Comprehensive 2D-LC-MS for oligonucleotide impurity profiling: real added value or unnecessary complexity?

Oligonucleotides are a promising class of therapeutics that selectively regulate gene expression. However, impurities can significantly affect their efficacy and safety, making their characterization and identification essential. Due to the high structural similarity between impurities and the full-length product, many impurities remain difficult to separate using conventional liquid chromatography methods, highlighting the need for more advanced analytical approaches. This study evaluates different combinations of chromatographic modes to identify the most effective 2D-LC configuration for oligonucleotide impurity profiling. A systematic screening of numerous chromatographic modes, including ion-pair reversed phase liquid chromatography (IP-RPLC), hydrophilic interaction chromatography (HILIC), anion exchange chromatography (AEX), size exclusion chromatography (SEC), and phenyl-based reversed phase chromatography (RPLC), was performed. The optimal combination was selected using a score based on a Derringer function that considered peak capacity, orthogonality, MS compatibility, and mobile phases compatibility between dimensions. The AEX combined with HILIC provided the best overall performance and was subsequently optimized in both the first and second dimensions to further improve peak capacity, compatibility and orthogonality when combined together. Fully comprehensive (LC×LC) and selective comprehensive (sLC×LC) 2D-LC strategies were then compared to identify the most suitable 2D-LC operation mode. The selected approaches were then compared with a conventional 1D-HILIC-MS method, to critically assess the practical benefits of multidimensional separations for oligonucleotide impurity profiling. This work provides valuable insights into the retention behavior of a fully phosphorothioated oligonucleotide and its impurities across various chromatographic modes. These findings improve our understanding of oligonucleotide retention mechanisms in both established and emerging chromatographic modes, for which retention behavior remains poorly understood. Furthermore, although LC×LC theoretically offers superior resolving power compared with one-dimensional LC or sLC×LC methods, our results demonstrate that the gain for oligonucleotide impurity profiling is less evident in practice. The numerous compromises required for LC×LC-MS, particularly regarding MS compatibility, appear to be not fully counterweighed by the gain in separation performance, because impurities extremely similar to the full-length product elute in a very narrow composition window. Alternative sLC×LC offers a more favorable balance between separation performance and method complexity.