title: Characterizing monoclonal antibody structure by carbodiimide/GEE footprinting.
abstract: Amino acid-specific covalent labeling is well suited to probe protein structure and macromolecular interactions, especially for macromolecules and their complexes that are difficult to examine by alternative means, due to size, complexity, or instability. Here we present a detailed account of carbodiimide-based covalent labeling (with GEE tagging) applied to a glycosylated monoclonal antibody therapeutic, which represents an important class of biologic drugs. Characterization of such proteins and their antigen complexes is essential to development of new biologic-based medicines. In this study, the experiments were optimized to preserve the structural integrity of the protein, and experimental conditions were varied and replicated to establish the reproducibility and precision of the technique. Homology-based models were generated and used to compare the solvent accessibility of the labeled residues, which include D, E, and the C-terminus, against the experimental surface accessibility data in order to understand the accuracy of the approach in providing an unbiased assessment of structure. Data from the protein were also compared to reactivity measures of several model peptides to explain sequence or structure-based variations in reactivity. The results highlight several advantages of this approach. These include: the ease of use at the bench top, the linearity of the dose response plots at high levels of labeling (indicating that the label does not significantly perturb the structure of the protein), the high reproducibility of replicate experiments (<2 % variation in modification extent), the similar reactivity of the 3 target probe residues (as suggested by analysis of model peptides), and the overall positive and significant correlation of reactivity and solvent accessible surface area (the latter values predicted by the homology modeling). 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. The results are also compared with data from hydroxyl radical-mediated oxidative footprinting on the same protein, showing that complementary information is gained from the 2 approaches, although the number of target residues in carbodiimide/GEE labeling is fewer. Overall, this approach is an accurate and precise method for assessing protein structure of biologic drugs.
authors.affiliation: a Center for Proteomics and Bioinformatics ; Case Western Reserve University ; Cleveland , OH USA.
authors.forename: Parminder
authors.initials: P
authors.lastname: Kaur
authors.forename: Sara
authors.initials: S
authors.lastname: Tomechko
authors.forename: Janna
authors.initials: J
authors.lastname: Kiselar
authors.forename: Wuxian
authors.initials: W
authors.lastname: Shi
authors.forename: Galahad
authors.initials: G
authors.lastname: Deperalta
authors.forename: Aaron T
authors.initials: AT
authors.lastname: Wecksler
authors.forename: Giridharan
authors.initials: G
authors.lastname: Gokulrangan
authors.forename: Victor
authors.initials: V
authors.lastname: Ling
authors.forename: Mark R
authors.initials: MR
authors.lastname: Chance
keywords: ACN, acetonitrile; CD, circular dichroism; CL, covalent labeling; DR, dose response; EDC, 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide; EIC, extracts the ion chromatogram; FPOP, fast photochemical oxidation of proteins; GEE; GEE, glycine ethyl ester; HC, heavy chain; HDX, hydrogen-deuterium exchange; HRF, hydroxyl radical footprinting; IT, ion trap; IgG, immunoglobulin gamma; LC, light chain; LysC, Lysyl endopeptidase; MS, mass spectrometry; NMR, nuclear magnetic resonance; RC, rate constant; SASA, solvent accessible surface area; SEC, size-exclusion chromatography; VEGF, vascular endothelial growth factor; covalent labeling; footprinting; mAb, monoclonal antibody; protein structure; structural proteomics