基于QbD理念的大肠埃希菌摇瓶阶段培养工艺开发  

Development of a shake-flask stage culture process for E.coli based on QbD concept

作　　者：徐世友 白彩虹 孙田华 易中恒 李薇 焦磊 雷清 XU Shiyou;BAI Caihong;SUN Tianhua;YI Zhongheng;LI Wei;JIAO Lei;LEI Qing(Lanzhou Institute of Biological Products Co.,Ltd.,Center for Gansu Provincial Vaccine Engineering Research,Lanzhou 730046,Gansu Province,China)

机构地区：[1]兰州生物制品研究所有限责任公司、甘肃省疫苗工程技术研究中心,甘肃兰州730046

出　　处：《中国生物制品学杂志》2024年第6期710-717,共8页Chinese Journal of Biologicals

基　　金：国家重点研发计划(2018YFC1200600)。

摘　　要：目的基于质量源于设计(Quality by Design,QbD)理念,开发一种具有较高生物量与较高菌体活力培养特点的大肠埃希菌摇瓶阶段培养工艺。方法以不同摇瓶配置为考察因素,菌体悬浮液A600值、培养物湿菌重和菌体活力值为培养结果考察指标,采用方差分析分析培养结果以获得具有高生物量和高菌体活力的第3次扩增摇瓶配置。以摇床温度和摇床转速为试验因子进行2因子2水平全因子试验,菌体悬浮液A值为响应值,培养累计时间为变量因素,摇床实时在线温度与摇床设备自测转速为补充变量因素,采用函数型主成分分析(functional principal component analysis,FPCA)方法进行广义回归,拟合生长曲线模型,通过生长曲线模型获得优化后的培养停止时间及培养工艺,对响应值添加随机噪声蒙特卡洛模拟(Monte Carlo simulation,MCS),再次优化获得摇床培养工艺设计空间,选择设计空间中最劣条件作为验证试验设定条件,以不同培养停止时间分阶段进行连续10批次验证试验。结果第3次扩增摇瓶配置:5 L一次性高效摇瓶,大面积透气膜盖。培养工艺设计空间:摇床温度36.5~37.5℃,摇床转速220~230 r/min,设计培养停止时间18 h。最劣条件验证试验表明,停止培养时间为16 h时,可获得较高菌体活力值且较低生物量的培养结果,停止培养时间为18 h时,可获得较高水平的生物量及菌体活力值。结论本研究设计的大肠埃希菌摇瓶阶段培养工艺具有较高生物量及较高菌体活力培养特点,同时可依据此培养工艺适应性调整以满足不同培养需求。Objective To develop a shake-flask stage culture process for E.coli with higher biomass and higher bacterial viability based on Quality by Design(QbD)concept.Methods Using different shake-flask configurations as the investigation factors,and A600value of the bacterial suspension,wet weight of the culture and viability value of the bacteria as the indicators for investigation of the culture results,ANOVA was used for the analysis of culture results to obtain the third amplification flask configurations with high biomass and high bacterial viability.The two-factor two-level full-factor test was carried out with the shaker temperature and shaker speed as the test factors,the A value of bacterial suspension as the response value,the culture accumulation time as a variable factor,and the real-time online temperature and self-test speed of the shaker as the supplemented variable factors.The functional principal component analysis(FPCA)method was used to perform a generalized regression model to model the quasi-growth curve,and the optimized culture stop time and culture process were obtained by the growth curve model.The design space of the shaker culture process was optimized again using Monte Carlo simulation(MCS)with random noise added to the response value.The worst condition in the design space was selected as the setting condition for verification test,and serial 10 batch verification tests were performed in stages with different culture stop time.Results The third amplification shake-flask configurations:5 L disposable high-efficiency shakeflask and large area breathable film cover.The culture process design space:shaker temperature of 36.5-37.5℃,shaker speed of 220-230 r/min,and the design culture stop time of 18 h.The worst condition verification test showed that when the culture was stopped for 16 h,the culture results of higher cell viability value and lower biomass could be obtained,and when the culture was stopped for 18 h,the results of higher biomass and bacterial viability value could be obtained.Conc

关 键 词：大肠埃希菌 培养工艺 摇瓶 方差分析 函数型主成分分析 质量源于设计 

分 类 号：Q815[生物学—生物工程]