Mucopolysaccharidosis type VI (Maroteaux–Lamy syndrome) is an orphan genetic disease caused by deficiency of the lysosomal enzyme arylsulfatase B (ASB). The need to develop a highly productive cell line for the production of recombinant ASB, is behind the concept and relevance of this study. The most promising approach seems to be the development of CHO producer cell lines coexpressing the target ASB enzyme and an auxiliary formylglycine-generating enzyme (FGE). At the same time, it is important from a practical perspective to have the possibility of cultivating producer cell lines as suspensions free of serum or other components of animal origin. The aim of the study was to develop highly productive cell lines for the production of recombinant ASB by coexpression of the auxiliary FGE. Materials and methods: a suspension CHO cell line was used in the study. CHO cells were transfected by electroporation using the MaxCyte STX system. Monoclonal cell lines were obtained with the help of the Cell Metric system. Enzyme-linked immunosorbent assay was used for determination of ASB concentration in the culture fluid. Culture fluid samples were analysed using polyacrylamide gel electrophoresis and Western blotting. The mRNA level was measured by real-time polymerase chain reaction. Results: producer cell lines coexpressing the target ASB enzyme and auxiliary FGE were obtained. An increase in the yield of the active target ASB enzyme from 2 to 100 mg/L was achieved by selecting the optimal ratio of plasmids during transfection. The highest yield of the target ASB enzyme was achieved at the 90:10 ratio (%) of plasmids encoding the ASB and FGE genes, respectively. Conclusions: the authors developed highly productive cell lines for the production of recombinant ASB, which coexpress the target and auxiliary enzymes. The coexpression of ASB and FGE improves the growth and production characteristics of the cell line, probably due to the modification of the ASB active site. The obtained results will help resolve the problem of low enzyme yield, which is typical of this class of medicines.

Fabry disease is a rare genetic multisystemic lysosomal disease characterized by progressive cellular accumulation of globotriaosylceramide (GL-3) in various organs with specific cutaneous (angiokeratoma), neurological (pain), renal (proteinuria, chronic kidney failure), cardiovascular (cardiomyopathy, arrhythmia), cochleovestibular and cerebrovascular manifestations (transient ischemic attacks, strokes). The phenotypic expression depends on the age of onset and, in females, on the level of X-inactivation. Enzyme replacement therapy is currently the standard therapy for the disease. JSC GENERIUM recently developed a potential biosimilar of Fabrazyme^®, GNR-062, and investigated its physicochemical and biological properties, pharmacokinetics and immunogenicity to assess its bioequivalence to Fabrazyme^®. Direct evaluation of the N-glycosylation profile by HPLC revealed differences mainly related to the distribution of complex sialylated glycans. The overall sialylation level was comparable between the two products. The same conclusion follows from the results of direct analysis of the sialic acid content. Despite a small difference of a few percent in biphosphorylated glycan content, the overall measured M6P values were comparable. Thus, the internalization of agalsidase β into the inner space of lysosomes by binding to key receptors appear to be the same for both products. In a single-blind, randomized comparative study of the pharmacokinetics and safety of a single intravenous infusion of GNR-062 (JSC GENERIUM, Russia) and Fabrazyme^® (Genzyme Europe B.V., The Netherlands) in a crossover design in healthy volunteers, the ratio of geometric means (90% CI) of AUCinf and Cmax for GNR-062 to agalsidase β was 0.9736 (0.9413–1.0070) and 0.9867 (0.9546–1.0199), respectively. These studies demonstrated pharmacokinetic bioequivalence of GNR-062 to agalsidase β. Immunogenicity findings are preliminary, as the study was not powered for formal immunogenicity equivalence, and the single-dose design in healthy volunteers does not reflect repeated long-term administration in patients with Fabry disease. In conclusion, GNR-062 demonstrated acceptable short-term tolerability in healthy volunteers and warrants further investigation as a potential therapeutic option for patients with Fabry disease.

INTRODUCTION . The development of a biopharmaceutical drug for enzyme replacement therapy based on recombinant acid α-1,4-glucosidase (α-glucosidase) is a pressing task, the solution of which could provide patients with Pompe disease in the Russian Federation with the necessary amount of the drug. This study demonstrates an effective method for developing, from the CHO-K1 cell line, a stable industrial producer clone that produces active α-glucosidase. AIM . This study aimed to develop monoclonal cell lines producing recombinant acid α-glucosidase and to evaluate the stability of growth characteristics and productivity during cultivation over 60 generations. MATERIALS AND METHODS . The suspension CHO-K1 cell line (ECACC) was cultured in BalanCD Growth A medium. Cell transfection was performed by electroporation using the MaxCyte system (according to the CHO protocol). Selection of producers was carried out using puromycin (5 μg/mL). Cloning was performed using a cell dispensing system based on microfluidic technology (C.SIGHT). The monoclonality of the cell lines was confirmed using an automated cell imaging system (Cell Metric CLD). The concentration of α-glucosidase in the culture fluid was determined by enzyme-linked immunosor­bent assay. Enzyme activity was measured by a colorimetric method using the substrate 4-nitrophenyl-α-D-glucopyranoside (pNP-α-D-Glc). RESULTS . Screening of 1000 producers was performed, resulting in the selection of 22 cell lines with a productivity of 0.14–0.65 g/L. The lead α-glucosidase producer GAA-14 was cloned, followed by confirmation of monoclonality. A panel of 20 monoclonal lines was obtained. The specific activity of the enzyme and the mannose-6-phosphate residue (М6Р) content did not differ from those of the reference drug (4.3±0.9 U/mg and 0.99±0.10 mol M6P/mol protein, respectively). When studying the stability of the lead clone over 60 generations, preservation of growth characteristics (viability, 98.5±1.5%; population doubling time, 20.0±1.3 h; and productivity, 450±20 mg/L) was demonstrated under batch cultivation over 7 days. CONCLUSIONS. A stable monoclonal α-glucosidase producer cell line based on CHO-K1 cells has been developed, yielding the active enzyme at 0.45 g/L on day 7. The resulting producer clone is suitable for scale-up and manufacture of the drug substance for preclini­cal studies.