High Throughput Screen Development And Optimization Of Serum-free Medium For CHO Cells

Antibody fusion protein has a remarkable curative effect for the treatment of many disease, has a capacious market prospect. However, currently the low antibody fusion protein productivity can’t meet the growing demands of the market. Besides, commercial medium due to the confidentiality composition, high cost, limiting the subsequent process optimization space. Thus, development the economic and high efficiency serum free medium for targeting CHO cells, and then optimization the fed-batch process, is one of the key step to improve the antibody fusion protein productivity, reduce the production cost, realize the industrialization.In this study, first of all, based in the8house-in serum free medium, through20days domestication, select6#medium as a basic medium named SFM-BM, in which CHO cells can grow stable, in batch culture the maximum viable cell density is20.6×105cells/ml. Metabolic analysis showed that partial amino acids depletion in advance, therefore, use the high-throughput screening technique combine with simple lattice design, to optimize the21amino acids concentrations and types in SFM-BM, then confirm the result through TPP tube culture. At last, successfully development the serum free medium SFM-OPT, to support CHO cell high density growth, and find three significant amino acids:glutamine, proline, methionine, by the component search method. In batch culture, process maintained for7days, the maximum viable cell density is52.0×105cells/ml, the average growth rate of logarithmic phase is0.82day-1, final antibody fusion protein concentrations is52.2mg/L. Compared to commercial medium Ex-cell302every aspect has improved. And study several characteristics of SFM-OPT from the cell morphology, cell cycle, the relationship between osmolarity and cell growth, wide adaptation. According to the osmolarity gradient survey, find that in growing stage osmolarity control in290-420mOsm/kg is a guarantee for the rapid growth, so adjustment osmolarity decreased to310mOsm/kg, as the basic medium of fed-batch culture.In the fed-batch culture process, cells need balanced nutrition both in basic media and in fed media to support rapid growth and effective productivity. So, using the central composite design to optimiz the trace elements, vitamins and other nutrients concentration in the feeding medium, find the appropriate proportion for adding3kinds of nutrients; according to the amino acids specific consumption rate, adjust the amino acids concentration increased to1.3times. Finally, the low osmolarity SFM-OPT as basic medium, and with the optimized feeding medium, glucose as the key control parameters, optimization of two stage dynamic fed-batch culture. The whole process maintain for10days, the maximum viable cell density is94.6×105cells/ml, finally antibody fusion protein yield600mg/L, raised11times compare to batch culture, antibody fusion protein antibody fusion protein specific production rate reached18.8mg/(109cells-day), increased by8times, achieved the same effect of commercial medium, cell growth rate increased by50%, sialic acids content also reached the industrial standard.Through this study, for the CHO cells which expression antibody fusion protein antibody fusion protein, successfully development of the serum free medium SFM-OPT, improved the economical, lay the foundation of antibody fusion protein industrialized production, and serve a reference for the other cell line in development serum-free medium.