Enabling online determination of the size-dependent RNA content of lipid nanoparticle-based RNA formulations

•It is the first description of a rapid online analytical method for size-dependent RNA payload distribution measurement in lipid nanoparticles (LNPs)•The online RNA distribution determination methodology was validated by size-based fractionation of various LNPs with subsequent offline analysis of the fractions.•This analytical advancement provides advanced characterization for LNPs, and could shed light on understanding the relationship between structure and in vivo performance of the RNA/DNA filled LNPs.•The reported method can also be used as part of quality control procedures to provide information on critical quality attributes, thus ensure consistent LNP-RNA therapeutic product. The potential of lipid nanoparticles (LNPs) as nucleic acid delivery vehicles has been demonstrated in recent years, culminating in the emergency use approval of LNP-based mRNA SARS-CoV-2 vaccines in late 2020. The determination of RNA content relative to LNP size can be important to the understanding of efficacy and adverse effects. This work presents the first description of a facile and rapid analytical method for online, size-dependent RNA payload distribution measurement using data from multi-angle light scattering, ultraviolet and refractive index detectors following separation of the LNPs by size-exclusion chromatography. The analysis was validated by size-based fractionation of the LNPs with subsequent offline analysis of the fractions. Four LNPs formulated with different PEG-lipids and different lipid compositions were tested. Good agreement was observed between the online and offline size-based RNA distributions among all four LNPs, demonstrating the utility of the online method for LNP-encapsulated RNA in general, and suggesting a means for simplified biophysical quantitation of a dosing-related critical quality attribute.