Validation of the Reverse Transcription-Quantitative Real-time PCR Method for the Detection of Retroviruses in Cell Cultures Used for Vaccine Production

Achira Namjan; Puthita  Chokreansukchai; Wipawee Wongchana; Sakalin  Trisiriwanich; Supaporn  Phumiamorn

Authors

Achira Namjan Institute of Biological Products, Department of Medical Sciences, Nonthaburi Province, Thailand
Puthita Chokreansukchai Institute of Biological Products, Department of Medical Sciences, Nonthaburi Province, Thailand
Wipawee Wongchana Institute of Biological Products, Department of Medical Sciences, Nonthaburi Province, Thailand
Sakalin Trisiriwanich สถาบันชีววัตถุ กรมวิทยาศาสตร์การแพทย์ กระทรวงสาธารณสุข จังหวัดนนทบุรี
Supaporn Phumiamorn Medical Sciences Technical Office, Department of Medical Sciences, Nonthaburi Province, Thailand

Keywords:

Retrovirus, RT-qPCR, Validation, Cell Culture, Vaccine Production

Abstract

The safety of vaccines is a critical issue in the management of infectious diseases. The significance of this is especially pronounced for vaccines generated in cell culture systems, which are vulnerable to unintended viral contamination. A major issue is the risk of contamination by retroviruses, which are RNA viruses that employ the enzyme reverse transcriptase to incorporate their genetic materials into the host cell genome. This contamination presents a potential health risk to vaccine recipients; therefore, rigorous retrovirus testing is required by regulatory agencies. This research aimed to validate a newly developed reverse transcription-quantitative PCR (RT-qPCR) assay for detecting retroviral contaminations in cell substrates utilized in vaccine manufacturing. The performance of the assay was assessed utilizing MS2 bacteriophage as a model system. The validation involved assessing key performance characteristics, including primer specificity, linearity, precision (intra-day, inter-day, and inter-operator), limit of detection (LOD), and accuracy. The in silico analysis conducted using Primer-BLAST demonstrated that the designed primers exhibited high specificity for MS2 bacteriophage, showing no predicted cross-reactivity with other viruses. The developed RT-qPCR assay exhibited high precision and consistency, effectively detecting low levels of enzyme activity. The detection limit was set at 500 µU/µL, and the accuracy, assessed through recovery study, satisfied the predetermined acceptance criteria. The results of this validation study indicate that the developed RT-qPCR assay is a robust, sensitive, and reliable method. This method is therefore appropriate for regular application for detecting retroviral contaminations in vaccine manufacturing.

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