Investigating the impact of trehalose crystallization on protein aggregation in lyophilized formulations

Abstract Trehalose is a commonly used lyoprotectant for stabilizing proteins in lyophilized formulations. Its physical state plays a critical role in maintaining both the physical and chemical stability of proteins. In this 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 60% monomers of bovine serum albumin (BSA) and 40% aggregates; BSA95, with about 95% monomers and 5% aggregates; and a monoclonal antibody (mAb), which had little aggregation (< 1%). Each was formulated with trehalose and lyophilized using varying annealing durations. Trehalose crystallized after 24 h of annealing in BSA60 formulations, whereas BSA95 and mAb formulations required 72 h of annealing to induce crystallization. Although crystalline trehalose was present in the 24-h annealed BSA60 samples after lyophilization, no additional aggregation was observed on storage. In contrast, BSA95 and mAb formulations, where crystalline trehalose was also observed after lyophilization, showed monomer loss at all time points (1 month–12 months) during storage at 50 °C. Overall, these results indicate that trehalose crystallization does not usually occur under typical lyophilization conditions, and generally requires prolonged annealing and a high trehalose-to-protein ratio. When crystallization does occur, it results in inhomogeneity, as shown by solid-state NMR spectroscopy, leading to reduced stability of the protein in the matrix.