scholarly journals Metabolic Analysis of the Asparagine and Glutamine Dynamics in an Industrial CHO Fed-Batch Process

2021 ◽  
Author(s):  
Brian Kirsch ◽  
Sandra Bennun ◽  
Adam Mendez ◽  
Amy Johnson ◽  
Hongxia Wang ◽  
...  
Keyword(s):  
Metabolic Flux ◽  
Chinese Hamster ◽  
Tca Cycle ◽  
Batch Process ◽  
Exponential Phase ◽  
Flux Analysis ◽  
Fed Batch ◽  
Cho Cell ◽  
Feed Composition ◽  
Trade Offs

Chinese Hamster Ovary (CHO) cell lines are grown in cultures with varying asparagine and glutamine concentrations, but further study is needed to characterize the interplay between these amino acids. By following 13C-glucose, 13C-glutamine, and 13C-asparagine tracers using metabolic flux analysis (MFA), CHO cell metabolism was characterized in an industrially relevant fed-batch process under glutamine supplemented and low glutamine conditions during early and late exponential growth. For both conditions MFA revealed glucose as the primary carbon source to the tricarboxylic acid (TCA) cycle followed by glutamine and asparagine as secondary sources. Early exponential phase CHO cells prefer glutamine over asparagine to support the TCA cycle under the glutamine supplemented condition, while asparagine was critical for TCA activity for the low glutamine condition. Overall TCA fluxes were similar for both conditions due to the trade-offs associated with reliance on glutamine and/or asparagine. However, glutamine supplementation increased fluxes to alanine, lactate and enrichment of glutathione, N-Acetyl-Glucosamine (NAG) and pyrimidine-containing-molecules. The late exponential phase exhibited reduced central carbon metabolism dominated by glucose, while lactate reincorporation and aspartate uptake were preferred over glutamine and asparagine. These 13C studies demonstrate that metabolic flux is process time dependent and can be modulated by varying feed composition.

scholarly journals Global Transcription and Metabolic Flux Analysis of Escherichia coli in Glucose-Limited Fed-Batch Cultivations

2008 ◽  
Vol 74 (22) ◽  
pp. 7002-7015 ◽  
Author(s):  
K. Lemuth ◽  
T. Hardiman ◽  
S. Winter ◽  
D. Pfeiffer ◽  
M. A. Keller ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Carbon Flux ◽  
Metabolic Flux ◽  
Genetic Regulation ◽  
Tca Cycle ◽  
Carbon Limitation ◽  
Oxidative Decarboxylation ◽  
Flux Analysis ◽  
Fed Batch ◽  
K 12

ABSTRACT A time series of whole-genome transcription profiling of Escherichia coli K-12 W3110 was performed during a carbon-limited fed-batch process. The application of a constant feed rate led to the identification of a dynamic sequence of diverse carbon limitation responses (e.g., the hunger response) and at the same time provided a global view of how cellular and extracellular resources are used: the synthesis of high-affinity transporters guarantees maximal glucose influx, thereby preserving the phosphoenolpyruvate pool, and energy-dependent chemotaxis is reduced in order to provide a more economic “work mode.” σS-mediated stress and starvation responses were both found to be of only minor relevance. Thus, the experimental setup provided access to the hunger response and enabled the differentiation of the hunger response from the general starvation response. Our previous topological model of the global regulation of the E. coli central carbon metabolism through the crp, cra, and relA/spoT modulons is supported by correlating transcript levels and metabolic fluxes and can now be extended. The substrate is extensively oxidized in the tricarboxylic acid (TCA) cycle to enhance energy generation. However, the general rate of oxidative decarboxylation within the pentose phosphate pathway and the TCA cycle is restricted to a minimum. Fine regulation of the carbon flux through these pathways supplies sufficient precursors for biosyntheses. The pools of at least three precursors are probably regulated through activation of the (phosphoenolpyruvate-)glyoxylate shunt. The present work shows that detailed understanding of the genetic regulation of bacterial metabolism provides useful insights for manipulating the carbon flux in technical production processes.

The impact of pH inhomogeneities on CHO cell physiology and fed-batch process performance - two-compartment scale-down modelling and intracellular pH excursion

2017 ◽  
Vol 12 (7) ◽  
pp. 1600633 ◽  
Author(s):  
Matthias Brunner ◽  
Philipp Braun ◽  
Philipp Doppler ◽  
Christoph Posch ◽  
Dirk Behrens ◽  
...  
Keyword(s):  
Intracellular Ph ◽  
Batch Process ◽  
Cell Physiology ◽  
Process Performance ◽  
Fed Batch ◽  
Cho Cell ◽  
Scale Down ◽  
The Impact

scholarly journals Metabolic Fluxes in Corynebacterium glutamicum during Lysine Production with Sucrose as Carbon Source

2004 ◽  
Vol 70 (12) ◽  
pp. 7277-7287 ◽  
Author(s):  
Christoph Wittmann ◽  
Patrick Kiefer ◽  
Oskar Zelder
Keyword(s):  
Carbon Source ◽  
Corynebacterium Glutamicum ◽  
Metabolic Flux ◽  
Tca Cycle ◽  
Pentose Phosphate ◽  
Flux Analysis ◽  
Phosphotransferase System ◽  
Lysine Production ◽  
Metabolic Fluxes ◽  
Fructose 1,6 Bisphosphate

