Abstract
Transient gene expression (TGE) bioprocesses have been difficult to scale up in large stirred tank bioreactors with volumes of more than 1.5 L. Low production levels are often observed, but the causes have not been investigated (Gutierrez-Granados et al. in Crit Rev Biotechnol 38:918–940, 2018). Chikungunya Virus-like particle (VLP), expressed by DNA–PEI transient transfection, is a representative case study for these difficulties. Clinical materials were produced in shake flasks, but the process suffered when transferred to large stirred tank bioreactors. The resulting process was not operationally friendly nor cost effective. In this study, a systematic approach was used to investigate the root causes of the poor scale up performance. The transfection conditions were first screened in ambr® 15 high throughput mini bioreactors then examined in 3 L stirred-tank systems. The studies found that production level was negatively correlated with inoculum cell growth status (P < 0.05). The pH range, DNA and PEI levels, order of the reagent addition, and gas-sparging systems were also studied and found to affect process performance. Further hydromechanical characterizations (Re, energy dissipation rates, and P/V, etc.) of shake flasks, ambr® 15, and 3-L stirred tank systems were performed. Overall, the study discovered that the shear stress (caused by a microsparger) and PEI toxicity together were the root causes of scale-up failure. Once the microsparger was replaced by a macrosparger, the scale-up was successful.
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We thank Robin Luedtke and Diane Wycuff (VPP/VRC/NIH) for reviewing the manuscript.
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PC designed the study, performed the experiments, analyzed the data, and wrote the manuscript. JD assisted part of the experiments. VBI performed the product titer analysis. JH, RS, and FA reviewed the manuscript/played management roles.
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Chen, P., Demirji, J., Ivleva, V.B. et al. The transient expression of CHIKV VLP in large stirred tank bioreactors. Cytotechnology 71, 1079–1093 (2019). https://doi.org/10.1007/s10616-019-00346-x
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DOI: https://doi.org/10.1007/s10616-019-00346-x