基于在线拉曼和在线活细胞检测的PAT控制PRV疫苗智能生产工艺研究

Intelligent Production Process for PAT Controlled PRV Vaccine Based on Online Raman and Online Live Cell Detection

减少乳酸和氨积累一直是动物细胞培养的一个目标,保持培养过程中较低浓度葡萄糖和谷氨酰胺的工艺控制法是最便捷和高效的优化策略。基于在线拉曼检测技术和比例-积分-微分(proportional-integral-derivative,PID)自动反馈控制技术,实现了BHK-21细胞伪狂犬病毒(pseudorabies virus,PRV)疫苗生产过程中低葡萄糖浓度(0.5 g/L左右)和低谷氨酰胺浓度(1.5 mmol/L左右)控制,乳酸和氨积累量分别降低了26.0%和30.3%,病毒接毒时pH维持在6.75以上,并利用在线活细胞检测技术确定了最佳补料时间、最佳接毒时间及最佳收毒时间。经过补料操作后,细胞比生长速率最大值延迟了12 h,且细胞高活性状态维持了更长时间(24~48 h),在保持单细胞产毒能力的同时,有效提高了接毒时的细胞密度,最大病毒滴度达到高糖对照组的10倍及手动流加组的1.4倍。替代传统手动补料操作,成功建立了基于低葡萄糖和谷氨酰胺浓度以及在线拉曼和在线活细胞检测的BHK-21细胞反应器过程分析技术(PAT)控制的PRV疫苗智能生产工艺,为实现生物药物的智能制造奠定了基础。

Reducing lactic acid and ammonia accumulation has long been a goal of the mammalian cell biotechnology industry,and process control is a more convenient and efficient way to achieve this.That is,the accumulation of lactic acid and ammonia is reduced by controlling lower concentrations of glucose and glutamine during culture.Based on online Raman monitoring technology and proportional-integral-derivative(PID)automatic feedback control technology,this research realized the control of low glucose concentration(about O.5 g/L)and low glutamine concentration(about 1.5 mmol/L)during the production of BHK-21 cell PRV vaccine.Lactic acid and ammonia accumulation were reduced by 26.0%and 30.3%,respectively.The pH at virus inoculation was maintained above 6.75,and the optimal feeding,inoculation,and harvest time of the virus were determined by online live cell detection technology.After the feeding operation,the maximum specific cell growth rate was delayed for 12 h and the high cell activity state was maintained for a longer time(24-48 h).While maintaining the virus-producing ability of single cells,the cell density at virus inoculation was effectively increased.The maximum virus titer was 10 times that of the high-glucose control group and 1.4 times that of the manual feeding group.Instead of the traditional manual feeding operation,the intelligent production process of PRV vaccine controlled by BHK-21 cell reactor PAT based on low glucose and glutamine concentration as well as online Raman and online live cell detection was successfully established,which laid the foundation for the intelligent production of biological drugs.