Understanding dynamics of virus propagation in plate and suspension cell cultures to improve upstream biomanufacturing performance
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Authors
Samuel, Shiny, Joan
Issue Date
2024-08
Type
Electronic thesis
Thesis
Thesis
Language
en_US
Keywords
Chemical engineering
Alternative Title
Abstract
Virus replication is pivotal in two broad aspects of biomanufacturing: detecting viral adventitious agents and producing therapeutics such as vaccines and viral vectors. Although rare, viral contamination poses significant risks to patient safety and has substantial economic consequences, necessitating routine virus testing at various production stages. The current state-of-the-art for general virus screening is the in vitro virus (IVV) assay, which is labor- and time-intensive. In the first aim, a discrete model coupled with a partial differential equation solver was employed to simulate a monolayer of CHO-K1 cells and reovirus-3 diffusion in an IVV system. Model predictions and analysis indicated that virus transport to the cell surface is a major limitation, resulting in a lower initial infection rate than previous works have modeled. Model output was used to enhance the sensitivity of laser force cytology (LFC), an alternative detection technique. Results suggested that the current sampling rate may be too low to distinguish infection accurately. The second aim focuses on biomarker discovery for adventitious virus detection by examining miRNA and mRNA expression in infected CHO-K1 cells. Nine miRNA biomarkers of infection were identified across a panel of viruses (Reo-3, MVM, EMCV, and PIV-2) for general detection. Gene prediction analysis of differentially expressed miRNAs in early infection suggested their involvement in pro-viral mechanisms, while miRNAs in late infection were implicated in cellular stress responses. Moreover, miR-484, miR-409-3p, miR-450b-5p, and miR-21-3p were differentially expressed as early as day 1 post-infection in some viruses. This is well ahead of the appearance of virus-induced morphological changes in cells that are typically used for detecting infection. The third aim addresses optimizing virus yields in suspension cell culture-based vaccine production via mathematical modeling. A model of recombinant measles virus production in Vero cells predicted the optimal time of harvest for five bioreactor runs despite significant run-to-run variation. Sensitivity analysis indicated that virus attachment parameters and the degradation rate of the virus influence infection dynamics across the bioreactor run. Moreover, the model underscores the importance of accurately characterizing seed virus quality, particularly for defective interfering particle (DIP) content, to reduce run-to-run variation in production and maximize yield. Overall, this work advances the understanding of viral kinetics, providing critical insights for enhancing adventitious virus detection methods and optimizing virus production processes in biomanufacturing.
Description
August 2024
School of Engineering
School of Engineering
Full Citation
Publisher
Rensselaer Polytechnic Institute, Troy, NY