Phase separation in solutions of monoclonal antibodies and the effect of human serum albumin

Abstract

We report the observation of liquid-liquid phase separation in a solution of human monoclonal antibody, IgG2, and the effects of human serum albumin, a major blood protein, on this phase separation. We find a significant reduction of phase separation temperature in the presence of albumin, and a preferential partitioning of the albumin into the antibody-rich phase. We provide a general thermodynamic analysis of the antibody-albumin mixture phase diagram and relate its features to the magnitude of the effective interprotein interactions. Our analysis suggests that additives (HSA in this report), which have moderate attraction with antibody molecules, may be used to forestall undesirable protein condensation in antibody solutions. Our findings are relevant to understanding the stability of pharmaceutical solutions of antibodies and the mechanisms of cryoglobulinemia.

Fig. 1.

Liquid-liquid phase separation of MAb solutions in 0.1 M Tris·HCl buffer at pH 7.4. The eye guide for the LLPS boundary is indicated by the dashed line. The crossed square is the critical point determined at the maximum of the phase boundary.

Fig. 2.

(A) Decrease of LLPS temperature, T_ph, vs. the HSA concentration, c₂, at fixed MAb concentration, c₁. Linear fitting of T_ph vs c₂ at each c₁ is shown by the dashed line. (B) LLPS boundaries at fixed c₂ shift to lower temperature as HSA concentration, c₂, increases (The data were obtained by interpolation of the data in SI Appendix; Fig. S1).

Fig. 3.

Partitioning of MAb and HSA upon LLPS at fixed temperature (A) T = -2.2 °C; and (B) T = -4.2 °C. The points representing the two coexisting phases are connected by the solid lines, i.e., the tie lines. Dashed lines are eye guides for the binodal curves fitted from both cloud-point measurements (open triangles) and partitioning measurements (solid circles). The critical points are represented by the crossed square.

Fig. 4.

Coexistence curves in the units of scaled phase separation temperatures, T_ph/T_c, and the scaled protein concentrations, c/c_c. Curve 1 (short dashed line) shows the theoretical coexistence curve of spherical particles using mean-field approximation of attraction and Carnahan-Starling expression for entropy. Curve 2 (long dashed line) shows the theoretical fit for the data of bovine γB crystallin (open squares) taken from (18). Curve 3 (solid line) shows the eye guide for the coexistence curve of MAb (solid circles). Data points on the coexistence curves of two other globular proteins (16, 24): human γD crystallin (open diamonds) and chicken egg white lysozyme (open triangles) are also shown.