ABSTRACT Metabolic fluxes in the central metabolism were determined for lysine-producing Corynebacterium glutamicum ATCC 21526 with sucrose as a carbon source, providing an insight into molasses-based industrial production processes with this organism. For this purpose, 13C metabolic flux analysis with parallel studies on [1-13CFru]sucrose, [1-13CGlc]sucrose, and [13C6 Fru]sucrose was carried out. C. glutamicum directed 27.4% of sucrose toward extracellular lysine. The strain exhibited a relatively high flux of 55.7% (normalized to an uptake flux of hexose units of 100%) through the pentose phosphate pathway (PPP). The glucose monomer of sucrose was completely channeled into the PPP. After transient efflux, the fructose residue was mainly taken up by the fructose-specific phosphotransferase system (PTS) and entered glycolysis at the level of fructose-1,6-bisphosphate. Glucose-6-phosphate isomerase operated in the gluconeogenetic direction from fructose-6-phosphate to glucose-6-phosphate and supplied additional carbon (7.2%) from the fructose part of the substrate toward the PPP. This involved supply of fructose-6-phosphate from the fructose part of sucrose either by PTSMan or by fructose-1,6-bisphosphatase. C. glutamicum further exhibited a high tricarboxylic acid (TCA) cycle flux of 78.2%. Isocitrate dehydrogenase therefore significantly contributed to the total NADPH supply of 190%. The demands for lysine (110%) and anabolism (32%) were lower than the supply, resulting in an apparent NADPH excess. The high TCA cycle flux and the significant secretion of dihydroxyacetone and glycerol display interesting targets to be approached by genetic engineers for optimization of the strain investigated.

Metabolic flux analysis of CHO cell metabolism in the late non-growth phase

2010 ◽  
Vol 108 (1) ◽  
pp. 82-92 ◽  
Author(s):  
Neelanjan Sengupta ◽  
Steven T. Rose ◽  
John A. Morgan
Keyword(s):  
Growth Phase ◽  
Metabolic Flux Analysis ◽  
Metabolic Flux ◽  
Cell Metabolism ◽  
Flux Analysis ◽  
Cho Cell

scholarly journals Metabolic flux analysis during galactose and lactate co-consumption reveals enhanced energy metabolism in continuous CHO cell cultures

2019 ◽  
Vol 205 ◽  
pp. 201-211 ◽  
Author(s):  
Mauro Torres ◽  
Julio Berrios ◽  
Yandi Rigual ◽  
Yesenia Latorre ◽  
Mauricio Vergara ◽  
...  
Keyword(s):  
Energy Metabolism ◽  
Cell Cultures ◽  
Metabolic Flux Analysis ◽  
Metabolic Flux ◽  
Flux Analysis ◽  
Cho Cell

scholarly journals Rational development of a serum-free medium and fed-batch process for a GS-CHO cell line expressing recombinant antibody

2012 ◽  
Vol 65 (3) ◽  
pp. 363-378 ◽  
Author(s):  
Huifeng Zhang ◽  
Haibin Wang ◽  
Mei Liu ◽  
Tao Zhang ◽  
Ji Zhang ◽  
...  
Keyword(s):  
Cell Line ◽  
Recombinant Antibody ◽  
Batch Process ◽  
Serum Free Medium ◽  
Free Medium ◽  
Fed Batch ◽  
Cho Cell ◽  
Serum Free ◽  
Rational Development ◽  
Cho Cell Line

scholarly journals Inclusion of maintenance energy improves the intracellular flux predictions of CHO

2020 ◽  
Author(s):  
Diana Széliová ◽  
Jerneja Štor ◽  
Isabella Thiel ◽  
Marcus Weinguny ◽  
Michael Hanscho ◽  
...  
Keyword(s):  
Growth Rates ◽  
High Throughput Screening ◽  
Energy Demand ◽  
Metabolic Flux ◽  
Chinese Hamster ◽  
Tca Cycle ◽  
Metabolic Model ◽  
Maintenance Energy ◽  
Uptake Rates ◽  
Wide Range

AbstractChinese hamster ovary (CHO) cells are the leading platform for the production of biopharmaceuticals with human-like glycosylation. The standard practice for cell line generation relies on trial and error approaches such as adaptive evolution and high-throughput screening, which typically take several months. Metabolic modeling could aid in designing better producer cell lines and thus shorten development times. The genome-scale metabolic model (GSMM) of CHO can accurately predict growth rates. However, in order to predict rational engineering strategies it also needs to accurately predict intracellular fluxes. In this work we evaluated the agreement between the fluxes predicted by pFBA using the CHO GSMM and a wide range of 13C metabolic flux data from literature. While glycolytic fluxes were predicted relatively well, the fluxes of tricarboxylic acid (TCA) cycle were vastly underestimated due to too low energy demand. Inclusion of computationally estimated maintenance energy significantly improved the overall accuracy of intracellular flux predictions. Maintenance energy was therefore determined experimentally by running continuous cultures at different growth rates and evaluating their respective energy consumption. The experimentally and computationally determined maintenance energy were in good agreement. Additionally, we compared alternative objective functions (minimization of uptake rates of seven nonessential metabolites) to the biomass objective. While the predictions of the uptake rates were quite inaccurate for most objectives, the predictions of the intracellular fluxes were comparable to the biomass objective function.

