Establishment, optimization and validation of a rapid titer detection method for freeze-dried live attenuated hepatitis A vaccine: cell culture-RT-qPCR method
RT-qPCR;Hepatitis A virus(HAV);Virus titer;Risk assessment;Quality by Design(QbD)
- VernacularTitle:冻干甲型肝炎减毒活疫苗滴度细胞培养-RT-qPCR快速检测方法的建立、优化及验证
- Author:
LI Yajing
- Publication Type:Journal Article
- Keywords:
RT-qPCR;
Hepatitis A virus(HAV);
Virus titer;
Risk assessment;
Quality by Design(QbD)
- From:
Chinese Journal of Biologicals
2025;38(09):1063-1071
- CountryChina
- Language:Chinese
-
Abstract:
Objective To establish a rapid cell culture-RT-qPCR method for detecting infectivity titer of freeze-dried live attenuated hepatitis A vaccine based on ICH Q2(R2) and Q14 guidelines, and to optimize and verify the method.Methods Using the Analytical Quality by Design(AQbD) concept, a cell culture-RT-qPCR platform was developed through analyzing Acceptance Test Procedure(ATP) and risk analysis and Design of Experiment(DoE). Key parameters such as cell density,culture temperature for virus, and incubation time were optimized with cycle threshold(Ct) as the response indicator.The specificity, linearity and intermediate precision of the method were verified, and the quantitative range was determined.The consistency between the developed method and traditional titer detection method was assessed by Bland-Altman analysis.Results A total of 13 main risk factors were identified, and the optimal reaction conditions were: cell density(1-2) ×10~5 cells/mL; virus dilution gradient spacing of 4 times; 96-well cell culture plate with edge effect; virus adsorption at 37 ℃ for 60 min, and virus culture at 35 ℃ for 72 h; PCR without location effect; denaturation at 90 ℃ for 63 s; reverse transcription at 60 ℃ for 15 min; calculation model of double logarithmic linear model. The method exhibited good specificity(Ct value > 28 for inactivated virus controls), linearity(R~2= 0. 966) and intermediate precision [geometric coefficient of variation(GCV) ≤ 9. 28%], within a quantitative range of 5. 0-8. 0 lgCCID_(50)/mL. The 95% limits of agreement(95% LoA) of the difference between this method and the traditional method ranged from-0. 06 to 0. 41 lgCCID_(50)/mL.Conclusion The established cell culture-RT-qPCR method shortens the detection cycle from 21-28 days to 3 days with the advantages of highthroughput and accurate quantification for titer detection, providing key technical support for improving the efficiency of process monitoring and hepatitis A virus(HAV) vaccine titer detection, and promoting the lot release.
