确定性筛选设计优化双特异性抗体细胞培养工艺

Optimization of bispecific antibody cell culture process by definitive screening design

[目的]旨在快速优化双特异性抗体细胞培养工艺参数,提高蛋白表达量,改善产品关键质量属性。[方法]应用确定性筛选实验设计研究工艺参数(接种密度、培养温度和流加策略)对细胞生长、蛋白表达及关键质量属性(电荷异质性、聚体和高甘露糖型)的影响,利用一步法找出最佳工艺条件,然后放大至2L生物反应器进行工艺验证。[结果]应用JMP软件对实验数据拟合分析找出了最佳工艺条件,接种密度:(1.3±0.2)×10^(6)cells/m L,培养温度:36.5±0.5℃,补料策略:第3 d、13 d流加初始培养体积3%,第5 d、7 d、9 d、11 d流加初始培养体积3.5%。通过2L生物反应器进行了工艺验证。活细胞密度峰值提高了11%,蛋白表达量提高了15%,质量合格。[结论]通过确定性筛选实验设计一步法快速找出了表达双特异性抗体的最佳细胞培养工艺条件,并且能在生物反应器中得到重复和验证。

[Objective]To optimize the process parameters of bispecific antibody cell culture to increase the protein expression and improve the critical quality attributes of the product. [Method]Definitive screening design( DSD) was applied to investigate the influence of inoculation density,culture temperature and feeding strategy on cell growth,protein expression and critical quality attributes of antibodies,using one-step method to find out optimal process conditions,then scaled up to a 2 litres bioreactor for process confirm.[Result]JMP was used to analyze the experimental data and find out the best process conditions,inoculation density is( 1. 3 ± 0. 2) × 10^(6)cells/m L,culture temperature is 36. 5 ± 0. 5 ℃ and feeding strategy is 3%( v/v) on the3 rd,13 th day and 3. 5%( v/v) on the 5 th,7 th,9 th,11 th day. The optimal process conditions were confirmed through 2 litres bioreactor. The peak viable cell density increased by 11%,protein concentration increased by 15%,and critical quality attributes meet the requirements.[Conclusion]The one-step method of definitive screening design can accurately find out the critical process parameters conditions,and can be repeated and confirmed in a bioreactor.