High-throughput Screening And Optimization Of Serum-free Media For Chinesehamster Ovary Cells Based On Tubespin Bioreactor

The large-scale cultivation of CHO cells is a key technique for commercialproduction of monoclonal antibodies which needs to be glycosylated or proteins withcomplicated structure, whereas screening and optimization of serum-free media hasbeen a hotspot in the field. We established a high-throughput screening platform forscreening serum-free media of CHO DG44cell cultivation by combining a novelbioreactor Tubespin with carbon dioxide box shaker ISF4-X. We optimized theculture conditions for the Tubespin bioreactor and developed a high performanceoriginal serum-free media formulations through CHO cells metabolism analysis andscreening on key added components. Our finding will provide technical support forthe establishment of a platform for large-scale production of monoclonal antibodies.The DF12media was first selected as a starting material for serum-free mediaoptimization after comparison of several media. The seeding density and rotatingspeed for CHO DG44culture were optimized with Tubespin respectively, and theoptimal culture conditions were obtained as following:180rpm/min,10ml,37℃,5%CO2, with the seeding density of0.3×105cells/ml. Usually, the cell aggregationswill occur when the cell density is more than8.117×106cells/ml in the suspensioncultivation and the problem was successfully overcome by adding25mg/L ofdextran sulfate in our study.The ITS (insulin, transferrin, sodium selenite) are the most important additivesfor CHO DG44cell cultivation. The optimal concentrations of ITS (insulin20mg/L,transferrin10mg/L, sodium selenite25μg/L) were obtained using the centralcomposite design method. Moreover, we successfully replaced the ITS using thelow-cost salt mixture containing16mg/L ferric citrate,1mg/L zinc sulfate and25μg/L sodium selenite. Eleven pivotal media components for the cell cultivation werescreened by Plackett-Burman method and the most important three components forpromoting cell growth are the reduced glutathione, putrescine and albuminsequentially. The alternations of17amino acids concentrations in media during cellscultivation from different media batch were measured using AQC precolumnderivatization RP-HPLC method, and the results showed that the amino acidsincluding aspartic acid, serine, arginine, histidine, cysteine, leucine, phenylalanine,and proline were consumed largely, among which cysteine was almost used out.According to the initial amino acid composition of the media and the amino acidconsumption pattern, we designed two original formula of amino acids for furtheroptimization. The cell density were enhanced using the new formula and the highestcell density was increased to70%compared with the control group. We also showedthat cell proliferation and survival can be significantly inhibited by hyperosmosisand the cell tolerance on hyperosmosis was increased when8mM Gln was addedinto the media.