Establishment and Validation of a Cell-Based Relative Potency Method for Respiratory Syncytial Virus mRNA Vaccine...

Background: An accurate, sensitive, and robust potency assay is essential for the quality control of mRNA drug substances, which are characterized by complex manufacturing processes, intricate molecular structures, and high susceptibility to degradation. Currently, mRNA vaccine manufacturers use a variety of biological potency assays, often without systematic method development or rigorous evaluation. As a result, these assays may lack sufficient accuracy and robustness, making it difficult to reliably distinguish mRNA drug substance samples with different potency levels. Therefore, there is a need for a standardized, robust, and reliable potency assay for the evaluation of mRNA drug substance samples across a range of potencies. Methods: In this study, we developed a cell-based relative potency assay for a respiratory syncytial virus (RSV) mRNA drug substance encoding an engineered prefusion (PreF) form of the RSV type A (RSV-A) F protein, a recognized target for RSV vaccine development. The RSV mRNA drug substance was complexed with transfection reagents and introduced into cells in vitro to enable expression of the RSV-A PreF protein, which was then quantified using a double-antibody sandwich ELISA. Results: Systematic optimization showed that cell line, cell density, transfection reagent, mRNA-to-transfection reagent ratios, and transfection duration all influenced assay performance. Under optimized conditions, the assay demonstrated acceptable accuracy and precision, with relative bias values ranging from -25% to 13% across the potency range of 44~156%, measured-to-expected ratios within 0.8~1.2, and relative standard deviations of 18% and 16% for intra- and inter-assay precision, respectively. Furthermore, the optimized potency assay effectively distinguished mRNA drug substance samples with varying potency levels. Conclusions: This study provides a useful functional complement to physicochemical characterization and supports quality control and batch-to-batch consistency of RSV mRNA drug substances. In addition, the development strategy may also serve as a useful reference for the establishment of in vitro potency assays for other mRNA drug substances.