Highly Efficient Production of an Influenza H9N2 Vaccine Using MDCK Suspension Cells

Author:  Yixiao Wu, Hanjing Jia, Hanzhang Lai, Xuping Liu*, Wen-Song Tan.
Bioresources and Bioprocessing, 2020, 7: 63

Abstract
The use of H9N2 subtype avian influenza vaccines is an effective approach for the control of the virus spread among poultry, and for the upgrading of vaccine manufacturing, cell culture-based production platform could overcome the limitations of conventional egg-based platform and alternate it. The development of serum-free suspension cell culture could allow even higher virus productivity, where a suspension cell line with good performance and proper culture strategies are required. In this work, an adherent Mardin–Darby canine kidney (MDCK) cell line was adapted to suspension growth to cell concentration up to 12 × 106 cells/mL in a serum-free medium in batch cultures. Subsequently, the H9N2 influenza virus propagation in this MDCK cell line was evaluated with the optimization of infection conditions in terms of MOI and cell concentration for infection. Furthermore, various feed strategies were tested in the infection phase for improved virus titer and a maximum hemagglutinin titer of 13 log2 (HAU/50 μL) was obtained using the 1:2 medium dilution strategy. The evaluation of MDCK cell growth and H9N2 virus production in bioreactors with optimized operating conditions showed comparable cell performance and virus yield compared to shake flasks, with a high cell-specific virus yield above 13,000 virions/cell. With the purified H9N2 virus harvested from the bioreactors, the MDCK cell-derived vaccine was able to induce high titers of neutralizing antibodies in chickens. Overall, the results demonstrate the promising application of the highly efficient MDCK cell-based production platform for the avian influenza vaccine manufacturing.

Efficient Production of H9N2 Influenza Vaccine Using MDCK Suspension Cells
The use of H9N2 subtype avian influenza vaccines is an effective approach for the control of the virus spread among poultry, and the cell culture production platform can overcome the limitations of the traditional egg-based platform and replace it, thereby upgrading the vaccine manufacturing process. The development of serum-free suspension cell culture technology, especially the development of high-yield suspension cell lines and the optimization of culture strategies, can help improve virus yield. In this study, a adherent MDCK cell line was acclimated to serum-free suspension culture, achieving a maximum cell density of 12×10^6 cells/mL under batch culture conditions. Subsequently, the amplification of H9N2 influenza virus in the MDCK suspension cell line was evaluated, and infection conditions were optimized in terms of MOI and cell density at the time of infection. In addition, to increase virus titer during the infection phase, different feeding strategies were investigated, and it was found that the 1:2 medium dilution strategy resulted in the highest HA titer of 13 log2 (HAU/50 μL). Further comparison between the bioreactor and shake flask culture systems, optimized for operational conditions, showed that the performance and virus yield of MDCK cells were comparable, but the virus yield per cell in the bioreactor was higher, reaching over 13,000 virions/cell. Finally, the H9N2 virus harvested and purified in the bioreactor, namely the vaccine produced by MDCK cells, was able to induce high-titer neutralizing antibodies in chickens. In summary, these results demonstrate the broad prospects of the MDCK cell high-efficiency production platform in the application of avian influenza vaccine production.