| Recombinant humanized anti-CD20 antibody was expressed by CHO,which has broad market prospects and great economic values in the treatment of the Non-Hodgkin's lymphoma(NHL)and other fields.In this thesis,in order to achieve high-level expression of high anti-CD20 antibody,the CHO cells culture process was improved in shake flask and bioreactor,respectively.In addition,the effect of different culture conditions on CHO cell culture performance and antibody glycosylation was investigated in bioreactor,which played a solid foundation for its production process scale-up.Culture process development in shake flask:1)Firstly,the combination of FortiCHO and Feed C was the optimum strategy for CHO cells growth and anti-CD20 antibody production through screening different basic media and feed media.The peak viable cell density and antibody concentration were 16.02×106cells/ml and 1.218g/L,respectively.2)The culture conditions such as seed cell density,feed concentration,first feed timing and temperature were optimized by DOE experiment.The first feed timing and seed cell density had no significant effects on the expression of the target antibody;however,the feed concentration of 10%was beneficial to improve antibody expression;the temperature was a major factor affecting antibody expression among the four factors,the temperature of 35? was obtained,where the cell viability was more than 80%and antibody concentration was 1.35g/L.3)Based on the optimized culture conditions,the effect of initial glutamine cncentration on cell culture performance was investigated as well.With increasing concentration of glutamine,the peak cell density was increased;however,the antibody expression and specific production rate of antibody were decreased,suggesting that the initial glutamine concentration should be controlled at 4mM/L in the culture.Culture process development in bioreactor:1)Based on the optimized culture conditions of shake flask culture,when cells were cultured in bioreactor,the maximum cell density and antibody concentration were 19.5×106cells/ml and 1.26g/L in bioreactor culture,respectively,antibody concentration was slightly lower than that obtained in shake flask culture.GOF,G1F and galactosylation were 53.5%,27.2%and 17.8%,respectively.2)Based on amino acids analysis,the limitation of tyrosine and cysteine concentrations could be the main reason that caused the decline in antibody expression in the late cell culture period.Antibody expression of 1.66g/L was reached by adding 1.5mM Tyr and 1mM Cys,improved by 24.1%;the specific production rate of antibody was increased from 1.73 pg/cell/day to 13.598 pg/cell/day in the last two days,improved by 87%.3)Based on the culture conditions of amino acids supplementation,the effect of pH,seed cell density and butyrate on the cell culture performance and glycosylation of antibody was investigated,respectively.Antibody expression was increased to 1.745g/L when pH was decreased to 6.8;however,the decrease in galactosylation was observed:G0F(67.4%)and G1F(19.2%),as well as a final ammonia concentration of 13mM/L.The seed cell density in the range of 0.5-0.8×106cells/ml had no significant effect on expression and glycoform of antibody.The cell density and cell cycle can be controlled in butyrate which improved antibody expression:the peak cell density was 16.565×106cells/ml,the expression of antibody was 1.828g/L.Besides,the butyrate had no significant effect on the glycoform:GOF(54.4%)and G1F(30.7%). |
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