Characterization of RNA using mass photometry: Method and technical perspectives

Developing analytical methods for RNA-based products, such as mRNA vaccines, requires tools that support release testing, stability assessment, and characterization. Mass photometry (MP) is a label-free, single-molecule technique increasingly applied to biotherapeutics and vaccines. Here, we outline best practices and technical considerations for using MP to analyze vaccine mRNA constructs and other RNA samples. With appropriate calibration, MP provides mRNA molecular weight and quantifies heterogeneous species, including those difficult to resolve using traditional techniques. We systematically evaluated experimental parameters influencing RNA measurements, including surface chemistry and buffer composition, and show that cationic-coated sample carriers and salt-containing buffers are essential for reproducible detection and mass resolution. Low-mass species were assessed using multiple mRNA constructs, kinetic analysis, and buffer controls to distinguish genuine sample heterogeneity from method artifacts. We further examined the effects of ionic additives on oligomeric species. MP results were compared with charge detection mass spectrometry and fragment analyzer measurements, showing good agreement when accounting for differences in measurement principles. Overall, MP provides a rapid, low-sample consumption, and information-rich approach for mRNA characterization and quality assessment, either as a standalone method or as an orthogonal complement to established RNA analytical techniques.