Error propagation from prime variables into specific rates and metabolic fluxes for mammalian cells in perfusion culture

Goudar, Chetan T.; Biener, Richard; Konstantinov, Konstantin; Piret, James M.

doi:10.1002/btpr.155

Cited by 38 publications

(38 citation statements)

References 25 publications

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“…6 analysis can also be extended to nutrient and metabolite specific rates for additional insights into optimizing nutrient feeding strategies. In addition, specific rates are inputs for metabolic flux analysis and given the error propagation from specific rates into metabolic fluxes (Goudar et al 2009), the logistic specific rates offer a robust input data set for accurate metabolic flux estimation in mammalian cell batch and fed-batch cultures.…”

Section: Specific Rate Time Coursesmentioning

confidence: 99%

Computer programs for modeling mammalian cell batch and fed-batch cultures using logistic equations

Goudar

2012

Cytotechnology

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A MATLAB Ò toolbox was developed for applying the logistic modeling approach to mammalian cell batch and fed-batch cultures. The programs in the toolbox encompass sensitivity analyses and simulations of the logistic equations in addition to cell specific rate estimation. The toolbox was first used to generate time courses of the sensitivity equations for characterizing the relationship between the logistic variable and the model parameters. Subsequently, the toolbox was used to describe CHO cell data from batch and fed-batch mammalian cell cultures. Cell density, product, glucose, lactate, glutamine, and ammonia data were analyzed for the batch culture while fedbatch analysis included cell density and product concentration. In all instances, experimental data were well described by the logistic equations and the resulting specific rate profiles were representative of the underlying cell physiology. The 6-variable batch culture data set was also used to compare the logistic specific rates with those from polynomial fitting and discrete derivative methods. The polynomial specific rates grossly misrepresented cell behavior in the initial and final stages of culture while those based on discrete derivatives had high variability due to computational artifacts. The utility of logistic specific rates to guide process development activities was demonstrated using specific protein productivity versus growth rate trajectories for the 3 cultures examined in this study. Overall, the computer programs developed in this study enable rapid and robust analysis of data from mammalian cell batch and fed-batch cultures which can help process development and metabolic flux estimation.

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Section: Specific Rate Time Coursesmentioning

confidence: 99%

Computer programs for modeling mammalian cell batch and fed-batch cultures using logistic equations

Goudar

2012

Cytotechnology

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“…Constrained weighted quadratic programming was used for consistency checking and flux variability analysis (Antoniewicz, Kelleher & Stephanopoulos 2006), while a Monte-Carlo approach was used to propagate errors in the primary concentration data into the calculated cell-specific rates (Goudar et al 2009, Quek et al 2014) (see also Appendix C). Estimated fluxes were also used for calculation of ATP production (Martínez, VS et al 2013).…”

Section: Metabolic Flux Analysismentioning

confidence: 99%

“…A Monte Carlo approach was used to propagate errors in primary data (growth data and extracellular nutrient and metabolite data) into the calculated cell-specific rates (Goudar et al 2009;Quek et al 2014). The average dry weight was measured to be 515 pg/cell.…”

Section: Metabolic Flux Analysismentioning

confidence: 99%

“…In short, the algorithm place a new knot in the "knot placement time interval", which is defined as a continuous segment Instead of the conventional MFA approach used in consistency checking (Goudar et al 2009;van der Heijden et al 1994;Wang & Stephanopoulos 1983), we used a Monte-Carlo approach for error propagation, and quadratic programming for error checking, flux calculation and sensitivity analysis. Cell number and cell culture concentrations were transformed into rates by a yield approach.…”

Section: Algorithm To Determine Knots Sequencementioning

confidence: 99%

“…Simple linear regression can be used for the transformation process by selectively performing MFA on growth phases that show steadystate metabolic behavior (Quek et al 2010). A Monte-Carlo approach was used to propagate errors in the primary concentration data into the calculated cell-specific rates (Goudar et al 2009). Constrained weighted quadratic programming was used for consistency checking and flux variability analysis (Antoniewicz, Kelleher & Stephanopoulos 2006).…”

Section: Algorithm To Determine Knots Sequencementioning

confidence: 99%

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Global metabolic effect of manipulating pyruvate dehydrogenase complex activity in mammalian cells

