Multi-Attribute mAb Variant Characterization with CE-MS and FabRICATOR® Digestion
Scientists at Aalen University and their collaborator present a detailed methodology for the rapid and thorough characterization of monoclonal antibody (mAb) variants using capillary electrophoresis-mass spectrometry (CE-MS) at the subunit level. mAbs are an important class of therapeutic molecules for treating various diseases, including cancer and autoimmune disorders. mAbs are large, complex molecules and can undergo a range of post-translational modifications (PTMs), which may affect their biological activity, stability, and therapeutic efficacy. Comprehensive characterization of these modifications is crucial to ensuring the safety and quality of therapeutic mAbs.
While numerous methods for analyzing mAb variants exists, many are time-consuming, labor-intensive, or limited to specific variants or antibodies. The challenge lies in creating a generic, efficient, and multi-attribute approach that can characterize a wide range of mAb variants with minimal preparation. This study addresses that challenge by introducing a CE-MS-based method for subunit-level analysis of mAbs that is faster, more versatile, and simpler than alternative techniques. The developed approach utilized the FabRICATOR (IdeS) protease to digest antibodies into subunits. FabRICATOR digests antibodies specifically below the hinge region, producing F(ab’)2 and Fc fragments, which are then reduced into Fd’, LC, and scFc subunits for more detailed analysis. Analysis at the subunit level reduces the overall complexity and time of analysis compared to more traditional methods like peptide mapping while providing more detailed and accurate detection of mAb variants compared to intact approaches.
Figure 1. FabRICATOR (IdeS) is an IgG-specific cysteine protease that digests antibodies at a single amino acid site below the hinge, generating homogenous F(ab’)2 and Fc fragments.
To demonstrate its broad applicability, the method was tested on nine different IgG1 antibodies and one stressed mAb. Following FabRICATOR digestion, the subunits were separated using a neutral-coated capillary optimized for the analysis of both size and charge variants, such as glycosylation, C-terminal lysine clipping, and oxidation. The separated subunits were then analyzed by mass spectrometry, revealing a range of mAb variants, which could be confirmed by MS/MS, including size variants (e.g., glycosylation profiles), charge variants (e.g., lysine clipping), modifications with small mass differences (e.g., deamidation) and other important proteoforms (e.g., glycation and oxidation). The technique proved capable of detecting low-abundance variants, identifying species present at less than 0.5% relative abundance.
The scientists here introduce an innovative, middle-level, multi-attribute method for mAb analysis, incorporating FabRICATOR digestion to improve overall speed and efficiency. The CE-MS/MS method offers a highly versatile and generic approach for characterizing mAb variants, with minimal sample preparation. Its broad applicability to different therapeutic antibodies, and ability to detect a wide range of modifications, makes it a powerful tool for biotherapeutic mAb characterization!
Reference
Schairer et al., 2025. CE-MS and CE-MS/MS for the multiattribute analysis of monoclonal antibody variants at the subunit level. Journal of Pharmaceutical and Biomedical Analysis.
FabRICATOR® – Below hinge digestion of IgG