Abstract
Similar to the recent COVID-19 pandemic, influenza A virus poses a constant threat to the global community. For the treatment of flu disease, both antivirals and vaccines are available with vaccines the most effective and safest approach. In order to overcome limitations in egg-based vaccine manufacturing, cell culture–based processes have been established. While this production method avoids egg-associated risks in face of pandemics, process intensification using animal suspension cells in high cell density perfusion cultures should allow to further increase manufacturing capacities worldwide. In this work, we demonstrate the development of a perfusion process using Madin-Darby canine kidney (MDCK) suspension cells for influenza A (H1N1) virus production from scale-down shake flask cultivations to laboratory scale stirred tank bioreactors. Shake flask cultivations using semi-perfusion mode enabled high-yield virus harvests (4.25 log10(HAU/100 μL)) from MDCK cells grown up to 41 × 106 cells/mL. Scale-up to bioreactors with an alternating tangential flow (ATF) perfusion system required optimization of pH control and implementation of a temperature shift during the infection phase. Use of a capacitance probe for on-line perfusion control allowed to minimize medium consumption. This contributed to a better process control and a more economical performance while maintaining a maximum virus titer of 4.37 log10(HAU/100 μL) and an infectious virus titer of 1.83 × 1010 virions/mL. Overall, this study clearly demonstrates recent advances in cell culture–based perfusion processes for next-generation high-yield influenza vaccine manufacturing for pandemic preparedness.
Key points
• First MDCK suspension cell–based perfusion process for IAV produciton was established.
• “Cell density effect” was overcome and process was intensified by reduction of medium use and automated process control.
• The process achieved cell density over 40 × 106 cells/mL and virus yield over 4.37 log10(HAU/100 μL).
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The datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request.
Change history
12 May 2021
A Correction to this paper has been published: https://doi.org/10.1007/s00253-021-11286-y
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Acknowledgments
The authors would like to thank Claudia Best, Nancy Wynerski, Susanna Koenig, and Heike Sperlich for their excellent technical assistance in terms of general lab work and virus sample analysis.
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Yixiao Wu acknowledges the financial support from the China Scholarship Council.
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YW, TB, YG, XL, UR, and W-ST conceived and designed the study. YW performed the experiments. YW, TB, YG, and XL analyzed the data. YW wrote the manuscript. All authors read, revised, and approved the manuscript.
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Wen-Song Tan and Xuping Liu are affiliated as directors with Shanghai BioEngine Sci-Tech and were involved in the development of the Xeno-CDM medium both for scientific and commercial purposes. The remaining authors declare that they have no conflict of interest.
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Wu, Y., Bissinger, T., Genzel, Y. et al. High cell density perfusion process for high yield of influenza A virus production using MDCK suspension cells. Appl Microbiol Biotechnol 105, 1421–1434 (2021). https://doi.org/10.1007/s00253-020-11050-8
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DOI: https://doi.org/10.1007/s00253-020-11050-8