Expanding the Analytical Toolbox for the Nondenaturing Analysis of siRNAs with Salt-Mediated Ion-Pair Reversed-Phase Liquid Chromatography

Short interfering RNA (siRNA) represents a rapidly expanding class of marketed oligonucleotide therapeutics. Due to its double-stranded nature, the characterization of siRNA is twofold: (i) at the single-strand (denaturing) level for impurity profiling and (ii) at the intact (nondenaturing) level to confirm duplex formation and quantify excess single strands (including single strand-derived impurities). While denaturing analysis can be carried out using conventional ion-pair reversed-phase liquid chromatography (IP-RPLC), nondenaturing characterization of siRNA is a significantly less straightforward task. Typical IP-RPLC conditions have an intrinsic denaturing effect on siRNA, thereby limiting the development of viable approaches for the intact duplex analysis. In this study, we demonstrate, through the design of experiments of siRNA melting temperatures and chromatography analyses, that the simple addition of salts, such as phosphate-buffered saline and ammonium acetate, to eluents enhances the suitability of IP-RPLC for the nondenaturing analysis of siRNA during both UV- and mass spectrometry-based analysis. This work represents a milestone in overcoming the challenges associated with nondenaturing analysis of siRNAs by IP-RPLC and offers a fresh angle for exploring IP-RPLC of siRNAs.