Development of In Vitro Potency Methods to Replace In Vivo Tests for Enterovirus 71 Inactivated Vaccine (Human Diploid Cell-Based/Vero Cell-Based)
Background: The three commercial Enterovirus 71 (EV71) inactivated vaccines which have effectively controlled the EV71 pandemic currently rely on inherent variable in vivo potency methods for batch release. To align with 3R (Replacement, Reduction, Refinement) principles and enhance quality control,...
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MDPI AG
2025-04-01
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| Online Access: | https://www.mdpi.com/2076-393X/13/4/404 |
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| author | Xuanxuan Zhang Li Yi Dan Yu Jun Li Xintian Li Xing Wu Fan Gao Qian He Wenhui Wang Kaiwen Wang Zejun Wang Zhengling Liu Yadong Li Yong Zhao Huiyi Li Xiao Ma Qingbing Zheng Longfa Xu Tong Cheng Rui Zhu Jing Guo Jing Li Qunying Mao Zhenglun Liang |
| author_facet | Xuanxuan Zhang Li Yi Dan Yu Jun Li Xintian Li Xing Wu Fan Gao Qian He Wenhui Wang Kaiwen Wang Zejun Wang Zhengling Liu Yadong Li Yong Zhao Huiyi Li Xiao Ma Qingbing Zheng Longfa Xu Tong Cheng Rui Zhu Jing Guo Jing Li Qunying Mao Zhenglun Liang |
| author_sort | Xuanxuan Zhang |
| collection | DOAJ |
| description | Background: The three commercial Enterovirus 71 (EV71) inactivated vaccines which have effectively controlled the EV71 pandemic currently rely on inherent variable in vivo potency methods for batch release. To align with 3R (Replacement, Reduction, Refinement) principles and enhance quality control, this study referred to WHO guidelines and the European Pharmacopoeia to develop in vitro relative potency (IVRP) methods. Methods: Working standards tracing to phase 3 clinical vaccines were established. Manufacture-specific IVRP methods were developed and validated per ICH Q14/Q2(R2), utilizing conformational epitope-targeting neutralizing monoclonal antibodies (MAbs). One of the MAbs (CT11F9) recognition sites was clarified with Cryo-EM. Subsequently, the performance of IVRP was assessed using varied concentrations and heat-treated vaccines. The correlation between IVRP and in vivo methods was analyzed, followed by setting IVRP specifications. Results: The manufacturer-specific working standard exhibited ED50 values comparable to those of related phase 3 clinical vaccines. All IVRP methods achieved a relative bias/precision/total error ≤ 15%. The IVRP methods correlated with in vivo methods (<i>p</i> < 0.05, r > 0.9) can discriminate EV71 antigen concentrations (<i>p</i> < 0.01, r > 0.99) and indicate the stability of the vaccines. Cryo-EM was adopted to identify the epitopes recognized by CT11F9, revealing that this neutralizing antibody recognizes a conformational epitope spanning VP1-3 of the same protomer. Using 31–47 batches of commercial vaccines, IVRP specifications were proposed as 0.56–1.35, 0.58–1.40, and 0.54–1.50. Conclusions: Based on conformational epitope-targeting neutralizing MAbs, manufacturer-specific IVRP methods, which were sensitive to process variations and correlated with in vivo results, have been established. IVRP methods provide a reliable, animal-free alternative for EV71 vaccine batch release. |
| format | Article |
| id | doaj-art-612ab32afb984e8a80d0f4f51ae86a57 |
| institution | DOAJ |
| issn | 2076-393X |
| language | English |
| publishDate | 2025-04-01 |
| publisher | MDPI AG |
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| series | Vaccines |
