Ilona Behrendt

A new MDCK suspension line cultivated in a fully defined medium in stirred-tank and wave bioreactor

An adherently growing MDCK cell line was adapted in a two-step process in a fully defined medium and in suspension. The resulting MDCK.SUS2 cells were subsequently evaluated for their potential as host cells for influenza vaccine production in two lab-scale bioreactors (wave and stirred-tank). Cell concentrations up to 2.3 x 10(6)cells/mL were obtained after 96 h, which is slightly higher than cell concentrations obtained with adherent MDCK cells cultivated on microcarriers (2g/L). Infections with influenza A/PR/8/34 and B/Malaysia resulted in high virus titers (2.90 and 2.75 log HA units/100 microL, respectively). The monitoring of extracellular metabolites, including amino acids, revealed a change in some of the metabolite consumption or release profiles, which indicates changes in metabolism during the adaptation process. Overall, the MDCK.SUS2 cell line represents a new cell substrate for a robust influenza vaccine production in a fully defined process.

High cell density cultivations by alternating tangential flow (ATF) perfusion for influenza A virus production using suspension cells

High cell densities in animal cell culture can be obtained by continuous perfusion of fresh culture medium across hollow fiber membranes that retain the cells. Careful selection of the membrane type and cut-off allows to control accumulation of target molecules and removal of low molecular weight compounds. In this report, perfusion with the scalable ATF (alternating tangential filtration, Refine Technology) system was evaluated for two suspension cell lines, the avian cell line AGE1.CR and the human cell line CAP. Both were cultivated in chemically defined media optimized for batch cell growth in a 1L stirred tank bioreactor connected to the smallest ATF unit (ATF2) and infected with cell line-adapted human influenza A virus (A/PR/8/34 (H1N1), typical diameter: 80-100 nm). At concentrations of about 25 million cells/mL three different membrane cut-offs (50 kDa, 0.2 μm and 0.5 μm) were tested and compared to batch cultivations performed at 5 million cells/mL. For medium and large cut-offs no cell-density effect could be observed with cell-specific virus yields of 1428-1708 virions/AGE1.CR cell (infected with moi 0.001) and 1883-4086 virions/CAP cell (moi of 0.025) compared to 1292 virions/AGE1.CR cell and 3883 virions/CAP cell in batch cultures. Even at a concentration of 48 million AGE1.CR cells/mL (cut-off: 0.2 μm) a cell-specific yield of 1266 virions/cell was reached. Only for the small cut-off (50 kDa) used with AGE1.CR cells a decrease in cell-specific yield was measured with 518 virions/cell. Surprisingly, the ratio of infectious to total virions seemed to be increased in ATF compared to batch cultures. AGE1.CR cell-derived virus particles were present in the permeate (0.2 and 0.5 μm cut-off), whereas CAP cell-derived virions were not, suggesting possible differences in morphology, aggregation or membrane properties of the virions released by the two cell lines. To our knowledge, this is the first study that illustrates the potential of ATF-based perfusion of chemically defined media across cell-retaining membranes for production of an influenza A vaccine.

Continuous Influenza Virus Production in Cell Culture Shows a Periodic Accumulation of Defective Interfering Particles

Influenza viruses are a major public health burden during seasonal epidemics and a continuous threat due to their potential to cause pandemics. Annual vaccination provides the best protection against the contagious respiratory illness caused by influenza viruses. However, the current production capacities for influenza vaccines are insufficient to meet the increasing demands. We explored the possibility to establish a continuous production process for influenza viruses using the duck-derived suspension cell line AGE1.CR. A two-stage bioreactor setup was designed in which cells were cultivated in a first stirred tank reactor where an almost constant cell concentration was maintained. Cells were then constantly fed to a second bioreactor where virus infection and replication took place. Using this two-stage reactor system, it was possible to continuously produce influenza viruses. Surprisingly, virus titers showed a periodic increase and decrease during the run-time of 17 days. These titer fluctuations were caused by the presence of defective interfering particles (DIPs), which we detected by PCR. Mathematical modeling confirmed this observation showing that constant virus titers can only emerge in the absence of DIPs. Even with very low amounts of DIPs in the seed virus and very low rates for de novo DIP generation, defective viruses rapidly accumulate and, therefore, represent a serious challenge for continuous vaccine production. Yet, the continuous replication of influenza virus using a two-stage bioreactor setup is a novel tool to study aspects of viral evolution and the impact of DIPs.

Production of high-titer human influenza A virus with adherent and suspension MDCK cells cultured in a single-use hollow fiber bioreactor.

Hollow fiber bioreactors (HFBRs) have been widely described as capable of supporting the production of highly concentrated monoclonal antibodies and recombinant proteins. Only recently HFBRs have been proposed as new single-use platforms for production of high-titer influenza A virus. These bioreactors contain multiple hollow fiber capillary tubes that separate the bioreactor in an intra- and an extra-capillary space. Cells are usually cultured in the extra-capillary space and can grow to a very high cell concentration. This work describes the evaluation of the single-use hollow fiber bioreactor PRIMER HF (Biovest International Inc., USA) for production of influenza A virus. The process was setup, characterized and optimized by running a total of 15 cultivations. The HFBRs were seeded with either adherent or suspension MDCK cells, and infected with influenza virus A/PR/8/34 (H1N1), and the pandemic strain A/Mexico/4108/2009 (H1N1). High HA titers and TCID₅₀ of up to 3.87 log₁₀(HA units/100 μL) and 1.8 × 10(10)virions/mL, respectively, were obtained for A/PR/8/34 influenza strain. Influenza virus was collected by performing multiple harvests of the extra-capillary space during a virus production time of up to 12 days. Cell-specific virus yields between 2,000 and 8,000 virions/cell were estimated for adherent MDCK cells, and between 11,000 and 19,000 virions/cell for suspension MDCK.SUS2 cells. These results do not only coincide with the cell-specific virus yields obtained with cultivations in stirred tank bioreactors and other high cell density systems, but also demonstrate that HFBRs are promising and competitive single-use platforms that can be considered for commercial production of influenza virus.