The avian cell line AGE1.CR.pIX characterized by metabolic flux analysis

Abstract

BackgroundIn human vaccine manufacturing some pathogens such as Modified Vaccinia Virus Ankara, measles, mumps virus as well as influenza viruses are still produced on primary material derived from embryonated chicken eggs. Processes depending on primary cell culture, however, are difficult to adapt to modern vaccine production. Therefore, we derived previously a continuous suspension cell line, AGE1.CR.pIX, from muscovy duck and established chemically-defined media for virus propagation.ResultsTo better understand vaccine production processes, we developed a stoichiometric model of the central metabolism of AGE1.CR.pIX cells and applied flux variability and metabolic flux analysis. Results were compared to literature dealing with mammalian and insect cell culture metabolism focusing on the question whether cultured avian cells differ in metabolism. Qualitatively, the observed flux distribution of this avian cell line was similar to distributions found for mammalian cell lines (e.g. CHO, MDCK cells). In particular, glucose was catabolized inefficiently and glycolysis and TCA cycle seem to be only weakly connected.ConclusionsA distinguishing feature of the avian cell line is that glutaminolysis plays only a minor role in energy generation and production of precursors, resulting in low extracellular ammonia concentrations. This metabolic flux study is the first for a continuous avian cell line. It provides a basis for further metabolic analyses to exploit the biotechnological potential of avian and vertebrate cell lines and to develop specific optimized cell culture processes, e.g. vaccine production processes.

A dynamic model linking cell growth to intracellular metabolism and extracellular by‐product accumulation

“…Interestingly, even after glutamine depletion, glutamate still accumulated. This phenomenon is not uncommon cell cultivations as glutamate is a nonessential amino acid and it is also produced from sources other than glutamine, for example, through lysine and proline catabolism (Lohr et al, 2014; Sonnewald, 2014). Although extracellular amino acids were not quantified in these experiments, batch cultivations with a similar medium composition performed for this cell line demonstrated that most amino acids were not depleted at the end of exponential cell growth phase (Priesnitz et al, 2012).…”

High-cell-density cultivations to increase MVA virus production

“…4B and D). Also, it has been reported that glutamine plays a minor role in energy supply for CR.pIX cells and that its absence might not influence the propagation of MVA in this cell line [29]. Therefore, the positive effect of a medium exchange at TOI on virus propagation might be also due to the dilution of inhibitory metabolites or signaling molecules rather than just a better supply of substrates.…”

“…Feeding was based on a constant cell-specific perfusion rate (CSPR) [27] taking into account the steady state mass balance for substrates as described by Kompala and Ozturk [28] with glucose as the major energy source for CR.pIX cells [29] as:…”

Designing cell lines for viral vaccine production: Where do we stand?

“…In the variant called AGE1.CR.pIX, the human adenovirus serotype 5 structural gene pIX is additionally added to increase possible virus titers by the action of the pIX protein [55]. The cells were grown in suspension first in serum‐free medium, and later in CDM [56], reaching up to 9×10 6 cells/mL in batch mode [57]. Cell numbers of up to 5×10 7 cells/mL in this CDM in perfusion have recently been reached [34, 35].…”