Supercharging design of an anti-lysozyme Fab antibody to regulate ligand-dependent reversible aggregation

Protein aggregation and liquid‒liquid phase separation (LLPS), as key physicochemical processes, orchestrate protein behavior and function, and engineering a protein surface charge offers a robust approach to modulate protein‒protein interactions and, consequently, aggregation and phase separation. Among protein surface engineering methods, supercharging leads to a drastic increase in the protein net charge by replacing surface residues with charged amino acid residues. Previous studies have reported that some physicochemical properties of proteins are improved by supercharging, and changing the surface charge is considered to affect intermolecular interactions. In this study, we designed a new supercharged antigen-binding fragment (Fab) antibody mutant and investigated its aggregation behavior. Upon examination of the physicochemical properties of the designed supercharged antibody, the thermal stability, structure, and ligand binding affinity of the antibody were retained despite having the same charge pairing of both the antibody and the antigen. Furthermore, we revealed that the antibody exhibited reversible ligand- and salt concentration-dependent aggregation. Our study demonstrated how supercharging can potentially modulate protein aggregation and LLPS. It is expected that this approach can be extended to other proteins, through which its applicability in various biological and biotechnological fields can be explored. Protein aggregation and liquid‒liquid phase separation (LLPS) orchestrate protein behavior and function. Engineering protein surface charge offers a robust approach to modulating these phenomena, and supercharging, which replaces surface residues with charged ones, leads to a drastic change in the protein net charge. In this study, we designed a new supercharged antigen-binding fragment antibody mutant and investigated its aggregation behavior. We revealed that the antibody exhibited reversible ligand- and salt concentration-dependent aggregation while retaining the physicochemical properties. Our study demonstrated how supercharging can potentially modulate protein aggregation and LLPS.