Biophysical tools to study preferred binding domains for protein interactions with chromatographic surfaces

Authors
Dhingra, Kabir
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Other Contributors
Royer, Catherine
Przybycien, Todd
Zha, Helen
Cramer, Steven
Issue Date
2023-08
Keywords
Chemical engineering
Degree
PhD
Terms of Use
This electronic version is a licensed copy owned by Rensselaer Polytechnic Institute (RPI), Troy, NY. Copyright of original work retained by author.
Full Citation
Abstract
Multimodal resins have proven to be quite successful in removing various process and product related impurities from protein therapeutics due to their ability to bind through multiple modes of interactions. Despite providing several advantages, the molecular interactions that take place at the protein-ligand interface in these resin systems and the preferred binding domains is still not thoroughly understood. The current work aims to address these challenges by employing a combination of chromatography and biophysical techniques. The initial part of this research focuses on investigating the chromatographic retention of a reference antibody (NIST mAb) on various multimodal cation exchanging resins. In addition, surface property maps were employed to elucidate the importance of charge and hydrophobic properties in these retention patterns. While useful, this initial work with the NIST mAb made it clear that other factors such as the protein structure and the surface topology could also significantly impact their binding to the resin surface. Therefore, we developed a novel technique based on diethylpyrocarbonate (DEPC) covalent labeling in conjunction with enzymatic digestion and mass spectrometry to investigate the protein retention on chromatographic surfaces. These experiments were carried out by performing the covalent labeling on the unbound and resin-bound proteins. The extent of modification of the amino acids present in these two states were then compared to identify the residues sterically hindered by the resin in the adsorbed protein and thus, likely in or near a region of the protein involved in the interaction to the resin. We employed this approach first on a set of model proteins to validate its applicability in determining the preferred binding patches on protein surfaces. Importantly, the results with the small model protein and the FC domain of an IgG1 corroborated the results previously obtained with the more complex NMR approach. We then extended this work to examine the retention behavior of two therapeutic antibodies on multimodal cation exchanging resins. We investigated the distinct domain contribution behavior of these two antibodies by highlighting their binding regions while interacting to the resin. Further, DEPC labeling was also employed to examine the impact of pH on the binding regions in monoclonal and bispecific antibodies in multimodal systems. Importantly, the results corroborated important chromatographic trends identified with this antibody set. While DEPC labeling is useful in identifying the binding regions on a wide range of proteins, it is still a time-consuming approach requiring several steps to obtain the modification percentages at the residue level. To address this, we developed a high-throughput approach based on DEPC labeling to examine the key interaction sites for several proteins using a 96-well plate format. We tested this technique on many model proteins with unique charge and hydrophobic characteristics on their surfaces. A separate chapter focuses on employing pH gradient experiments to investigate the selectivity of a library of Fab variants with single- or double-point mutations in multimodal anion exchanging resins. These studies in concert with protein surface analyses, shed light on the contributions of different binding regions on the Fabs to the selectivities achieved in these MMA systems. Future studies will utilize all the techniques developed and employed in this research in combination with several other biophysical and computational approaches to more deeply understand key phenomenon in multimodal separations such as selectivity, peak shape and binding capacity. The insights gained from this research will help in the development of next generation manufacturing processes for a range of biotherapeutic products.
Description
August2023
School of Engineering
Department
Dept. of Chemical and Biological Engineering
Publisher
Rensselaer Polytechnic Institute, Troy, NY
Relationships
Rensselaer Theses and Dissertations Online Collection
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