Therapeutic siRNA quantification in lipid nanoparticles using ion-pair reversed-phase chromatography

The clinical development of lipid nanoparticles encapsulating small interfering RNA (siRNA-LNP) requires robust and efficient analytical methods for quality assessment. In this study, we investigated the chromatographic profiles of siRNAs using Ion-Pair Reversed-Phase (IP-RP) HPLC at six different temperatures, including the melting temperature of duplexes, on two RP columns. The column temperature was identified as a critical parameter influencing chromatographic profiles, with characteristic profiles evolving in a temperature-dependent manner, corresponding to the melting temperatures (T_m) of the two siRNA duplexes analyzed, across both tested columns. Moreover, the siRNA dissociation observed on the columns was consistent with the melting temperatures of the duplexes measured by UV-Vis spectroscopy under HPLC mobile phase conditions. An IP-RPHPLC method using 100 mM TEAA with ACN and a column temperature of 80 °C, above the T_m of the analyzed duplexes, was successfully applied for the quantitative determination of siRNAs in LNP formulations. A simple, one-step sample preparation was employed by directly adding a neutral surfactant to the LNP samples before injection. This method proved highly accurate, as shown by a 100 % recovery of siRNA immediately following microfluidic formulation. Additionally, the impact of the dialysis purification process on siRNA recovery was assessed, revealing a 30 % loss of siRNA. Importantly, the results obtained from this method were consistent with those from the fluorescence reference method. This method helps improve the quality control of siRNA-LNP therapeutics, thereby enhancing their clinical translation potential and supporting the development of next-generation RNA-based therapies.