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Introduction
Modeling supports the quantitative understanding of complex biological processes. A quantitative model helps to understanding and it can be used to improve and engineer process conditions. Here metabolic flux analysis is applied to analyze the metabolism of an adherent MDCK cell line used in influenza vaccine production. The cultivation of adherent cell lines can hardly be performed under strict stationary conditions; batch and fed-batch cultivations are instationary. Therefore we define quasi stationary phases where the fluxes can be assumed to be constant. Three phases are used: In the first phase the cells attach to the surface and already show some moderate metabolism. In the second phase we assume exponential growth, in the last phase inhibited growth. The metabolic network model is applied to these phases at standard process conditions, with additional pyruvat in the media and compared to some theoretical scenarios.
Metabolic network model and theoretical calculations
The metabolic model applied to the different phases is derived from literature and online databases. For clarity some linear reaction sequences were lumped. Two compartments (cytosol and mitochondria) are taken into account. In contrast to most metabolic networks, the transport reactions and its energy needs are considered.
For the interpretation of the measured flux distributions the calculated values are compared to theoretically optimal flux distributions. Minimal input fluxes are calculated that will provide enough energy and precursors to reach the observed growth rates.
The model suggests optimal biomass yields with glucose as the main energy source, isoleucine and lysine as secondary sources. Instead of excreting lactate (to reduce the excess of NADH in the cytosol), the optimal flux distribution suggests the transport of glycolytic NADH into the mitochondria via malate (originating from PEP) that can pass the membrane via the dicarboxylate carrier. In the mitochondria the malate dehydrogenase will be used to regenerate NADH in the mitochondria. The respiratory chain will then provide enough ATP for protein assembly. However, the measured fluxes differ significantly. Already from glycolysis ATP is generated in abundance though the TCA reactions would regenerate ATP more effective. Thus, substrates that enter in the lower part of glycolysis (as shown for pyruvate) or into the TCA (e.g. isoleucine) should be taken to more effectively use the carbon source and finally reduce lactate excretion from overflow metabolism.
