Chromatographic Separation and Stability Analysis of Small Interfering RNA and Lipid Vehicles Using Ion-Pair Reversed Phase Liquid Chromatography
Authors:   , Li Li , Foley Joe Preston 
Published: July 2021

Chromatographic methods were developed capable of separating and quantitating siRNA, lipids, and their potential breakdown products due to oxidation or hydrolysis. Such methods are essential to developing lipid nanoparticles (LNPs) as a formulation delivery system for siRNAs. Separation of siRNAs was achieved using ion-pair reversed-phase liquid chromatography. Part 1 of the thesis describes the development of an ion-pair reversed-phase HPLC method for the separation of closely related stereoisomers of a chemically modified siRNA duplex. A systematic evaluation of key chromatographic parameters showed that a BEH C18 column with sub-2 [mu]-m particle size, coupled with the use of triethylammonium acetate as the ion-pair reagent and acetonitrile as the strong solvent of the hydro-organic mobile phase, achieved baseline resolution of siRNA stereoisomers and their desulfurization products. A high column temperature, creating a denaturing condition for siRNA, is critical to the separation of stereoisomers. An aprotic organic modifier, such as acetonitrile, can effectively disrupt the hydrogen bonding interaction between the duplex and enable the separation of stereoisomers by promoting hydrophobic interactions between the C18 stationary phase and the stereoisomers. Part 2 of the thesis expands the utility of the ion-pair reversed-phase liquid chromatography to include a simultaneous separation of the main lipid components of an LNP system. Ion-pair reversed-phase separation conditions were developed that can reduce the retention gap between siRNAs and lipids that have significant differences in their physical and chemical properties. Studies showed that a BEH phenyl column could significantly retain siRNA due to a combination of both hydrophobic and [pi] - [pi] interactions. In contrast, the lipids experienced a reduced retention with the phenyl column, a key advantage attributed to the presence of a short alkyl chain component in the stationary phase compared to octyl- or octadecyl-derivatized silica. While the ion-pair reagents had virtually no impact on the separation of the lipids, the retention times of the siRNAs showed a quantitative correlation with the structure of the ion-pair reagents in the mobile phase. The chromatographic separation conditions with a phenyl stationary phase, particularly with dibutylammonium acetate as the ion-pair reagent, markedly reduced the retention gap between the siRNAs and the lipids, achieving a baseline resolution of a complex matrix containing five siRNAs and six lipids in a 20-minute gradient elution method. Finally, the ion-pair reversed-phase method was applied to the degradation products of a model siRNA system. Stress testing showed that the model siRNA developed minimal hydrolysis products at neutral pH. This indicated the importance of chemical modification at the 2'-position in the ribose unit of siRNA molecules. In contrast, the siRNA was prone to oxidation by hydrogen peroxide, with or without trace levels of a transition metal, and to oxidation by a radical initiator. Desulfurization and phosphodiester strand scission were the likely main degradation pathways contributing to the observed oxidative reactivity.