Metabolite-balancing techniques vs.13C tracer experiments to determine metabolic fluxes in hybridoma cells

Bonarius, H.P.J.; Timmerarends, Bram; Gooijer, C.D. de; Tramper, J.

doi:10.1002/(sici)1097-0290(19980420)58:2/3<258::aid-bit20>3.0.co;2-7

Cited by 54 publications

(30 citation statements)

References 16 publications

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“…However, if the pseudo steady state assumption during a phase is justified, it can give a useful overview of its average metabolism [55]. The information obtained by flux balance analysis can be validated [56] or further enriched by specific labelling information [57] to resolve reversible, cyclic or parallel fluxes as for example pentose phosphate pathway split [58, 59]. …”

Section: Discussionmentioning

confidence: 99%

Quantitative characterization of metabolism and metabolic shifts during growth of the new human cell line AGE1.HN using time resolved metabolic flux analysis

Niklas

Schrader

Sandig³

et al. 2010

Bioprocess Biosyst Eng

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For the improved production of vaccines and therapeutic proteins, a detailed understanding of the metabolic dynamics during batch or fed-batch production is requested. To study the new human cell line AGE1.HN, a flexible metabolic flux analysis method was developed that is considering dynamic changes in growth and metabolism during cultivation. This method comprises analysis of formation of cellular components as well as conversion of major substrates and products, spline fitting of dynamic data and flux estimation using metabolite balancing. During batch cultivation of AGE1.HN three distinct phases were observed, an initial one with consumption of pyruvate and high glycolytic activity, a second characterized by a highly efficient metabolism with very little energy spilling waste production and a third with glutamine limitation and decreasing viability. Main events triggering changes in cellular metabolism were depletion of pyruvate and glutamine. Potential targets for the improvement identified from the analysis are (i) reduction of overflow metabolism in the beginning of cultivation, e.g. accomplished by reduction of pyruvate content in the medium and (ii) prolongation of phase 2 with its highly efficient energy metabolism applying e.g. specific feeding strategies. The method presented allows fast and reliable metabolic flux analysis during the development of producer cells and production processes from microtiter plate to large scale reactors with moderate analytical and computational effort. It seems well suited to guide media optimization and genetic engineering of producing cell lines.Electronic supplementary materialThe online version of this article (doi:10.1007/s00449-010-0502-y) contains supplementary material, which is available to authorized users.

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Section: Discussionmentioning

confidence: 99%

Quantitative characterization of metabolism and metabolic shifts during growth of the new human cell line AGE1.HN using time resolved metabolic flux analysis

Niklas

Schrader

Sandig³

et al. 2010

Bioprocess Biosyst Eng

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“…The network includes 8 extracellular substrates (glucose, arginine, asparagine, cysteine, glutamine, isoleucine, leucine, and serine), 6 metabolic products (biomass, lactate, alanine, glutamate, glycine, and lipids) and 35 balanced intracellular metabolites. Constraints from cofactor metabolites such as ATP and NAD(P)H were excluded because these balances have been shown to produce unreliable results in mammalian systems (Bonarius et al, 1998). Refer to Supplementary Materials for a detailed description of the model formulation and assumptions.…”

Section: Methodsmentioning

confidence: 99%

Isotopically nonstationary 13C flux analysis of Myc-induced metabolic reprogramming in B-cells

Murphy

Dang

Young

2013

Metabolic Engineering

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We assessed several methods of 13C metabolic flux analysis (MFA) and found that isotopically nonstationary MFA achieved maximum flux resolution in cultured P493-6 B-cells, which have been engineered to provide tunable expression of the Myc oncoprotein. Comparison of metabolic flux maps obtained under oncogenic (High) and endogenous (Low) Myc expression levels revealed network-wide reprogramming in response to ectopic Myc expression. High Myc cells relied more heavily on mitochondrial oxidative metabolism than Low Myc cells and globally upregulated their consumption of amino acids relative to glucose. TCA cycle and amphibolic mitochondrial pathways exhibited 2- to 4-fold flux increases in High Myc cells, in contrast to modest increases in glucose uptake and lactate excretion. Because our MFA approach relied exclusively upon isotopic measurements of protein-bound amino acids and RNA-bound ribose, it is readily applicable to more complex tumor models that are not amenable to direct extraction and isotopic analysis of free intracellular metabolites.

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“…While initially mostly applied to microorganisms, 13 C-metabolic flux analysis have now been conducted on many relevant animal cell lines, including hybridoma cells [27][28][29], CHO cells [30], A549 lung carcinoma cells [31], hepatic cells [32] and brown adipocytes [33]. One key point emerging from some of these studies are the significant discrepancies observed between fluxes determined from labeling and the ones calculated from metabolite balancing [32,34], highlighting the need for additional independent constraints in comprehensive metabolic studies.…”

mentioning

confidence: 99%

Unraveling the metabolism of HEK-293 cells using lactate isotopomer analysis

Henry

Jolicœur

Kamen

2010

Bioprocess Biosyst Eng

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HEK-293 is the most extensively used human cell line for the production of viral vectors and is gaining increasing attention for the production of recombinant proteins by transient transfection. To further improve the metabolic characterization of this cell line, we have performed cultures using ¹³C-labeled substrates and measured the resulting mass isotopomer distributions in lactate by LC/MS. Simultaneous metabolite and isotopomer balancing allowed improvement and validation of the metabolic model and quantification of key intracellular pathways. We have determined the amounts of glucose carbon channeled through the PPP, incorporated into the TCA cycle for energy production and lipids biosynthesis, as well as the cytosolic and mitochondrial malic enzyme fluxes. Our analysis also revealed that glutamine did not significantly contribute to lactate formation. An improved and quantitative understanding of the central carbon metabolism is greatly needed to pursue the rational development of engineering approaches at both the cellular and process levels.

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Metabolite-balancing techniques vs.13C tracer experiments to determine metabolic fluxes in hybridoma cells

Cited by 54 publications

References 16 publications

Quantitative characterization of metabolism and metabolic shifts during growth of the new human cell line AGE1.HN using time resolved metabolic flux analysis

Quantitative characterization of metabolism and metabolic shifts during growth of the new human cell line AGE1.HN using time resolved metabolic flux analysis

Isotopically nonstationary 13C flux analysis of Myc-induced metabolic reprogramming in B-cells

Unraveling the metabolism of HEK-293 cells using lactate isotopomer analysis

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