Therapeutic Antibody Engineering To Improve Viscosity and Phase Separation Guided by Crystal Structure
| Autor: | Chi-Kin Chow, Barrett Allan, Shane Atwell, Jirong Lu, Qing Chai |
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| Rok vydání: | 2016 |
| Předmět: |
0301 basic medicine
Work (thermodynamics) Low protein Protein Conformation Liquid-Liquid Extraction Analytical chemistry Pharmaceutical Science Crystal structure Flory–Huggins solution theory Protein Engineering 030226 pharmacology & pharmacy law.invention 03 medical and health sciences Viscosity 0302 clinical medicine Dynamic light scattering law Drug Discovery Humans Crystallization Chemistry Osmolar Concentration Antibodies Monoclonal Hydrogen-Ion Concentration Pharmaceutical Solutions 030104 developmental biology Amino Acid Substitution Virial coefficient Immunoglobulin G Molecular Medicine |
| Zdroj: | Molecular Pharmaceutics. 13:915-923 |
| ISSN: | 1543-8392 1543-8384 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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