Large scale gene expression profiling of metabolic shift of mammalian cells in culture

Korke, Rashmi; Gatti, Marcela De Leon; Lau, Ally; Lim, Jeremy; Seow, Teck Keong; Chung, Maxey C. M.; Hu, Wei Shou

doi:10.1016/j.jbiotec.2003.09.007

Cited by 80 publications

(56 citation statements)

References 24 publications

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“…The yield of lactate on glucose (ΔL/ΔG) decreased from 1.16 to 0.16 mol/mol, which dramatically reduced by-product accumulation and improved process performance [47,50]. Similar metabolic shifts were investigated at the transcriptional and translational levels [51,52]. Comparison of high and low ΔL/ΔG conditions revealed that glucose-related genes were down-regulated when lactate production was low.…”

Section: Glycolysis and Lactate Productionmentioning

confidence: 94%

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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Section: Glycolysis and Lactate Productionmentioning

confidence: 94%

Towards dynamic metabolic flux analysis in CHO cell cultures

Ahn

Antoniewicz

2011

Biotechnology Journal

112

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“…Consequently, there is now a clear relationship established between the cellular energetics and anti-apoptosis proteins in the mitochondria and other cellular compartments including the endoplasmic reticulum. Additional data from Korke et al (2004) also suggests a link between apoptosis and metabolism; differential proteomic analyses comparing a normal cell line with its metabolically 'less-active' sister cell line shows that, among other proteins, the receptor of the glycolytic enzyme triosephosphate-isomerase that associates with Bcl-XL and VDAC was differentially modulated.…”

Section: Intertwining Apoptosis and Metabolismmentioning

confidence: 99%

Expression of anti‐apoptosis genes alters lactate metabolism of Chinese Hamster Ovary cells in culture

Dorai

Kyung

Ellis

et al. 2009

Biotech & Bioengineering

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ABSTRACT:In an effort to develop robust Chinese Hamster Ovary host cell lines, a variety of anti-apoptotic genes were over-expressed, either singly or in combination, followed by screening of transfectants for improved cell growth, extended longevity, reduced caspase 3/7 activity, and enhanced mitochondrial membrane potential (MMP). Two particular cell lines, one containing two anti-apoptotic genes, E1B-19K and Aven (EA167), and another containing three, E1B-19K, Aven, and a mutant of XIAP (EAX197), exhibited a reduction in caspase 3 activity of at least 60% and a 170% enhancement in mitochondrial membrane potential compared to controls when treated with staurosporine. In batch cell growth experiments, the peak viable cell densities and viabilities were higher resulting in a 186% increase in integrated viable cell densities. Analyses of metabolite utilization and formation of waste products indicated that the apoptotic resistant cell lines depleted all the lactate when grown in commercially available CD-CHO medium while significant levels (>1.8 g/L) accumulated in the host cell lines. When the lactate level was replenished daily in the apoptotic resistant cell lines, the cell lines consumed lactate and the culture longevity was extended up to four additional days compared to control cell lines. Furthermore, the anti-apoptosis cell lines also accumulated lower levels of ammonia. The ability of the apoptotic resistant cell lines to consume lactate was exploited by cultivating them in a ''high'' glucose medium containing 15 g/L (60 mM glucose) in which apoptotic resistant cell lines exhibited lower maximum lactate (1.8 g/L) compared to control cell lines which accumulated concentrations of lactate (2.2 g/L) that appeared to be deleterious for growth. The shaker flask titer of a therapeutic antibody product expressed in an apoptotic resistant cell line in ''high'' glucose medium reached 690 mg/ L compared to 390 mg/L for a cell line derived from a control host cell line. These results represent to our knowledge the first example in the literature in which manipulation of the apoptosis pathway has altered the nutrient consumption profile of mammalian cells in culture; findings that underscore the interdependence of the apoptotic cellular machinery and metabolism and provide greater flexibility to mammalian bioreactor process development.

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“…(Newholme et al 2003). Thus, it appears that in the glutamine limited CFB-D4 culture medium there is a definite metabolic shift from proliferation to protein synthesis (Korke et al 2004;Kumar et al 2007), which may account for the ''goodness of fit'' (R 2 = 0.91) not being as good as in the V-PB and V-SB models for B-D4. In contrast, BFB-D2 and CFB-D2 cultures (R 2 C 0.95) are grown in glucose limiting media where cells are not subjected to metabolic shift.…”

Section: Verhulst Modelsmentioning

confidence: 96%

“…As discussed before, the reason for the lower value of growth factor r for the fed-batch culture is the periodic additions of soy hydrolysate, which may cause metabolic shift from proliferation to protein synthesis (Korke et al 2004;Kumar et al 2007). This is evident from the data for 50 ml bioreactor which showed an increase in MAb production (B-D1 546 lg/ml, BFB-D1: 841 lg/ml) and a reduction in lactate production (B-D1: 24 mM/l, BFB-D1: 17 mM/l), a sign of slowing down of growth.…”

Section: Verhulst Modelsmentioning

confidence: 99%

Revisiting Verhulst and Monod models: analysis of batch and fed-batch cultures

et al. 2014

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The paper re-evaluates Verhulst and Monod models. It has been claimed that standard logistic equation cannot describe the decline phase of mammalian cells in batch and fed-batch cultures and in some cases it fails to fit somatic growth data. In the present work Verhulst, population-based mechanistic growth model was revisited to describe successfully viable cell density (VCD) in exponential and decline phases of batch and fed-batch cultures of three different CHO cell lines. Verhulst model constants, K, carrying capacity (VCD/ml or lg/ml) and r, intrinsic growth factor (h -1 ) have physical meaning and they are of biological significance. These two parameters together define the course of growth and productivity and therefore, they are valuable in optimisation of culture media, developing feeding strategies and selection of cell lines for productivity. The Verhulst growth model approach was extended to develop productivity models for batch and fed-batch cultures. All Verhulst models were validated against blind data (R 2 [ 0.95). Critical examination of theoretical approaches concluded that Monod parameters have no physical meaning. Monodhybrid (pseudo-mechanistic) batch models were validated against specific growth rates of respective bolus and continuous fed-batch cultures (R 2 & 0.90). Thedescribes specific growth rate during metabolic shift (R 2 & 0.95). Verhulst substrate-based growth models compared favourably with Monod-hybrid models. Thus, experimental evidence implies that the constants in the Monod-hybrid model may not have physical meaning but they behave similarly to the biological constants in Michaelis-Menten enzyme kinetics, the basis of the Monod growth model.List of symbols K Carrying capacity for VCD K V-PB Carrying capacity, Verhust-population based for VCD K V-SB Carrying capacity, Verhust-substrate eased for VCD K V-MAb Carrying capacity, Verhust for MAb r Intrinsic growth factor r X Intrinsic productivity factor r V-PB Intrinsic growth factor, Verhulst population based for VCD r V-SB Intrinsic growth factor, Verhulst substrate based for VCD r V-MAb

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Large scale gene expression profiling of metabolic shift of mammalian cells in culture

Cited by 80 publications

References 24 publications

Towards dynamic metabolic flux analysis in CHO cell cultures

Towards dynamic metabolic flux analysis in CHO cell cultures

Expression of anti‐apoptosis genes alters lactate metabolism of Chinese Hamster Ovary cells in culture

Revisiting Verhulst and Monod models: analysis of batch and fed-batch cultures

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