Characterization of Antisense Oligonucleotide Impurities by Ion-Pairing Reversed-Phase and Anion Exchange Chromatography Coupled to Hydrophilic Interaction Liquid Chromatography/Mass Spectrometry Using a Versatile Two-Dimensional Liquid Chromatography Setup

Determination of phosphorothioate oligonucleotide purity and impurity profile is commonly performed by ion-pairing reversed-phase liquid chromatography (IPRP) with a mobile phase containing triethylamine (TEA) and hexafluoro-2-propanol (HFIP). However, ion-suppressing effects of TEA hamper mass spectrometry (MS) instrumentation sensitivity and HFIP can affect the robustness of the mass spectrometer due to its corrosive nature. Anion exchange chromatography (AEX) is an orthogonal separation mode to IPRP but typically cannot be directly coupled to MS. In this work, we developed a multiple heart-cutting IPRP-, AEX-hydrophilic interaction liquid chromatography(HILIC)/MS method for quantification and high sensitivity identification of antisense oligonucleotide (ASO) impurities using a Q-Exactive mass spectrometer. Notably, both AEX-HILIC and IPRP-HILIC modes could be operated on a versatile two-dimensional liquid chromatography (2D-LC) setup including several column selectors. The HILIC mobile phase contained 25 mM ammonium acetate and allowed identifying impurities at levels down to 0.3%. Careful selection of the sample loop volume and the 2D HILIC column dimension allowed straightforward coupling of HILIC for both IPRP and AEX without the need to use any solvent modulation. Overall, the 2D HILIC allowed online desalting of AEX and IPRP modes and further separation of additional impurities.