Anomalous Clouding Behavior of Polysorbate 80─Deciphering the Role of Nonesterified Components.

Polysorbates (PSs) are key excipients for the colloidal stability of biopharmaceuticals with unique properties. A comprehensive understanding of the physicochemical properties of these multicomponent products is essential to address potential stability issues without compromising their functionality. Here, we demonstrate that polysorbate 80 HP (PS80) shows an anomalous clouding, i.e., a thermotropic liquid-liquid phase separation behavior, which cannot adequately be explained by the conventional interpretation assuming a pseudobinary system. In a binary two-phase system of surfactant and buffer, an increase in the total surfactant concentration increases the fraction of the surfactant-rich phase in the respective proportion (lever rule). PS80 within about 7 K of the lower critical solution temperature fails to comply with this; concentrations and compositions of the coexisting phases change with the total concentration. This renders the phases more alike and, at some point, eliminates phase separation. This significant deviation from the pseudobinary phase behavior can be resolved by conceptually dividing the numerous chemical species in PS80 into two independent pseudocomponents, PS80-I and -II. Ternary phase diagrams derived from this approach successfully explain the observed anomalous behavior. RP-UPLC-MS analysis indicated a concentration-dependent redistribution of the nonesterified components (NECs), suggesting, along with other evidence, that NECs are key constituents of component II. Specifically, free polyethylene glycol (PEG) and/or PEG-sorbitans seem to function as intrinsic cosurfactant(s) within PS80, modulating its wetting and clouding properties. The latter is important for interaction, association, and phase separation properties in biologics.