scholarly journals Glycosylation flux analysis reveals dynamic changes of intracellular glycosylation flux distribution in Chinese hamster ovary fed-batch cultures

2017 ◽  
Author(s):  
Sandro Hutter ◽  
Thomas K. Villiger ◽  
David Brühlmann ◽  
Matthieu Stettler ◽  
Hervé Broly ◽  
...  
Keyword(s):  
Chinese Hamster ◽  
Specific Productivity ◽  
Specific Factor ◽  
Glycan Structure ◽  
Flux Analysis ◽  
Fed Batch ◽  
Dynamic Changes ◽  
Consumption Rates ◽  
The Media ◽  
Mab Production

AbstractN-linked glycosylation of proteins has both functional and structural significance. Importantly, the glycan structure of a therapeutic protein influences its efficacy, pharmacokinetics, pharmacodynamics and immunogenicity. In this work, we developed glycosylation flux analysis (GFA) for predicting intracellular production and consumption rates (fluxes) of glycoforms, and applied this method to CHO fed-batch monoclonal antibody (mAb) production using two different media compositions, with and without additional manganese feeding. The GFA is based on a constraint-based modelling of the glycosylation network, employing a pseudo steady state assumption. While the glycosylation fluxes in the network are balanced at each time point, the GFA allows the fluxes to vary with time by way of two scaling factors: (1) an enzyme-specific factor that captures the temporal changes among glycosylation reactions catalyzed by the same enzyme, and (2) the cell specific productivity factor that accounts for the dynamic changes in the mAb production rate. The GFA of the CHO fed-batch cultivations showed that regardless of the media composition, the fluxes of galactosylation decreased with the cultivation time in comparison to the other glycosylation reactions. Furthermore, the GFA showed that the addition of Mn, a cofactor of galactosyltransferase, has the effect of increasing the galactosylation fluxes but only during the beginning of the cultivation period. The results thus demonstrated the power of the GFA in delineating the dynamic alterations of the glycosylation fluxes by local (enzyme-specific) and global (cell specific productivity) factors.

TAMI-80. CELLULAR METABOLISM IN DIFFUSE INTRINSIC PONTINE GLIOMA

2021 ◽  
Vol 23 (Supplement_6) ◽  
pp. vi215-vi215
Author(s):  
Omkar Ijare ◽  
Jeanne Manalo ◽  
Martyn Sharpe ◽  
David Baskin ◽  
Kumar Pichumani
Keyword(s):  
Metabolic Flux ◽  
Treatment Strategies ◽  
Cellular Metabolism ◽  
Tca Cycle ◽  
Pediatric Brain Tumors ◽  
Diffuse Intrinsic Pontine Glioma ◽  
Glutamine Metabolism ◽  
Flux Analysis ◽  
Pontine Glioma ◽  
Carboxylation Pathway

Abstract Diffuse intrinsic pontine glioma (DIPG) is an aggressive form of brain tumor in children, comprising >10% of all pediatric brain tumors. The median survival after diagnosis is < 1 year. Since DIPG tumors infiltrate brainstem and pons, they are inoperable. Currently radiotherapy is the mainstay of treatment, and there is a great need for novel therapies for the treatment of DIPG. Cellular metabolism plays a key role in carcinogenesis, unravelling active metabolic pathways in DIPG would help in developing targeted therapies. Glucose and glutamine are the two major nutrients necessary for the growth and proliferation of cancer cells. In this study, we have investigated the glucose and glutamine metabolism in SF8628 DIPG cells using 1H/13C NMR and GC-MS based metabolic flux analysis. SF8628 cells were grown in DMEM containing 11.0 mM glucose, supplemented with 10% FBS, and 2.0 mM glutamine at 37 °C under humidified air and 5% CO2. When cells reached confluency (replicates = 4), treated with 11.0 mM [U-13C]glucose or 4.0 mM glutamine in DMEM (supplemented with 10% FBS). After 24 h, cells were harvested for NMR/GC-MS analysis. The 13C-isotopomer analysis revealed that SF8628 cells produced 25.26 ± 10.63% acetyl-CoA from [U-13C]glucose which is ~3.7 times higher than that produced from GBM cells (6.83 ± 0.76%; our previous work), suggesting that DIPGs are metabolically very active. [U-13C]glutamine metabolism showed that DIPG cells also have an active TCA cycle metabolism (citrate M+4; 40.07 ± 1.06%) and moderately active reductive carboxylation pathway (citrate M+5; 10.59 ± 1.13%). Inhibition of both glycolytic and glutaminolysis pathways will be valuable in developing treatment strategies for DIPGs and these studies are in progress.

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