运用氨基酸分析法和DOE法优化抗PD-1单克隆抗体生产用培养基

Optimiztion of anti-programmed death-1 monoclonal antibody medium using amino acid analysis and design of experiment

本研究采用氨基酸分析法结合DOE设计法优化并获得高表达抗PD-1单克隆抗体生产用基础和补料培养基。通过对市售多种基础和补料培养基进行筛选,获得细胞生长状况较优的基础培养基和抗体表达较高的补料培养基,利用氨基酸分析法检测较优基础培养基和补料培养基中氨基酸消耗情况,确定影响细胞生长和抗体表达的关键氨基酸种类,利用DOE分析软件设计分别在较优基础和补料培养基中添加不同浓度的氨基酸种类及浓度,根据细胞生长及抗体表达,优化得到抗PD-1单克隆抗体的基础和补料培养基组合。最终优化后基础培养基配方为:Hycell CHO培养基中添加1.04 mmol/L L-天冬酰胺和0.76 mmol/L L-谷氨酰胺。最终优化后补料培养基配方为:OPM CHOCD Feed1补料培养基中添加38.7 mmol/L L-组氨酸,75.0 mmol/L L-酪氨酸,64.0 mmol/L L-丝氨酸,49.2 mmol/L L-谷氨酰胺和18.7 mmol/L L-半胱氨酸。经过3 L反应器培养验证,优化后的培养基比未优化时,最大活细胞密度(PVCD)提高了62.7%,抗PD-1单克隆抗体表达量提高了71.5%,且活性无明显差异。

The purpose of this study was to optimize anti-PD-1 monoclonal antibody basic and feed medium by using amino acid analysis and design of experiment. The better basic and feed medium was obtained according to the cell growth and antibody expression through single factor screening a variety of basic and feed medium from different vendors. The kinds of key amino acids affecting cell culture and antibody expression through analysis amino acid consumption of better basic and feed medium were determined using amino acid analysis method. The anti-PD-1 monoclonal antibody basic and feed medium were optimized according to cell growth and antibody expression through adding different concentrations of amino acids using design of experiment analysis software. The ultimately determined optimal basic medium formula were as follows ： Hycell CHO Medium, 1.04 mmol/L L-asparagine, 0.76 mmol/L L-glutamine. The optimal feed medium formula were the following： OPM CHOCD Feed 1, 38.7 mmol/L L-histidine, 75.0 mmol/L L-tyrosine, 64.0 mmol/L L-serine, 49.2 mmol/L L-glutamine, 18.7 mmol/L L-cysteine. Through the 3 L test validation, the optimized medium peak viable cell density （PVCD）increased by 62.7% than that of the unoptimized and the anti-PD-1 monoclonal antibody expression increased by 71.5% than that of unoptimized, respectively. The biological activity of anti-PD-1 monoclonal antibody in optimized medium was not significantly reduced if compared with that of the unoptimized medium.