Coupling Mixed-Mode Size Exclusion Chromatography with Native Mass Spectrometry for Sensitive Detection and Quantitation of Homodimer Impurities in Bispecific IgG.

Detection and quantitation of homodimer impurities in therapeutic bispecific antibody (bsAb) drug products is essential to support development and quality control (QC) release. LC-MS-based techniques have been frequently applied for this analysis. However, sensitive detection of low-abundance homodimer impurities can still be challenging for regular workflows, which is largely due to the lack of chromatographic resolution between the impurities and the main bsAb species. Here, we report the development of a novel analytical method, which couples mixed-mode size exclusion chromatography (mmSEC) with online native MS detection (mmSEC-MS) for highly sensitive detection and quantitation of homodimer impurities in bsAb samples. Secondary interactions between the protein analytes and the column matrix, which are typically unwanted in SEC applications, are utilized to separate mAb species with similar hydrodynamic volume but different surface characteristics. Using four different bsAbs as testing standards, we demonstrated the versatility of this method in separating homodimer species from bsAb based on either electrostatic interaction or hydrophobic interaction, which was easily achieved by utilizing SEC columns with different properties as well as modulating the salt concentrations. The chromatographic separation between homodimer impurities and bsAb, as achieved by the mmSEC method, was demonstrated to be critical for the improved sensitivity in detecting low-abundance homodimer impurities (LOD from 0.01% to 0.1%). To the best of our knowledge, this newly developed mmSEC-MS method represents the most sensitive MS-based technique in both detection and quantitation of homodimer impurities in bsAb samples.