Water Mobility and Clustering in Lyophilized Human Serum Albumin

Water plays a critical role in the stability of lyophilized protein therapeutics by stabilizing protein structures while also contributing to degradation processes like deamidation. Previous studies suggest a critical water threshold where degradation rates change, possibly due to shifts in water distribution between bound and free forms. However, the use of human serum albumin (HSA) as a model to understand the role of water clustering in protein stability has not been systematically explored. Our study examines water distribution, mobility, and clustering in lyophilized HSA using dynamic vapor sorption (DVS), solid-state NMR (ssNMR) spectroscopy, and molecular dynamics (MD) simulations. The water sorption isotherm follows Type II behavior, with Brunauer-Emmett-Teller (BET) analysis indicating a monolayer water content of 6 wt %. MD simulations also show water clusters forming at a similar threshold (∼5.5%). ssNMR analysis reveals increased water mobility between 7.4 and 11.5% water content, marking the transition from bound to free water. Our findings suggest that there is a critical water threshold for lyophilized HSA at 5-6%, where initial clustering occurs, leading to free water with increased mobility (7.4-11.5%). This indicates progressive water interactions and dynamics. This alignment of experimental and modeling data provides a useful insight into the role of water distribution in protein stability in lyophiles. In addition, these results offer a basis for further studies to explore the relationship between water thresholds and protein degradation, with potential applications for optimizing the stability of lyophilized pharmaceutical products.