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

Abstract

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.

Figure 1

Effect of column temperature and mobile-phase ionic strength on HILIC analysis of as-202PS, that possess four PS modifications, indicated by a purple V. EICs showing the comparison of 90 (red), 40 (orange), and 5 °C (blue) as column temperature and 10 mM (i) and 100 mM (ii) as AA concentration in the mobile phases. Circled nucleotides indicate sugar modifications (2′-OMe indicated by a solid line and 2′-F by a dotted line).

Figure 2

(A) Melting curves of as-202PS (blue traces) and ss-202PS (green traces) measured over the temperature range of 70–10 °C and using either 1× PBS, H₂O, or 100 mM AA in 35:65 H₂O/ACN as the diluent. (B) Corresponding EICs of as-202PS and ss-202PS using the generic gradient and 100 mM as AA concentration in the mobile phases and 5 °C as column temperature.

Figure 3

EICs of highly structured (HS1) and unstructured (L1) ONs using 100 mM as the AA concentration in mobile phases and 5 °C as the column temperature.

Figure 4

Analysis of intact double-stranded siRNAs. (A) EICs of ds-4PS and ds-8PS using 100 mM as AA concentration in mobile phases and 5 °C as column temperature. Selectivities were calculated between the first and last detectable peaks (marked by an orange dot) in the apparent cluster of chromatographic peaks. For more information about the sequences, please refer to Table S1.

Figure 5

EIC of as-202PS under favorable chromatographic conditions (100 mM AA, 5 °C) using mobile phases containing MeOH as the organic modifier.