实验设计法在优化表达Fc融合蛋白的CHO细胞发酵培养工艺中的应用

Application of Design of Experiments in optimization of fermentation culture process of CHO cells for expression of Fc fusion protein

目的通过实验设计(Design of Experiments,DOE)分析优化重组CHO细胞在生物反应器中的培养工艺。方法以2 L生物反应器控制参数温度(T)、pH及溶氧(dissolved oxygen,DO)作为变量,以蛋白表达量、蛋白纯度及蛋白唾液酸含量为响应变量,应用Design Expert 10软件进行3因素2水平DOE拟合分析,筛选最适表达Fc融合蛋白的CHO细胞发酵培养工艺参数,并采用5 L生物反应器进行验证。结果蛋白表达量仅与T有关,且呈成正相关,pH和DO对其无显著影响,T与pH对其有交互作用;蛋白纯度受T和pH影响显著,T呈负相关,pH呈正相关,DO对其无显著影响,T与pH对其有交互作用;唾液酸含量受T和pH影响显著,T呈正相关,pH呈负相关,DO对其无显著影响。最适工艺参数确定T为(34±0.5)℃,pH为(7.05±0.02),DO为(45±5)%。采用最适工艺参数的反应器与优化前工艺比较,活细胞密度(viable cell density,VCD)增加;蛋白表达量、蛋白纯度、唾液酸含量分别提高了29.7%、8%和87.9%。结论通过DOE方法快速优化了生物反应器培养表达Fc融合蛋白的CHO细胞的工艺参数,在不降低蛋白产量及纯度的前提下,极大提高了融合蛋白唾液酸含量。

Objective To optimize the culture process of recombinant CHO cells in bioreactor by Design of Experiments(DOE)and increase the sialic acid content.Methods The fermentation culture process of CHO cells for Fc fusion protein in a 2 L bioreactor was optimized by three-factor and two-level DOE fitting analysis using Design Expert 10 software,serving the temperature(T),pH and dissolved oxygen(DO)as variables while protein expression level,purity and sialic acid content as a response variables,of which the results were verified in a 5 L bioreactor.Results The protein expression level was only positively related to the T.Neither pH nor DO showed significant effect on the protein expression.However,both T and pH showed interactions with protein expression.Protein purity was significantly affected by T and pH,which was negatively related to the T while positively related to the pH.DO showed no significant effect on,while T and pH showed interaction with,the protein purity.Sialic acid content was significantly affected by T and pH,which was positively related to the T while negatively related to the pH.However,DO showed no significant effect on the sialic acid content.The process parameters were optimized as T(34±0.5)℃,pH(7.05±0.02)and DO(45±5)%.The optimized process was verified in a 5 L bioreactor,and the result showed that the VCD increased,while the expression level,purity and sialic acid content of Fc fusion protein increased by 29.7%,8%and 87.9%respectively.Conclusion The technological parameters of culture of CHO cells for expression of Fc fusion in bioreactor was rapidly optimized by DOE method,which increased the sialic acid content significantly without decrease of yield and purity of Fc fusion protein.