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*

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。

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

Fed-batch culture has been one of main bioprocesses for monoclonal antibody (mAb) 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 mAb yield. However, the impact of pH on mAb production by CHO cells was not significant and there were no interaction between pH 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 0.1mmol/L at late stage of cultivation process under the optimal culture condition. As such, additional 1.5mmol/L tyrosine and 1mmol/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.