Recombinant monoclonal antibodies (mAbs) against tumor necrosis factor alpha are widely used in the biopharmaceutical therapy of autoimmune diseases. Currently, a large number of drugs based on these antibodies are available. Accordingly, the development of these products for the Russian market is an important goal. The aim of the current study is to describe the development of one such technology. CHO-DG44-derived cell lines producing mAb were developed using two strategies, one based on individual clones and the other based on cell pools. To obtain recombinant cell lines with highly amplified genes of interest, the clones underwent dihydrofolate reductase-mediated gene amplification. Using the best strategy for the selection and amplification of mAb-producing clones, we achieved the production of more than 1 g/L in small scale, non-optimized conditions.

In a previous study we found that in chickweed the expression level of the pro-SmAMP2 gene was comparable or even higher to that of the β-actin gene. This high level of the gene expression has attracted our attention as an opportunity for the identification of novel strong promoters of plant origin, which could find its application in plant biotechnology. Therefore, in the present study we focused on the nucleotide sequence identification and the functional characteristics of the pro-SmAMP2 promoter in transgenic plants. In chickweed (Stellaria media), a 2120 bp promoter region of the pro-SmAMP2 gene encoding antifungal peptides was sequenced. Six 5′-deletion variants −2120, −1504, −1149, −822, −455, and −290 bp of pro-SmAMP2 gene promoter were fused with the coding region of the reporter gene gusA in the plant expression vector pCambia1381Z. Independent transgenic plants of tobacco Nicotiana tabacum were obtained with each genetic structure. GUS protein activity assay in extracts from transgenic plants showed that all deletion variants of the promoter, except −290 bp, expressed the gusA gene. In most transgenic plants, the GUS activity level was comparable or higher than in plants with the viral promoter CaMV 35S. GUS activity remains high in progenies and its level correlates positively with the amount of gusA gene mRNA in T3 homozygous plants. The activity of the рro-SmAMP2 promoter was detected in all organs of the transgenic plants studied, during meiosis and in pollen as well. Our results show that the рro-SmAMP2 promoter can be used for target genes expression control in transgenic plants.

Recombinant tissue plasminogen activator (international nonproprietary name — alteplase) which was developed by «GENERIUM» (Russia) and received a marketing authorisation in Russia is completely analogous to Actilyse® which is used to treat medical conditions accompanied by thrombosis, such as acute myocardial infarction, pulmonary embolism, and ischemic stroke. The aim of the study was to carry out a comprehensive comparison of physico-chemical and biological properties of Revelyse® and the reference product Actilyse® in order to assess their biosimilarity. Materials and Methods: comparative peptide mapping and determination of comparability of chromatographic profiles of tryptic hydrolysates was performed using RP-HPLC and massspectrometry; the molecular weight distribution was determined by mass-spectrometry and polyacrylamide gel electrophoresis (Laemmli method). The purity and homogeneity of products as well as the content of related impurities (oligomers and fragments) were determined using gel filtration; N-glycosylation profile was analysed by hydrophilic HPLC, total sialic acid was quantified by the Svennerholm resorcinol method. Protein binding to fibrin and human fibrinogen was assessed by surface plasmon resonance, and the specific activity was compared by fibrin clot lysis. Results: the research demonstrated a complete overlap of the products’ peptide maps, which indicates the identity of аlteplase amino acid sequences in the two medicines being compared. The authors of the study also determined the molecular weight and the content of the intact single-stranded form of the protein, and quantified post-translational modifications, the content of sialic acids and neutral sugars. The analysis of the N-glycosylation profile revealed insignificant differences in the percentage of multiantenna complex glycans. The specificity of alteplase was evaluated by analysing the formation of protein complexes with natural alteplase ligands – fibrin and plasminogen activator inhibitor-1, but no significant differences were found. The comparison of specific activation of plasminogen fibrinolytic activity was performed based on the results of the assay analysing the fibrin clot lysis rate, and it demonstrated comparability of Revelyse® and Actilyse®. Conclusions: comparative experimental studies have shown no differences in the structure, charge distribution heterogeneity, impurities content, and specific activity of alteplase as a component of Revelyse® and the reference product Actilyse®, which leads to the conclusion that they are similar in terms of physicochemical and biological properties.

Objectives. In this biosimilar research, we compare the monoclonal antibody eculizumab obtained from different drugs [original Soliris^® (Alexion Pharmaceuticals) and candidate Elizaria^® (Generium)] by intact mass measurement and middle-up mass spectrometry analysis to enhance the role of mass spectrometry methods in biopharmaceutical development processes.Methods. The intact mass measurement is performed using a high-resolution ESI-MS. The middle-up analysis is performed by reversed-phase high-performance liquid chromatography with ESI-MS detection, subsequent IdeS treatment of antibodies, and disulfide bond reduction.Results. We have shown some small differences between the original and candidate drugs in the minor glycans level. Man5 glycan is only found in the original Soliris, and G0 is only found in the Elizaria. Glycation sites are also found in the light chain and Fd subunits of the original Soliris. The glycation level does not exceed 4.4%. The non-clipped C-end lysine level and G0F glycan levels are slightly lower in the original Soliris. All registered differences are not crucial for eculizumab’s quality and do not affect its effectiveness and preclinical safety. Generally, the results show a high level of similarity between the original and candidate drugs.Conclusions. The comparative mass spectrometry analysis of eculizumab in the original Soliris and Elizaria allows us to estimate their high degree of similarity by molecular masses and major modification profiles.