Abstract: The mRNA vaccine platform has emerged as a revolutionary technology in global public health and precision medicine, driven by its high immunoprotective efficacy, and flexible target design. As a critical quality attribute in industrial-scale mRNA production, capping efficiency directly impacts drug safety, efficacy, and manufacturing costs. However, current analytical methods suffer from limitations in accuracy, throughput, cost-effectiveness, and scalability. To address these challenges, this study innovatively optimized RNase H-based analytical strategies through rational probe design and streamlined purification processes. Seventeen sequence-specific probes with varied lengths and hybridization sites were systematically evaluated through RNase H digestion followed by LC-MS characterization. Three optimized probes demonstrated precise RNase H-mediated cleavage at the －2 position with over 99% efficiency. Subsequent silica-based purification effectively eliminated interference from uncut mRNA fragments, achieving consistent capping rates of 97% across multiple batches. This refined methodology establishes a robust, cost-effective platform for capping efficiency assessment, significantly advancing quality control paradigms for mRNA vaccine development.

Key words: mRNA vaccine, quality control, cap rate detection, RNase H, LC-MS