| spelling | doaj-art-612ab32afb984e8a80d0f4f51ae86a572025-08-20T03:13:51ZengMDPI AGVaccines2076-393X2025-04-0113440410.3390/vaccines13040404Development of In Vitro Potency Methods to Replace In Vivo Tests for Enterovirus 71 Inactivated Vaccine (Human Diploid Cell-Based/Vero Cell-Based)Xuanxuan Zhang0Li Yi1Dan Yu2Jun Li3Xintian Li4Xing Wu5Fan Gao6Qian He7Wenhui Wang8Kaiwen Wang9Zejun Wang10Zhengling Liu11Yadong Li12Yong Zhao13Huiyi Li14Xiao Ma15Qingbing Zheng16Longfa Xu17Tong Cheng18Rui Zhu19Jing Guo20Jing Li21Qunying Mao22Zhenglun Liang23NHC Key Laboratory of Research on Quality and Standardization of Biotech Products, NMPA Key Laboratory for Quality Research and Evaluation of Biological Products, State Key Laboratory of Drug Regulatory Science, Research Units of Innovative Vaccine Quality Evaluation and Standardization, Chinese Academy of Medical Sciences, National Institutes for Food and Drug Control, Beijing 102629, ChinaInstitute of Medical Biology, Chinese Academy of Medical Sciences, Kunming 650118, ChinaSinovac Biotech, Beijing 100085, ChinaSinovac Biotech, Beijing 100085, ChinaSinovac Biotech, Beijing 100085, ChinaNHC Key Laboratory of Research on Quality and Standardization of Biotech Products, NMPA Key Laboratory for Quality Research and Evaluation of Biological Products, State Key Laboratory of Drug Regulatory Science, Research Units of Innovative Vaccine Quality Evaluation and Standardization, Chinese Academy of Medical Sciences, National Institutes for Food and Drug Control, Beijing 102629, ChinaNHC Key Laboratory of Research on Quality and Standardization of Biotech Products, NMPA Key Laboratory for Quality Research and Evaluation of Biological Products, State Key Laboratory of Drug Regulatory Science, Research Units of Innovative Vaccine Quality Evaluation and Standardization, Chinese Academy of Medical Sciences, National Institutes for Food and Drug Control, Beijing 102629, ChinaNHC Key Laboratory of Research on Quality and Standardization of Biotech Products, NMPA Key Laboratory for Quality Research and Evaluation of Biological Products, State Key Laboratory of Drug Regulatory Science, Research Units of Innovative Vaccine Quality Evaluation and Standardization, Chinese Academy of Medical Sciences, National Institutes for Food and Drug Control, Beijing 102629, ChinaWuhan Institute of Biological Products Co., Ltd., Wuhan 430207, ChinaWuhan Institute of Biological Products Co., Ltd., Wuhan 430207, ChinaWuhan Institute of Biological Products Co., Ltd., Wuhan 430207, ChinaInstitute of Medical Biology, Chinese Academy of Medical Sciences, Kunming 650118, ChinaInstitute of Medical Biology, Chinese Academy of Medical Sciences, Kunming 650118, ChinaInstitute of Medical Biology, Chinese Academy of Medical Sciences, Kunming 650118, ChinaKey Laboratory of Research on Quality and Standardization of Biotech Products, Chinese Pharmacopoeia Commission, Beijing 100061, ChinaNHC Key Laboratory of Research on Quality and Standardization of Biotech Products, NMPA Key Laboratory for Quality Research and Evaluation of Biological Products, State Key Laboratory of Drug Regulatory Science, Research Units of Innovative Vaccine Quality Evaluation and Standardization, Chinese Academy of Medical Sciences, National Institutes for Food and Drug Control, Beijing 102629, ChinaState Key Laboratory of Vaccines for Infectious Diseases, Xiang An Biomedicine Laboratory, School of Public Health, Xiamen University, Xiamen 361005, ChinaState Key Laboratory of Vaccines for Infectious Diseases, Xiang An Biomedicine Laboratory, School of Public Health, Xiamen University, Xiamen 361005, ChinaState Key Laboratory of Vaccines for Infectious Diseases, Xiang An Biomedicine Laboratory, School of Public Health, Xiamen University, Xiamen 361005, ChinaState Key Laboratory of Vaccines for Infectious Diseases, Xiang An Biomedicine Laboratory, School of Public Health, Xiamen University, Xiamen 361005, ChinaWuhan Institute of Biological Products Co., Ltd., Wuhan 430207, ChinaSinovac Biotech, Beijing 100085, ChinaNHC Key Laboratory of Research on Quality and Standardization of Biotech Products, NMPA Key Laboratory for Quality Research and Evaluation of Biological Products, State Key Laboratory of Drug Regulatory Science, Research Units of Innovative Vaccine Quality Evaluation and Standardization, Chinese Academy of Medical Sciences, National Institutes for Food and Drug Control, Beijing 102629, ChinaNHC Key Laboratory of Research on Quality and Standardization of Biotech Products, NMPA Key Laboratory for Quality Research and Evaluation of Biological