Decoupling liquid-liquid phase separation and opalescence from stress-induced aggregation in therapeutic mAb formulations
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Liquid-liquid phase separation (LLPS) and high opalescence resulting from attractive interactions in the absence of large-scale self-association are commonly encountered in liquid monoclonal antibody (mAb) formulations and often associated with aggregation. Here, we evaluated the formulation dependent self-assembly behavior of a therapeutic mAb (termed mAb1) and provide a link to physical stability after applied stress. Using a combinatorial microdroplet platform to delineate the phase space of mAb1, we highlight its unusual solution properties. Specifically, mAb1 undergoes LLPS in a narrow window of low NaCl concentrations, in a formulation relevant design space, at room temperature (RT), without crowding agents and more than one pH unit lower than its isoelectric point. Using time-resolved small-angle X-ray scattering, we measured the attractive interactions underpinning LLPS to be on the sub second timescale and leading to the formation of small clusters, which altogether could be rapidly inhibited by L-arginine HCl. The elevated opalescence of mAb1 solutions extended beyond the LLPS regime, where a highly similar interaction fingerprint to the LLPS condition was observed but no microscopic association was detected. This suggests that LLPS and opalescence differ in the distribution of interactions within their networks, dictating their ability to form long-range assemblies (phase-separated droplets) or not. Finally, our physical stability assessment highlights that mAb1 LLPS and molecular assemblies giving rise to high opalescence both resist stirring stress and that the observed aggregation is uncorrelated with ionic strength at our selected timescale. This observation challenges an unequivocal relationship between LLPS/opalescence and reduced physical stability of mAb solutions.
Significance Statement
The development of formulations maximizing the stability of therapeutic monoclonal antibodies is a crucial part of their successful development. However, their proclivity for self-association often results in increased opalescence, which has been shown to indicate a propensity for liquid-liquid phase separation (LLPS), involving the de-mixing of solutions into two phases with different concentrations of solute. Both phenomena are commonly tied to a high propensity for aggregation. However, given the paucity of experimental studies, evidence for a general correlation between these solution properties and aggregation remains scant. In this work, we decouple stress-induced aggregation from LLPS and high opalescence without large-scale self-association through the fundamental study of these phenomena and solution integrity after applied stress.
