Development And Optimization Of Protein-free Medium For CHO Cells Producing Monoclonal Antibody

Monoclonal antibody is extensively exploited for use in the fields of medicine, thus holding great social and economic values. However, current limited production capability based on animal cells does not meet the ever-increasing demand. For animal cell culture, developing culture medium represents one of the most important steps. Traditional serum-free media usually contain animal-originated proteins, which are the potential source of contamination. On the other hand, the medium components of current available commercialized protein-free media are complicated and kept as a trade secret, which presents a big hurdle for process development of animal cell culture. It is therefore a prerequisite to develop protein-free medium supporting high cell density and high protein expression level, in order to meet the requirements of low cost and high yield for the industrialization of antibody production, which is the major objective of the present work.In this study, based on a serum-free medium (SF) with independent intellectual property rights owned by our lab, a protein-free medium named as PF1 was developed firstly, which supported the growth and stable-passaging of CHO cells. The maximum viable cell concentration and antibody concentration of the batch culture in PF1 were 35.0×105 cells/mL and 160 mg/L, corresponding to an increase of 21% and 36%, respectively, in comparison with those in SF medium. In addition, as the major nutrient components, the effects of amino acids, vitamins and glucose on cell growth, metabolism and antibody production were investigated. It was found that supplementation of those most consumed amino acids promoted cell viability in the later stage, thus increasing the antibody productivity by 25%, though the cell concentration remained unchanged. The addition of three types of vitamins improved cell growth and extended culture duration and the antibody productivity was increased by 22%,53% and 24%, respectively. As an important carbon and energy source, an extremely low or high concentration of glucose in culture medium is inhibitory for cell growth, and thereby was disadvantageous for antibody production. It was demonstrated that by maintaining the glucose concentration in an optimal range (5-30 mmol/L), cell growth was improved, culture duration were prolonged, and the antibody productivity increased by 126% and 67% in comparison with that at low and high glucose concentration, respectively.Based on the above results, an optimized protein-free medium named PF-Opt was developed with optimized nutrient components in the basal PF1 medium. The maximum viable cell concentration and antibody productivity were 52.6×105 cells/mL and 274 mg/L, respectively. As a matter of fact, the maximum viable cell concentration in PF-Opt medium was 1.3 and 1.9 times of that in commercialized medium HyQ SFM4CH0 and Ex-cel1302, and the antibody productivity was 1.1 and 2.3 times, respectively.Taken together, a protein-free medium for CHO cells producing antibody was successfully developed and optimized, which laid a solid foundation for the industrialized production of the antibody. Meanwhile, it will certainly facilitate the subsequent process development and optimization, and provide a guide for developing protein-free media for other animal cells.