Recent biophysical investigations into protein-ligand interactions carried out in our lab have helped us to develop a deeper molecular level understanding of the binding of biological products to Multimodal (MM) chromatographic systems. These biophysical investigations employed Nuclear Magnetic Resonance (NMR) spectroscopy to get the residue specific binding information for various proteins. However, NMR spectroscopy requires the use of <sup>15</sup>N-labeled and assigned proteins that can be challenging for large proteins like monoclonal antibodies (mAbs) or viral capsid proteins, which are generally greater than 50kDa. Hence, there is a need to employ a different technique that can be applied to a broader range of proteins as well as binding conditions. To address this, we used a combination of covalent labeling and Liquid Chromatography/Mass Spectrometry (LC/MS) to identify the regions on the protein surface that are involved in binding to the chromatographic resin. In this study, we evaluated the binding region on small model protein Ubiquitin (Ubq) using Diethyl pyrocarbonate (DEPC) as a covalent label for binding to various MM chromatographic resin systems. DEPC is a versatile covalent label that can readily react with several nucleophilic amino acid residues on the protein surface. First, a DOE study was designed to identify the optimum DEPC:Protein ratio as well as the reaction time for covalent labeling of Ubq in solution. These optimized conditions were then used for the on-resin labeling experiments of Ubq. By a comparative study of the in-solution and the on-resin labeling reaction, the residues involved in binding with the chromatographic resin were identified. The binding region identified using this technique was confirmed with the results obtained from NMR spectroscopy. This combined covalent labeling and LC/MS approach allowed us to develop a molecular level understanding of binding of Ubq to different MM chromatographic systems. This work, which requires very small amounts of samples, has potential applications for identifying the residues on surfaces of complex biological products like mAbs, bispecific antibodies, viral capsid proteins, etc. for binding to various chromatographic systems. This rich binding region information would guide us to design efficient separation processes <i>a priori</i> for new biological products that are associated with product-related variants.

Identifying preferred binding regions in multimodal chromatography using covalent labeling and liquid chromatography/mass spectrometry

Macromolecular Chemistry: The Second Century

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ACS Spring 2020 National Meeting & Expo

The official archive and repository for all ACS Meetings & Expositions abstracts and select posters or presentations.