Products, State Key Laboratory of Drug Regulatory Science, Research Units of Innovative Vaccine Quality Evaluation and Standardization, Chinese Academy of Medical Sciences, National Institutes for Food and Drug Control, Beijing 102629, ChinaBackground: The three commercial Enterovirus 71 (EV71) inactivated vaccines which have effectively controlled the EV71 pandemic currently rely on inherent variable in vivo potency methods for batch release. To align with 3R (Replacement, Reduction, Refinement) principles and enhance quality control, this study referred to WHO guidelines and the European Pharmacopoeia to develop in vitro relative potency (IVRP) methods. Methods: Working standards tracing to phase 3 clinical vaccines were established. Manufacture-specific IVRP methods were developed and validated per ICH Q14/Q2(R2), utilizing conformational epitope-targeting neutralizing monoclonal antibodies (MAbs). One of the MAbs (CT11F9) recognition sites was clarified with Cryo-EM. Subsequently, the performance of IVRP was assessed using varied concentrations and heat-treated vaccines. The correlation between IVRP and in vivo methods was analyzed, followed by setting IVRP specifications. Results: The manufacturer-specific working standard exhibited ED50 values comparable to those of related phase 3 clinical vaccines. All IVRP methods achieved a relative bias/precision/total error ≤ 15%. The IVRP methods correlated with in vivo methods (<i>p</i> < 0.05, r > 0.9) can discriminate EV71 antigen concentrations (<i>p</i> < 0.01, r > 0.99) and indicate the stability of the vaccines. Cryo-EM was adopted to identify the epitopes recognized by CT11F9, revealing that this neutralizing antibody recognizes a conformational epitope spanning VP1-3 of the same protomer. Using 31–47 batches of commercial vaccines, IVRP specifications were proposed as 0.56–1.35, 0.58–1.40, and 0.54–1.50. Conclusions: Based on conformational epitope-targeting neutralizing MAbs, manufacturer-specific IVRP methods, which were sensitive to process variations and correlated with in vivo results, have been established. IVRP methods provide a reliable, animal-free alternative for EV71 vaccine batch release.https://www.mdpi.com/2076-393X/13/4/404enterovirus 71 inactivated vaccinein vitro relative potencyin vivo potencycorrelationreplacement |
| spellingShingle | Xuanxuan Zhang Li Yi Dan Yu Jun Li Xintian Li Xing Wu Fan Gao Qian He Wenhui Wang Kaiwen Wang Zejun Wang Zhengling Liu Yadong Li Yong Zhao Huiyi Li Xiao Ma Qingbing Zheng Longfa Xu Tong Cheng Rui Zhu Jing Guo Jing Li Qunying Mao Zhenglun Liang Development of In Vitro Potency Methods to Replace In Vivo Tests for Enterovirus 71 Inactivated Vaccine (Human Diploid Cell-Based/Vero Cell-Based) Vaccines enterovirus 71 inactivated vaccine in vitro relative potency in vivo potency correlation replacement |
| title | Development of In Vitro Potency Methods to Replace In Vivo Tests for Enterovirus 71 Inactivated Vaccine (Human Diploid Cell-Based/Vero Cell-Based) |
| title_full | Development of In Vitro Potency Methods to Replace In Vivo Tests for Enterovirus 71 Inactivated Vaccine (Human Diploid Cell-Based/Vero Cell-Based) |
| title_fullStr | Development of In Vitro Potency Methods to Replace In Vivo Tests for Enterovirus 71 Inactivated Vaccine (Human Diploid Cell-Based/Vero Cell-Based) |
| title_full_unstemmed | Development of In Vitro Potency Methods to Replace In Vivo Tests for Enterovirus 71 Inactivated Vaccine (Human Diploid Cell-Based/Vero Cell-Based) |
| title_short | Development of In Vitro Potency Methods to Replace In Vivo Tests for Enterovirus 71 Inactivated Vaccine (Human Diploid Cell-Based/Vero Cell-Based) |
| title_sort | development of in vitro potency methods to replace in vivo tests for enterovirus 71 inactivated vaccine human diploid cell based vero cell based |
| topic | enterovirus 71 inactivated vaccine in vitro relative potency in vivo potency correlation replacement |
| url | https://www.mdpi.com/2076-393X/13/4/404 |
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