Ion exclusion chromatography of RNA-lipid nanoparticles using polyethyleneimine-modified spongy monoliths

Over-adsorption of RNA-lipid nanoparticles (LNPs) to separation media requires sample pre-saturation and hinders accurate characterization of LNP heterogeneity. To evaluate this effect, LNP adsorption during size exclusion chromatography (SEC) was examined as a representative method. To overcome these challenges, ion exclusion chromatography (IEC) on polyethyleneimine-modified spongy monoliths was developed for rapid LNP separation. The macroporous monoliths were derivatized with branched 2 kDa polyethyleneimine and cross-linked to create a highly permeable, charge-repulsive surface. The modified columns were confirmed to retain their high permeability, and physical appearance was assessed by scanning electron microscopy (SEM). The chromatographic retention mechanism was elucidated, and method conditions were optimized for partial resolution of two distinct LNP populations. Integration of IEC into a two-dimensional liquid chromatography mass spectrometry (2D-LC/MS) workflow, using reversed-phase separation in the second dimension for lipid quantitation, highlighted compositional differences between populations. Later-eluting LNPs were found to have lower molar ratios of the ionizable lipid SM-102. The method was applied in a stability-indicating assay to evaluate the effects of repeated freeze-thaw cycles on surface charge and compositional heterogeneity.