Efficient influenza vaccine manufacturing: Single MDCK suspension cells in chemically defined medium

Facing the constant global high demand for influenza vaccines, improving production capacity is most important. For influenza vaccine production, cell culture-based processes have advantages regarding flexibility, efficiency, and safety in comparison with the traditional egg-based processes. To avoid problems related to microcarrier-based approaches and serum containing media, growth of suspension cells in chemically-defined media is favoured. In addition, such a process has advantages regarding the improvement of virus titers, the scale-up of the production process, and overall productivity in up- and downstream processing. In this study, a previously developed MDCK suspension cell line [1] was cultivated in an in-house chemically defined medium to evaluate cell growth and virus production. For the purpose of process intensification, virus adaptation and infection strategies were investigated to achieve high cell densities and to maximize virus titers. Therefore, an adapted influenza virus strain (A/PR/8/34 H1N1 RK1) was generated by a series of virus passages with low multiplicity of infection (MOI). Virus infections were carried out by supplementing 100% of fresh medium, infecting cells with a MOI of 10-3, and with trypsin addition at 72 h of cell cultivations in shake flasks and bioreactors. For scale-up, MDCK cells were cultivated in a DASGIP bioreactor system, optimizing stirring speed, time of infection, pH and DO levels both for cell growth and virus infection. Cell count, viability, main extracellular metabolites, and virus titers were measured to compare productivity between shake flasks and bioreactors. In batch culture (shake flasks and bioreactors), single MDCK cells were grown to maximum cell densities of 1.2 x107 cells/ml with cell viabilities exceeding 98% at high cell specific growth rates of 0.036 h-1. Virus adaptation to the MDCK suspension cell line led to a fast infection and stable virus titers over time. Regarding process optimization, optimal pH (cell growth: 7.00, infection: 7.20), DO (40%) and agitation speed (80 rpm) were chosen for influenza A virus production in three parallel bioreactors. Cell densities of 1.0 x107 cells/ml were achieved at time of infection (72 h) before performing a dilution step. Post infection, similar virus infection dynamics were observed in shake flasks and bioreactors. For both cultivation systems maximal HA titers of 3.6 log10(HAU/100µl) were achieved without reduction of cell-specific virus titer (12,000 virions/cell). Overall, a highly efficient and scalable upstream process was realized by cultivation of MDCK suspension cells as single cells in chemically defined medium. This is a strong basis for a promising application in large-scale influenza vaccine manufacturing and potential process intensification towards high cell density virus production. [1] Huang D. et al., PloS One 10 (2015): e0141686. doi: 10.1371/journal.pone.014168