Deformulating Size Exclusion Chromatography for LNP Payload Quantitation

This application note discusses the use of a GTxResolve™ Premier BEH™ SEC 450 Å Column for the online deformulation of LNPs and size exclusion separation of the mRNA payload. Common detergent disruption protocols have been tested and found to lead to incomplete deformulation of the sample, as both intact LNPs and freed nucleic acids can be still observed in the SEC chromatograms. While pursuing alternative options, we discovered that a detergent and organic solvent could be applied as mobile phase additives to achieve complete disruption of intact LNP samples upon their injection into the LC system, thus removing the need for a sample preparation step. With the developed method, nucleic acid payloads can be effectively liberated from the lipid vehicle, size separated and quantified in a rapid and robust assay requiring only a minimal amount of sample. Such an assay provides instant insights into an otherwise challenging characterization of LNP payloads and is especially suitable to samples with multiple payloads important for emerging gene editing therapies.