Characterizing and Minimizing Aggregation and Particle Formation of Three Recombinant Fusion-Protein Bulk Antigens for Use in a Candidate Trivalent Rotavirus Vaccine.

In a companion paper, the structural integrity, conformational stability, and degradation mechanisms of 3 recombinant fusion-protein antigens comprising a non-replicating rotavirus (NRRV) vaccine candidate (currently being evaluated in early-stage clinical trials) are described. In this work, we focus on the aggregation propensity of the 3 NRRV antigens coupled to formulation development studies to identify common frozen bulk candidate formulations. The P2-VP8-P[8] antigen was most susceptible to shaking and freeze-thaw-induced aggregation and particle formation. Each NRRV antigen formed aggregates with structurally altered protein (with exposed apolar regions and intermolecular β-sheet) and dimers containing a non-native disulfide bond. From excipient screening studies with P2-VP8-P[8], sugars or polyols (e.g., sucrose, trehalose, mannitol, sorbitol) and various detergents (e.g., Pluronic F-68, polysorbate 20 and 80, PEG-3350) were identified as stabilizers against aggregation. By combining promising additives, candidate bulk formulations were optimized to not only minimize agitation-induced aggregation, but also particle formation due to freeze-thaw stress of P2-VP8-P[8] antigen. Owing to limited material availability, stabilization of the P2-VP8-P[4] and P2-VP8-P[6] was confirmed with the lead candidate P2-VP8-P[8] formulations. The optimization of these bulk NRRV candidate formulations is discussed in the context of subsequent drug product formulations in the presence of aluminum adjuvants.