Emerging trends in the characterization of oligonucleotide diastereomers

Chemical modifications have enabled the use of oligonucleotides as therapeutic drugs. However, they also complicate their analytical characterization. Phosphorothioate (PS)-modified oligonucleotides exist as complex mixtures of diastereomers. Health authorities have emphasized the need for suitable state-of-the-art methods to provide, at a minimum, an estimate of diastereomeric distribution (ratio of isomers), and the batch-to-batch reproducibility. The number of PS linkages varies by the oligonucleotide classes, with antisense oligonucleotides (ASOs) containing a higher number of PS linkages and >8000 diastereomers. The Food and Drug Administration (FDA)-approved small interfering RNAs (siRNAs) have received more attention for analytical research of diastereomers, as their limited number of PS linkages per strand (≤ 5) can enable complete separation of all diastereomers. While past chromatographic method development focused on minimizing the impact of diastereomers on the separation of impurities, a recent surge in research focused on the characterization of diastereomers has been observed to bridge the analytical gap. Ion mobility mass spectrometry (IM-MS) and hydrophilic interaction liquid chromatography (HILIC) have only recently been applied to the characterization oligonucleotide diastereomers. However, challenges remain in the identification of the PS linkage chiral configuration (Sp/Rp) and the maximal number of diastereomers that can be separated by one technique has not improved (maximum of 32 so far). This review critically examines recent technologies, especially those developed since 2023, and the long-standing challenges.