Therapeutic Antibody Engineering To Improve Viscosity and Phase Separation Guided by Crystal Structure

Antibodies at high concentrations often reveal unanticipated biophysical properties suboptimal for therapeutic development. The purpose of this work was to explore the use of point mutations based on crystal structure information to improve antibody physical properties such as viscosity and phase separation (LLPS) at high concentrations. An IgG4 monoclonal antibody (Mab4) that exhibited high viscosity and phase separation at high concentration was used as a model system. Guided by the crystal structure, four CDR point mutants were made to evaluate the role of hydrophobic and charge interactions on solution behavior. Surprisingly and unpredictably, two of the charge mutants, R33G and N35E, showed a reduction in viscosity and a lower propensity to form LLPS at high concentration compared to the wild-type (WT), while a third charge mutant S28K showed an increased propensity to form LLPS compared to the WT. A fourth mutant, F102H, had reduced hydrophobicity, but unchanged viscosity and phase separation behavi...