediates after adding free cofactors, purified assembly proteins, or P. denitrificans membranes.