Protein‐free fed‐batch culture of non‐GS NS0 cell lines for production of recombinant antibodies

Burky, John E.; Wesson, Mark C.; Young, Amy J.; Farnsworth, Sharyn; Dionne, Ben; Zhu, Ying; Hartman, Taymar; Qu, Limin; Zhou, Weichang; Sauer, Paul W.

doi:10.1002/bit.21060

Cited by 36 publications

(20 citation statements)

References 34 publications

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“…It may be the case that the variant is simply more efficient metabolically and thus the stress response as a consequence of nutrient depletion is delayed. There is certainly a significant metabolic difference with consumption of lactate in the variant at the decline phase-a feature that has been previously reported in mammalian cell lines (Altamirano et al, 2006;Burky et al, 2007;Zhou et al, 1997) and notably associated with the onset of stationary/death phase. It has also been shown to occur in cell lines overexpressing the anti-apoptotic genes e1b-19k, aven, and xiap (Dorai et al, 2009), thus it is possible that it represents some form of survival strategy.…”

Section: Discussionmentioning

confidence: 89%

Analysis of an artificially selected GS‐NS0 variant with increased resistance to apoptosis

Browne

Al‐Rubeai

2010

Biotech & Bioengineering

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Heterogeneity in gene expression and phenotypic traits is an inherent feature within the "clonal" cell lines used for biopharmaceutical production. This feature can allow the selection of cell lines with improved phenotypes and adaptation to growth under preferential conditions to improve productivity or provide a platform to study the molecular basis of improved characteristics. A repeated process of extended batch culture of a recombinant antibody producing GS-NS0 myeloma cell line generated a stable variant cell line displaying increased resistance to both environmental stresses and chemical apoptosis inducers, and resulted in extended culture viability and increased antibody production. An interesting feature of the variant cell line was an altered metabolic state with consumption of lactate as the culture progressed. The variant cell line also showed altered expression of proteins associated with autophagy suggesting a role for this process in extending cell survival in culture. Targeted transcriptomic analysis was carried out on the parental and variant cell lines using a qRT-PCR array containing a panel of apoptosis-associated genes to elucidate both the predominant apoptotic pathways in the NS0 cell line with batch culture progression, and the altered gene expression contributing to increased survival in the variant line. Results indicated a balance between pro- and anti-apoptotic signaling is triggered with the onset of cell death in the NS0 cell line. Pro-survival pathways such as NFκB signaling and the unfolded protein response were implicated along with death receptor, endoplasmic reticulum stress and p53 mediated apoptotic pathways. The identification of altered gene expression in the variant cell line also provides several potential targets for cell engineering strategies to create improved cell lines for production.

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Section: Discussionmentioning

confidence: 89%

Analysis of an artificially selected GS‐NS0 variant with increased resistance to apoptosis

Browne

Al‐Rubeai

2010

Biotech & Bioengineering

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“…In control culture, this metabolism shift was coupled to a decrease of the cell growth rate (from 0.04 to 0.01 h -1 ) ( Table 1). In fact, the shift is often correlated with the end of the exponential cell growth phase (Burky et al 2007;Wilkens et al 2011). Interestingly, the addition of hydrolysates maintained the maximal cell specific growth rate (0.04 h -1 ), despite the metabolism shift.…”

Section: Glycolysismentioning

confidence: 99%

“…It can be noticed that the pH decrease has been already shown to decrease glucose uptake and to promote the cell oxidative metabolism (Yoon et al 2005;Kuwae et al 2005;Burky et al 2007). The pH decrease occurring after 45 h in Erlenmeyer flask culture could have induced this metabolism shift.…”

Section: Glycolysismentioning

confidence: 99%

Combination of yeast hydrolysates to improve CHO cell growth and IgG production

et al. 2012

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Many studies underlined the great benefits of hydrolysates used as additives in animal free media on cell culture performances. However, to precisely define hydrolysate supplementation strategies, a deeper understanding of their effect on cell growth and protein production is required. In the present study, the effect of addition of one yeast extract (YE) and two yeast peptones (named YP.A and YP.B) in a chemically defined medium was first assessed on cell culture performances. Interestingly, specific effects were found depending on the degree of degradation of yeast hydrolysates. The YE at 1 g L -1 increased the maximal cell density by 70 %, while a mixture of YE (1 g L -1 ) and YP.A (4 g L -1 ) increased IgG production by 180 %. These conditions were then evaluated on the CHO cell kinetics all over cultures. Hydrolysates extended the cell growth phase in Erlenmeyer flask and increased the maximal growth rate in bioreactor up to 20 %. Cell growth stimulation induced by hydrolysates addition was linked with energetic metabolism improvement suggesting that they promote oxidative pathway. Furthermore, hydrolysates provided an additional source of substrate that supported cell growth despite glutamine limitation.

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“…Previous researchers have detected lactate consumption in hybridoma, NS0 myeloma and CHO cultures (Burky et al, 2008;deZengotita et al, 2000;Pascoe et al, 2007;Zhou et al, 1995Zhou et al, , 1997. However, these cells typically exhibit lactate production in the exponential phase and transition to consumption in the stationary phases to suggest that lactate can represent both an intermediate by-product and a carbohydrate fuel source (Brooks, 1985;Burky et al, 2008).…”

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

confidence: 99%

“…However, these cells typically exhibit lactate production in the exponential phase and transition to consumption in the stationary phases to suggest that lactate can represent both an intermediate by-product and a carbohydrate fuel source (Brooks, 1985;Burky et al, 2008). In the current study, the control CHO cultures appeared to consume some lactate in the stationary phase, particularly in the ''high'' glucose medium (Fig.…”

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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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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Protein‐free fed‐batch culture of non‐GS NS0 cell lines for production of recombinant antibodies

Cited by 36 publications

References 34 publications

Analysis of an artificially selected GS‐NS0 variant with increased resistance to apoptosis

Analysis of an artificially selected GS‐NS0 variant with increased resistance to apoptosis

Combination of yeast hydrolysates to improve CHO cell growth and IgG production

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

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