Evaluation of preferred binding regions on ubiquitin and IgG1 F_C for interacting with multimodal cation exchange resins using DEPC labeling/mass spectrometry

Abstract

There is significant interest in identifying the preferred binding domains of biological products to various chromatographic materials. In this work, we develop a biophysical technique that uses diethyl pyrocarbonate (DEPC) based covalent labeling in concert with enzymatic digestion and mass spectrometry to identify the binding patches for proteins bound to commercially available multimodal (MM) cation exchange chromatography resins. The technique compares the changes in covalent labeling of the protein in solution and in the bound state and uses the differences in this labeling to identify residues that are sterically shielded upon resin binding and, therefore, potentially involved in the resin binding process. Importantly, this approach enables the labeling of many amino acids and can be carried out over a pH range of 5.5-7.5, thus enabling the protein surface mapping at conditions of interest in MM cation exchange systems. The protocol is first developed using the model protein ubiquitin and the results indicate that lysine residues located on the front face of the protein show dramatic changes in DEPC labeling while residues present on other regions have minimal or no reductions. This indicates that the front face of ubiquitin is likely involved in resin binding. In addition, surface property maps indicate that the hypothesized front face binding region consists of overlapping positively charged and hydrophobic patches. The technique is then employed with | © 2023 Wiley Periodicals LLC.

Comparative Analysis of Protein Surface Hydrophobicity Maps Determined by Sparse Sampling INDUS and Spatial Aggregation Propensity

“…Protein surface hydrophobicity plays an important role in a range of biological processes that involve protein–protein interactions and molecular recognition. Protein hydrophobicity is crucial in therapeutic developability where pharmaceutical product quality is strongly impacted by protein solubility, viscosity, and aggregation propensity. − The presence of protein aggregates in therapeutics is highly undesirable, leading to loss in product quality as well as increasing the chances of induced immunogenicity and allergic responses. − The hydrophobic surfaces of proteins also play an important role in determining their preferred binding domains to multimodal chromatographic surfaces and can impact their retention in a range of chromatographic separation systems. − Clearly, it is important to properly characterize and quantify the hydrophobicity of protein surfaces for a number of applications.…”

Exploring preferred binding domains of IgG1 mAbs to multimodal adsorbents using a combined biophysics and simulation approach

“…[43][44][45][46][47][48][49] While DEPC covalent labeling in concert with enzymatic digestion and mass spectrometry has been employed to study protein-protein and protein-ligand interactions, 39,[50][51][52][53][54][55][56] our group has recently expanded the use of this technique to study binding regions of the proteins ubiquitin and the IgG1 F C domain for interacting with MM cation exchange resins. 57 In the present work, we have expanded the DEPC approach to study the binding of more complex biomolecules with multiple binding domains (e.g., full mAbs) to multimodal surfaces. This study builds upon previous chromatographic work from our group that employed the retention behavior of mAb fragments to elucidate selectivity trends observed with these mAbs in MMCEX chromatography.…”

“…In contrast, DEPC covalent labeling of proteins can be carried out at pH 5.5 to pH 7.5 and can modify up to six amino acids, providing increased protein surface coverage 43–49 . While DEPC covalent labeling in concert with enzymatic digestion and mass spectrometry has been employed to study protein–protein and protein–ligand interactions, 39,50–56 our group has recently expanded the use of this technique to study binding regions of the proteins ubiquitin and the IgG1 F C domain for interacting with MM cation exchange resins 57 …”