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.

Thermostability of proteins: role of metal binding and pH on the stability of the dinuclear CuA site of Thermus thermophilus.

The dinuclear copper center (TtCuA) forming the electron entry site in the subunit II of the cytochrome c oxidase in Thermus thermophilus shows high stability toward thermal as well as denaturant-induced unfolding of the protein at ambient pH. We have studied the effect of pH on the stability of the holo-protein as well as of the apo-protein by UV-visible absorption, far-UV, and visible circular dichroism spectroscopy. The results show that the holo-protein both in the native mixed-valence state as well as in the reduced state of the metal ions and the apo-protein of TtCuA were extremely stable toward unfolding by guanidine hydrochloride at ambient pH. The thermal unfolding studies at different values of pH suggested that decreasing pH had almost no effect on the thermal stability of the protein in the absence of the denaturant. However, the stability of the proteins in presence of the denaturant was considerably decreased on lowering the pH. Moreover, the stability of the holo-protein in the reduced state of the metal ion was found to be lower than that in the mixed-valence state at the same pH. The denaturant-induced unfolding of the protein at different values of pH was analyzed using a two-state unfolding model. The values of the free energy of unfolding were found to increase with pH. The holo-protein showed that the variation of the unfolding free energy was associated with a pKa of approximately 5.5. This is consistent with the model that the protonation of a histidine residue may be responsible for the decrease in the stability of the holo-protein at low pH. The results were interpreted in the light of the reported crystal structure of the protein.

The menaquinol-oxidizing cytochrome bc complex from Thermus thermophilus: protein domains and subunits.

A recently resolved respiratory complex III, isolated from the extreme thermophile Thermus thermophilus, is discussed in terms of cofactor and subunit composition, and with respect to the origin of its protein modules. The four polypeptides, encoded by a single operon, share general homologies to canonical complexes both of the bc and b6f type, but exhibit some unexpected features as well. Evidence for high thermostability of the isolated protein and for its quinol substrate specificity is derived from EPR and kinetic measurements. A functional integration of this complex into an aerobic electron transfer scheme, connecting known dehydrogenase activities to the terminal oxidase branches of Thermus is outlined, as well as the specific principles of redox protein interactions prevailing at high temperature. Findings from this enzyme are linked to present knowledge on other menaquinol oxidizing bc complexes.

The electron transfer complex between cytochrome c552 and the CuA domain of the Thermus thermophilus ba3 oxidase. A combined NMR and computational approach.

The structural analysis of the redox complex between the soluble cytochrome c552 and the membrane-integral cytochrome ba3 oxidase of Thermus thermophilus is complicated by the transient nature of this protein-protein interaction. Using NMR-based chemical shift perturbation mapping, however, we identified the contact regions between cytochrome c552 and the CuA domain, the fully functional water-soluble fragment of subunit II of the ba3 oxidase. First we determined the complete backbone resonance assignments of both proteins for each redox state. Subsequently, two-dimensional [15N,1H]TROSY spectra recorded for each redox partner both in free and complexed state indicated those surface residues affected by complex formation between the two proteins. This chemical shift analysis performed for both redox states provided a topological description of the contact surface on each partner molecule. Remarkably, very pronounced indirect effects, which were observed on the back side of the heme cleft only in the reduced state, suggested that alterations of the electron distribution in the porphyrin ring due to formation of the protein-protein complex are apparently sensed even beyond the heme propionate groups. The contact residues of each redox partner, as derived from the chemical shift perturbation mapping, were employed for a protein-protein docking calculation that provided a structure ensemble of 10 closely related conformers representing the complex between cytochrome c552 and the CuA domain. Based on these structures, the electron transfer pathway from the heme of cytochrome c552 to the CuA center of the ba3 oxidase has been predicted.

A four-subunit cytochrome bc(1) complex complements the respiratory chain of Thermus thermophilus.

