Particulate adjuvants increase vaccine immunogenicity and are included in a substantial portion of the multi-billion dollar global vaccine sector. Adjuvanted vaccines often require resuspension prior to administration and as formulation complexity increases, the need to understand mechanisms leading to resuspension difficultly also increases. The addition of antigens to adjuvanted systems can lead to large, persistent clumps in the sediment that do not resuspend after prolonged storage in clinical settings. A mechanistic understanding of the complex interactions that influence particle packing would be a first step to systematically improving adjuvant robustness for various antigens. Here, we discuss two analytical methods developed in our group to characterize the resuspendability of aluminum phosphate adjuvants bound to bovine serum albumin (BSA) and lysozyme. First, bulk rheological measurements were used to quantify the flowability and microstructure of sedimented adjuvants, which were formulated by centrifuging compositions with varying ionic strengths (0 and 150 mM sodium chloride), protein types (BSA and lysozyme), and protein concentrations (0 to 1 mg/mL). We find an exponential correlation between the weight fraction of sediments and number of inversions required for complete resuspension of sediments. In particular, salt-free formulations containing BSA are the least resuspendable—they pack densely into homogenous sediments with low yield strength but require >100 inversions to resuspend. These sediments are likely to have a greater inter-aggregate bond density, caused potentially by protein bridging between adjuvant particles, which strengthen the overall microstructure and resist fluidization into homogenous suspensions. Second, confocal microscopy, fluorescent tagging, and image processing showed that adding BSA to aluminum phosphate created large subpopulations of dense secondary aggregates (i.e. large clumps of sediment) that are > 50 μm in size. In contrast, lysozyme samples yielded a metastable gel-like state with few secondary aggregates. These secondary aggregates differed from those formed by changing salt concentration and persisted despite repeated inversion attempts. Increasing sediment density also correlated to a higher frequency of dense secondary aggregates. These findings highlight the roles of protein bridging and sediment microstructural properties in determining the resuspendability of sediments, providing correlations and mechanistic insights relevant to optimizing adjuvant formulation stability.
