Decoupling liquid-liquid phase separation and opalescence from stress-induced aggregation in therapeutic mAb formulations

Abstract

Liquid-liquid phase separation (LLPS) and high opalescence are two self-association phenomena commonly encountered in monoclonal antibody (mAb) formulations. Because of their impact on colloidal stability, they are commonly avoided, due to a suspected link with aggregation and reduced product shelf-life. However, the molecular underpinnings and interrelation between these phenomena remain unclear, complicating predictions of their occurrence. By combining light and X-ray scattering techniques with microscopy and advanced microfluidic setups, we here report the delicate phase behavior of a model mAb, named mAb1. This is characterized by rapid clustering and LLPS in a narrow NaCl range, above which it transitions into an opalescent state devoid of micron-sized assemblies, yet retaining a similar interaction fingerprint. Using Monte Carlo simulations, we report that the macroscopic solution state of mAb1 is controlled by a positive patch, whose degree of charge screening determines whether LLPS or opalescence will take place. Specifically, neutralization of this patch via counterion interactions diminishes intermolecular repulsion and favors the concerted action of weaker dipole-dipole/hydrophobic interactions, amounting to the creation of a new solution phase, via LLPS. Further NaCl addition distributes ions more uniformly across the surface, attenuating these attractive interactions, leading to the dismantling of droplets while preserving solution opalescence. Finally, we show that LLPS and opalescence are decoupled from stirring-induced aggregation, challenging an unequivocal relationship between these phenomena. Significance StatementTailoring formulations to maximize the stability of therapeutic antibodies is crucial for their development. This is complicated by their tendency for self-association at high concentrations, where increased opalescence and phase separation, that are thought to precede irreversible aggregation, are routinely observed. Here, we studied the molecular underpinnings of mAb opalescence versus liquid-liquid phase separation. We report the mechanisms determining the two phenomena and provide a foundation for their prediction, which may guide the rational development of mAb formulations. We further show that LLPS and opalescence can be decoupled from stress-induced aggregation. We hypothesize that excluding mAbs from the bulk solvent via LLPS may even be harnessed to enhance drug product stability.