Formulations That Suppress Aggregation During Long-Term Storage of a Bispecific Antibody are Characterized by High Refoldability and Colloidal Stability

Abstract

Understanding the formulation features that ensure sufficient stability during long-term storage is critical for developing next-generation therapeutic proteins. In this work, we investigate the physical stability of a bispecific antibody (Bis-mAb) in 12 different formulation conditions. Isothermal chemical denaturation with urea indicates a higher resistance to denaturant-induced unfolding when pH is increased from 5.0 to 6.5 but shows minor influence from the buffer type and ionic strength. Dynamic and static light scattering are used to derive the interaction parameter (kD) and second virial coefficient (A2), respectively. These two parameters indicate that Bis-mAb exhibits highest colloidal stability in formulations containing 10 mM histidine buffer without added sodium chloride. Further, we observe that the highest relative monomer yield (RMY) after isothermal refolding, that is the highest refoldability, from urea is measured for the low ionic strength histidine formulations. Finally, we show long-term stability data on all 12 Bis-mAb formulations after storage at 4 °C and 25 °C for 12 months. The least amount of soluble aggregates and subvisible particles were detected in the Bis-mAb formulations with the highest colloidal stability and refoldability from urea. We suggest that the optimization of these two features is crucial for obtaining physically stable formulations of Bis-mAb.