Towards dynamic metabolic flux analysis in CHO cell cultures

Ahn, Woo Suk; Antoniewicz, Maciek R.

doi:10.1002/biot.201100052

Cited by 112 publications

(79 citation statements)

References 132 publications

(167 reference statements)

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“…Statistical inference can then be used to contrast the flux control coefficient distributions under different scenarios and constraints. In particular, for comparing the medians of control coefficient distributions under different conditions, we used the nonparametric Wilcoxon rank sum test to test whether two samples have equal medians [34].…”

Section: 6statistical Analysismentioning

confidence: 99%

A probabilistic framework for the exploration of enzymatic capabilities based on feasible kinetics and control analysis

Saa

Nielsen

2016

Biochimica et Biophysica Acta (BBA) - General Subjects

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BackgroundAnalysis of limiting steps within enzyme-catalyzed reactions is fundamental to understand their behavior and regulation. Methods capable of unravelling control properties and exploring kinetic capabilities of enzymatic reactions would be particularly useful for protein and metabolic engineering. While single-enzyme control analysis formalism has previously been applied to well-studied enzymatic mechanisms, broader application of formalism is limited in practice by the limited amount of kinetic data and the difficulty of describing complex allosteric mechanisms. MethodsTo overcome these limitations, we present here a probabilistic framework enabling control analysis of previously unexplored mechanisms under uncertainty. By combining a thermodynamically consistent parameterization with an efficient Sequential Monte Carlosampler embedded in a Bayesian setting, this framework yields insights into the capabilities of enzyme-catalyzed reactions with modest kinetic information, provided that the catalytic mechanism and a thermodynamic reference point are defined. ResultsThe framework was used to unravel the impact of thermodynamic affinity, substrate saturation levels and effector concentrations on the flux control and response coefficients of a diverse set of enzymatic reactions. ConclusionsOur results highlight the importance of the metabolic context in the control analysis of isolated enzymes as well as the use of statistically sound methods for their interpretation. General SignificanceThis framework significantly expands our current capabilities for unravelling the control properties of general reaction kinetics with limited amount of information. This framework will be useful for both theoreticians and experimentalists in the field.

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Section: 6statistical Analysismentioning

confidence: 99%

A probabilistic framework for the exploration of enzymatic capabilities based on feasible kinetics and control analysis

Saa

Nielsen

2016

Biochimica et Biophysica Acta (BBA) - General Subjects

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“…The biopharmaceutical industry has substantially progressed since the approval of recombinant insulin, the first recombinant drug, in the early 1980s by the Food and Drug Administration (FDA) 1,2 . More than 50% of the remarkable number of approved recombinant therapeutics on the market today are being manufactured in mammalian cell lines 1,6 .…”

Section: Cell Culture Process Optimizationmentioning

confidence: 99%

“…In the early 1980s the pharmaceutical industry embarked on a remarkable journey when the first recombinant drug, recombinant insulin produced in E. coli, was approved by the Food and Drug Administration (FDA) 1,2 . Patients have benefited ever since from safer and more efficacious drug products due to recombinant analogs rather than proteins of animal origin, which enabled substantial purity improvements 3 .…”

Section: Introductionmentioning

confidence: 99%

Tailoring recombinant protein quality by rational media design

Brühlmann

Jordan

Hemberger³

et al. 2015

Biotechnology Progress

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Clinical efficacy and safety of recombinant proteins are closely associated with their structural characteristics. The major quality attributes comprise glycosylation, charge variants (oxidation, deamidation, and C‐ & N‐terminal modifications), aggregates, low‐molecular‐weight species (LMW), and misincorporation of amino acids in the protein backbone. Cell culture media design has a great potential to modulate these quality attributes due to the vital role of medium in mammalian cell culture. The purpose of this review is to provide an overview of the way both classical cell culture medium components and novel supplements affect the quality attributes of recombinant therapeutic proteins expressed in mammalian hosts, allowing rational and high‐throughput optimization of mammalian cell culture media. A selection of specific and/or potent inhibitors and activators of oligosaccharide processing as well as components affecting multiple quality attributes are presented. Extensive research efforts in this field show the feasibility of quality engineering through media design, allowing to significantly modulate the protein function. © 2015 American Institute of Chemical Engineers Biotechnol. Prog., 31:615–629, 2015

