Development of cell lines producing recombinant therapeutic proteins, with denosumab as a case study

INTRODUCTION. A key factor in the creation of biotechnological medicinal products is to establish cell lines for high-yield production of recombinant proteins. The development of selection protocols and highly efficient screening approaches for cell lines producing target proteins is a necessary step in the development of recombinant technology for high-yield target protein production.

AIM. This study aimed to derive producer cell lines from a CHO suspension cell line for high-yield production of the recombinant monoclonal antibody denosumab.

MATERIALS AND METHODS. A CHO-K1 suspension cell line was cultured using serum- and animal component-free media and feeds. The cells were transfected with plasmids containing light and heavy chains of denosumab by electroporation using a MaxCyte STX system. The transfected cells were selected under antibiotic pressure (hygromycin and geneticin). Monoclonal cell lines were obtained using a ClonePix FL system. Leader monoclonal cell lines were identified by determining denosumab concentrations by enzyme-linked immunosorbent assay (ELISA) following fed-batch culture.

RESULTS. The optimum concentrations of antibiotics for the selection of CHO-derived denosumab-producing cell lines were 600 mg/L for hygromycin and 600 mg/L for geneticin. The selection process following transfection was successful in 1041 (about 54%) of 1920 minipools. Denosumab-producing minipools were identified by screening culture fluid samples from 96-, 24-, and 6-well plates using ELISA. Then, 23 leader minipools were chosen and adapted to suspension culture in shaker flasks. The growth and production characteristics of these 23 minipools indicated the leader minipool for cloning (mp-19). This minipool provided a denosumab yield of 1.92 g/L on day 7 of fed-batch culture. Using mp-19, the authors obtained monoclonal cell lines providing up to 6.5 g/L denosumab yields on day 9 of fed-batch culture.

CONCLUSIONS. The authors obtained monoclonal cell lines for high-yield denosumab production. The offered approach to producer cell line development can be applied to the production of various recombinant proteins, including monoclonal antibodies, enzymes, and blood coagulation factors.

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