Characterizing monoclonal antibody structure by carboxyl group footprinting |
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Authors: | Parminder Kaur Sara E Tomechko Janna Kiselar Wuxian Shi Galahad Deperalta Aaron T Wecksler Giridharan Gokulrangan Victor Ling Mark R Chance |
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Institution: | 1.Center for Proteomics and Bioinformatics; School of Medicine; Case Western Reserve University; Cleveland, OH, USA;2.NeoProteomics, Inc.; Cleveland, OH, USA;3.Pulmonary, Critical Care, and Sleep Medicine; School of Medicine; Case Western Reserve University; Cleveland, OH, USA;4.Center for Synchrotron Biosciences; School of Medicine; Case Western Reserve University; Cleveland, OH, USA;5.Protein Analytical Chemistry; Genentech; South San Francisco, CA, USA |
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Abstract: | Structural characterization of proteins and their antigen complexes is essential to the development of new biologic-based medicines. Amino acid-specific covalent labeling (CL) is well suited to probe such structures, especially for cases that are difficult to examine by alternative means due to size, complexity, or instability. We present here a detailed account of carboxyl group labeling (with glycine ethyl ester (GEE) tagging) applied to a glycosylated monoclonal antibody therapeutic (mAb). The experiments were optimized to preserve the structural integrity of the mAb, and experimental conditions were varied and replicated to establish the reproducibility of the technique. Homology-based models were generated and used to compare the solvent accessibility of the labeled residues, which include aspartic acid (D), glutamic acid (E), and the C-terminus (i.e., the target probes), with the experimental data in order to understand the accuracy of the approach. Data from the mAb were compared to reactivity measures of several model peptides to explain observed variations in reactivity. Attenuation of reactivity in otherwise solvent accessible probes is documented as arising from the effects of positive charge or bond formation between adjacent amine and carboxyl groups, the latter accompanied by observed water loss. A comparison of results with previously published data by Deperalta et al using hydroxyl radical footprinting showed that 55% (32/58) of target residues were GEE labeled in this study whereas the previous study reported 21% of the targets were labeled. Although the number of target residues in GEE labeling is fewer, the two approaches provide complementary information. The results highlight advantages of this approach, such as the ease of use at the bench top, the linearity of the dose response plots at high levels of labeling, reproducibility of replicate experiments (<2% variation in modification extent), the similar reactivity of the three target probes, and significant correlation of reactivity and solvent accessible surface area. |
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Keywords: | acetonitrile circular dichroism covalent labeling dose response 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide extracted ion chromatogram glycine ethyl ester heavy chain hydrogen-deuterium exchange hydroxyl radical footprinting immunoglobulin gamma ion trap light chain lysyl endopeptidase monoclonal antibody mass spectrometry rate constant solvent accessible surface area size-exclusion chromatography |
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