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“…In mammalian cell cultures, L-glutamine is commonly added to the media as a nitrogen source (1). For medical applications, it is useful for treating injuries and gastric ulcers (2).…”

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confidence: 99%

Metabolic Engineering for l -Glutamine Overproduction by Using DNA Gyrase Mutations in Escherichia coli

Hayashi

Tabata

2013

Appl Environ Microbiol

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An L-glutamine-overproducing mutant of an Escherichia coli K-12-derived strain was selected from randomly mutagenized cells in the course of L-alanyl-L-glutamine strain development. Genome-wide mutation analysis unveiled a novel mechanism for Lglutamine overproduction in this mutant. Three mutations were identified that are related to the L-glutamine overproduction phenotype, namely, an intergenic mutation in the 5=-flanking region of yeiG and two nonsynonymous mutations in gyrA (Gly821Ser and Asp830Asn). Expression of yeiG, which encodes a putative esterase, was enhanced by the intergenic mutation. The nonsynonymous mutations in gyrA, a gene that encodes the DNA gyrase ␣ subunit, affected the DNA topology of the cells. Gyrase is a type II topoisomerase that adds negative supercoils to double-stranded DNA. When the opposing DNA-relaxing activity was enhanced by overexpressing topoisomerase I (topA) and topoisomerase IV (parC and parE), an increase in L-glutamine production was observed. These results indicate that a reduction of chromosomal DNA supercoils in the mutant caused an increase in L-glutamine accumulation. The mechanism underlying this finding is discussed in this paper. We also constructed an L-glutamine-hyperproducing strain by attenuating cellular L-glutamine degradation activity. Although the reconstituted mutant (with yeiG together with gyrA) produced 200 mM L-glutamine, metabolic engineering finally enabled construction of a mutant that accumulated more than 500 mM L-glutamine. L-Glutamine, which is a semiessential amino acid in humans, is involved in biosynthesis of amino acids, nucleotides, and cofactors as an amido donor for glutamine amidotransferase in archaeal, bacterial, and eukaryotic cells. In mammalian cell cultures, L-glutamine is commonly added to the media as a nitrogen source (1). For medical applications, it is useful for treating injuries and gastric ulcers (2). We have already reported a novel method using L-amino acid ␣-ligase for fermentative production of L-alanyl-Lglutamine (Ala-Gln), an L-glutamine-containing dipeptide (3). In the course of development of the Ala-Gln-producing Escherichia coli strain, we found that enhancement of the metabolic flux with respect to substrate amino acids (i.e., L-alanine and L-glutamine) was essential for Ala-Gln production. In order to increase the Lglutamine supply in E. coli cells, two genes responsible for the regulation of L-glutamine biosynthesis were deleted from chromosome. One such gene, glnE, encodes glutamine synthetase (GS) adenylyltransferase, and the other, glnB, encodes PII protein. Deletion of glnE resulted in enhanced GS activity by preventing inactive GS-AMP formation. Furthermore, transcription of the glnA gene, encoding GS, was increased via the NtrB/NtrC regulatory system as a result of deleting glnB. We sought an alternative way of enhancing the metabolic flux for L-glutamine and have isolated an L-glutamine-overproducing mutant by random mutation and selection from the parental strain, JKYP9 (3). The genetic background t...

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Towards dynamic metabolic flux analysis in CHO cell cultures

Cited by 112 publications

References 132 publications

A probabilistic framework for the exploration of enzymatic capabilities based on feasible kinetics and control analysis

A probabilistic framework for the exploration of enzymatic capabilities based on feasible kinetics and control analysis

Tailoring recombinant protein quality by rational media design

Metabolic Engineering for l -Glutamine Overproduction by Using DNA Gyrase Mutations in Escherichia coli

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