Development and Validation of a qPCR Assay for Quantitative Evaluation of Residual Host Cell DNA for Rabies Vaccines Produced in Vero Cells

Residual host cell DNA in biological products poses potential risks of tumorigenicity and infectivity. Therefore, current regulatory authorities including the World Health Organization (WHO), the Chinese Pharmacopoeia, and the U.S. Food and Drug Administration (FDA) have limited the acceptance amounts of residual DNA (less than 10 ng or 3 ng/dose). Among various detection methods for residual DNA, quantitative PCR (qPCR) is recognized as the most practical method for its high sensitivity, accuracy, precision, and efficiency. This study aimed to develop a qPCR method for detecting residual Vero cell DNA to facilitate downstream purification and ensure quality control during vaccine manufacturing. We selected two highly repetitive Vero genomic DNA sequences as amplification targets: the "172 bp" sequence and the Alu repetitive sequence. The qPCR method was meticulously optimized and validated for linearity, quantitation limit, specificity, accuracy, repeatability, intermediate precision, and robustness. The "172 bp" sequence qPCR assay showed excellent linearity and amplification efficiency, establishing a dependable approach for quantifying residual Vero DNA in pharmaceuticals and for regulatory compliance monitoring.