基于DOE设计和氨基酸补加策略提高CHO细胞表达抗CD20单克隆抗体

Enhancement of Anti-CD20 Monoclonal Antibody Expression by CHO based on DOE and Amino Acid Supplemental Strategy

中国仓鼠卵巢细胞(CHO)流加培养生产单克隆抗体是目前主流培养方式,其中环境参数(pH和温度)和营养成分均影响细胞生长、碳氮源代谢和外源蛋白表达,是培养过程中关键的控制参数。采用实验设计(design of experiment,DOE)方法研究培养参数(温度、pH)对CHO细胞生长和抗CD20抗体表达的影响,建立营养限制型氨基酸流加策略,实现抗CD20抗体的高表达。结果表明,温度是影响蛋白质表达的显著因素,35℃有助于提高细胞密度和目标抗CD20抗体表达,而pH对抗CD20表达影响不显著,且温度和pH无交互作用,经DOE预测分析最佳培养条件是温度35℃和pH7.0。在该最佳培养条件下,在培养后期酪氨酸和半胱氨酸的浓度都低于0.1mmol/L。在培养的第2天通过补加1.5mmol/L酪氨酸和1mmol/L半胱氨酸避免营养限制,抗CD20抗体表达水平提高了24.1%,且对蛋白糖型无影响。

Fed-batch culture has been one of main bioprocesses for monoclonal antibody( m Ab) production by Chinese hamster ovary( CHO) cells.It has been reported that environmental parameters like temperature and pH and nutritional ingredients are key factors that can influence cell growth,carbon and/or nitrogen source metabolism and foreign protein expression in CHO cell suspension culture.Objective: Effects of culture process parameters( temperature and pH) on cell growth and anti-CD20 antibody expression by CHO cells were investigated based on design of experiment( DOE),and a fed-batch strategy was successfully developed to improve anti-CD20 antibody expression level with amino acid supplementation.The results show that temperature was a key factor for anti-CD20 antibody expression: 35℃ was the optimal temperature with an increased cell density and target m Ab yield.However,the impact of pH on m Ab production by CHO cells was not significant and there were no interaction between p H and temperature.The optimal culture conditions were 35℃ and pH7.0 according to the analysis of DOE predictive profiler.In addition,it found that residual concentrations of tyrosine and cysteine in culture was below 0.1 mmol/L at late stage of cultivation process under the optimal culture condition.As such,additional 1.5 mmol/L tyrosine and 1 mmol/L cysteine were fed on day 2,led to an increase in anti-CD20 antibody titer by 24.1% and no changes in glycosylation of anti-CD20 antibody.