Separation of Plasmid DNA Topological Forms, Messenger RNA, and Lipid Nanoparticle Aggregates Using an Ultrawide Pore Size Exclusion Chromatography Column

[Image: see text] Health authorities have highlighted the need to determine oligonucleotide aggregates. However, existing technologies have limitations that have prevented the reliable analysis of size variants for large nucleic acids and lipid nanoparticles (LNPs). In this work, nucleic acid and LNP aggregation was examined using prototype, low adsorption ultrawide pore size exclusion chromatography (SEC) columns. A preliminary study was conducted to determine the column’s physicochemical properties. A large difference in aggregate content (17.8 vs 59.7 %) was found for a model messenger RNA (mRNA) produced by different manufacturers. We further investigated the nature of the aggregates via a heat treatment. Interestingly, thermal stress irreversibly decreased the amount of aggregates from 59.7 to 4.1% and increased the main peak area 3.3-fold. To the best of our knowledge, for the first time, plasmid DNA topological forms and multimers were separated by analytical SEC. The degradation trends were compared to the data obtained with an anion exchange chromatography method. Finally, unconjugated and fragment antigen-binding (Fab)-guided LNPs were analyzed and their elution times were plotted against their sizes as measured by DLS. Multi-angle light scattering (MALS) was coupled to SEC in order to gain further insights on large species eluting before the LNPs, which were later identified as self-associating LNPs. This study demonstrated the utility of ultrawide pore SEC columns in characterizing the size variants of large nucleic acid therapeutics and LNPs.