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

Abstract

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.

Interaction of Aluminum-adjuvanted Recombinant P[4] Protein Antigen With Preservatives: Storage Stability and Backbone Flexibility Studies

“…1a, no changes in antibodyantigen binding curves were observed for each of AH-adsorbed P [4] ± preservative samples compared to a control at time zero, with the exception of TH. At time 0, we observed an initial increase in antibody binding in the presence of TH, which has been reported previously 23 (see Discussion). During 4°C storage over 12 weeks (Fig 1c), in the absence of preservative, a small to no notable change in antibody binding to AH-adsorbed P [4] was seen.…”

“…The structural integrity of the AH-adsorbed P [4] protein (± preservatives) was first assessed during storage using competitive ELISA assay (i.e., P [4] binding to an anti-P [4] specific mAb that recognizes a conformational epitope). 23 As shown in Fig. 1a, no changes in antibodyantigen binding curves were observed for each of AH-adsorbed P [4] ± preservative samples compared to a control at time zero, with the exception of TH.…”

“…Experimental details of physicochemical methods and immunochemical methods used in this study with AH-adsorbed P [4] protein including UV-visible spectroscopy, SDS-PAGE, differential scanning calorimetry (DSC), and competitive ELISA have been adapted from previously described reports 23,24,26 and described in supplemental methods. The BCA protein assay method is described in the supplemental section.…”

“…5b, d, f). Significant differences in uptake were observed across the entire P [4] molecule except for the N-terminal region (residues M 1 -E 15 , peptide #1-5) and residues L 16 -L 23 (peptide #6) (Fig. 5b), which were already saturated with deuterium (see butterfly plot, HX-MS supplemental information).…”

“…[20][21][22] In addition, we recently established different stability profiles of site-directed mutants of the P [4] and P [8] protein, both in solution and AH-adsorbed, especially in the presence thimerosal (TH). 23 The destabilizing effects of TH were mitigated by preparing site-directed mutants of P [4] and P [8] to replace the single cysteine residue, albeit the mutants had lower inherent conformational stability. The molecular nature of the interaction of TH with the P [4] protein in solution leading to subsequent structural alterations and destabilization was also described, 24 in which an aqueous solution degradation byproduct of TH (ethylmercury) reacts with the single free cysteine residue in P [4] antigen (via Hg-S coordinate bond as described in literature with other model proteins).…”