A Middle-Up Approach with Online Capillary Isoelectric Focusing-Mass Spectrometry for In-depth Characterization of Cetuximab Charge Heterogeneity.

Previously, we reported a new online capillary isoelectric focusing/mass spectrometric (CIEF/MS) method for intact monoclonal antibody (mAb) charge variant analysis that uses an electrokinetically pumped sheath-flow nanospray ion source on a time-of-flight (TOF) MS with a pressure-assisted chemical mobilization. The direct online CIEF/MS method exhibited excellent resolution of charge variants conforming to those of imaged capillary isoelectric focusing with ultraviolet detection (iCIEF/UV). However, for complex mAbs, CIEF/MS spectra of the intact charge variant peaks may be too convoluted to be effectively interpreted. In the present study, we implemented a middle-up approach to enhance the capability of the CIEF/MS method for characterizing complex mAb charge variants by reducing sample complexity. To demonstrate such a strategy, we fragmented cetuximab through IdeS enzymatic cleavage and dithiothreitol (DTT) reduction. For the first time, online CIEF/MS resolved the complex charge variants of cetuximab at subunit level, corroborating the profiles obtained by iCIEF/UV. Furthermore, high-resolution TOF mass spectra with high mass accuracy were obtained for the eight charge variants separated by CIEF/MS after IdeS cleavage and for the 11 charge variants after IdeS digestion with subsequent DTT reduction. In-depth analyses revealed the identities of all charge variants and pinpointed the causes of charge heterogeneity, which are in accord with those reported in the literature. The main sources of charge heterogeneity of cetuximab were identified as terminal lysine on the Fc domain (up to one on each single-chain Fc), glycolylneuraminic acid residues on the Fd' domain (up to two on each Fd'), and likely several deamidation species on the Fd' domain. No charge heterogeneity contribution was found from light chain. The in-depth characterization of complex charge variants for cetuximab demonstrates the remarkable capability of this middle up CIEF/MS approach. This novel workflow holds great potential for detecting and elucidating charge variants to help understand proteins with complex charge heterogeneity.