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通过试验设计(DOE)方法和JMP软件分析快速优化重组CHO细胞在生物反应器中的培养工艺,保证抗体高表达量的同时降低五聚甘露糖(Man5)水平。以培养基CD-CIM1为基础培养基,在细胞培养过程中添加补料培养基流加培养,利用3 L小型生物反应器,对反应器pH值,溶氧(DO),温度(T)3个控制参数进行3因子2水平比较,以抗体表达量(Titer)、产物蛋白Man5水平为响应变量,应用JMP软件进行拟合模型最小二乘法分析,找出最优工艺参数。用优化的工艺参数进行验证,并且放大到50 L生物反应器中,先后培养两个批次,进一步验证优化结果。通过分析,参数T、pH值及3因素间两两交互作用对Titer均有显著影响,DO对Titer影响不显著;pH对Man5有显著影响,T、DO及3因素间两两交互作用对Man5的影响均不显著。结果预测,为获得高表达量及可接受范围(0%~10%)内的Man5水平,最优工艺参数为pH 7.10,DO 60%,T 34.5℃。用优化工艺在小型生物反应器内进行验证,收获时抗体表达量分别为1 152,901 mg/L;Man5水平分别为8.60%,7.49%;两个批次的50 L生物反应器放大培养,抗体表达量分别为840,849 mg/L,Man5水平分别为6.03%,9.45%,结果均符合预期。成功运用DOE方法快速高效的建立了降低Man5水平的细胞培养工艺,为后期放大提供依据。
Through the DOE method and JMP software analysis, the culture process of recombinant CHO cells in bioreactor was rapidly optimized to ensure the high expression level of antibody and reduce the Man5 level. The medium of CD-CIM1 was used as the basal medium. The culture medium was fed with culture medium supplemented with 3-L mini-bioreactor. The pH, dissolved oxygen (DO), temperature The control parameters were compared by 3 factors and 2 levels. The titer and Man5 level of the antibody were used as response variables. The least square method was used to analyze the fitting parameters to find out the optimal process parameters. Validated with optimized process parameters and scaled up to a 50 L bioreactor, two batches were grown in succession to further verify the optimization results. Through the analysis, there are significant effects on the Titer between the two parameters of the parameter T, the pH value and the three factors, and the DO has no significant effect on the Titer. The pH has a significant effect on the Man5. The interactions between T, DO and three factors on Man5 The impact is not significant. The results predicted that the optimum process parameters were pH 7.10, DO 60% and T 34.5 ℃ for high expression level and Man5 level in acceptable range (0% -10%). The optimum conditions were optimized in a small bioreactor. The antibody expression levels at harvest were 1 152 and 901 mg / L, respectively. The Man5 levels were 8.60% and 7.49%, respectively. Two batches of 50 L bioreactor were expanded and cultured, Respectively, 840, 849 mg / L, and Man5 levels were 6.03% and 9.45% respectively. The results were in line with expectations. The DOE method has been successfully used to rapidly and efficiently establish a cell culture process that reduces Man5 levels and provides a basis for later amplification.