“Bigger, the Better?”─The Influence of Sugar Size and Residual Moisture on Protein Stability and Accessibility in Lyophilizates

Lyophilization is a key technology to improve the long-term stability of protein drug products, traditionally using the disaccharides sucrose and trehalose for cryo- and lyoprotection. Monosaccharides are less favored due to the low glass transition temperature and Maillard reaction potential. Additionally, trisaccharides and tetrasaccharides typically do not play significant roles, as they are often not approved for parenteral use and have been associated with lower protein stabilization. Key stability parameters include the preserved protein structure, solid-state accessibility, and monomer content. This study explores the long-term monomer retention of human serum albumin (HSA) in lyophilizates at 2–8, 25, and 40 °C by investigating the effect of a series of mono- to tetrasaccharides based on glucose (glucose, maltose, maltotriose, and maltotetraose) as well as glucose maltose and glucose maltotriose mixtures. We varied the residual moisture (RM) content (1, 1.5, and 2%) postlyophilization to understand the effects of water replacement, vitrification, and matrix mobility on protein stability. The molar ratios of maltose to HSA were set at 360:1 and 180:1 to investigate the impact of the sugar concentration at overall low sugar ratios. Solid-state hydrogen–deuterium exchange mass spectrometry (ssHDX MS) was performed on a QDa benchtop mass spectrometer to evaluate protein accessibility and structural preservation using RMs of 1% D 2 O, 2% D 2 O, and 1% D 2 O + 1% H 2 O. The larger the sugar, the lower its stabilizing potential and the higher the protein accessibility, indicating insufficient water replacement. Increasing the RM from 1 to 1.5 and 2% enhanced stability, highlighting the superiority of residual water molecules, which was especially the case for the tri- and tetrasaccharides. Mixtures of small and large sugars showed stabilization benefits in maintaining the monomer content and structural preservation, indicating a good balance of water replacement and vitrification. Overall, the ssHDX MS findings of samples with headspace-spiked D 2 O did correspond with monomer retention, indicating that it could be a valuable tool for characterization and understanding the stabilizing capacity of lyophilized formulations. Our findings highlight the importance of RM control for optimal stability as well as the importance of the sugar size on lyoprotection based on water replacement and the potential of sugar mixtures to optimize the stability of lyophilized proteins.