Two-Dimensional Liquid Chromatography Method for Assembly Homogeneity Analysis and Mass Spectrometric Characterization of RNA Nanoparticles

RNA nanoparticles (RNA NPs) have emerged as a class of delivery vehicles for therapeutic oligonucleotides with the advantages of versatile design and programmable pharmacology properties. Composed of chemically modified oligonucleotide strands that self-assemble into nanostructures exceeding 100 kDa, their complexity poses significant challenges during early discovery and chemistry, manufacturing, and control (CMC) development. This paper introduced a two-dimensional liquid chromatography (2D-LC) platform for characterizing RNA therapeutics delivery vehicles using four-way junction RNA NPs carrying hypoxanthine phosphoribosyl transferase siRNAs as a model molecule. Size-exclusion chromatography (SEC) was used to understand RNA NPs assembly and separate fully assembled molecules from partially assembled intermediates and single strand impurities. SEC × SEC 2D-LC results demonstrated the thermodynamic stability of RNA NPs under physiological conditions. Ion-pairing reverse phase (IPRP) chromatography identified component strands and key impurities within the peak of interest as well as to quantify antisense strands. SEC × IPRP 2D-LC data showed the uniformity of the assembly and strand composition. The distribution of molecular weight, as shown by SEC coupled with multiangle light scattering (MALS), was consistent with the findings on assembly homogeneity from the SEC × IPRP method. The native mass of RNA NPs was obtained with SEC coupled to high-resolution mass spectrometry, achieving a mass error of less than 0.0025%. <i>In vitro</i> gene knockdown analysis demonstrated that the presence of partially assembled intermediates did not have a significant effect on the potency of RNA NPs. These findings enhance the understanding of RNA NPs assembly and structural integrity, crucial for their development and clinical application.