Engineering of a mammalian cell line for reduction of lactate formation and high monoclonal antibody production

Chen, Keqin; Liu, Qian; Xie, Liangzhi; Sharp, Phillip A.; Wang, Daniel I. C.

doi:10.1002/1097-0290(20010105)72:1<55::aid-bit8>3.0.co;2-4

Cited by 147 publications

(72 citation statements)

References 22 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%

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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%

“…7A and B). An added process benefit is that lower levels of lactate in cultures of apoptotic R cell lines will result in lower osmolarity and higher pH, both of which are highly desirable for superior product quality (Chen et al, 2001).…”

Section: Comparison Of Metabolic Profiles Of Apoptotic R and Control mentioning

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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“…To increase productivity, many cellular pathways including energy metabolism, cell cycle, apoptosis and protein secretion have been extensively studied (Chen et al 2001;Kim and Lee 2002;Watanabe et al 2002;Ifandi and Al-Rubeai 2005;Seth et al 2006;Jayapal et al 2007;Kim et al 2012). Increasing cell density by either reducing apoptosis (Meents et al 2002;Figueroa et al 2007) or altering cell metabolism (Chen et al 2001) can lead to increase in product titers.…”

Section: Introductionmentioning

confidence: 99%

“…Increasing cell density by either reducing apoptosis (Meents et al 2002;Figueroa et al 2007) or altering cell metabolism (Chen et al 2001) can lead to increase in product titers. Alternatively, the protein synthesis and secretion machinery can be targeted to increase the specific productivity of a cell.…”

Section: Introductionmentioning

confidence: 99%

Dynamics of unfolded protein response in recombinant CHO cells

Prashad

Mehra

2014

Cytotechnology

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Genes in the protein secretion pathway have been targeted to increase productivity of monoclonal antibodies in Chinese hamster ovary cells. The results have been highly variable depending on the cell type and the relative amount of recombinant and target proteins. This paper presents a comprehensive study encompassing major components of the protein processing pathway in the endoplasmic reticulum (ER) to elucidate its role in recombinant cells. mRNA profiles of all major ER chaperones and unfolded protein response (UPR) pathway genes are measured at a series of time points in a high-producing cell line under the dynamic environment of a batch culture. An initial increase in IgG heavy chain mRNA levels correlates with an increase in productivity. We observe a parallel increase in the expression levels of majority of chaperones. The chaperone levels continue to increase until the end of the batch culture. In contrast, calreticulin and ERO1-L alpha, two of the lowest expressed genes exhibit transient time profiles, with peak induction on day 3. In response to increased ER stress, both the GCN2/PKR-like ER kinase and inositol-requiring enzyme-1alpha (Ire1a) signalling branch of the UPR are upregulated. Interestingly, spliced X-Box binding protein 1 (XBP1s) transcription factor from Ire1a pathway is detected from the beginning of the batch culture. Comparison with the expression levels in a low producer, show much lower induction at the end of the exponential growth phase. Thus, the unfolded protein response strongly correlates with the magnitude and timing of stress in the course of the batch culture.

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“…A key early success was achieved with the insertion and amplification of the glutamine synthetase gene in CHO and myeloma cells, allowing growth in glutamine-free medium and thereby significantly reducing ammonia formation (Bebbington et al, 1992;Birch et al, 1994;Cockett et al, 1990). Partial disruption of the gene encoding lactate dehydrogenase A in hybridoma cells was also shown to lower the production of lactate, translating into an increase in cell concentration and antibody production (Chen et al, 2001). Another approach consisted of the expression of a yeast pyruvate carboxylase (PYC) gene in mammalian cells to compensate for the apparent lack of enzymatic activities linking glycolysis and the TCA cycle.…”

Section: Introductionmentioning

confidence: 99%

Enhanced glycoprotein production in HEK-293 cells expressing pyruvate carboxylase

Henry

Durocher

2011

Metabolic Engineering

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Engineering of a mammalian cell line for reduction of lactate formation and high monoclonal antibody production

Cited by 147 publications

References 22 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

Dynamics of unfolded protein response in recombinant CHO cells

Enhanced glycoprotein production in HEK-293 cells expressing pyruvate carboxylase

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