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2012, Number 4

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Biotecnol Apl 2012; 29 (4)

Mathematical model for the application of Metabolic Flux Analysis to CHO cells producing recombinant human erythropoietin

Fernández O, Dustet JC, Chico E

Language: Spanish
References: 34
Page: 246-252
PDF size: 208.59 Kb.


ABSTRACT

Mathematical modeling of metabolism meets several important applications in the context of bioprocess engineering, such as the interpretation of cell physiology. Metabolic Flux Analysis, one of the tools of this discipline, was used in the present work to characterize the biosynthesis of recombinant human erythropoietin in CHO cells. In order to apply this method, we built a matrix of stoichiometric numbers representing the major metabolic pathways for the generation of energy and the synthesis of essential precursors for product and biomass accumulation. Equations representing the biosynthesis of recombinant human erythropoietin and the growth of CHO were also derived, conferring an advantage to the proposed model over other existing designs. The dimensions of the obtained matrix were 47 × 44, with a rank of 44 and a condition number of 83; therefore, the model has a unique solution and is not sensitive. The metabolic flux map obtained by solving the mathematical model using experimental data showed results consistent with the known biochemistry of CHO cells and with the findings of other reports on this and other mammalian cell lines. The general steps of the methodology used to obtain the proposed mathematical model are also outlined.


REFERENCES

  1. Nyberg GB, Balcarcel RR, Follstad BD, Stephanopoulos G, Wang DI. Metabolism of peptide amino acids by Chinese hamster ovary cells grown in a complex medium. Biotechnol Bioeng. 1999;62(3):324-35.

  2. Kauffman KJ, Prakash P, Edwards JS. Advances in flux balance analysis. Curr Opin Biotechnol. 2003;14(5):491-6.

  3. Stephanopoulos GN, Aristidou AA, Nielsen J. Metabolic Flux Analysis. In: Metabolic Engineering: Principles and methodologies. California: Academic Press; 1998:309-51.

  4. Altamirano C, Illanes A, Casablancas A, Gamez X, Cairo JJ, Godia C. Analysis of CHO cells metabolic redistribution in a glutamate-based defined medium in continuous culture. Biotechnol Prog. 2001;17(6):1032-41.

  5. Forbes NS, Clark DS, Blanch HW. Using isotopomer path tracing to quantify metabolic fluxes in pathway models containing reversible reactions. Biotechnol Bioeng. 2001;74(3):196-211.

  6. Gòdia C, Cairó JJ. Metabolic engineering of animal cells. Bioprocess Eng. 2002;24(5):289-98.

  7. Srivastava S, Chan C. Application of metabolic flux analysis to identify the mechanisms of free fatty acid toxicity to human hepatoma cell line. Biotechnol Bioeng. 2008;99(2):399-410.

  8. Wahl A, Sidorenko Y, Dauner M, Genzel Y, Reichl U. Metabolic flux model for an anchorage-dependent MDCK cell line: characteristic growth phases and minimum substrate consumption flux distribution. Biotechnol Bioeng. 2008;101(1):135-52.

  9. Goudar C, Biener R, Boisart C, Heidemann R, Piret J, de Graaf A, et al. Metabolic flux analysis of CHO cells in perfusion culture by metabolite balancing and 2D [13C, 1H] COSY NMR spectroscopy. Metab Eng. 2010;12(2):138-49.

  10. Zupke C, Stephanopoulos G. Intracellular flux analysis in hybridomas using mass balances and in vitro (13)C nmr. Biotechnol Bioeng. 1995;45(4):292-303.

  11. Schmidt K, Marx A, de Graaf AA, Wiechert W, Sahm H, Nielsen J, et al. 13C tracer experiments and metabolite balancing for metabolic flux analysis: comparing two approaches. Biotechnol Bioeng. 1998;58(2-3):254-7.

  12. Deshpande RR. Mammalian Cell Culture: High throughput applications of oxygen sensor plates and cellular physiological studies using 13C-labeling [dissertation]. Saarbrücken: Universität des Saarlandes; 2008.

  13. Savinell JM, Palsson BO. Optimal selection of metabolic fluxes for in vivo measurement. II. Application to Escherichia coli and hybridoma cell metabolism. J Theor Biol. 1992;155(2):215-42.

