Abstract
The analysis of metabolites is an important prerequisite for the optimization of many biotechnological processes, especially in animal cell culture. Apart from monitoring the parameters pH, temperature, oxygen, and carbon dioxide, the medium composition is analyzed in most cases and concentrations of substrates like glucose and glutamine as well as product and other metabolites like lactate and ammonia are measured. However, to get further insights into the metabolic network, it is essential to determine intracellular intermediates of different pathways. In this work, an adherent MDCK cell line, used for the production of influenza vaccine, is examined for intracellular intermediates of the energy metabolism, especially glycolysis and citric acid cycle. This cell line is usually cultivated in batch mode and after a growth period of about one week, cells are infected with virus. Clear differences in the metabolite profiles are expected before and after infection, but also with regard to media composition, e.g. with and without serum. However, in advance to these measurements, a suitable procedure for quenching and extraction has to be identified to assure an acceptable accuracy of the method. In this presentation, first results of our experimental approach are shown. Quantative measurements are performed on two different anion exchange chromatography systems. One system (DX-320, Dionex, Idstein, Germany) is designed for the separation of inorganic ions, organic acids, and energy phosphates using conductivity and UV for detection. The other system (DX-600, Dionex) is suitable for the analysis of sugars and sugar phosphates using a Pulsed Amperometric Detector (PAD). In standard runs, a wide range of intracellular metabolites could be separated and quantified even in concentrations below the micromolar range. However, different chemical and physical properties of the intermediates to measure, as well as the subsecond in-vivo enzyme kinetics for some reactions complicate the finding of a single quenching, washing, and extraction procedure for both chromatography systems. Various strategies and methods are under systematic investigation. After the elucidation of important parameters influencing metabolite pools during extraction, further work will include the validation of the quantification method and identification of not yet revealed peaks. Moreover, variations in metabolite composition in different physiological states e.g. during growth, before and after virus infection, medium depletion, will be investigated.
