The transient expression of CHIKV VLP in large stirred tank bioreactors
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.
KeywordsChikungunya virus Virus-like particle vaccine Transient gene expression DNA and PEI pH Shear stress Scale-up challenge ambr® 15 bioreactor Large stirred tank bioreactor
We thank Robin Luedtke and Diane Wycuff (VPP/VRC/NIH) for reviewing the manuscript.
Compliance with ethical standards
Conflict of interest
The authors declare no competing financial interest.
- Backliwal G, Hildinger M, Chenuet S, Wulhfard S, De Jesus M, Wurm FM (2008a) Rational vector design and multi-pathway modulation of HEK 293E cells yield recombinant antibody titers exceeding 1 g/l by transient transfection under serum-free conditions. Nucleic Acids Res 36:e96. https://doi.org/10.1093/nar/gkn423 CrossRefPubMedPubMedCentralGoogle Scholar
- Cervera L, Gutiérrez-Granados S, Martínez M, Blanco J, Gòdia F, Segura MM (2013) Generation of HIV-1 Gag VLPs by transient transfection of HEK 293 suspension cell cultures using an optimized animal-derived component free medium. J Biotechnol 166:152–165. https://doi.org/10.1016/j.jbiotec.2013.05.001 CrossRefPubMedGoogle Scholar
- de los Milagros Bassani Molinas M, Beer C, Hesse F, Wirth M, Wagner R (2014) Optimizing the transient transfection process of HEK-293 suspension cells for protein production by nucleotide ratio monitoring. Cytotechnology 66:493–514. https://doi.org/10.1007/s10616-013-9601-3 CrossRefPubMedGoogle Scholar
- Delouvroy F, Siriez G, Tran A-V, Mukankurayija L, Kochanowski N, Malphettes L (2015) ambr™ Mini-bioreactor as a high-throughput tool for culture process development to accelerate transfer to stainless steel manufacturing scale: comparability study from process performance to product quality attributes. BMC Proc 9:P78. https://doi.org/10.1186/1753-6561-9-s9-p78 CrossRefPubMedCentralGoogle Scholar
- Janakiraman V, Kwiatkowski C, Kshirsagar R, Ryll T, Huang YM (2015) Application of high-throughput mini-bioreactor system for systematic scale-down modeling, process characterization, and control strategy development. Biotechnol Prog 31:1623–1632. https://doi.org/10.1002/btpr.2162 CrossRefPubMedGoogle Scholar
- Muller N, Derouazi M, Van Tilborgh F, Wulhfard S, Hacker DL, Jordan M, Wurm FM (2007) Scalable transient gene expression in Chinese hamster ovary cells in instrumented and non-instrumented cultivation systems. Biotechnol Lett 29:703–711. https://doi.org/10.1007/s10529-006-9298-x CrossRefPubMedGoogle Scholar
- Pham PL, Perret S, Doan HC, Cass B, St-Laurent G, Kamen A, Durocher Y (2003) Large-scale transient transfection of serum-free suspension-growing HEK293 EBNA1 cells: peptone additives improve cell growth and transfection efficiency. Biotechnol Bioeng 84:332–342. https://doi.org/10.1002/bit.10774 CrossRefPubMedGoogle Scholar
- Rajendra Y, Hougland MD, Alam R, Morehead TA, Barnard GC (2015) A high cell density transient transfection system for therapeutic protein expression based on a CHO GS-knockout cell line: process development and product quality assessment. Biotechnol Bioeng 112:977–986. https://doi.org/10.1002/bit.25514 CrossRefPubMedGoogle Scholar
- Shaddeau AW, Schneck NA, Li Y, Ivleva VB, Arnold FJ, Cooper JW, Lei QP (2019) Development of a new tandem ion exchange and size exclusion chromatography method to monitor vaccine particle titer in cell culture media. Anal Chem 91:6430–6434. https://doi.org/10.1021/acs.analchem.9b00095 CrossRefPubMedGoogle Scholar
- Tait AS, Brown CJ, Galbraith DJ, Hines MJ, Hoare M, Birch JR, James DC (2004) Transient production of recombinant proteins by Chinese hamster ovary cells using polyethyleneimine/DNA complexes in combination with microtubule disrupting anti-mitotic agents. Biotechnol Bioeng 88:707–721. https://doi.org/10.1002/bit.20265 CrossRefPubMedGoogle Scholar