Highly efficient influenza virus production: A MDCK-based high-cell-density process
June 17-22, 2018
Seasonal vaccination campaigns for influenza in developed and developing countries create a massive demand for 500 million (2015) vaccine doses every year . Besides egg-based vaccine manufacturing, production platforms based on animal cell culture increasingly contribute to this overall growing market. In order to intensify cell culture-based influenza virus production, high-cell-density (HCD) cultivation of suspension cells can be applied to improve virus titer, process productivity and production costs . For process optimization and evaluation of HCD conditions, cells cultivated using semi-perfusion approaches in small shakers can be used as a scale-down model for bioreactors operating in full perfusion mode .
In this study, a previously developed MDCK suspension cell line  was adapted to a new serum free medium  to facilitate higher growth rate, cell density and virus titer both in batch and in HCD. Therefore, MDCK cells cultivated in Smif-8 medium were slowly adapted to a new cultivation medium (Xeno™) by stepwise increasing the Xeno content. Fully adapted cells were cultivated in shaker flasks to evaluate the performance of influenza A virus production in batch and HCD. Cell densities exceeding 2∙107 cells/mL were achieved in shakers using semi-perfusion, where cell free medium was manually replaced with fresh medium. Volume and time interval of media replacement were chosen to achieve a constant cell-specific perfusion rate of 2.5 pL/(cell h). Cell cultures were infected with influenza virus (A/PR/8/34 H1N1 RKI) with trypsin addition. Cell count, viability, main metabolites and virus titer (HA-assay & TCID50) were monitored pre and post infection.
Medium adaptation resulted in a MDCK suspension cell line with morphological, growth, and metabolic characteristics different from parental cells. Cells fully adapted to Xeno medium were growing to higher cell densities (1.4∙107 vs 6∙106 cells/mL) with higher specific growth rate (µmax: 0.036 vs 0.026 1/h), cells were bigger (15-16 vs 13-14 µm) and grew without aggregate formation. Due to higher cell densities at time of infection, virus titers up to 3.6 log10(HAU/100µL) were reached. In semi-perfusion, adapted MDCK cells were grown up to 6∙107 cells/mL, however, maximum virus titer and productivity were observed with 4∙107 cells/mL. In multiple harvests, very high virus titer exceeding 4 log10(HAU/100µL) and up to 9∙109 virions/mL (TCID50) were measured, which corresponded to an accumulated titer of 4.5 log10(HAU/100µL). Cell-specific virus titer was similar or higher compared to the reference batch infections, depending on perfusion and infection strategy.
Overall, results in this semi-perfusion scale-down model for influenza A virus production suggest a highly efficient and productive upstream process for influenza virus production, with an up to six-fold improved space time yield compared to batch mode.
 Palache A. et al., Vaccine 35 (2017): 4681–4686. doi: 10.1016/j.vaccine.2017.07.053
 Genzel Y. et al., Vaccine 32 (2014): 2770–2781. doi: 10.1016/j.vaccine.2014.02.016
 Vázquez-Ramírez D. et al., Vaccine (2018): article in press. doi: 10.1016/j.vaccine.2017.10.112
 Lohr V. et al., Vaccine 28 (2010): 6256–6264. doi: 10.1016/j.vaccine.2010.07.004
 Xeno™-S001S MDCK Cell Serum Free Medium (#FG0100402), Bioengine, Shanghai, China
Thomas Bissinger, Udo Reichl, Yvonne Genzel, Yixiao Wu, Xuping Liu, and Wen-Song Tan, "Highly efficient influenza virus production: A MDCK-based high-cell-density process" in "Vaccine Technology VII", Amine Kamen, McGill University Tarit Mukhopadhyay, University College London Nathalie Garcon, Bioaster Charles Lutsch, Sanofi Pasteur Eds, ECI Symposium Series, (2018). http://dc.engconfintl.org/vt_vii/13