Characterization of CHO-GS system and metabolic waste reduction through cell engineering for efficient therapeutic protein production

Abstract

Chinese hamster ovary (CHO) cells have been the most widely used host cell line for the production of therapeutic proteins in the biopharmaceutical industry. To establish a stable recombinant CHO (rCHO) cell line with high productivity, two expression systems are dominantly used: dihydrofolate reductase (DHFR) system and glutamine synthetase (GS) system. Compared to the DHFR system which has been studied and characterized for a prolonged period of CHO cell history, there has not been much study which characterizes the GS system regarding cell line generation process and production stability. In the first part of the study, GS knockout host cell line (GS KO) was established by transcription activator-like effect nuclease technology to improve selection stringency of GS system. Then, the whole process of rCHO cell line generation using GS system was characterized using two host cell lines (CHO-K1 and GS KO) and three selection conditions (0, 25, 50 μM of methionine sulfoximine (MSX, a GS inhibitor)). Clones selected at each MSX concentration were analyzed with respect to specific productivity, relative gene copy number, relative mRNA level, metabolites and amino acids utilization. To see the long-term production stability, clones were cultured in the presence and absence of MSX during 30 passages. Furthermore, clones generated at 0 μM MSX condition were subjected to a higher MSX concentration to evaluate a potential of GS-mediated gene amplification by multiple rounds of selection In the second part of the study, addition to the use of the GS system which mitigates ammonia accumulation in the culture media, lactate dehydrogenase-A (LDH-A) gene was downregulated with shRNA in a monoclonal antibody (mAb)-producing CHO cell line to reduce lactate production. The engineered cell lines showed not only reduced production of lactate but also further reduced production of ammonia. Waste reduction increased the galactosylation level of N-glycosylation, which improved antibody quality.