Ion-pairing hydrophilic interaction chromatography for impurity profiling of therapeutic phosphorothioated oligonucleotides

Therapeutic oligonucleotides (ONs) are short synthetic DNA or RNA strands used to modulate protein expression in disease treatment. Besides the full-length product (FLP), ON samples contain many closely related impurities that must be profiled to ensure drug quality. Separation techniques, such as ion-pairing reversed-phase liquid chromatography (IP-RPLC), anion-exchange chromatography (AEX), and hydrophilic interaction chromatography (HILIC) are used for ON impurity profiling. However, it is challenging to resolve impurities comprising the same number of nucleotides as the FLP. Here, we investigated the performance of ion-paring HILIC (IP-HILIC) as an alternative, mass-spectrometry (MS) compatible LC mode for ON impurity profiling. In IP-HILIC, ion pair reagents (IPRs) are expected to reduce the relative contribution of the phosphate moiety on retention, potentially increasing separation selectivity based on the nature of nucleobases and conjugated groups. A fully phosphorothioated, N-acetylgalactosamine-conjugated 16-mer antisense ON served as an FLP model compound, along with shortmer, longmer, PS-PO converted, deaminated and non-conjugated products, which are potential impurities. We describe the effect of several parameters on the IP-HILIC performance, such as IPR hydrophobicity, eluent pH, and column temperature. The addition of IPRs to the eluent resulted in a unique separation selectivity compared to IP-RPLC, AEX, and HILIC. In particular, IP-HILIC successfully separated the deamination impurity (DA) from the FLP. This is noteworthy as current MS-compatible, one-dimensional LC methods cannot resolve this impurity and MS resolution is often insufficient to differentiate the FLP and DA due to a 1 Da mass difference. The proposed IP-HILIC method shows potential for ON impurity profiling.