The therapeutic potential of lipid nanoparticles (LNPs) as delivery vehicles has been demonstrated in recent years, culminating in the emergency use approval of mRNA-based SARS-CoV-2 vaccines in late 2020. Research has shown that the tissue-penetrating ability and on-target effectiveness of LNPs are related to particle size. The determination of RNA content relative to LNP size can be important to the understanding of efficacy and adverse effects. Size-exclusion chromatography (SEC) coupled with multi-angle light scattering (MALS) and UV and refractive Index (dRI) detectors is often used for the characterization of large molecules or complex macro-entities such as LNPs. However, for particles larger than 50 nm in diameter, size based RNA content determination with the MALS-UV-dRI multi-detectors is confounded by the UV scattering of particles at 260 nm, characteristic absorption wavelength of RNA . This work presents the first description of a novel facile and rapid analytical method for online, size-dependent RNA payload distribution measurement using data from MALS, UV and dRI detectors following separation of the LNPs by SEC. 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 molar 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.
Learning Objectives:
Apply the new capability in their research and therapeutic development
Use it as a tool to monitor LNP manufacturing process
Have better understanding of LNP internal structure
