Relationship of PEG-induced precipitation with protein-protein interactions and aggregation rates of high concentration mAb formulations at 5 °C.

Native protein-protein interactions can play an important role in determining the tendency of monoclonal antibodies (mAbs) to aggregate under storage conditions. In this context, phase separation of mAb solutions induced by the addition of neutral polymers such as poly(ethylene glycol) (PEG) represents a simple method to assess the tendency of proteins to self-associate in the native state. Here, we investigated their relationships between PEG-induced phase separation, protein-protein interactions and long-term aggregation rate of several formulations of four mAbs at 100 mg/mL and 5 °C over 12 weeks of storage. We observed that the location of the phase boundary correlated well with the osmotic second virial coefficient B22 determined in absence of the polymer, indicating that for our solutions PEG primarily leads to depletion forces between protein molecules, which are additive to protein-protein interactions. However, limited correlation between aggregation rate at 5 °C and phase behavior was observed across different mAbs, pH values and ionic strengths, indicating that colloidal stability is not the only determinant of aggregation even at such low temperature and high protein concentration. Our results contribute to the growing realization that aggregation propensity in the context of antibody developability is a complex feature, which depends on a variety of biophysical properties rather than one single parameter.