Analytical and functional similarity of biosimilar Elizaria® with eculizumab reference product.

A recombinant humanized monoclonal antibody (mAb) Eculizumab, C5-complement cascade inhibitor, is an important treatment of complement-based diseases recommended by international guidelines. Elizaria® Drug Product (DP), developed by IBC Generium, Russia, is the world's first registered biosimilar of eculizumab (Soliris®, Alexion Pharmaceuticals). Using sensitive state-of-the-art analytical techniques extensive similarity assessment has been conducted to demonstrate the structural and functional similarity of original Soliris® (Eculizumab Reference Product, RP) and the biosimilar Elizaria®, focusing on the physicochemical and biological quality attributes, including those known to affect the mechanisms of action. A multitude of analyses revealed that amino acid sequence is identical, the higher order structures, post-translational modifications, purity, and product variants are highly similar between Elizaria® DP and Eculizumab RP, except for minor differences in the relative abundance of the charge variants and glycan moieties considered not clinically significant. The results demonstrate that Elizaria® is highly analytically similar to Eculizumab RP in terms of physicochemical properties and biological activities.

Physicochemical and Biological Characterization of rhC1INH Expressed in CHO Cells.

The disfunction or deficiency of the C1 esterase inhibitor (C1INH) is associated with hereditary or acquired angioedema (HAE/AAE), a rare life-threatening condition characterized by swelling in the skin, respiratory and gastrointestinal tracts. The current treatment options may carry the risks of either viral infection (plasma-derived Berinert®) or immune reaction (human recombinant C1INH from rabbit milk, Ruconest®). This study describes the physicochemical and biological characterization of a novel recombinant human C1 esterase inhibitor (rhC1INH) from Chinese hamster ovary (CHO) cells for the treatment of hereditary angioedema compared to the marketed products Berinert® and Ruconest®. The mass spectrometry results of total deglycosylated rhC1INH revealed a protein with a molecular mass of 52,846 Da. Almost full sequence coverage (98.6%) by nanoLC-MS/MS peptide mapping was achieved. The purity and C1s inhibitory activity of rhC1INH from CHO cells are comparable with Ruconest®, although we found differences in charge isoforms distribution, intact mass values, and N-glycans profile. Comparison of the specific activity (IC50 value) of the rhC1INH with human C1 esterase inhibitor from blood serum showed similar inhibitory properties. These data allow us to conclude that the novel rhC1INH molecule could become a potential therapeutic option for patients with HAE/AAE.

Control of therapeutic IgG antibodies galactosylation during cultivation process and its impact on IgG1/FcγR interaction and ADCC activity.

Therapeutic monoclonal antibodies (mAbs) are within the fastest growing group of pharmaceuticals on the global market. IgG1 subclass is the most potent effector in Fc-related functions. The N-linked glycosylation of mAbs Fc-domain significantly influences its therapeutic activity and the presence of this modification is largely dependent on producer cell and parameters of manufacturing process. Here we examined and characterized cell culture conditions that determine during cultivation selective changes in galactose content of a model therapeutic mAb IgG1, trastuzumab biosimilar. We demonstrated that such in cultivation process shift of galactosylation does not affect binding of the mAb to its antigen yet modifies interaction of the mAb with Fcγ receptors and therefore enhances antibody dependent cellular cytotoxicity (ADCC).

Roles of the minor capsid protein P7 in the assembly and replication of double-stranded RNA bacteriophage phi6.

The polymerase complexes of double-stranded RNA (dsRNA) viruses are multifunctional RNA processing machineries that carry out viral genome packaging, replication, and transcription. The polymerase complex forms the innermost virion shell and is structurally related in dsRNA viruses infecting a diversity of host organisms. In this study, we analyzed the properties and functions of the minor polymerase complex protein P7 of dsRNA bacteriophage phi6 using terminally truncated P7 polypeptides and an in vitro self-assembly system established for the phi6 polymerase complex. The N-terminally truncated P7 failed to dimerize, whereas C-terminally truncated P7 polypeptides formed functional dimers that were incorporated into the polymerase complex. Nevertheless, the polymerase complex assembly kinetics and stability were altered by the incorporation of the C-terminally truncated P7. Using the in vitro assembly system for phi6 nucleocapsids and subsequent infectivity assays, we confirmed that full-length P7 is necessary for the formation of infectious viral particles. Contrary to previous results, we found that P7 must be incorporated into polymerase complexes during shell assembly.

Crystallization and preliminary X-ray diffraction analysis of bacteriophage varphi12 packaging factor P7.

Bacteriophage varphi12 protein P7 is a structural component of the polymerase complex and ensures stable packaging of the genomic RNA. varphi12 P7 has been cloned, purified and crystallized. Crystals belong to space group P3(2)21, with unit-cell parameters a = 75.7, b = 75.7, c = 45.2 A, alpha = 90, beta = 90, gamma = 120 degrees , and diffract beyond 2.0 A. Multiple anomalous dispersion data have been collected from crystals of selenomethionylated P7. Mass spectroscopy showed proteolysis of the crystallized protein and a truncated form, P7DeltaC, gave crystals of similar morphology. Cross-linking experiments implicated the N-terminal domain of P7 as being essential for dimerization.