Article Dans Une Revue Journal of Pharmaceutical and Biomedical Analysis Open Année : 2025

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

Résumé

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 temperaturedependent 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.

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hal-05048019 , version 1 (26-04-2025)

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Ludivine Ferey, Sandy Al Bardawil, Emilie Dols, Aurore Guédin, Viet-Ha Phan, et al.. Therapeutic siRNA quantification in lipid nanoparticles using ion-pair reversed-phase chromatography. Journal of Pharmaceutical and Biomedical Analysis Open, 2025, 5, pp.100072. ⟨10.1016/j.jpbao.2025.100072⟩. ⟨hal-05048019⟩
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