Ion-pair-free reversed-phase liquid chromatographic profiling of oligonucleotides through selective phosphorothioate...

Phosphorothioate (PS) oligonucleotides typically contain critical impurities, including phosphodiester linkages and shortmer variants. Owing to their high structural similarity, these species are difficult to resolve by reversed-phase liquid chromatography, without the addition of ion-pairing agents. In this study, a selective alkylation strategy targeting the sulfur atom was investigated, enabling differentiation of oligonucleotides with varying numbers of PS modifications and thereby facilitating comprehensive impurity profiling using ion-pair-free RPLC. Whereas conventional oligonucleotides display a pronounced on-off retention mechanism, alkylation leads to a weakened on-off retention mechanism. Diastereomeric separations of oligonucleotides containing multiple PS modifications were achieved using an optimized method, employing two 150 mm columns in series and applying 5 °C column temperature. For oligonucleotide sequences containing three and four PS modifications, 6 out of 8 and 11 out of 16 diastereomers could be resolved, respectively.