Investigation into the thermodynamic and molecular basis of protein-ligand interactions in multimodal chromatography
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Authors
Gudhka, Ronak Bharat
Issue Date
2020-08
Type
Electronic thesis
Thesis
Thesis
Language
ENG
Keywords
Chemical engineering
Alternative Title
Abstract
The drive towards process intensification and the increased complexity of new classes of biological products has catalyzed significant interest in new bioseparation modalities. Multimodal chromatography has been shown to exhibit enhanced selectivities as compared to the traditional single mode systems resulting from the combination of electrostatic, hydrogen bonding, hydrophobic and/or aromatic interactions within a single ligand. Even though there are several studies demonstrating the successful application of multimodal chromatography for challenging separations, there is still a lack of fundamental understanding of how proteins interact with these surfaces. To this end, the work presented in this thesis has focused on investigating the thermodynamic and molecular basis of protein binding in these multimodal chromatographic systems using a combination of chromatographic, biophysical and computational tools.
Since these NMR studies were limited by the size of the protein as well as the need to use assigned isotopically labeled protein, we developed an alternative platformable approach to identify the preferred binding region on proteins. Specifically, we successfully demonstrated a proof of concept study employing covalent labeling and liquid chromatography/mass spectrometry to identify the preferred binding regions on ubiquitin in multimodal systems.
The combined chromatographic-biophysical tool set and workflow developed in this thesis has improved our understanding of protein binding in multimodal systems and has significant potential for elucidating the nature of selectivity and for the development of more efficient downstream bioprocessing for next generation biotherapeutics.
In order to obtain molecular level insights into the interacting residues on the FC domain, we performed NMR experiments with 15N-labeled FC and multimodal ligands in solution. The results from NMR experiments indicated that the hinge region and the interface of the CH2 and CH3 domains were the preferred multimodal binding sites on the FC and interacted with weak millimolar binding affinities. In order to incorporate the co-operativity and avidity effects during binding, we developed a gold nanoparticles based pseudo solid-state resin system that mimicked the chromatographic resin while also being amenable to NMR experiments with large biomolecules. The results from NMR experiments suggested that the FC residues exhibited “high” micromolar binding affinities for interactions with multimodal functionalized surfaces. Further, the results from NMR experiments, performed in both the presence and absence of salt, provided some insights into the binding mechanisms of FC with the two multimodal systems that differed only in the surface presentation of the functional groups. Although interactions of the FC with the two multimodal surfaces were localized in regions of overlapping charge and hydrophobicity, Capto MMC and Nuvia cPrime surfaces appeared to interact primarily via hydrophobic and electrostatic interactions, respectively. Importantly, NMR experiments were extended to various ligand density surfaces where we experimentally demonstrated the effect of ligand density on interacting residues, with important implications for recently hypothesized ligand clustering effects on chromatographic surfaces.
We then examined the chromatographic behavior and domain contributions in multimodal chromatography of an important NIST reference mAb. The chromatographic evaluation of the NIST mAb revealed interesting selectivity patterns for retention in multimodal systems as a function of pH. These results also provided insights into how ligand chemistry and surface presentation of functional groups on resin surface affects protein retention in multimodal systems. Further, the chromatographic retention of domains of the NIST mAb in concert with protein surface property maps suggested that, while the FC domain appeared to contain important interaction sites on mAb at pH 5, the Fab domain contained dominant binding sites at pH 7.
First, we employed a van’t Hoff analysis of isocratic retention data at a range of salt and temperature conditions to determine the governing thermodynamic driving forces towards mAb binding in multimodal cation exchange systems. While the mAb binding to various multimodal resins showed favorable enthalpic contributions at low salts, the entropic contributions appeared to be important for binding at higher salts. Further, the heat capacity data provided some insights into the nature of interactions in these systems.
Since these NMR studies were limited by the size of the protein as well as the need to use assigned isotopically labeled protein, we developed an alternative platformable approach to identify the preferred binding region on proteins. Specifically, we successfully demonstrated a proof of concept study employing covalent labeling and liquid chromatography/mass spectrometry to identify the preferred binding regions on ubiquitin in multimodal systems.
The combined chromatographic-biophysical tool set and workflow developed in this thesis has improved our understanding of protein binding in multimodal systems and has significant potential for elucidating the nature of selectivity and for the development of more efficient downstream bioprocessing for next generation biotherapeutics.
In order to obtain molecular level insights into the interacting residues on the FC domain, we performed NMR experiments with 15N-labeled FC and multimodal ligands in solution. The results from NMR experiments indicated that the hinge region and the interface of the CH2 and CH3 domains were the preferred multimodal binding sites on the FC and interacted with weak millimolar binding affinities. In order to incorporate the co-operativity and avidity effects during binding, we developed a gold nanoparticles based pseudo solid-state resin system that mimicked the chromatographic resin while also being amenable to NMR experiments with large biomolecules. The results from NMR experiments suggested that the FC residues exhibited “high” micromolar binding affinities for interactions with multimodal functionalized surfaces. Further, the results from NMR experiments, performed in both the presence and absence of salt, provided some insights into the binding mechanisms of FC with the two multimodal systems that differed only in the surface presentation of the functional groups. Although interactions of the FC with the two multimodal surfaces were localized in regions of overlapping charge and hydrophobicity, Capto MMC and Nuvia cPrime surfaces appeared to interact primarily via hydrophobic and electrostatic interactions, respectively. Importantly, NMR experiments were extended to various ligand density surfaces where we experimentally demonstrated the effect of ligand density on interacting residues, with important implications for recently hypothesized ligand clustering effects on chromatographic surfaces.
We then examined the chromatographic behavior and domain contributions in multimodal chromatography of an important NIST reference mAb. The chromatographic evaluation of the NIST mAb revealed interesting selectivity patterns for retention in multimodal systems as a function of pH. These results also provided insights into how ligand chemistry and surface presentation of functional groups on resin surface affects protein retention in multimodal systems. Further, the chromatographic retention of domains of the NIST mAb in concert with protein surface property maps suggested that, while the FC domain appeared to contain important interaction sites on mAb at pH 5, the Fab domain contained dominant binding sites at pH 7.
First, we employed a van’t Hoff analysis of isocratic retention data at a range of salt and temperature conditions to determine the governing thermodynamic driving forces towards mAb binding in multimodal cation exchange systems. While the mAb binding to various multimodal resins showed favorable enthalpic contributions at low salts, the entropic contributions appeared to be important for binding at higher salts. Further, the heat capacity data provided some insights into the nature of interactions in these systems.
Description
August 2020
School of Engineering
School of Engineering
Full Citation
Publisher
Rensselaer Polytechnic Institute, Troy, NY