Engineering cell metabolism for high-density cell culture via manipulation of sugar transport

Wlaschin, Katie F.; Hu, Wei Shou

doi:10.1016/j.jbiotec.2007.06.006

Cited by 94 publications

(58 citation statements)

References 23 publications

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“…Numerous strategies have been devised to address the accumulation of excessive lactate build-up including (1) maintaining low medium glucose concentrations (Kurokawa et al, 1994;Xie and Wang, 1993;Zhang et al, 2004;Zhou et al, 1995), (2) feeding alternative sugars, including fructose (Martinelle et al, 1998, Altamirano et al, 2004Wlaschin and Hu, 2007), (3) partially knocking out lactate dehydrogenase (LDH) expression by homologous recombination or siRNA technology (Chen et al, 2001;Kim and Lee, 2007a); (4) over-expression of pyruvate carboxylase (Kim and Lee, 2007b); (5) use of dichloracetate (DCA), a pyruvate dehydrogenase (PDH) activator (via PDH kinase inhibition) (Stacpoole et al, 2003) and (6) oxamic acid, an LDH competitive inhibitor (Mothersill and Seymour, 1986). While the above strategies have been partially successful in reducing the lactate concentration, an alternative approach to consider is stimulating mitochondrial respiration in order to enhance culture performance.…”

Section: Discussionmentioning

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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Section: Discussionmentioning

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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“…Up to now, there have been many approaches to overcome apoptotic cell death during batch cultures by (1) genetically modifying important genes in cell growth, 8,[10][11][12][13][14][15][16][17][18] (2) supplementing essential nutrients, [19][20][21] or (3) adding chemical inhibitors. 22,23 We address each of these approaches below.…”

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confidence: 99%

“…8,[10][11][12] Moreover, manipulation of genes that are important for metabolism such as the fructose transporter also increase cell survival. 13 In addition, an increasing number of reports support the view that engineering pro-survival genes such as E1B-19K, Aven, Fadd, and Faim, is a potential method to achieve higher cell viability. 14,15 Also, reports on downregulation of caspases have shown to inhibit the execution of apoptosis.…”

mentioning

confidence: 99%

Autophagy and apoptosis in Chinese hamster ovary cell culture

Hwang

Lee²

2008

Autophagy

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“…Wlaschin and Hu (2007) reported that CHO cells became proliferative in fructose-based medium by transfection of GLUT5 gene and some clones of them exhibited a significant increase in the final cell concentration in fed-batch culture. Thus our co-expression system may be applicable to CHO cells.…”

Section: Discussionmentioning

confidence: 99%

Efficient production of recombinant IgG by metabolic control and co-expression with GLUT5 in a fructose-based medium

et al. 2010

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A fructose-based cell culture is suitable for the process control of protein production because of slow sugar consumption rate and low lactate accumulation. The fructose transporter, GLUT5, mediates its incorporation into cells and is required for the fructosebased culture. In order to produce efficiently recombinant IgG by metabolic control and co-expression with GLUT5 in a fructose-based medium, an IgG and GLUT5 co-expression vector was constructed and transfected into the human myeloma derived cell line, SC-01MFP, which produced stably recombinant proteins. The cell proliferation in the fructose-based medium was improved by the GLUT5 gene transfection. The recombinant IgG production of the cells cultured in the fructose-based medium exhibited about two-fold increase of that in the glucose-based medium. Flow cytometoric analysis indicated that the GLUT5 protein expression level in cell surface was increased in the fructose-based medium. An exogenous but not endogenous GLUT5 transcription activator remarkably raised IgG productivity in the fructose-based medium when compared to that in the glucose-based medium, suggesting that exogenous GLUT5 expression may be involved in it. The GLUT5 co-expression system may be useful for efficient production of recombinant proteins by the fructose-based cell culture.

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Engineering cell metabolism for high-density cell culture via manipulation of sugar transport

Cited by 94 publications

References 23 publications

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

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

Autophagy and apoptosis in Chinese hamster ovary cell culture

Efficient production of recombinant IgG by metabolic control and co-expression with GLUT5 in a fructose-based medium

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