Vaccines, along with most other protein biologics, are currently manufactured in large-scale, centralized facilities. Such manufacturing paradigms lack the flexibility and agility required to quickly address global epidemics and have led to stagnant coverage and missed immunization targets due to failures in campaigns or supply chains. Multivalent vaccines are particularly challenging since each subunit is manufactured individually, requiring multiple campaigns to produce a single batch of drug product. New manufacturing methods, and associated process development strategies, are needed to improve the flexibility and dependability of vaccine manufacturing at low costs. We have previously demonstrated a small-scale, integrated and automated manufacturing platform capable of rapidly producing a variety of recombinant proteins with phase-appropriate quality for early development. Our platform comprises three modules for fermentation via perfusion, straight-through chromatographic purification, and formulation combined with a holistic strategy for process design. Here, we demonstrate the application of our platform to manufacture a trivalent rotavirus vaccine in a single campaign through co-expression and co-purification. We first developed and deployed processes for the production of each individual subunit antigen, while maintaining similarity in process operating conditions. Our initial processes showed a 40% reduction in the number of steps as compared to the original manufacturing method for these molecules. While process-related variants were removed, product-related variants including <i>N</i>-terminal truncation, glycosylation, and aggregation persisted. In order to maintain process conditions compatible with all three subunits, we addressed product-related variants at the molecular level by strategically altering specific amino acids. We then created a single <i>Pichia pastoris</i> strain that expressed all three engineered subunits. Finally, a single manufacturing campaign was executed on our system. Successful co-expression and co-purification of all three subunits was confirmed by RPLC, SEC and LC-MS. Process-related impurities were reduced below typical regulatory guidelines. We believe that this work demonstrates the feasibility of manufacturing a multivalent vaccine in a single campaign. We hope that such process intensification will enable more flexible and agile manufacturing of vaccines to meet global needs.

Manufacturing of a trivalent subunit vaccine in a single campaign

ACS Fall 2020 Virtual Meeting

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