Study On N-1 Perfusion & High Density Culture Technology Of TIM-3 Monoclonal Antibody Based On CHO Cell

Antibody drugs have gradually become one of the main therapeutic weapons in the field of modern medicine because of their strong specificity,high efficacy and small toxic and side effects,and are widely used in cancer,autoimmune,transplant rejection,infectious diseases and other diseases.Immunotherapy has become a popular way to improve the therapeutic effect of tumor by reactivating anti-tumor immune cells and overcoming the immune escape mechanism of tumor.TIM-3(T cell immunoglobulin domain and mucin domain-3)is a kind of T cell surface inhibitory molecule,mainly expressed in CD4+T helper cells 1(Th1)and CD38+CTL cells,as well as Treg cell subpopulation with enhanced inhibitory function.Multiple scientific studies have shown that anti-TIM-3monoclonal antibodies induce dendritic cells to produce a series of chemical immune signals.These immune molecules further activate T cells,causing a series of anti-tumor immune responses that significantly inhibit tumor growth.TIM-3 antibody production is a very complicated process.At present,the ovary is mainly expressed through mammalian cells such as CHO(chinese hamster Ovary).The preparation process can be divided into upstream cell culture and downstream separation and purification.However,the whole process of mammalian cell culture requires absolute asepsis,the correlation of technological parameters is complex,and the regulatory technical barriers are extremely high.The cell culture medium is CD(chemical defined)type animal-free medium,and the material cost is high.Moreover,the cell culture duration is long,the expression efficiency is low,the production cost of TIM-3 antibody drugs is high,the production capacity is very low,and the drug penetration and accessibility is much lower than that of developed countries,and the patients faced with the extremely severe challenges of clinical demand.By developing a recombinant humanized monoclonal antibody cell culture process targeting TIM-3 with high expression level and short culture duration,the production cost of TIM-3antibody can be significantly reduced,production facility utilization can be improved,production capacity can be improved,and drug market penetration and patient accessibility can be improved.In this study,a pilot process study was conducted on CHO cell growth curve,N-1 seed perfusion culture process,conventional feeding batch culture of CHO cells and high density feeding batch culture process of TIM-3 monoclonal antibody production,and a 1000L commercial scale process scale-up study was conducted based on the principle of linear scale-up.Finally,efficient cell culture antibody production was realized.Based on the perfusion process of alternating Tangential flow filtration(ATF),the N-1 seed cell density reached 53×10~6 cells/m L on the 10th day after 7 days of perfusion culture.N-1 seed cells were inoculated into the final reactor and fed with ultra-high inoculation density(u HSD)batch production.The cells skipped the adaptation period and directly entered the logarithmic phase for rapid cell division and proliferation.After 2 days,the cells multiplied to the highest density of 27×10~6 cells/m L,and then entered the stable phase for high protein expression.The total expression of TIM-3 mab reached 5.68 g/L after 8 days of culture.The process of conventional feeding batch culture and ultra-high density feeding batch culture was studied.The cell culture cycle was shortened from 14 days to8 days,the product expression was increased from 3.51 g/L to 5.68 g/L,the key quality parameters of the product were similar to the original process,and the production efficiency was increased about 3 times in the same production line and time.The comparative study of the small scale and commercial scale processes showed that the cell growth trend,product expression level and key quality parameters were highly consistent,and the process had good scaleability.The preliminary results have showed that the high-efficiency antibody production of CHO cell culture has been achieved by the small test and scale-up study of N-1 seed perfusion culture and batch culture technology of ultra-high density feeding.