Development of a HILIC-WAX two-dimensional liquid chromatography method for UV-based quantitation of antisense
Xiao Zhou1, Tai Nguyen1, Xiaoqing Kong1
1Pharmaceutical Operations & Technology, Biogen, 225 Binney Street, Cambridge, MA 02142, USA.
Abstract:
Chromatographic separation of product-related impurities in antisense oligonucleotides (ASOs) is inherently challenging due to the structure similarity among closely related species. Coupling mass spectrometry (MS) to chromatography and UV detection has been a successful strategy to overcome some of the challenges; however, it does not address the need to implement orthogonal chromatographic methods to ensure comprehensive characterization of ASO impurities through distinct separation techniques. In this study, we developed a two-dimensional liquid chromatography (2D-LC) method for the analysis and quantitation of major product-related impurities in ASOs. The method integrates a hydrophilic interaction chromatography (HILIC) in the first dimension with a weak anion exchange (WAX) separation in the second, thus providing high orthogonality based on differences in polarity and charge. We employed Design of Experiments (DoE) to guide method development and systematically optimized key chromatographic parameters in both dimensions. The final optimized method was configured in selective comprehensive mode, and the main peak region from the first dimension was fully sampled in multiple cuts and analyzed by fast WAX separation. Solvent compatibility between dimensions was confirmed, and no additional solvent modulation was necessary. The method demonstrated excellent linearity (R² > 0.999), low limits of quantitation (0.3-0.6 µg/mL, or 0.3 %-0.6 %), and detection limits down to 0.1 % for all impurities tested, well within regulatory requirements for impurity profiling. This fully UV-based 2D-LC platform offers a practical solution for ASO impurity analysis without the need for MS detection and analysis, suitable for both development and routine quality control. The ability to switch between HILIC-WAX and HILIC-MS modes enhances the method's versatility in oligonucleotide characterization and complex impurity profiling.
