|Although live attenuated, orally delivered rotavirus (RV) vaccines are available globally to provide protection against enteric RV disease, efficacy is substantially lower in low to middle income settings leading to interest in the development of alternate RV vaccines. Moreover, the high cost and limited supply of current RV vaccines are prohibitive to their successful introduction in the developing countries where the need for RV vaccines is most. One promising new RV vaccine candidate is a trivalent non-replicating rotavirus vaccine (NRRV), comprised of three recombinant fusion proteins (truncated rotavirus VP8 subunit proteins fused to the P2 CD4+ T cell epitope from tetanus toxoid). The three NRRV antigens are referred to as P2-VP8-P, P2-VP8-P, and P2-VP8-P. A monovalent P2-VP8-P adsorbed to Alhydrogel adjuvant was found to be safe and immunogenic in the early phase clinical trials in infants and toddlers in South Africa. Consequently, the trivalent vaccine consisting of all three P2-VP8-P[x] antigens adsorbed to Alhydrogel is currently being evaluated in Phase I/II clinical trials in South Africa. Successful development and eventual commercialization of this recombinant subunit RV vaccine candidate will not only depend on clinical safety and efficacy results, but also the ability to produce the NRRV vaccine at low cost and abundant supply for use in the developing world. This dissertation work contributes towards developing analytical tools and formulation strategies (for both bulk drug substance and adjuvanted final drug product) to support the pharmaceutical development of this subunit RV vaccine candidate. A stable formulation should maintain the structural integrity, physicochemical stability, and ultimately the safety and efficacy of this vaccine throughout its shelf-life. First, a wide variety of analytical techniques were employed to compare the physicochemical properties of the three NRRV recombinant fusion proteins. Various environmental stresses were used to evaluate antigen stability and elucidate their degradation pathways. The P2-VP8-P and P2-VP8-P antigens displayed similar conformational stability profiles while P2-VP8-P was more stable. Forced degradation studies with each NRRV antigen revealed Met1 was most susceptible to oxidation, the single Cys residue (at position 172 in P and 173 in P and P) formed inter-molecular disulfide bonds (P2-VP8-P was most susceptible), and Asn7 showed the highest increased levels of deamidation. These results are visualized in a structural model of the NRRV antigens. Although the potential impact of physicochemical structural alterations on immunogenicity is unknown at this time, the stability-indicating analytical tools developed and the structural knowledge gained in this work will be useful to (1) set critical manufacturing process parameters to ensure consistency, (2) monitor key structural attributes during comparability assessments, and (3) develop stable formulations for the bulk drug substance and adjuvanted final drug product. Second, we focused on the aggregation propensity of the three NRRV antigens and developed stable formulations for long-term storage of frozen liquid bulks of each antigen in a common formulation buffer. The P2-VP8-P antigen was most susceptible to shaking and freeze-thaw-induced aggregation. Each NRRV antigen formed aggregates with structurally altered protein (with exposed apolar regions and inter-molecular β-sheet) and dimers containing a non-native disulfide bond. From excipient screening studies with P2-VP8-P, sugars/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 excipients, candidate bulk formulations were optimized to not only minimize agitation-induced aggregation, but also particle formation due to freeze-thaw stress. Third, we explored the use of an aluminum adjuvant and two preservatives to develop an adjuvanted multi-dose formulation with goal to further reduce the NRRV vaccine cost. The compatibility and stability of monovalent P2-VP8-P antigen with Alhydrogel, with and without the vaccine preservative thimerosal, was examined using a wide variety of physicochemical and immunochemical methods. Antigen structural integrity was intact upon Alhydrogel binding as measured by ELISA, fluorescence spectroscopy, differential scanning calorimetry (DSC) and SDS-PAGE combined with LC-MS peptide mapping. An immediate destabilizing effect of thimerosal was observed upon heating by DSC. Over three months of storage, the aluminum adsorbed P2-VP8-P antigen was stable at 4°C, while instability was observed at 25°C and 37°C which was greatly accelerated by thimerosal addition. Compatibility of aluminum-adsorbed P2-VP8-P antigen with an alternative preservative (2-phenoxyethanol) was also evaluated and similar incompatibility was observed. Due to limited availability of P2-VP8-P and P2-VP8-P antigens, key assays from P2-VP8-P studies were performed with these monovalent aluminum-adsorbed antigens. Varying levels of preservative incompatibility were observed depending on the antigen, temperature, and analytical method. In summary, these results demonstrate good overall stability of the monovalent aluminum-adsorbed NRRV antigens at 4°C for three months in the absence of preservative. However, additional formulation development efforts are required to produce a stable multi-dose formulation of the NRRV vaccine.