Comprehensive characterization of monoclonal antibody by Fourier transform ion cyclotron resonance mass spectrometry |
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Authors: | Yutong Jin Ziqing Lin Qingge Xu Cexiong Fu Zhaorui Zhang Qunying Zhang |
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Institution: | 1. Department of Chemistry, University of Wisconsin-Madison, Madison, Wisconsin, USA;2. Human Proteomics Program, School of Medicine and Public Health, University of Wisconsin-Madison, Madison, Wisconsin, USA;3. Department of Cell and Regenerative Biology, University of Wisconsin-Madison, Madison, Wisconsin, USA;4. Process Analytical, AbbVie Inc, North Chicago, Illinois, USA |
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Abstract: | The pharmaceutical industry’s interest in monoclonal antibodies (mAbs) and their derivatives has spurred rapid growth in the commercial and clinical pipeline of these effective therapeutics. The complex micro-heterogeneity of mAbs requires in-depth structural characterization for critical quality attribute assessment and quality assurance. Currently, mass spectrometry (MS)-based methods are the gold standard in mAb analysis, primarily with a bottom-up approach in which immunoglobulins G (IgGs) and their variants are digested into peptides to facilitate the analysis. Comprehensive characterization of IgGs and the micro-variants remains challenging at the proteoform level. Here, we used both top-down and middle-down MS for in-depth characterization of a human IgG1 using ultra-high resolution Fourier transform MS. Our top-down MS analysis provided characteristic fingerprinting of the IgG1 proteoforms at unit mass resolution. Subsequently, the tandem MS analysis of intact IgG1 enabled the detailed sequence characterization of a representative IgG1 proteoform at the intact protein level. Moreover, we used the middle-down MS analysis to characterize the primary glycoforms and micro-variants. Micro-variants such as low-abundance glycoforms, C-terminal glycine clipping, and C-terminal proline amidation were characterized with bond cleavages higher than 44% at the subunit level. By combining top-down and middle-down analysis, 76% of bond cleavage (509/666 amino acid bond cleaved) of IgG1 was achieved. Taken together, we demonstrated the combination of top-down and middle-down MS as powerful tools in the comprehensive characterization of mAbs. |
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Keywords: | monoclonal antibodies top-down mass spectrometry middle-down mass spectrometry proteoforms post-translational modifications |
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