At high concentrations, therapeutic monoclonal antibodies (mAbs) often display increased self-association, which may result in liquid-liquid phase separation (LLPS) or high opalescence alone. Due to a suspected link with aggregation, formulation strategies aim at preventing their occurrence. However, the molecular underpinnings of LLPS and opalescence remain unclear, complicating their forecasting at early stages. By combining light and X-ray scattering with microscopy and microfluidics, we report the phase behavior of a model mAb (mAb1). This is characterized by clustering and LLPS in a narrow NaCl range, above which it transitions into an opalescent state devoid of micron-sized assemblies, yet retaining similar self-interaction fingerprints. Using Monte Carlo simulations, we show that the behavior of mAb1 is controlled by a positive patch in its Fab domain, whose degree of charge screening determines solution fate. Finally, we show that LLPS and opalescence are decoupled from stirring-induced aggregation.
Brakti et al. (Tue,) studied this question.