Intact mass analysis of monoclonal antibodies by capillary electrophoresis—Mass spectrometry

• Intact mass analysis of monoclonal antibody provides important information on sequence integrity and post-translational modifications.
• Although liquid chromatography–mass spectrometry (MS) is widely used in antibody characterization, capillary electrophoresis (CE) combines with MS provides high separation efficiency and accurate mass characterization in protein analysis and has been a long sought-after approach.
• However, the intrinsic technical challenge in CE-MS has hindered its broad application.
• Recently, a new CE-MS interface has been developed. Herein, we report implementation of this technology for coupling CE to time-of-flight mass spectrometer for intact mass analysis of antibodies.

Characterization of monoclonal antibody (mAb) therapeutics by intact mass analysis provides important information on sequence integrity and post-translational modifications. In order to obtain domain specific information, monoclonal antibodies are reduced to heavy and light chain components or enzymatically digested into smaller portions or peptides. Liquid chromatography (LC) is widely used for separation of the antibody fragments in line with mass spectrometry (MS) for characterization. Capillary electrophoresis (CE) is an analytical technique with high separation efficiency, high sensitivity, and minimal inter-run sample carryover. Combining the resolving power of CE with electrospray ionization (ESI) MS has great potential in regards to accurate mass characterization of protein therapeutics and has been a long sought-after approach. However, the intrinsic technical difficulty in coupling CE to MS has hindered the broad application of CE-MS across the biopharmaceutical industry. Recently, a CE-MS interface has been developed [1] and commercialized. Herein, we report implementation of this technology for coupling CE to an Agilent time-of-flight (TOF) mass spectrometer. CE-MS provides an attractive complement to LC–MS for separation and intact mass determination of mAbs and antibody-based therapeutics.