title: Therapeutic Antibody Engineering To Improve Viscosity and Phase Separation Guided by Crystal Structure.
abstract: 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 behavior. We further evaluated the correlation of various biophysical measurements including second virial coefficient (A2), interaction parameter (kD), weight-average molecular weight (WAMW), and hydrodynamic diameters (DH), at relatively low protein concentration (4 to 15 mg/mL) to physical properties, such as viscosity and liquid-liquid phase separation (LLPS), at high concentration. Surprisingly, kD measured using dynamic light scattering (DLS) at low antibody concentration correlated better with viscosity and phase separation than did A2 for Mab4. Our results suggest that the high viscosity and phase separation observed at high concentration for Mab4 are mainly driven by charge and not hydrophobicity.
authors: Biotechnology Discovery Research, Lilly Research Laboratories, Eli Lilly and Company , Indianapolis, Indiana 46285, United States.
authors: Chi-Kin
authors: CK
authors: Chow
authors: Eli Lilly Biotechnology Center , San Diego, California 92121, United States.
authors: Barrett W
authors: BW
authors: Allan
authors: Eli Lilly Biotechnology Center , San Diego, California 92121, United States.
authors: Qing
authors: Q
authors: Chai
authors: Eli Lilly Biotechnology Center , San Diego, California 92121, United States.
authors: Shane
authors: S
authors: Atwell
authors: Biotechnology Discovery Research, Lilly Research Laboratories, Eli Lilly and Company , Indianapolis, Indiana 46285, United States.
authors: Jirong
authors: J
authors: Lu
mesh_terms: D019943:Amino Acid Substitution; D000911:Antibodies, Monoclonal / Q000737:chemistry* / Q000235:genetics / Q000627:therapeutic use; D003460:Crystallization; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007074:Immunoglobulin G / Q000276:immunology*; D059625:Liquid-Liquid Extraction / Q000379:methods*; D009994:Osmolar Concentration; D019999:Pharmaceutical Solutions; D011487:Protein Conformation; D015202:Protein Engineering / Q000379:methods*; D014783:Viscosity
chemical_list: D000911:Antibodies, Monoclonal; D007074:Immunoglobulin G; D019999:Pharmaceutical Solutions
keywords: electrostatic interaction; interaction parameter (kD); liquid−liquid phase separation (LLPS); monoclonal antibody (Mab or mAb); second virial coefficient (A2); viscosity