A Bioreactor-Based Yellow Fever Virus-like Particle Production Process with Integrated Process Analytical Technology Based on Transient Transfection.

Yellow Fever (YF) is a severe disease that, while preventable through vaccination, lacks rapid intervention options for those already infected. There is an urgent need for passive immunization techniques using YF-virus-like particles (YF-VLPs). To address this, we successfully established a bioreactor-based production process for YF-VLPs, leveraging transient transfection and integrating Process Analytical Technology. A cornerstone of this approach was the optimization of plasmid DNA (pDNA) production to a yield of 11 mg/L using design of experiments. Glucose, NaCl, yeast extract, and a phosphate buffer showed significant influence on specific pDNA yield. The preliminary work for VLP-production in bioreactor showed adjustments to the HEK cell density, the polyplex formation duration, and medium exchanges effectively elevated transfection efficiencies. The additive Pluronic F-68 was neutral in its effects, and anti-clumping agents (ACA) adversely affected the transfection process. Finally, we established the stirred-tank bioreactor process with integrated dielectric spectroscopy, which gave real-time insight in relevant process steps, e.g., cell growth, polyplex uptake, and harvest time. We confirmed the presence and integrity of YF-VLP via Western blot, imaging flow cytometry measurement, and transmission electron microscopy. The YF-VLP production process can serve as a platform to produce VLPs as passive immunizing agents against other neglected tropical diseases.