From screen to selectivity: bespoke liquid chromatography strategies for separating siRNA phosphorothioate diastereomers across diverse sequences and conjugates.

Phosphorothioate (PS) linkages are widely incorporated into therapeutic siRNAs to improve stability and pharmacokinetics, but each PS site introduces chirality, generating complex mixtures of Rp/Sp diastereomers that are difficult to resolve. Although liquid chromatography (LC) methods for PS diastereomer separation have attracted increasing interest, practical development strategies applicable across the structural diversity of therapeutic siRNA sequences and conjugates remain limited. Here, we describe an LC workflow beginning with an orthogonal screen of chromatography mode (anion exchange, AEX; ion-pair reversed-phase, IPRP), pH, and temperature, followed by targeted optimisation of gradient slope, flow rate, and, for IPRP, ammonium acetate concentration and stationary-phase pore size. Applied to a panel of single strands representative of therapeutic siRNAs (19-36 nucleotide; varied PS patterns; 2'-F/2'-OMe/MOE; unconjugated and GalNAc, peptide, or lipid-conjugated), the workflow showed that diastereomer selectivity is strongly sequence- and structure-dependent, requiring strand-specific method development. Fine temperature control was essential in both modes, although no single temperature was universally optimal. In AEX, pH 9 often maximised coverage, while pH 11 provided complementary selectivity in some cases. In IPRP, ammonium acetate, shallow gradients, and C18 stationary phases provided a robust starting point, with larger-pore stationary phases beneficial for challenging separations. Conjugation affected the two modes differently: AEX generally outperformed IPRP for GalNAc and peptide-conjugated strands, while highly hydrophobic conjugates required an organic modifier to elute from the AEX column. Using this approach, we achieved complete separation of 16/16 diastereomers for multiple single strands, demonstrating that bespoke PS diastereomer methods can be developed systematically for therapeutic siRNAs.