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Metabolic flux analysis of CHO cells at growth and non-growth phases using isotopic tracers and mass spectrometry

Paper ID Volume ID Publish Year Pages File Format Full-Text
31619 44824 2011 12 PDF Available
Title
Metabolic flux analysis of CHO cells at growth and non-growth phases using isotopic tracers and mass spectrometry
Abstract

Chinese hamster ovary (CHO) cells are the main platform for production of biotherapeutics in the biopharmaceutical industry. However, relatively little is known about the metabolism of CHO cells in cell culture. In this work, metabolism of CHO cells was studied at the growth phase and early stationary phase using isotopic tracers and mass spectrometry. CHO cells were grown in fed-batch culture over a period of six days. On days 2 and 4, [1,2-13C] glucose was introduced and the labeling of intracellular metabolites was measured by gas chromatography-mass spectrometry (GC–MS) at 6, 12 and 24 h following the introduction of tracer. Intracellular metabolic fluxes were quantified from measured extracellular rates and 13C-labeling dynamics of intracellular metabolites using non-stationary 13C-metabolic flux analysis (13C-MFA). The flux results revealed significant rewiring of intracellular metabolic fluxes in the transition from growth to non-growth, including changes in energy metabolism, redox metabolism, oxidative pentose phosphate pathway and anaplerosis. At the exponential phase, CHO cell metabolism was characterized by a high flux of glycolysis from glucose to lactate, anaplerosis from pyruvate to oxaloacetate and from glutamate to α-ketoglutarate, and cataplerosis though malic enzyme. At the stationary phase, the flux map was characterized by a reduced flux of glycolysis, net lactate uptake, oxidative pentose phosphate pathway flux, and reduced rate of anaplerosis. The fluxes of pyruvate dehydrogenase and TCA cycle were similar at the exponential and stationary phase. The results presented here provide a solid foundation for future studies of CHO cell metabolism for applications such as cell line development and medium optimization for high-titer production of recombinant proteins.

► CHO cells were grown in fed-batch culture with [1,2-13C]glucose as tracer. ► Time profiles of intracellular metabolite labeling measured by GC–MS. ► Intracellular metabolic fluxes quantified using non-stationary 13C-MFA. ► Pentose phosphate pathway is inactive during growth phase, but active in stationary phase. ► Anaplerosis from pyruvate to oxaloacetate is active during growth phase.

Keywords
3PG, 3-phosphoglycerate; AcCoA, acetyl coenzyme A; AKG, α-ketoglutarate; CHO, Chinese hamster ovary; FBA, flux balance analysis; MFA, metabolic flux analysis; EMU, elementary metabolite unit; GAP, glyceraldehyde 3-phosphate; GC–MS, gas chromatography mass
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Metabolic flux analysis of CHO cells at growth and non-growth phases using isotopic tracers and mass spectrometry
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Publisher
Database: Elsevier - ScienceDirect
Journal: Metabolic Engineering - Volume 13, Issue 5, September 2011, Pages 598–609
Authors
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Subjects
Physical Sciences and Engineering Chemical Engineering Bioengineering
Get Full-Text Now
Don't Miss Today's Special Offer
Price was $35.95
You save - $31
Price after discount Only $4.95
100% Money Back Guarantee
Full-text PDF Download
Online Support
Any Questions? feel free to contact us