Growth of Clusters toward Liquid-Liquid Phase Separation of Monoclonal Antibodies as Characterized by Small-Angle...

In concentrated protein solutions, short-range attractions (SRAs) contribute to liquid-liquid phase separation (LLPS) as a function of temperature and salinity, particularly when the charge and thus long-range repulsions are low near the isoelectric point pI. Herein, we study how SRA and solution morphology vary with the approach to LLPS from increased SRA for two monoclonal antibodies (mAbs) as salt concentration is reduced near the pI. These properties are quantified using small-angle X-ray scattering (SAXS) interpreted via coarse-grained (CG) molecular dynamics (MD) simulations and compared with less descriptive properties from static and dynamic light scattering. Experimental structure factors are fit with a library of MD simulations for a CG 12-bead mAb model to determine the SRA strength (K) and cluster size distributions. Proximity to LLPS and clustering characteristics in mAb solutions are impacted by both net charge, which are modified by pH, and the strength of anisotropic electrostatic SRA (charge-charge, charge-dipole, hydrogen bonding, etc.), which are screened and weakened by added salts. The trends in LLPS are consistent with the reduced diffusion interaction parameter kD/B₂₂^ex for dilute solutions. However, greater insight is provided with SAXS along with CG-MD simulations; in particular, the growth of clusters is observed with the approach to LLPS with decreasing salinity over a wide range of concentrations.