biostudies-literatureDesligniere EDutch Research Council (NWO)101454https://www.ebi.ac.uk/biostudies/studies/S-EPMC11999443biostudies-literatureUnknown33(2)The impressive success of mRNA-based vaccines to combat COVID-19 has encouraged biopharmaceutical companies to invest in broader applications of alike vaccines for various diseases. Analytical approaches must keep pace to support this surge in the development of mRNA-based therapies. Intact mass analysis of mid- to large mRNA molecules (>1,000 nt) poses significant analytical challenges due to mRNA size, heterogeneity, and instability. Here, we demonstrate how single-particle Orbitrap-based charge detection mass spectrometry (CDMS) and mass photometry (MP) approaches can rapidly measure the mass of various intact high-mass capped mRNAs, up to 9,400 nt (∼3 MDa) in size. While ensemble MS yielded approximate masses for mRNAs <2,000 nt, it failed to provide information on samples of longer seMolecular therapy. Methods & clinical developmentCharacterization of intact mRNA-based therapeutics by charge detection mass spectrometry and mass photometry.PMC11999443SPI.2017.028Heck AJRBarnes LFPowers TWFriese OVDesligniere EfalseCharacterization of intact mRNA-based therapeutics by charge detection mass spectrometry and mass photometry.The impressive success of mRNA-based vaccines to combat COVID-19 has encouraged biopharmaceutical companies to invest in broader applications of alike vaccines for various diseases. Analytical approaches must keep pace to support this surge in the development of mRNA-based therapies. Intact mass analysis of mid- to large mRNA molecules (>1,000 nt) poses significant analytical challenges due to mRNA size, heterogeneity, and instability. Here, we demonstrate how single-particle Orbitrap-based charge detection mass spectrometry (CDMS) and mass photometry (MP) approaches can rapidly measure the mass of various intact high-mass capped mRNAs, up to 9,400 nt (∼3 MDa) in size. While ensemble MS yielded approximate masses for mRNAs <2,000 nt, it failed to provide information on samples of longer se2025-01-01T00:00:00Z2025 Jun2025-07-07T03:10:13.694Z2025-07-07T03:10:13.694ZS-EPMC119994434023649710.1016/j.omtm.2025.101454