Liquid Chromatography–Mass Spectrometry-Based Qualitative Profiling of mRNA Therapeutic Reagents Using Stable Isotope-Labeled Standards Followed by the Automatic Quantitation Software Ariadne

[Image: see text] mRNA-based medicines are a promising modality for preventing virus-caused illnesses, including COVID-19, and treating various types of cancer and genetic diseases. To develop such medicines, methods to characterize long mRNA molecules are needed for quality control and metabolic analysis. Here, we developed an analytical platform based on isotope-dilution liquid chromatography–mass spectrometry (LC–MS) that quantitatively characterizes long, modified mRNAs by comparing them to a stable isotope-labeled reference with an identical sequence to that of the target medicine. This platform also includes database searching using the mass spectra as a query, which allowed us to confirm the primary structures of 200 to 4300 nt mRNAs including chemical modifications, with sequence coverage at 100%, to detect/identify defects in the sequences, and to define the efficiencies of the 5′-capping and integrity of the polyadenylated tail. Our findings indicated that this platform should be valuable for quantitatively characterizing mRNA vaccines and other mRNA medicines.