Counting the water: Characterize the hydration level of aluminum adjuvants using contrast matching small-angle neutron scattering

Adjuvants are often included in vaccine formulations to enhance and modulate immune responses against pathogenic antigens. Aluminum-based adjuvants are prepared as hydrated colloids and are widely used due to their high efficacy and strong safety record. Previous studies have shown that freezing and thawing of aluminum adjuvants can lead to the aggregation of adjuvant particles, resulting in reduced vaccine potency. In this study, we demonstrated the use of contrast-matching small-angle neutron scattering (SANS) as a tool to directly probe hydration states of aluminum adjuvants in solution. Our study shows that aluminum hydroxide and aluminum phosphate adjuvants could be represented with the hydrated forms of Al(OH) 3 •2 H 2 O and AlPO 4 •1.5 H 2 O respectively. Moreover, our results demonstrate that freeze-thaw stress could lead to the dehydration of aluminum adjuvants, followed by aggregation of dehydrated adjuvant particles. Including sucrose in samples reduced freeze-thaw-induced dehydration of aluminum adjuvants, which in turn, inhibited the formation of large aggregates. The relationship between freeze-thaw-induced dehydration and aggregation of aluminum adjuvants could be used to explain the loss of adjuvanticity upon accidental freezing and thawing, and thus enable the more efficient development of thermostable vaccine products.