Institution: | 1.Key Lab of Industrial Fermentation Microbiology of the Ministry of Education & Tianjin Key Lab of Industrial Microbiology, College of Biotechnology,Tianjin University of Science and Technology,Tianjin,People’s Republic of China;2.State Key Laboratory of Food Nutrition and Safety,Tianjin,People’s Republic of China;3.Lidzix Biotechnology Tianjin Co., Ltd.,Tianjin,People’s Republic of China |
Abstract: | ObjectivesTo establish stable infliximab-expressing Chinese hamster ovary (CHO) cells with high tolerance to serum-free culture.ResultsBcl-2 antagonist/killer 1 (BAK1), which is a key mediator of the apoptosis pathway, was disrupted, and infliximab, which is a broadly used monoclonal antibody for the treatment of rheumatoid arthritis and other autoimmune diseases, was incorporated into the BAK1 locus of the CHO chromosome using the clustered regularly interspaced short palindromic repeats (CRISPR)/Cas genome-editing technique. The activating effects of serum starvation on BAK1 and cytochrome C (CytC) were suppressed in the genome-edited cells, and the ability of the cells to resist the serum starvation-induced loss of mitochondrial membrane potential and apoptosis was increased, as indicated by the results of polymerase chain reaction (PCR), flow cytometry, enzyme-linked immunosorbent assay (ELISA) and high-performance liquid chromatography (HPLC) analysis. In addition, during subsequent passages, infliximab could be stably produced in the genome-edited CHO cells, and the recombinant antibody could effectively antagonize the cytotoxic effect of tumor necrosis factor α (TNFα).ConclusionsA CHO cell line capable of stably expressing infliximab and adapting to serum-free culture was constructed. This work lays the foundation for the development of infliximab biosimilars. |