CHO细胞强化流加培养过程理性设计与建立

Rational Design and Establishment of CHO Cell Intensified Fed-batch Culture Process

目的:由于强化流加培养工艺(intensified fed batch of ultra-high seeding density,uHSD-IFB)的接种密度与运行密度较高,传统低密度培养工艺的流加策略往往不能提供充足的营养物质用于该过程的细胞维持与产物表达,最终导致过程产率低、经济性下降;通过优化流加培养基以及补料方案,成功建立CHO细胞强化流加培养过程,从而提高目的蛋白产量。方法:以一株表达单克隆抗体的CHO-K1细胞株为研究对象,通过代谢动力学与化学计量学分析,设计出以葡萄糖为控制模型的两阶段动态反馈流加策略,并结合实验设计(design of experiment,DoE)筛选并优化流加培养基中关键微量元素的营养浓度。结果:优化设计后的uHSD-IFB过程有效缓解了uHSD-IFB过程营养物质的耗竭与代谢副产物累积之间的矛盾,实现了超高接种密度培养工艺的细胞生长与产物合成的目的;累积产量相较于优化前提高了95%,日体积产量提高了约97%。结论:该补料策略有助于快速建立高细胞密度、高产物表达的高接种密度强化流加培养过程。

Objective:Due to the higher seeding density and running density of the intensified fed-batch of ultra-high seeding density(uHSD-IFB)process,the feeding strategy of the traditional low-density culture process often does not provide sufficient nutrients for cell maintenance and product expression of the process,which ultimately leads to low process yield and lower production efficiency.By optimizing the feed medium and feeding strategy,the uHSD-IFB process will be successfully established and the target protein yield will be increased.Methods:A monoclonal antibody-expressing CHO-K1 cell line was studied in this paper,a two-stage dynamic feedback feeding strategy with glucose as the control indicator was designed by metabolic kinetics and stoichiometry analysis,and the nutrient concentrations for key trace elements in the feed medium were screened and optimized in combination with design of experiment(DoE).Results:The optimized process effectively alleviated the contradiction between nutrient depletion and accumulation of metabolic by-products in the uHSD-IFB process and achieved the purpose of cell growth and product synthesis in the ultra-high inoculation density culture process.The cumulative titer of the optimized design of the uHSD-IFB process was increased by 95% and the daily yield was increased by about 97% compared to that before optimization.Conclusion:This proposed feeding strategy can provide help to rapidly establish an enhanced fed-batch culture process of ultra-high seeding density with high cell density and high product expression.