Covalent Labeling/Mass Spectrometry of Monoclonal Antibodies with Diethylpyrocarbonate: Reaction Kinetics for Ensuring Protein Structural Integrity

Abstract

Diethylpyrocarbonate (DEPC)-based covalent labeling together with mass spectrometry is a promising tool for the higher-order structural analysis of antibody therapeutics. Reliable information about antibody higher-order structure can be obtained, though, only when the protein's structural integrity is preserved during labeling. In this work, we have evaluated the applicability of DEPC reaction kinetics for ensuring the structural integrity of monoclonal antibodies (mAbs) during labeling. By monitoring the modification extent of selected proteolytic fragments as a function of DEPC concentration, we find that a common DEPC concentration can be used for different monoclonal antibodies in formulated samples without perturbing their higher-order structure. Under these labeling conditions, we find that the antibodies can accommodate up to four DEPC modifications without being structurally perturbed, indicating that multidomain proteins can withstand more than one label, which contrasts to previously studied single-domain proteins. This more extensive labeling provides a more sensitive measure of structure, making DEPC-based covalent labeling-mass spectrometry suitable for the higher-order structural analyses of mAbs.

Complementary Structural Information for Stressed Antibodies from Hydrogen–Deuterium Exchange and Covalent Labeling Mass Spectrometry

“…Labeling was conducted at a DEPC:protein molar ratio of 4:1 at 37 °C for 5 min. We have found that a 5 min reaction a 4:1 molar ratio leads to sufficient labeling of antibodies without perturbing the protein’s structure . Imidazole was then added at a 1:50 DEPC:imidazole molar ratio to quench the reaction.…”

“…We have found that a 5 min reaction a 4:1 molar ratio leads to sufficient labeling of antibodies without perturbing the protein's structure. 30 Imidazole was then added at a 1:50 DEPC:imidazole molar ratio to quench the reaction. For each stress conditions, at least three replicates were performed on the rituximab samples.…”

Mass Spectrometry Methods for Measuring Protein Stability

“…Footprinting techniques such as covalent labeling, HDX, and FPOP provide a direct and granular assessment of antibody stabilities . Covalent labeling measurements can reveal subtle changes to HOS, structural integrity, and antigen interactions . HDX exchange methodologies are well established for the assessment of mAb stability and are increasingly used to determine elements of biotherapeutic HOS. , FPOP methods have also demonstrated their general utility in the characterization of mAb HOS , and have specifically excelled in the area of epitope mapping. , …”

“…Footprinting MS. Footprinting techniques such as covalent labeling, HDX, and FPOP provide a direct and granular assessment of antibody stabilities. 331 Covalent labeling measurements can reveal subtle changes to HOS, 332 structural integrity, 333 and antigen interactions. 162 HDX exchange methodologies are well established for the assessment of mAb stability 334 and are increasingly used to determine elements of biotherapeutic HOS.…”

Complementary Structural Information for Antibody–Antigen Complexes from Hydrogen–Deuterium Exchange and Covalent Labeling Mass Spectrometry

“…Another CL reagent that has been used extensively to study protein interactions is diethylpyrocarbonate (DEPC). − Unlike FPOP, which requires a special experiment setup, DEPC can simply be added to solution whereupon it labels nucleophilic residues such as Cys, Lys, His, Ser, Thr, and Tyr. DEPC reacts with protein side chains on the seconds to minutes time scale and is therefore affected by protein dynamics in a different manner than FPOP.…”