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Anion solvation enhanced by positive supercharging mutations preserves thermal stability of an antibody in a wide pH range

Keisuke Kasahara, , Aki Tanabe, Raiji Kawade, ,

Abstract: Proteins function through interactions with other molecules. In protein engineering, scientists often engineer proteins by mutating their amino acid sequences on the protein surface to improve various physicochemical properties. “Supercharging” is a method to design proteins by mutating surface residues with charged amino acids. Previous studies demonstrated that supercharging mutations conferred better thermal resistance, solubility, and cell penetration to proteins. Likewise, antibodies recognize antigens through the antigen-binding site on the surface. The genetic and structural diversity of antibodies leads to high specificity and affinity toward antigens, enabling antibodies to be versatile tools in various applications. When assessing therapeutic antibodies, surface charge is an important factor to consider because the isoelectric point plays a role in protein clearance inside the body. In this study, we explored how supercharging mutations affect physicochemical properties of antibodies. Starting from a crystal structure of an antibody with the net charge of −4, we computationally designed a supercharged variant possessing the net charge of +10. The positive-supercharged antibody exhibited marginal improvement in thermal stability, but the secondary structure and the binding affinity to the antigen (net charge of +8) were preserved. We also used physicochemical measurements and molecular dynamics simulations to analyze the effects of supercharging mutations in sodium phosphate buffer with different pH and ion concentrations, which revealed preferential solvation of phosphate ions to the supercharged surface relative to the wild-type surface. These results suggest that supercharging would be a useful approach to preserving thermal stability of antibodies in a wide range of pH, which may enable further diversification of antibody repertoires beyond natural evolution.
Keywords: Antibody / Fab / Computational design / Thermal stability / Supercharging / Preferential solvation

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