Development And Optimization Of The Serum-free Medium For CHO DG44Stable Cell Line

Recently monoclonal antibody therapeutics has entered a rapid development period, and its market potential is extremely huge. But in our country large-scale mammalian cell culture manufacture has become the curdle of antibody drug industrialization which includes cell culture medium, cell culture process optimization and bioreactor scaling-up technology. Due to batch-to-batch variations, potential risk of contaminations, false operations in products separation and purification in traditional media containing serum, meanwhile the commercial serum-free medium is expensive and related components are kept secret, it is necessary for us to develop homemade serum-free medium which can support high density cell culture and high level expression to meet the ends of large-scale antibody production and industrialization.In this study, we developed a serum-free basal medium named BM, which supported the CHO DG44host cell growth and transfection selected from rapid screening of medium library. Then we obtained Opti-BM after optimization of the concentration of dextran sulfate, yeast hydrolyzate, ferric citrate, initial glucose and amino acids relevant to ammonia metabolism for CHO DG44-10G11stable cell line which expressed chimeric antibody E6F6. Using this medium, the maximum viable cell density and antibody production were able to exceed7.0×106cells/mL and70mg/L respectively. Also, we created the feed medium called FM and FM plus of which these combination cell culture time was extended to15days, and the maximum viable cell density and antibody production reached1.2×107cells/mL and150mg/L in the fed-batch culture system.Finally, we made a preliminary exploration on aeration and agitation of multi-bioreactors BIOSTAT(?) B-DCUⅡ2L. Cell culture performance was accordant with the one cultured in the shake flask, realizing the scale-up manufacture from shake flask to bioreactor culture. In summary, we successfully developed a serum-free medium for CHO DG44-10G11cell line, and carry out the work of process optimization and cell scaling up. Meanwhile we built a serum-free medium screening method and optimization platform, which provide experience for other stable cell lines, and laid a foundation for the development of large-scale mammalian cell culture process.