FabRICATOR in Efficient Structural Characterization of mAbs

In contrast to small generic molecules, therapuetic monoclonal antibodies (mAbs) exhibit inherent heterogeneity that may arise during production and formulation or due to the storage conditions. Therefore, it is essential to characterize the structural heterogeneity of mAbs with respect to properties including conformational changes, aggregation and post-translational modifications. In this work, Zhu et al. at the Chinese Academy of Medical Sciences & Peking Union Medical College present an integration strategy for structural characterization of mAbs by combining intact mass and middle-down analysis using only a high-resolution Q-TOF mass spectrometer.


The mAbs trastuzumab and adalimumab were analyzed at intact level using native SEC-MS and denatured RPLC-MS to measure the molecular mass, detect heterogenous modified protein species and to obtain a relative quantification of all the major Fc glycoproteoforms. In order to obtain a more detailed structural confirmation of the protein sequence and glycosylation profile, antibodies were digested using the FabRICATOR® (IdeS) enzyme and reduced to generate homogenous LC, Fd’ and Fc/2 fragments for middle-down analysis.


The optimized native and denatured methods were suitable for rapidly assessing the structural heterogeneity while the combined CID and ETD middle-down analysis enhanced the sequence coverage of the fragments from both mAbs. The integrated workflow resulted in quantitative and qualitative glycosylation profiling while better resolving the overall heterogeneity caused both N-glycosylation and other modifications such as C-terminal processing. This integrated strategy can easily be implemented for in-depth structural characterization of mAbs during pharmaceutical development and quality control.


Zhu et al., 2020. Integrating Intact Mass Analysis and Middle-Down Mass Spectrometry Approaches to Effectively Characterize Trastuzumab and Adalimumab Structural Heterogeneity. J. Proteome Res. doi: 10.1021/acs.jproteome.0c00373




FabRICATOR (Ides) 
Cysteine protease for below hinge digestion of IgG