Buchsteiner¹

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Aerobic glycolysis is an inefficient metabolic phenotype displayed by many rapidly proliferating cells during growth. It is characterized by high glycolytic activity and only partial oxidation of glucose resulting in the production of high amounts of lactate. This phenotype was originally reported by Otto Warburg in 1927 as a hallmark of cancer andwhile it is now known to occur in other fast growing cells as well -it remains an interesting target for cancer therapy. Aerobic glycolysis also has major implications for biopharmaceutical production, since lactate accumulation can be growth inhibiting, limiting the cell density that can be achieved in culture.Due to its association with various diseases and being an unfavorable metabolic phenotype in industrial applications, reducing the Warburg effect and analyzing accompanying effects on the cell as a whole are of great interest. Whereas in cancer therapy the objective is to kill cells relying on aerobic glycolysis, the aim in industrial applications is to reduce aerobic glycolysis without inducing cell death or inhibiting cell growth. Pyruvate dehydrogenase complex (PDC) is a mitochondrial gatekeeping enzyme determining how much pyruvate is converted to acetyl-CoA and subsequently enters the TCA cycle. PDC activity is regulated by reversible phosphorylation catalyzed by pyruvate dehydrogenase kinase (PDK) (phosphorylation → inactivation) and pyruvate dehydrogenase phosphatase (dephosphorylation → activation). PDC activity can be increased by inhibiting PDK using dichloroacetate (DCA) a known PDK inhibitor, hereby reducing aerobic glycolysis.The objective in this thesis was twofold; i) analyzing metabolic as well as growth inhibitory effects of DCA in human embryonic kidney 293 (HEK293) cells, and ii) investigating the effects of a non growth inhibiting DCA concentration using Chinese hamster ovary (CHO) cells in industrial relevant bioprocesses. In both studies aerobic glycolysis decreased with increasing DCA concentration characterized by reduced glucose consumption and lactate production. At lower DCA concentrations cell growth was unaffected. Furthermore, no increase in oxidative metabolism was detected at low DCA concentration indicating that the cells adopt a more energy efficient metabolism without directing more pyruvate into the TCA cycle. However, it appears that the cytoplasmic pyruvate fraction is reduced as not only less lactate but also less alanine is produced. The metabolic changes observed were mostly attributable to post-translational regulation since transcriptomics and proteomics analyses revealed only minor changes to metabolic enzymes. However, in the absence of iv increased TCA cycle activity, allosteric regulation of glycolytic enzymes did not readily explain reduced glycolysis.Cell growth in HEK293 cells was reduced only at higher DCA concentration when increased cellular stress and TCA cycle activity were detected. Since DCA was found to depolarize mitochondria the increased TCA cycle activity may not result in higher ATP productio...

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Towards dynamic metabolic flux analysis in CHO cell cultures

Ahn

Antoniewicz

2011

Biotechnology Journal

112

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Chinese hamster ovary (CHO) cells are the most widely used mammalian cell line for biopharmaceutical production, with a total global market approaching $100 billion per year. In the pharmaceutical industry CHO cells are grown in fed-batch culture, where cellular metabolism is characterized by high glucose and glutamine uptake rates combined with high rates of ammonium and lactate secretion. The metabolism of CHO cells changes dramatically during a fed-batch culture as the cells adapt to a changing environment and transition from exponential growth phase to stationary phase. Thus far, it has been challenging to study metabolic flux dynamics in CHO cell cultures using conventional metabolic flux analysis techniques that were developed for systems at metabolic steady state. In this paper we review progress on flux analysis in CHO cells and techniques for dynamic metabolic flux analysis. Application of these new tools may allow identification of intracellular metabolic bottlenecks at specific stages in CHO cell cultures and eventually lead to novel strategies for improving CHO cell metabolism and optimizing biopharmaceutical process performance.

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Error propagation from prime variables into specific rates and metabolic fluxes for mammalian cells in perfusion culture

Cited by 38 publications

References 25 publications

Computer programs for modeling mammalian cell batch and fed-batch cultures using logistic equations

Computer programs for modeling mammalian cell batch and fed-batch cultures using logistic equations

Global metabolic effect of manipulating pyruvate dehydrogenase complex activity in mammalian cells

Towards dynamic metabolic flux analysis in CHO cell cultures

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