Development of a two-dimensional liquid chromatography/mass spectrometry workflow for characterization of phosphorodiamidate morpholino oligomers

A sensitive two-dimensional liquid chromatography/mass spectrometry (2D-LC/MS) workflow was developed for characterization of phosphorodiamidate morpholino oligomers (PMOs) for the first time. This workflow employed strong cation exchange chromatography (SCX) as the first dimension (1D), and hydrophilic interaction chromatography (HILIC) as the second dimension (2D), with ammonium formate as MS-compatible salt in both dimensions. Peaks of interest were collected using multiple heart cutting (MHC) and transferred to 2D HILIC for online desalting and orthogonal separation prior to high resolution MS (HR-MS) analysis for impurity identification. Active solvent modulation (ASM) was utilized to address the elution strength incompatibility between 1D SCX and 2D HILIC, allowing for improved peak shape leading to enhanced resolution, and increased peak capacity by the 2D-LC approach. This workflow was initially demonstrated by analyzing a 30-mer PMO spiked with n-1 deletion impurity markers and was subsequently applied to effectively elucidate the impurity profiles of three PMO drug substances (ranging from 22-mer to 30-mer), affording identification of impurities at low levels. The impurity profiles suggested common impurity classes were formed in the PMOs, despite varying lengths or sequences, and were structurally related to their parent PMOs. This 2D-LC/MS workflow improves PMO impurity characterization by increasing peak capacity, providing high MS sensitivity, and enabling streamlined impurity analysis with short data turnaround time.