Process Optimization For Gs-Cho Cells Culture

The human tumor necrosis factor receptor linked to the Fc portion of human IgGI, named TNFR-Fc, has been used for the treatment of rheumatoid arthritis successfully. The demand for protein products for therapeutic applications by mammalian cell culture was rising constantly. However, the limitation in the production capacity was unable to meet the huge demands in the world. Thus, there was an urgent need to improve the protein products efficieney by mammalian cell large-scale culture. The complexity of the nutritional needs and process control was considered as the principal problem limiting cell growth and the final product concentration, resulting in the low production capacity. The purpose of mammalian cell large-scale culture was to improve mammalian cell culture production capacity through solving the above problems. Therefore, the development of an economic and efficient dynamic fed-batch cultivation for GS-CHO cells producing TNFR-Fc was necessary.The concentration of glucose and glutamine in the biphasic dynamic fed-batch cultivation should be maintained about10and0.5mM, according to the result of glucose, glutamine limiting culture.An efficient biphasic dynamic fed-batch model was established based on nutrients analysis of biphasic culture. The balanced and well-formulated feed medium was designed based on rational design for the substances that could be measured and shake-flask cultures for the substances that could not be measured to add nutrients at appropriate stoichiometric rates equal to their consumption rates. The robust, metabolically responsive feeding strategy was intermittent feeding based on the offline measurement of glucose with the aim of supplying sufficient nutrients to match their consumption, simultaneously minimize the accumulation of byproducts (lactic acidn and ammonia). In this process, the maximal viable cell and antibody concentration reached3.415×106cells/mL and800.3mg/L, respectively. Compared with the biphasic cultures, the newly developed dynamic fed-batch technology longered the culture duration (18days) and increased the cell yield by29%and the final antibody concentration by92%. In addition, the concentrations of lactate and ammonia in the last FB-B culture supernatants were just6.2and3.07mmol/L, respectively. The test for biological activity of rhTNFR-Fc and the determination of sialic acid in Etanercept were qualified. The optimization results show that the problem of the nutrient limitation/inhibition and metabolic byproducts accumulation was solved by controlling the concentration of glucose and glutamine in the culture and the design of a rational and balanced feed medium.In addition, even though the total amount of the recombinant protein rhTNFR-Fc increased by1times compared to the control through alternate inducing methods of0.5mM sodium butyrate (NaBu) when the culture temperature was37℃, it was not improved with the addition of NaBu when the cells were cultured in biphasic dynamic fed-batch culture process. It didnot exert a synergistic effect on the biosynthesis of recombinant protein rhTNFR-Fc with the temperature. The beneficial effect of NaBu on specific protein productivity appears to be not only dependent on the cell line or gene expression system but also culture environment.In this research, the importance of biphasic dynamic fed-batch process optimization to the nutrient limitation elimination in the culture, the cell concentration improvement and the rhTNFR-Fc production improvement was highlighted, thereby facilitating the manufacture of therapeutic antibody by GS-CHO cells.