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.
Highlights
The combined Stage 1 and 2 developability assessments were completed in ~4 months, the timeline in this case study benefited from previous work in our lab on the physicochemical characterization and formulation development of three different E. coli expressed recombinant nonreplicating rotavirus (NRRV) protein antigens (P[8], P[6] and P[4]),9e11 especially in terms of availability of immunological reagents required for antigen-antibody binding studies
Developability assessments have been widely applied to the design of small molecule and monoclonal antibodies drug candidates, such considerations are just beginning as part of antigen design for vaccine development, especially in terms of optimizing vaccine antigens for improved stability as formulated drug products
A two-step developability assessment workflow is presented for screening variants of recombinant protein vaccine antigens using small amounts of material
Summary
There is a promising trend towards developing new vaccine candidates using recombinant subunit protein antigens designed by reversed vaccinology, which offers advantages of improved safety and efficacy, and enhanced manufacturing scalability and lower costs compared to the traditional approaches (i.e., live, attenuated or inactivated versions of viral or bacterial pathogens).. The major cost categories associated with manufacturing such recombinant protein antigens include production facilities and their maintenance, labor, equipment and quality control.. We sought to develop a framework to rapidly and cost-efficiently address challenges in developing low-cost formulations of recombinant protein vaccine candidates including using (1) adjuvants to augment immune responses resulting in antigen sparing (i.e., more doses per mg of antigen),5e7 and (2) preservatives to develop multi-dose presentations (i.e., more doses per vial of vaccine)
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