Structural Elucidation of Fc- and Fab-Associated N-Glycans in Cetuximab Using Protein A-Assisted Domain-Resolved Glycan Profiling Using Mass Spectrometry.

Glycosylation of monoclonal antibodies is a key determinant of their efficacy, stability, and safety, requiring analytical methods capable of resolving site-specific heterogeneity with high sensitivity. In this work, we present a Protein A-assisted, domain-resolved glycan profiling approach for the mass spectrometry-based structural elucidation and quantification of N-glycans linked to Fc and Fab in cetuximab. Antibody domains were selectively extracted using Protein A affinity separation, allowing for independent enzymatic release of N-glycans from Fc and Fab sections, followed by high-resolution mass spectrometry for detailed glycan profiling and comparative characterization of Fc- and Fab-associated glycan populations. In comparison to conventional total glycan analysis, our technique decreases spectral complexity and allows for obvious distinction of domain-specific glycosylation patterns. Crucially, domain-specific released glycan profiling improves the detection and precise measurement of low-abundance glycoforms, such as sialylated, high-mannose, and afucosylated species that frequently remain hidden in conventional approaches. The proposed approach facilitates domain-specific characterization of glycan distributions across antibody regions by reducing analytical complexity and improving interpretation of Fc- and Fab-associated glycosylation patterns. Our results show that a reliable and repeatable platform for thorough, domain-level glycan characterization and quantification is available, supporting applications in quality control and biosimilar comparability studies, and in general during biopharmaceutical development.