Phase separation of an IgG1 antibody solution under a low ionic strength condition

Area of Science:

• Biophysical Chemistry
• Protein Science
• Pharmaceutical Formulation

Background:

• Monoclonal antibodies (mAbs) are critical therapeutics, but their formulation can be challenging.
• Understanding protein self-association and phase separation is crucial for stable mAb formulations.
• Liquid-liquid phase separation (LLPS) can impact mAb solution properties and efficacy.

Purpose of the Study:

• To investigate the phase separation of monoclonal antibody A (MAb A) solutions.
• To determine the relationship between MAb A self-association and phase separation.
• To elucidate the factors influencing MAb A phase behavior.

Main Methods:

• Constructed a phase diagram for MAb A, examining ionic strength and pH effects.
• Characterized protein self-associations using dynamic light scattering (DLS) and analytical ultracentrifugation (AUC).
• Measured solution viscosity under varying conditions.

Main Results:

• MAb A solutions exhibited liquid-liquid phase separation (LLPS) at low ionic strength, forming distinct light and heavy phases.
• Phase separation was reversible with increased ionic strength or temperature.
• DLS and AUC revealed MAb A self-association above 16.5 mg/mL at low ionic strength, driven by attractive electrostatic interactions.

Conclusions:

• Low ionic strength induces LLPS in MAb A solutions via reversible self-association.
• Attractive electrostatic interactions are the primary drivers of MAb A self-association in the heavy phase.
• These findings provide insights into mAb behavior and formulation strategies.