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Abstract:
The avian designer cells AGE1.CR and AGE1.CR.pIX (duck) were evaluated over the last years for production of influenza and vaccinia virus. With regards to vaccinia replication, the modified cell line AGE1.CR.pIX shows higher specific productivity [1,2], which was expected because the pIX protein is known to stabilize the virus capsid. Both cells grow in suspension in a chemically defined medium. Along with the design of an efficient production process, efforts were made regarding media optimization for cell proliferation and production. Therefore, the metabolism during cell growth was examined carefully to reveal differences between both producer cell lines as well as differences between cultivation systems using systems biology approaches.
Here, we present results for cultivations of AGE1.CR and AGE1.CR.pIX cells in 1 L stirred tank reactor and 1 L wave bioreactor. Focus was on metabolic flux analysis during batch growth. Biomass and elemental composition of avian cells were determined experimentally and showed to be comparable to mammalian cells. On the basis of networks describing metabolism of other eukaryotic cells and several databases, a stoichiometric model of the central metabolism of duck cells was derived. It comprises 70 metabolites and 101 reactions, including the avian-specific uric acid synthesis. All reactions included were checked against annotated genomes of avian species in the KEGG database.
Cell concentration, cell diameter and concentrations of extracellular metabolites and amino acids were measured in the exponential growth phase to characterize the metabolic profile of the cells. A Monte Carlo based approach was used to compute intracellular flux distributions based on extracellular rates taking into account standard deviations of assays [3]. The computed flux distributions were (despite some specifics of avian metabolism) comparable with published data of mammalian cell lines. Comparison of wave bioreactor and stirred tank reactor cultivations revealed some differences in the flux distribution. In particular, shear stress and aeration conditions seem to lead to changes in metabolism. Differences between both cell lines cultivated in wave bioreactor were observed for example for the consumption rate of glucose which was higher in the modified AGE1.CR.pIX cells. The intracellular rates of these cells also reflected an overall increased metabolism. This indicates that the modified cell line needs slightly more substrates to constituently produce the pIX protein. Therefore, when using the AGE1.CR.pIX cells, the higher metabolic activity has to be taken into account for medium design and process optimization.
