Unravelling the Link between Oligonucleotide Structure and Diastereomer Separation in Hydrophilic Interaction Chromatography.

Therapeutic oligonucleotides (ONs) commonly incorporate phosphorothioate (PS) modifications. These introduce chiral centers and generate ON diastereomers. The increasing number of ONs undergoing clinical trials and reaching the market has led to a growing interest to better characterize the ON diastereomer composition, especially for small interfering ribonucleic acids (siRNAs). In this study, and for the first time, we identify higher-order structures as the major cause of ON diastereomer separation in hydrophilic interaction chromatography (HILIC). We have used conformational predictions and melting profiles of several representative full-length ONs to first analyze ON folding and then run mass spectrometry and HILIC to underpin the link between their folding and diastereomer separation. On top, we show how one can either enhance or suppress diastereomer separation depending on chromatographic settings, such as column temperature, pore size, stationary phase, mobile-phase ionic strength, and organic modifier. This work will significantly facilitate future HILIC-based characterization of PS-containing ONs; e.g., enabling monitoring of batch-to-batch diastereomer distributions in full-length siRNAs, a complex task that is now for the first time shown as possible on this delicate class of therapeutic double-stranded ONs.