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.
Chinese hamster ovary (CHO) 세포는 바이오의약품 시장에서 치료용 단백질 생산을 위하여 가장 널리 쓰이는 세포주이다. 고생산 재조합CHO세포주를 만들기 위해서는 dihydrofolate reductase (DHFR) 시스템과 glutamine synthetase (GS) 시스템이 널리 쓰인다. 오랜 기간 동안 사용되어왔고 자세히 분석되어온 DHFR 시스템과는 달리 GS 시스템은 최근 들어 많이 사용되기 시작하였으며 세포주생성공정과 생산안정성에 관한 연구가 부족한 실정이다.
본 연구의 첫 번째 부분에서는 GS시스템의 선별과정을 강화하기 위해 GS 유전자를 knockout 시킨 세포주를 개발하였다. 재조합 CHO세포주를 만드는 전체과정을 두 가지 숙주세포주와 세가지 선별조건을 이용하여 묘사하였고 이를 통해 만들어진 클론들을 분석하였다. 생산안정성을 확인하기 위해 클론들을 3개월동안 계대 배양 하였으며, GS를 이용한 유전자 증폭가능성을 시험하기 위해 GS 억제제인 methionine sulfoximine (MSX)의 농도를 높여 유전자 증폭을 관찰하였다.
두 번째 부분에서는 암모니아 생산을 감소시키는 GS시스템의 타고난 장점에 더해 젖산을 추가로 감소시키기 위해 항체를 생산하는 재조합 CHO세포주에 lactate dehydrogenase-A (LDH-A) 유전자를 저발현하였다. LDH-A 단백질의 발현이 저하된 CHO세포주들에게서 젖산과 암모니아의 발현이 저하된 것을 관찰할 수 있었으며, 이러한 세포부산물의 감소는 항체 당쇄의 galactosylation 정도를 높이게 되어 항체의 품질을 향상시켰다.
