Abstract
To meet the challenges of global health, vaccine design and development must be reconsidered to achieve cost of goods as low as 15¢ per dose. A new recombinant protein‐based rotavirus vaccine candidate derived from non‐replicative viral subunits fused to a P2 tetanus toxoid CD4(+) T cell epitope is currently under clinical development. We have sought to simplify the existing manufacturing process to meet these aims. To this end, we have taken a holistic process development approach to reduce process complexity and costs while producing a product with the required characteristics. We have changed expression system from Escherichia coli to Pichia pastoris, to produce a secreted product, thereby reducing the number of purification steps. However, the presence of proteases poses challenges to product quality. To understand the effect of fermentation parameters on product quality small‐scale fermentations were carried out. Media pH and fermentation duration had the greatest impact on the proportion of full‐length product. A novel acidic pH pulse strategy was used to minimize proteolysis, and this combined with an early harvest time significantly increased the proportion of full‐length material (60–75%). An improved downstream process using a combination of CIEX and AIEX to further reduce proteases, resulted in maintaining product quality (95% yield).
Highlights
A Non Replicative Rotavirus Vaccine (NRRV) subunit vaccine was expressed in P. pastoris using an α-mating factor secretion signal, resulting in the protein of interest being secreted from the cells
Molecules of 20,517 and 20,386 Da were both considered as full-length P2-VP8-P[4], as the methionine (131 Da) on the N-terminal was an artifact of cloning from E. coli to P. pastoris (Figure 2b and d)
Proteases can be problematic during the production of secreted heterologous proteins in P. pastoris
Summary
The Ultra Low-cost Transferable Automated (ULTRA) Platform for Vaccine Manufacturing seeks to combine integrated, automated, modular manufacturing to achieve the aims of the Bill & Melinda Gates. Foundation Grand Challenge—“Innovations in Vaccine Manufacturing for Global Markets”. The stated aim of which is “to create a new manufacturing platform to enable ultra-low cost, high quality, recombinant subunit vaccines at 15¢ (USD) a dose for global health initiatives.”. Our initial model system to demonstrate this approach is the production of a recombinant subunit rotavirus vaccine candidate The stated aim of which is “to create a new manufacturing platform to enable ultra-low cost, high quality, recombinant subunit vaccines at 15¢ (USD) a dose for global health initiatives.” Our initial model system to demonstrate this approach is the production of a recombinant subunit rotavirus vaccine candidate
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