Characterizing monoclonal antibody structure by carboxyl group footprinting

Abstract

Structural characterization of proteins and their antigen complexes is essential to the development of new biologicbased 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.

Localized conformational interrogation of antibody and antibody-drug conjugates by site-specific carboxyl group footprinting

“…It was generally established that the EDC-mediated carboxyl group labeling in a protein solution is a pseudo first-order reaction. 32,35,36,40 To ensure the structural integrity of proteins in the labeling experiments, the extent of modification has been well controlled and optimized to ensure that deviation from first-order reaction kinetics, which would indicate chemical modification-induced protein unfolding, 18,32 does not occur. The RCs of some D/E residues vary from 0.01 hr ¡1 to as high as 1.1 hr ¡1 (shown in Fig.…”

“…These data confirm that structural perturbation induced by the chemical labeling in our experiments is negligible. 18,32 To facilitate the comparison of RCs between samples, a RC difference (DRC %) of individual carboxyl residues between mAb-A and deglycosylated mAb-A was plotted (Fig. 5B).…”

“…The specificity of labeling on Asp and Glu residues makes data analysis and interpretation relatively simple and straightforward, assuming that there are sufficient D/E residues distributed throughout the primary sequence. In the case of mAbs, approximately 10% of the sequence is composed of D/E residues, so the CGF approach can provide reasonable coverage, 32 and the subsequent trypsin peptide mapping used for detecting and quantitating the extent of labeling does not have to vary significantly from standard methodologies used for in-depth PTM characterization within the industry.…”

“…The approach makes use of simple bench-top chemistry that introduces GEE tags on solvent-accessible side chains of carboxyl residues, 32,35,36,40 and requires no special equipment and can be done in any analytical lab. The specificity of labeling on Asp and Glu residues makes data analysis and interpretation relatively simple and straightforward, assuming that there are sufficient D/E residues distributed throughout the primary sequence.…”

“…In principle, covalent labeling coupled with MS (termed "footprinting" technology) [15][16][17][18][31][32][33][34][35][36][37][38] is orthogonal method to HDX MS because covalent labeling utilizes reagents that target and react with amino acid side chains instead of protein backbone. Solvent-exposed side chains are labeled readily in a protein solution and those buried in the protein higher-order structure or interacted with other partners/molecules are more protected and less-labeled.…”