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.

Efficient Transfection via an Unexpected Mechanism by Near Neutral Polypiperazines with Tailored Response to Endosomal pH.

Cationic pH-responsive polymers promise to overcome critical challenges in cellular delivery. Ideally, the polymers become selectively charged along the endosomal pathway disturbing only the local membrane and avoiding unintended interactions or cytotoxic side effects at physiological conditions. Polypiperazines represent a novel, hydrophilic class of pH-responsive polymers whose response can be tuned within the relevant pH range (5-7.4). The authors discovered that the polypiperazines are effectively binding plasmid DNA (pDNA) and demonstrate high efficiency in transfection. By design of experiments (DoE), a wide parameter space (pDNA and polymer concentration) is screened to identify the range of effective concentrations for transfection. An isopropyl modified polypiperazine is highly efficient over a wide range of concentrations outperforming linear polyethylenimine (l-PEI, 25 kDa) in regions of low N*/P ratios. A quantitative polymerase chain reaction (qPCR) surprisingly revealed that the pDNA within the piperazine-based polyplexes can be amplified in contrast to polyplexes based on l-PEI. The pDNA must therefore be more accessible and bound differently than for other known transfection polymers. Considering the various opportunities to further optimize their structure, polypiperazines represent a promising platform for designing effective soluble polymeric vectors, which are charge-neutral at physiological conditions.

Statistical experimental designs to optimize the transient transfection of HEK 293T cells and determine a transfer criterion from adherent cells to larger-scale cell suspension cultures.

The transient transfection of mammalian cells is a rapid and versatile platform for the manufacture of recombinant proteins, but industrial processes depend on reliable scalability and efficient conversion from adherent to suspension cell cultures. Here we describe the optimized transfection of HEK 293T cells in both culture formats. DMEM was the best transfection medium for adherent HEK 293T cells, so we determined the kinetics of linear polyethyleneimine (LPEI) polyplex formation with plasmid DNA (pDNA) and subsequent cellular uptake. Statistical experimental designs revealed optimal transfection efficiency using 0.7 pg pDNA and 4.5 pg LPEI per cell. We used the amount of pDNA and LPEI per cell as the transfer criterion for HEK 293T/17 SF cell suspension cultures in FreeStyle 293 medium and confirmed optimal transfection at 1.1 pg pDNA and 6.6 pg LPEI per cell. We observed a strong correlation between polyplex size, transfection efficiency and post-transfection cell viability. Suspension cell transfection could be scaled to a 100-mL working volume without loss of efficiency. We conclude that pg pDNA and pg LPEI per cell is a suitable transfer criterion allowing the optimization of transient transfection using statistical experimental designs, thus minimizing the amount of pDNA and LPEI used without sacrificing transfection efficiency.

Upstream and Downstream Processes for Viral Nanoplexes as Vaccines.

The increasing medical interest in viral nanoplexes, such as viruses or virus-like particles used for vaccines, gene therapy products, or oncolytic agents, raises the need for fast and efficient production processes. In general, these processes comprise upstream and downstream processing. For the upstream process, efficiency is mainly characterized by robustly achieving high titer yields, while reducing process times and costs with regard to the cell culture medium, the host cell selection, and the applied process conditions. The downstream part, on the other hand, should effectively remove process-related contaminants, such as host cells/cell debris as well as host cell DNA and proteins, while maintaining product stability and reducing product losses. This chapter outlines a combination of process steps to successfully produce virus particles in the controlled environment of a stirred tank bioreactor, combined with a platform-based purification approach using filtration-based clarification and steric exclusion chromatography. Additionally, suggestions for off-line analytics in terms of virus characterization and quantification as well as for contaminant estimation are provided.