Metabolic flux analysis of CHO cells at growth and non-growth phases using isotopic tracers and mass spectrometry |
| |
Authors: | Ahn Woo Suk Antoniewicz Maciek R |
| |
Affiliation: | Department of Chemical Engineering, Metabolic Engineering and Systems Biology Laboratory, University of Delaware, 150 Academy St, Newark, DE 19716, USA |
| |
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. |
| |
Keywords: | Abbreviations: 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&ndash MS, gas chromatography mass spectrometry GDH, glutamate dehydrogenase Glyc3P, glycerol-3-phosphate ME, malic enzyme MID, mass isotopomer distribution oxPPP, oxidative pentose phosphate pathway PC, pyruvate carboxylase PDH, pyruvate dehydrogenase PEP, phophoenolpyruvate PPP, pentose phosphate pathway SIR, selected ion recording TCA, tricarboxylic acid cycle SSR, variance-weighted sum of squared residuals |
本文献已被 ScienceDirect PubMed 等数据库收录! |
|