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

In this work, we employ a recently developed biophysical technique that uses diethylpyrocarbonate (DEPC) covalent labeling and mass spectrometry for the identification of mAb binding patches to two multimodal cation exchange resins at different pH. This approach compares the labeling results obtained in the bound and unbound states to identify residues that are sterically shielded and thus located in the mAb binding domains. The results at pH 6 for one mAb (mAb B) indicated that while the complementarity determining region (CDR) had minimal interactions with both resins, the FC domain was actively involved in binding. In contrast, DEPC/MS data with another mAb (mAb C) indicated that both the CDR and FC domains were actively involved in binding. These results corroborated chromatographic retention data with these two mAbs and their fragments and helped to explain the significantly stronger retention of both the intact mAb C and its Fab fragment. In contrast, labeling results with mAb C at pH 7, indicated that only the CDR played a significant role in resin binding, again corroborating chromatographic data. The binding domains identified from the DEPC/MS experiments were also examined using protein surface hydrophobicity maps obtained using a recently developed sparse sampling molecular dynamics (MD) approach in concert with electrostatic potential maps. These results demonstrate that the DEPC covalent labeling/mass spectrometry technique can provide important information about the domain contributions of multidomain proteins such as monoclonal antibodies when interacting with multimodal resins over a range of pH conditions.

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

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 an IgG1 FC and the results indicate that residues on the CH 2-CH 3 interface and the hinge are significantly sterically shielded upon binding to the resin. Further, these regions are again associated with significant overlap of positively charged and hydrophobic patches. On the other hand, while, residues on the CH 2 and the front face of the IgG1 FC also exhibited some changes in DEPC labeling upon binding, these regions have less distinct charged and hydrophobic patches. Importantly, the hypothesized binding patches identified for both ubiquitin and FC using this approach are shown to be consistent with previously reported NMR studies. In contrast to NMR, this new approach enables the identification of preferred binding regions without the need for isotopically labeled proteins or chemical shift assignments. The technique developed in this work sets the stage for the evaluation of the binding domains of a wide range of biological products to chromatographic surfaces, with important implications for designing biomolecules with improved biomanufacturability properties.