Impact of mobility and homogeneity on biologic’s stability in lyophilized matrices

Biologics like proteins have become crucial mode of treatment for various ailment. They have advantages over small molecules for being target specific and minimized side effects. Despite the benefits of biologic drugs, they are difficult to stabilize. Lyophilization is one of the ways to formulate stable solid protein formulation. Lyophilization can cause many freezing and drying related stresses. Cryoprotectants are a major excipient class of excipients that are used to protect the biologics during lyophilization and storage. Disaccharides such as trehalose and sucrose are commonly used as cryoprotectants or stabilizers. For both the disaccharides to be good stabilizers, they must form a rigid, immobile matrix where proteins are homogenously distributed. We have used ssNMR as a tool to assess mobility and homogeneity to understand long term stability of the protein formulations. We investigated the relationship between molecular mobility, miscibility, and long-term stability in lyophilized formulations of a mAb with trehalose or sucrose. We used solid-state nuclear magnetic resonance (ssNMR) to measure 1H T1 and 1H T1 relaxation times, probing the mobility and miscibility of the components at 20-50 nm and 2-5 nm scales, respectively. Formulations with higher disaccharide content showed longer 1H T1 relaxation times and greater monomer retention, consistent with a more rigid glassy matrix and enhanced stability. Relaxation values of mAb and disaccharide were closely matched in most formulations, indicating good miscibility, which was essential for stability. However, exceptions were observed in samples where disaccharide crystallization occurred, leading to phase separation and inhomogeneity. Despite having long 1H T1 values, these samples showed reduced physical stability, underscoring the importance of maintaining both low mobility and homogeneous amorphous structure. Overall, this study demonstrates that ssNMR relaxation measurements are effective predictors of lyophilized protein stability as it provides information on both matrix rigidity and component miscibility in a formulation. In another study, three types of protein/trehalose systems were studied to understand the impact of trehalose crystallization on protein aggregation in the lyophilized state. The three systems included BSA60, which contains approximately 40 % aggregates and 60 % monomer of bovine serum albumin (BSA); BSA95, with about 5 % aggregates and 95 % monomer; and 14 a monoclonal antibody (mAb), which had little pre-aggregation (