  14. Bonarius H, Hatzimanikatis V, Meesters K, Gooijer C, Schmid G, Tramper J. Metabolic Flux Analysis of Hybridoma Cells in Different Culture Media Using Mass Balances. Biotechnol Bioeng. 1996;50:299-318.

  15. Gambhir A, Korke R, Lee J, Fu PC, Europa A, Hu WS. Analysis of cellular metabolism of hybridoma cells at distinct physiological states. J Biosci Bioeng. 2003;95(4):317-27.

  16. Europa AF, Gambhir A, Fu PC, Hu WS. Multiple steady states with distinct cellular metabolism in continuous culture of mammalian cells. Biotechnol Bioeng. 2000;67(1):25-34.

  17. Nelson DL, Cox MM. Lehninger. Principios de Bioquímica. 4th ed. Barcelona: Ediciones Omega S.A.; 2005.

  18. Zubay GL, Parson WW, Vance DE. Principles of Biochemistry. New York: William C Brown; 1995.

  19. Okayasu T, Ikeda M. The amino acids composition of mammalian and bacterial cells. Amino Acids. 1997;13:379-91.

  20. Voet D, Voet JG. Bioquímica. 3rd ed. Buenos Aires: Médica Panamericana; 2006.

  21. Metzler DE, Metzler CM. Biochemistry. The chemical reactions of living cells. 2nd ed. San Diego: Academic Press; 2003.

  22. Koolman J, Roehm KH. Color Atlas of Biochemistry. 2nd ed. Stuttgart: Thieme; 2005.

  23. Xie L, Wang DI. Material balance studies on animal cell metabolism using a stoichiometrically based reaction network. Biotechnol Bioeng. 1996;52(5):579-90.

  24. Xie L, Nyberg G, Gu X, Li H, Mollborn F, Wang DI. Gamma-interferon production and quality in stoichiometric fed-batch cultures of Chinese hamster ovary (CHO) cells under serum-free conditions. Biotechnol Bioeng. 1997;56(5):577-82.

  25. Lee DE, Ha BJ, Kim SJ, Park JS. Carbohydrate structure of N - and O - linked oligosaccharides of human erythropoietin expressed in Chinese hamster ovary cells. J Biochem Mol Biol. 1996;29:266-71.

  26. Sasaki H, Bothner B, Dell A, Fukuda M. Carbohydrate structure of erythropoietin expressed in Chinese hamster ovary cells by a human erythropoietin cDNA. J Biol Chem. 1987;262(25):12059-76.

  27. Elkin RG, Wasynczuk AM. Amino acid analysis of feedstuff hydrolysates by precolumn derivatization with phenylisothiocyanate and reversed-phase high-performance liquid chromatography. Cereal Chem. 1987;64(4):226-9.

  28. Doran PM. Bioprocess Engineering Principles. London: Academic Press; 1995.

  29. van der Heijden RT, Heijnen JJ, Hellinga C, Romein B, Luyben KC. Linear constraint relations in biochemical reaction systems: I. Classification of the calculability and the balanceability of conversion rates. Biotechnol Bioeng. 1994;43(1):3-10.

  30. van der Heijden RT, Romein B, Heijnen JJ, Hellinga C, Luyben KC. Linear constraint relations in biochemical reaction systems: II. Diagnosis and estimation of gross errors. Biotechnol Bioeng. 1994;43(1):11-20.

  31. Calik P, Ozdamar T. Metabolic flux analysis for human therapeutic protein productions and hypothesis for new therapeutical strategies in medicine. Biochem Eng J. 2002;11(1):49-68.

  32. Ahn WS, Antoniewicz MR. Metabolic flux analysis of CHO cells at growth and non-growth phases using isotopic tracers and mass spectrometry. Metab Eng. 2011;13(5):598-609.

  33. Mulukutla BC, Khan S, Lange A, Hu WS. Glucose metabolism in mammalian cell culture: new insights for tweaking vintage pathways. Trends Biotechnol. 2010;28(9):476-84.

  34. Fernández-Oliva O, Dustet-Mendoza JC, Chico-Véliz E. Dos aplicaciones de la técnica de análisis de flujos metabólicos. Rev Cubana Quím. 2012;24(1):70-82.




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