摘要
目的基于质量源于设计(Quality by Design,QbD)理念,优化柯萨奇病毒A6型(Coxsackievirus A6,CV-A6)培养工艺,建立关键工艺参数设计空间,提高CV-A6产量。方法结合石川图和失效模式效应分析(failure mode effects analysis,FMEA)进行工艺参数的风险评估,评估需进行试验研究的潜在重要或关键工艺参数。在确认的缩小模型中,应用完全析因试验设计研究潜在关键工艺参数对关键质量属性的影响,筛选有显著效应的关键工艺参数。使用中心复合表面设计对关键工艺参数进行优化,基于蒙特卡洛模拟确定关键工艺参数的设计空间,并在缩小和放大规模中对设计空间进行验证。结果病毒培养规模、培养基种类、培养基碳酸氢钠(NaHCO_(3))浓度、培养基血清浓度、接种感染复数(multiplicity of infection,MOI)、培养温度和培养时间风险优先值(risk priority number,RPN)>27,为潜在重要或关键工艺参数。6孔细胞培养板和5层细胞工厂病毒增殖曲线保持一致,抗原含量和病毒滴度等价。完全析因试验设计结果显示,病毒培养温度、培养基DMEM占比、培养基NaHCO_(3)浓度对抗原含量和病毒滴度具有显著效应(P<0.05),病毒接种MOI和培养基血清浓度无显著效应(P>0.05)。基于蒙特卡洛模拟确定的病毒培养温度、培养基DMEM占比和培养基NaHCO_(3)浓度设计空间分别为32.33~34.33℃、83%~100%、2~4 g/L。缩小和放大规模设计空间的抗原含量和病毒滴度验证值均满足规格限,且均落于拟合值的95%置信区间和95%预测区间内。工艺优化后,CV-A6培养时间为11~12 d,相比于工艺优化前,抗原含量提高约5倍,病毒滴度提高0.5~1.0 LgCCID_(50)/mL。结论基于QbD理念,优化了CV-A6培养工艺,建立了关键工艺参数设计空间,且显著提高了CV-A6产量。
Objective To optimize the production process of Coxsackievirus A6(CV-A6),establish the design space for critical process parameters and improve the yields of CV-A6 virus,based on the concept of Quality by Design(QbD).Methods Risk assessment analysis of process parameters was performed using Ishikawa diagram combined with failure mode effects analysis(FMEA)to identify potential key or critical process parameters that require experimental research.The full factorial experiment design was applied to study the effects of potential critical process parameters on critical quality attributes and to screen out the critical process parameters in the validated scale-down model.The central composite facecentered design was used to optimize the critical process parameters,and the design space of critical process parameters was determined based on Monte Carlo simulation.The design space was verified in both scale-down scale and expanded scale.Results The CV-A6 virus production scale,types of virus culture medium,sodium bicarbonate(NaHCO_(3))concentration in virus culture medium,serum concentration in virus culture medium,multiplicity of infection(MOI),virus culture temperature and virus harvest time were considered to be the potential key or critical process parameters based on risk priority number(RPN)of above 27.The virus proliferation curves of the 6-well cell culture plate and 5-layer cell factory were consistent,and the antigen yields and virus titers were equivalent.The results of full factorial experiment design showed that virus culture temperature,DMEM ratio and NaHCO_(3)concentration in virus culture medium had significant effects on antigen yields and virus titers(P<0.05),while MOI and serum concentration in virus culture medium had no significant effects(P>0.05).The design space of virus culture temperature,DMEM ratio and NaHCO_(3)concentration in virus culture medium determined by Monte Carlo simulation was 32.33-34.33℃,83%-100%and 2-4 g/L,respectively.The validated values of antigen yields and virus titers for both scale-down scale and expanded scale design space met the specification limits and fell within the 95%confidence interval and 95%prediction interval of the fitted values.After the process optimization,the harvest time of CV-A6 was 11-12 d.Compared with the yields before optimization,the antigen yields had increased by approximately five times and the virus titers had increased by 0.5-1.0 LgCCID_(50)/mL.Conclusion The production process of CV-A6 was optimized,the design space for critical process parameters was established and the yield of CV-A6 virus was significantly increased,based on the concept of QbD.
作者
万鑫
戴旱雨
肖奥
李伟
孟胜利
王泽鋆
郭靖
申硕
WAN Xin;DAI Hanyu;XIAO Ao;LI Wei;MENG Shengli;WANG Zejun;GUO Jing;SHEN Shuo(Viral Vaccine Research Laboratory,Wuhan Institute of Biological Products Co.,Ltd.,National Engineering Technology Research Center of Combined Vaccines,Vaccine Technology Innovation Center of Hubei Province,National Key Laboratory for Novel Vaccines Research and Development of Emerging Infectious Diseases,Wuhan 430207,Hubei Province,China)
出处
《中国生物制品学杂志》
2026年第3期314-325,共12页
Chinese Journal of Biologicals
基金
国家科技重大专项“重大新药创制”(2015ZX09102021)
湖北省技术创新重大专项(2017ACA078)。
关键词
柯萨奇病毒A6型
缩小模型
风险评估
完全析因试验设计
中心复合表面设计
设计空间
Coxsackievirus A6(CV-A6)
Scale-down model
Risk assessment
Full factorial experiment design
Central composite face-centered design
Design space
