Biophysical investigation for elucidating complex chromatographic behavior in downstream processing of biologics

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Ranjan, Swarnim
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Electronic thesis
Chemical engineering
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Monoclonal antibodies (mAbs) have emerged as highly important protein therapeutics in the recent years. In spite of targeting a wide array of targets, mAbs have a conserved amino acid sequence and structure and share a high degree of similarity in biophysical properties, which facilitates development of a platform process for antibody manufacturing. Chinese Hamster Ovary (CHO) cells are the most commonly used expression systems to produce mAbs due to their ability to express antibodies with human-compatible glycosylation patterns. One of the challenges with these expression systems is the over-expression of a large number of host cell proteins (HCPs) that are released into the cell culture fluid during processing along with the mAb product. Removal of these HCPs from the antibody feedstocks, particularly those that are associated with the mAb product, is a major impediment in establishing robust platforms for two-column downstream bioprocesses. A fundamental understanding of the impurities and their interactions with the product would help in designing efficient separation process. The aim of this study was to delve into the nature of mAb-HCP complex formation and obtain mechanistic insights using a combination of biophysical techniques and computational tools, and to understand the impact of various process variables on the strength of these interactions. The first part of the work involved identification of model proteins that interact with the mAb using cross interaction chromatography. The proteins that demonstrated highest strength of interaction were then utilized to explore the mAb-model protein interactions in more detail using fluorescence polarization. Binding studies under varying salt concentrations provided insights into the driving force of these interactions. The energetics of interactions as well as enthalpic and entropic contributions to the binding were obtained by employing fluorescence polarization at different temperatures. Computational tools like protein-protein docking and protein surface property maps further aided in identifying residues or clusters on the protein surface that are important for these interactions. The insights obtained from this study were then applied to an industrially relevant host cell protein that has been identified to be challenging to remove, Cathepsin D, to identify potential mechanism of interaction with different mAbs. Surface plasmon resonance was employed to obtain the kinetics of these interactions under a range of fluid phase conditions with varying salt concentrations and pH, and it was observed that higher pH had a significant effect on mitigating these interactions. The data obtained from SPR experiments was used to evaluate the effectiveness of the wash conditions in disrupting mAb-Cathepsin D interactions using protein A chromatography and Cathepsin D enzymatic activity assay. Protein surface property maps and protein-protein docking calculations were then used to elucidate the mechanism of interaction by identifying potential residues or clusters of residues involved in the binding. The sites of interaction on the mAb surface and Cathepsin D were also identified using covalent crosslinking coupled with mass spectrometry, and the importance of the CDR regions in mediating these interactions was established. Importantly, a strong agreement was shown between experimental and simulation data. This work establishes various experimental and computational approaches for studying mAb-host cell protein interactions that are encountered in downstream bioprocessing of monoclonal antibodies, which would be applied in the future to create predictive tools for mAb-host cell protein interactions to improve the bio-separation processes.
School of Engineering
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Rensselaer Polytechnic Institute, Troy, NY
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