Nie Jianqi, Sun Yang, Han Fei, Yang Yankun, Liu Xiuxia, Liu Chunli, Li Ye, Bai Zhonghu
The Key Laboratory of Industrial Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi, 214122, China.
National Engineering Laboratory for Cereal Fermentation Technology, Jiangnan University, Wuxi, 214122, China.
Cytotechnology. 2020 Apr;72(2):283-293. doi: 10.1007/s10616-020-00377-9. Epub 2020 Feb 21.
This study described a successful application of the Quality by Design (QbD) approach to pseudorabies virus (PRV) production process development in a fixed-bed bioreactor using the serum-free medium (SFM). The innovated tube-fixed-bed bioreactor was used as a scale-down model of the fixed-bed bioreactor for process development. Risk analysis was performed using Ishikawa diagram combined with failure mode effects analysis (FMEA). The comparative experiment was performed to screen proper medium for adherent African green monkey kidney (Vero) cells from three commercially available SFMs (VP-SFM, ProVERO-1 and Vero-A). The Vero-A medium showed as an outstanding one for further study. The PRV titer in harvest medium was consider as Critical Quality Attribute (CQA) and the Critical Process Parameters (CPPs) [time of infection (TOI), multiplicity of infection (MOI) and initial inoculation cell density] ranked high with risk priority number (RPN) were taken into design of experiment (DoE) methodology. Then prediction model of PRV production process was established and a robust PRV production process was explored. Under the robust setpoint conditions, the Xcell 1 L laboratory-scale fixed-bed bioreactor yielded PRV titer up to 7.87 log TCID/mL at 3 dpi, which was comparable with that in the tube-fixed-bed bioreactor. Combination of the tube-fixed-bed bioreactor and QbD approach could further accelerate the development of a robust virus production process.
本研究描述了质量源于设计（QbD）方法在使用无血清培养基（SFM）的固定床生物反应器中伪狂犬病病毒（PRV）生产工艺开发中的成功应用。创新的管式固定床生物反应器用作固定床生物反应器的缩小模型以进行工艺开发。使用石川图结合失效模式影响分析（FMEA）进行风险分析。进行了比较实验，从三种市售的SFM（VP-SFM、ProVERO-1和Vero-A）中筛选适合贴壁非洲绿猴肾（Vero）细胞的培养基。Vero-A培养基表现出色，可用于进一步研究。将收获培养基中的PRV滴度视为关键质量属性（CQA），并将风险优先数（RPN）排名较高的关键工艺参数（CPPs）[感染时间（TOI）、感染复数（MOI）和初始接种细胞密度]纳入实验设计（DoE）方法。然后建立了PRV生产工艺的预测模型，并探索了稳健的PRV生产工艺。在稳健的设定条件下，Xcell 1 L实验室规模的固定床生物反应器在3 dpi时产生的PRV滴度高达7.87 log TCID/mL，与管式固定床生物反应器中的滴度相当。管式固定床生物反应器和QbD方法的结合可以进一步加速稳健病毒生产工艺的开发。
