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

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 performance, with IPRs reducing the relative contribution of the phosphate moiety on retention, thereby increasing separation selectivity based on the nature of nucleobases and conjugated groups. For a poly(dT) ladder, the effective peak capacity was reduced from 35 to 22 when introducing triethylamine as IPR; however, improved separations were observed for PS ONs. By employing an eluent containing 25 mM triethylamine acetate (pH 6.3) and a column temperature of 80 °C, IP-HILIC successfully resolved the DA impurities from both the FLP and the NC-FLP. This is noteworthy, as current MS-compatible, one-dimensional LC methods cannot resolve the DA impurity from the FLP, and MS resolution is often insufficient to differentiate the FLP and DA due to a mass difference of less than 1 Da. The proposed IP-HILIC method shows the potential for ON impurity profiling.