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Metabolic flux analysis of CHO cells in perfusion culture by metabolite balancing and 2D [13C, 1H] COSY NMR spectroscopy

Paper ID Volume ID Publish Year Pages File Format Full-Text
31809 44840 2010 12 PDF Available
Title
Metabolic flux analysis of CHO cells in perfusion culture by metabolite balancing and 2D [13C, 1H] COSY NMR spectroscopy
Abstract

The physiological state of CHO cells in perfusion culture was quantified by determining fluxes through the bioreaction network using 13C glucose and 2D-NMR spectroscopy. CHO cells were cultivated in a 2.5 L perfusion bioreactor with glucose and glutamine as the primary carbon and energy sources. The reactor was inoculated at a cell density of 8×106 cells/mL and operated at ∼10×106 cells/mL using unlabeled glucose for the first 13 days. The second phase lasted 12 days and the medium consisted of 10% [U-13C]glucose, 40% labeled [1-13C]glucose with the balance unlabeled. After the culture attained isotopic steady state, biomass samples from the last 3 days of cultivation were considered representative and used for flux estimation. They were hydrolyzed and analyzed by 2D [13C, 1H] COSY measurements using the heteronuclear single quantum correlation sequence with gradients for artifacts suppression. Metabolic fluxes were determined using the 13C-Flux software package by minimizing the residuals between the experimental and the simulated NMR data. Normalized residuals exhibited a Gaussian distribution indicating good model fit to experimental data. The glucose consumption rate was 5-fold higher than that of glutamine with 41% of glucose channeled through the pentose phosphate pathway. The fluxes at the pyruvate branch point were almost equally distributed between lactate and the TCA cycle (55% and 45%, respectively). The anaplerotic conversion of pyruvate to oxaloacetate by pyruvate carboxylase accounted for 10% of the pyruvate flux with the remaining 90% entering the TCA cycle through acetyl-CoA. The conversion of malate to pyruvate catalyzed by the malic enzyme was 70% higher than that for the anaplerotic reaction catalyzed by pyruvate carboxylase. Most amino acid catabolic and biosynthetic fluxes were significantly lower than the glycolytic and TCA cycle fluxes. Metabolic flux data from NMR analysis validated a simplified model where metabolite balancing was used for flux estimation. In this reduced flux space, estimates from these two methods were in good agreement. This simplified model can routinely be used in bioprocess development experiments to estimate metabolic fluxes with much reduced analytical investment. The high resolution flux information from 2D-NMR spectroscopy coupled with the capability to validate a simplified metabolite balancing based model for routine use make 13C-isotopomer analysis an attractive bioprocess development tool for mammalian cell cultures.

Keywords
2D-NMR spectroscopy; Mammalian cells; Metabolic flux analysis; Perfusion culture
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Metabolic flux analysis of CHO cells in perfusion culture by metabolite balancing and 2D [13C, 1H] COSY NMR spectroscopy
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Publisher
Database: Elsevier - ScienceDirect
Journal: Metabolic Engineering - Volume 12, Issue 2, March 2010, Pages 138–149
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