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

Phase separation of monoclonal antibody A (MAb A) solution and its relation to protein self-association are studied.

A phase diagram of MAb A and its dependence on ionic strength and pH were investigated. The protein self-associations were characterized by dynamic light scattering (DLS), analytical ultracentrifugation analysis (AUC) and viscosity measurement.

MAb A solution with a clear appearance in an isotonic ionic strength condition turned opalescent in a low ionic strength condition, followed by liquid-liquid phase separation (LLPS) into light and heavy phases. The protein concentrations of the two phases were dependent on the ionic strength and pH. The two phases became reversibly miscible when the ionic strength or temperature was increased. DLS and AUC showed that MAb A under a low ionic strength condition self-associates at a protein concentration above the critical concentration of 16.5 mg/mL. The viscosity of the heavy phase was high and dependent on the shear rate. These results indicate that attractive protein-protein interaction in the heavy phase induces LLPS.

LLPS was induced in MAb A solution in a low ionic strength condition due to reversible protein self-association mediated mainly by attractive electrostatic interaction among the MAb A molecules in the heavy phase.