The Relationship between Protein-Protein Interactions and Liquid-Liquid Phase Separation for Monoclonal Antibodies

Area of Science:

• Biophysical chemistry
• Protein aggregation
• Therapeutic protein formulation

Background:

• Predicting and controlling concentrated monoclonal antibody (mAb) solution properties is crucial for therapeutic formulation development.
• High protein concentrations can lead to undesirable properties like high viscosity, opalescence, particle formation, and precipitation.
• Understanding the relationship between dilute solution parameters and liquid-liquid phase separation (LLPS) is essential.

Purpose of the Study:

• To investigate the relationship between dilute solution parameters (reduced osmotic second virial coefficient, b22, and diffusion interaction parameter, kD) and LLPS in mAb solutions.
• To determine if b22 can reliably predict LLPS for mAbs, considering their potential for reversible self-association.
• To map coexistence curves for three mAbs (COE-01, COE-07, COE-19) under varying salt conditions.

Main Methods:

• Mapping liquid-liquid coexistence curves for three mAbs (COE-01, COE-07, COE-19) using b22 and kD parameters.
• Conducting measurements under low salt conditions (near pI) to promote anisotropic interactions and high ammonium sulfate concentrations (salting-out) to screen electrostatic interactions.
• Analyzing deviations from the linear correlation between b22 and kD as an indicator of reversible self-association.

Main Results:

• Under salting-out conditions, all mAbs followed the b22-kD correlation, suggesting nonspecific protein-protein interactions.
• Deviations from the correlation under low salt conditions for COE-01 and COE-19 indicated reversible self-association.
• For most conditions, LLPS onset (b22c) occurred within a narrow window (-1.6 > b22c > -2.3), similar to globular proteins, except for COE-19 under low salt (b22c ≈ -5.1).

Conclusions:

• When mAb interactions are weakly anisotropic (e.g., high salt), phase separation initiates within a specific b22 window.
• Deviations from this window can signal reversible self-association, though this depends on the nature of oligomer formation.
• Fitting LLPS data to simplified interaction models for mAbs can offer insights into protein-protein interactions and aid in predicting concentrated solution properties.