Chemical cleavage coupled with LCMS to report on the true diastereomeric distributions of phosphorothioate linkages in gapmer ASO.

Phosphorothioate (PS) modifications are commonly employed to enhance the performance of antisense oligonucleotides (ASOs). Each phosphorus atom at a PS linkage acts as a chiral center, typically resulting in thousands or more diastereomers in a therapeutic PS ASO. Since the synthesis of most PS ASOs under development is not stereo-selective, establishing stereochemical comparability between batches is crucial for ensuring the consistency of the drug substance. In this study, we evaluate a nuclease-free approach based on the Maxam-Gilbert sequencing and alkylation sequencing method to chemically cleave gapmer PS ASOs into smaller fragments to facilitate the chromatographic separation of diastereomers. Compared to our previous report using nuclease P1 coupled with liquid chromatography-mass spectrometry analysis, the chemical cleavage approach demonstrated superior repeatability and sensitivity in analyzing gapmer PS ASOs. Beyond comparability, this method can accurately report on the true diastereomeric distributions at certain PS linkages, which significantly benefits the comparison of stereochemistry pre- and post-purification.