Several components of the respiratory chain of the eubacterium Thermus thermophilus have previously been characterized to various extent, while no conclusive evidence for a cytochrome bc(1) complex has been obtained. Here, we show that four consecutive genes encoding cytochrome bc(1) subunits are organized in an operon-like structure termed fbcCXFB. The four gene products are identified as genuine subunits of a cytochrome bc(1) complex isolated from membranes of T. thermophilus. While both the cytochrome b and the FeS subunit show typical features of canonical subunits of this respiratory complex, a further membrane-integral component (FbcX) of so far unknown function copurifies as a subunit of this complex. The cytochrome c(1) carries an extensive N-terminal hydrophilic domain, followed by a hydrophobic, presumably membrane-embedded helical region and a typical heme c binding domain. This latter sequence has been expressed in Escherichia coli, and in vitro shown to be a kinetically competent electron donor to cytochrome c(552), mediating electron transfer to the ba(3) oxidase. Identification of this cytochrome bc(1) complex bridges the gap between the previously reported NADH oxidation activities and terminal oxidases, thus, defining all components of a minimal, mitochondrial-type electron transfer chain in this evolutionary ancient thermophile.

Interaction of cytochrome c with cytochrome oxidase: two different docking scenarios.

Cytochrome c is the specific and efficient electron transfer mediator between the two last redox complexes of the mitochondrial respiratory chain. Its interaction with both partner proteins, namely cytochrome c(1) (of complex III) and the hydrophilic Cu(A) domain (of subunit II of oxidase), is transient, and known to be guided mainly by electrostatic interactions, with a set of acidic residues on the presumed docking site on the Cu(A) domain surface and a complementary region of opposite charges exposed on cytochrome c. Information from recent structure determinations of oxidases from both mitochondria and bacteria, site-directed mutagenesis approaches, kinetic data obtained from the analysis of isolated soluble modules of interacting redox partners, and computational approaches have yielded new insights into the docking and electron transfer mechanisms. Here, we summarize and discuss recent results obtained from bacterial cytochrome c oxidases from both Paracoccus denitrificans, in which the primary electrostatic encounter most closely matches the mitochondrial situation, and the Thermus thermophilus ba(3) oxidase in which docking and electron transfer is predominantly based on hydrophobic interactions.

Different interaction modes of two cytochrome-c oxidase soluble CuA fragments with their substrates.

Cytochrome-c oxidase is the terminal enzyme in the respiratory chains of mitochondria and many bacteria and catalyzes the formation of water by reduction of dioxygen. The first step in the cytochrome oxidase reaction is the bimolecular electron transfer from cytochrome c to the homobinuclear mixed-valence CuA center of subunit II. In Thermus thermophilus a soluble cytochrome c552 acts as the electron donor to ba3 cytochrome-c oxidase, an interaction believed to be mainly hydrophobic. In Paracoccus denitrificans, electrostatic interactions appear to play a major role in the electron transfer process from the membrane-spanning cytochrome c552. In the present study, soluble fragments of the CuA domains and their respective cytochrome c electron donors were analyzed by stopped-flow spectroscopy to further characterize the interaction modes. The forward and the reverse electron transfer reactions were studied as a function of ionic strength and temperature, in all cases yielding monoexponential time-dependent reaction profiles in either direction. From the apparent second-order rate constants, equilibrium constants were calculated, with values of 4.8 and of 0.19, for the T. thermophilus and P. denitrificans c552 and CuA couples, respectively. Ionic strength strongly affects the electron transfer reaction in P. denitrificans indicating that about five charges on the protein interfaces control the interaction, when analyzed according to the Brønsted equation, whereas in the T. thermophilus only 0.5 charges are involved. Overall the results indicate that the soluble CuA domains are excellent models for the initial electron transfer processes in cytochrome-c oxidases.

Interaction of cytochrome c with cytochrome c oxidase: an NMR study on two soluble fragments derived from Paracoccus denitrificans.

The functional interactions between the various components of the respiratory chain are relatively short-lived, thus allowing high turnover numbers but at the same time complicating the structural analysis of the complexes. Chemical shift mapping by NMR spectroscopy is a useful tool to investigate such transient contacts, since it can monitor changes in the electron-shielding properties of a protein as the result of temporary contacts with a reaction partner. In this study, we investigated the molecular interaction between two components of the electron-transfer chain from Paracoccus denitrificans: the engineered, water-soluble fragment of cytochrome c(552) and the Cu(A) domain from the cytochrome c oxidase. Comparison of [(15)N,(1)H]-TROSY spectra of the [(15)N]-labeled cytochrome c(552) fragment in the absence and in the presence of the Cu(A) fragment showed chemical shift changes for the backbone amide groups of several, mostly uncharged residues located around the exposed heme edge in cytochrome c(552). The detected contact areas on the cytochrome c(552) surface were comparable under both fully reduced and fully oxidized conditions, suggesting that the respective chemical shift changes represent biologically relevant protein-protein interactions.