Evaluating the Compatibility of New Recombinant Protein Antigens (Trivalent NRRV) with a Mock Pentavalent Combination Vaccine Containing Whole-Cell Pertussis: Analytical and Formulation Challenges.

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.

Mechanism of Thimerosal-Induced Structural Destabilization of a Recombinant Rotavirus P[4] Protein Antigen Formulated as a Multi-Dose Vaccine.

In a companion paper, a two-step developability assessment is presented to rapidly evaluate low-cost formulations (multi-dose, aluminum-adjuvanted) for new subunit vaccine candidates. As a case study, a non-replicating rotavirus (NRRV) recombinant protein antigen P[4] was found to be destabilized by the vaccine preservative thimerosal, and this effect was mitigated by modification of the free cysteine (C173S). In this work, the mechanism(s) of thimerosal-P[4] protein interactions, along with subsequent effects on the P[4] protein's structural integrity, are determined. Reversible complexation of ethylmercury, a thimerosal degradation byproduct, with the single cysteine residue of P[4] protein is demonstrated by intact protein mass analysis and biophysical studies. A working mechanism involving a reversible S-Hg coordinate bond is presented based on the literature. This reaction increased the local backbone flexibility of P[4] within the helical region surrounding the cysteine residue and then caused more global destabilization, both as detected by HX-MS. These effects correlate with changes in antibody-P[4] binding parameters and alterations in P[4] conformational stability due to C173S modification. Epitope mapping by HX-MS demonstrated involvement of the same cysteine-containing helical region of P[4] in antibody-antigen binding. Future formulation challenges to develop low-cost, multi-dose formulations for new recombinant protein vaccine candidates are discussed.

Rapid Developability Assessments to Formulate Recombinant Protein Antigens as Stable, Low-Cost, Multi-Dose Vaccine Candidates: Case-Study With Non-Replicating Rotavirus (NRRV) Vaccine Antigens.

A two-step developability assessment workflow is described to screen variants of recombinant protein antigens under various formulation conditions to rapidly identify stable, aluminum-adjuvanted, multi-dose vaccine candidates. For proof-of-concept, a series of sequence variants of the recombinant non-replicating rotavirus (NRRV) P[8] protein antigen (produced in Komagataella phaffii) were compared in terms of primary structure, post-translational modifications, antibody binding, conformational stability, relative solubility and preservative compatibility. Based on these results, promising P[8] variants were down-selected and the impact of key formulation conditions on storage stability was examined (e.g., presence or absence of the aluminum-adjuvant Alhydrogel and the preservative thimerosal) as measured by differential scanning calorimetry (DSC) and antibody binding assays. Good correlations between rapidly-generated developability screening data and storage stability profiles (12 weeks at various temperatures) were observed for aluminum-adsorbed P[8] antigens. These findings were extended and confirmed using variants of a second NRRV antigen, P[4]. These case-study results with P[8] and P[4] NRRV variants are discussed in terms of using this vaccine formulation developability workflow to better inform and optimize formulation design with a wide variety of recombinant protein antigens, with the long-term goal of rapidly and cost-efficiently identifying low-cost vaccine formulations for use in low and middle income countries.

Application of three-photon excitation FCS to the study of protein oligomerization.

Three-photon excitation fluorescence correlation spectroscopy was used to detect oligomerization equilibria of rat liver phosphofructokinase. The fluorescence intensity produced by the three-photon excitation of tryptophan was collected using the DIVER microscope. In this home-built upright microscope, a large area photomultiplier, placed directly below the sample, is used as the detector. The lack of optical elements in the microscope detection path results in a significantly improved detection efficiency in the UV region down to about 300 nm, which encompasses the fluorescence emission from tryptophan. The three-photon excitation autocorrelation decays obtained for phosphofructokinase in the presence of F6P showed the presence of large oligomers. Substitution of F6P with ATP in the buffer medium results in dissociation of the large oligomers, which is reported by the decreased autocorrelation amplitude. The three-photon excitation process was verified from the slope of the log-log plot of intensity against laser power.