Development of acid alpha-glucosidase-producing clones based on the CHO-K1 cell line

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.

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