Project Summary Influenza A affects 5-30% of the world’s population annually, resulting in 3 to 5 million cases of serious illness and 250,000 to 500,000 deaths each year. Vaccination is the best way to prevent disease. >80% of the influenza vaccines are made in eggs, and this makes the process slow and not able to quickly change as the influenza virus mutates. The vaccine is optimized to grow in eggs, which makes it a less like the circulating virus, and thus reduces the effectiveness of the vaccine. One way to improve the vaccine is to use cell-based vaccines. However, cells-based vaccines are expensive to produce and the manufacturing facilities are expensive. After 10 years on the market, cell-based vaccines are not the dominate vaccine used in the US. We propose to develop a novel manufacturing process of a cell-based influenza virus like particle (VLP). The process will continuously produce the VLP, which will allow for smaller equipment and thus reduce the cost of building a new manufacturing plant. Continuous processing also reduces operating costs, allowing for the vaccine to be sold at a lower cost and likely competing in price with egg-produced vaccines. The VLP will elicit a stronger immune response than egg-based vaccines and the modular design will allow for quick adaption of the vaccine to the circulating influenza strains. Our team has designed a novel, end to end, continuous process to manufacture an influenza VLP. First, we will continuous produce the VLP in a unique, three-reactor bioreactor cascade that will allow for continuous processing using a baculovirus production system in Sf9 insect cells. There is not currently a continuous baculovirus production system in use. The continuous downstream will use aqueous two-phase extraction and other polishing steps to purify the VLP. Chromatography and other periodic operations will be avoided. The VLP will produce HA protein antigenically identical to the chosen circulating strain with no potential for selection of HA mutations and the HA protein can easily be changed to another circulating influenza strain, as needed. Process analytics will be conducted to confirm the purity and antigenicity of the produced influenza VLP. At the completion of this project, the team will run the first end to end continuous process for one month to produce an influenza VLP and the process economics will be evaluated to determine the economic feasibility of the process. The VLP will have superior immunogenicity to egg-derived vaccines and subunit vaccines. This will be the first ever demonstration of a truly end-to-end continuous VLP production process that will revolutionize biologics manufacturing for multiple products, including other vaccines and gene therapy vectors.