Ion-Pairing Hydrophilic Interaction Chromatography for Impurity Profiling of Therapeutic Phosphorothioated Oligonucleotides.

ABSTRACT: Therapeutic oligonucleotides (ONs) may contain many closely related impurities. Using conventional liquid chromatography (LC) modes, the separation of impurities comprising the same number of nucleotides as the ON product remains a challenge. In this study, we investigated the performance of ion-pairing HILIC (IP-HILIC) as an alternative mass-spectrometry (MS)-compatible LC mode for ON impurity profiling. A fully phosphorothioated, N-acetylgalactosamine-conjugated 16-mer antisense ON (full-length product; FLP) served as a model compound, along with shortmer, longmer, PS-PO converted, deaminated (DA) and nonconjugated (NC) products, which are potential impurities. We describe the effect of ion-pairing reagent (IPR) hydrophobicity, eluent pH, and column temperature on IP-HILIC perform

SUBMITTER: Tutis L 

PROVIDER: S-EPMC12311887 | biostudies-literature | 2025 Jul

REPOSITORIES: biostudies-literature