Introducing new recombinant protein antigens to existing pediatric combination vaccines is important in improving coverage and affordability, especially in low- and middle-income countries (LMICs). This case-study highlights the analytical and formulation challenges encountered with three recombinant non-replicating rotavirus vaccine (NRRV) antigens (t-NRRV formulated with Alhydrogel® adjuvant, AH) combined with a mock multidose formulation of a pediatric pentavalent vaccine used in LMICs. This complex formulation contained (1) vaccine antigens (i.e., whole-cell pertussis (wP), diphtheria (D), tetanus (T), Haemophilus influenza (Hib), and hepatitis B (HepB), (2) a mixture of aluminum-salt adjuvants (AH and Adju-Phos®, AP), and (3) a preservative (thimerosal, TH). Selective, stability-indicating competitive immunoassays were developed to monitor binding of specific mAbs to each antigen, except wP which required the setup of a mouse immunogenicity assay. Simple mixing led to the desorption of t-NRRV antigens from AH and increased degradation during storage. These deleterious effects were caused by specific antigens, AP, and TH. An AH-only pentavalent formulation mitigated t-NRRV antigen desorption; however, the Hib antigen displayed previously reported AH-induced instability. The same rank-ordering of t-NRRV antigen stability (P[8] > P[4] > P[6]) was observed in mock pentavalent formulations and with various preservatives. The lessons learned are discussed to enable future multidose, combination vaccine formulation development with new vaccine candidates.

This study evaluated the formulation and stability of a quadrivalent glycoconjugate Shigella vaccine candidate based on four predominant strains (S. flexneri; 2a, 3a, and 6, and S. sonnei) covering ~64% of global Shigella infections. Each glycoconjugate antigen consists of a strain-specific O-polysaccharide (O-PS) covalently linked to the carrier protein IpaB, a component of the Shigella type III secretion system. First, selective competitive ELISAs were developed to measure antigenicity of the four O-PS-IpaB conjugates formulated with different adjuvants (i.e., Alhydrogel^®, AH; Adju-phos^®, AP; and CpG-1018^®, CpG). Next, the monovalent S. sonnei O-PS-IpaB conjugate was studied to elucidate interactions with aluminum salt adjuvants (AH, AP) under different solution conditions. Third, the stability profiles of AH- or AP-adjuvanted S. sonnei O-PS-IpaB conjugate in various formulations (±CpG) were determined at different temperatures. Interestingly, incubation at 25 °C for 2 weeks resulted in increased antigenicity values when the antigen was bound to AP or AH, suggesting increased epitope exposure upon adjuvant binding. When bound to AP adjuvant at pH 5.8, the best glycoconjugate antigen stability was observed at elevated temperatures. The CpG adjuvant under these conditions, however, displayed incompatibility (i.e., material loss), presumably from precipitation due to lack of interaction with AP and presence of the detergent LDAO from the bulk antigen buffer. In contrast, the glycoconjugate antigen and CpG adjuvant were both bound to the AH adjuvant and stable at 2–8 °C, pH 7.0. This AH-CpG formulation of the O-PS-IpaB conjugate antigens was identified as a promising candidate for future animal immunogenicity testing.