Fed-batch culture of recombinant NS0 myeloma cells with high monoclonal antibody production

Zhou, Weichang; Chen, Chun-Chiang; Buckland, Barry C.; Auniņš, John G.

doi:10.1002/(sici)1097-0290(19970905)55:5<783::aid-bit8>3.0.co;2-7

Cited by 120 publications

(92 citation statements)

References 28 publications

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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%

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: 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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“…Glucose, lactate, glutamine and glutamate concentrations in the culture broth were measured on YSI systems (YSI Life Sciences). The concentration of IgG1 antibody protein was assayed by Protein A affinity chromatography method (Zhou et al 1997), while that of IgG4 was determined by either BIAcore mass assay method (Mullett et al 2000) or the Protein A method. Both IgG1 and IgG4 concentrations are expressed as unit/ml for proprietary reasons.…”

Section: Methodsmentioning

confidence: 99%

“…In addition, NS0 cells are well-suited for use of the glutamine synthetase (GS) expression system because they lack endogenous GS activity, thus a plasmid containing both GS and the antibody gene of interest can be constructed to allow easy isolation of MAb-secreting transfectants in glutamine-free medium (Bebbington et al 1992). Through process optimization, MAb production from the GS-NS0 system has reached over 2.5 g/l in fed-batch reactor cultures (Zhou et al 1997;Varma et al 2003;Schmelzer et al 2004).…”

Section: Introductionmentioning

confidence: 99%

Development of Animal-free, Protein-Free and Chemically-Defined Media for NS0 Cell Culture

Zhang

Robinson

2005

Cytotechnology

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There has been a recent boom of monoclonal antibodies on the market, and a significant portion of them were produced by NS0 cell lines. As regulations become more stringent in ensuring production processes are free of potential contamination by adventitious agents, it is highly desirable to further develop serumfree media into ones that do not contain any components of animal origin, or 'animal-free media'. Using a shake-flask batch culture system, recombinant proteins (human albumin and human insulin) and synthetic compounds (tropolone and ferric ammonium citrate) were identified to be capable of replacing the animalsourced proteins commonly found in serum-free media for NS0 cell culture, namely bovine albumin, insulin and transferrin. The cholesterol requirement of NS0 cells was satisfied by the use of a commercially available non-proteinaceous, non-animal sourced cholesterol/fatty acid mix in place of bovine lipoproteins, which in effect also eliminated the need for recombinant albumin. In the animal-free medium thus formulated, NS0 cell lines, either the host or recombinant constructs, were all able to grow in batch culture to 1$ 3 · 10 6 viable cells/ml for multiple passages, with no requirement for gradual adaptation even when seeded from 10% serum-containing cultures. It was surprising to observe that the recombinant insulin was essentially ineffective as sodium salt compared to its zinc salt. Studies showed that the zinc deficiency in the former resulted in a rapid decline of cell viabilities. Supplementation of zinc ions greatly improved growth, and even led to the total replacement of recombinant insulin and hence the formulation of a protein-free medium. When the cell lines were adapted to cholesterol-independent growth which eliminated the need for any lipid source, a completely chemically-defined animal-free medium was formulated. In all cases, antibody production by various GS-NS0 constructs in animal-free media was stable for multiple passages and at least similar to the original serum-free medium containing the animal-sourced proteins. The medium also served well for cryopreservation of NS0 cells in the absence of serum.

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“…Production of recombinant Ab by stably transfected cell lines depends on the presence of effective genetic regulatory elements in vectors, selective agents, selection of parental cell line, and culture medium (Page and Sydenham 1991). For some Abs the Ab production can be as high as 2 g/L (Zhou et al 1997). The highest production in HEK293 cells grown in a serum-free medium ranged from 20 to 1,000 mg/L (Barnes et al 2001).…”

Section: Introductionmentioning

confidence: 99%

Production of anti TNF-α antibodies in eukaryotic cells using different combinations of vectors carrying heavy and light chains

et al. 2014

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Tumor necrosis factor-a (TNF-a) plays a key role in rheumatoid arthritis and some other autoimmune diseases. Therapy with anti-TNF-a recombinant antibodies (Ab) appears to be highly effective. Production of new hyper-producing eukaryotic cell lines can decrease the treatment cost, which currently is very high. However, due to the complexity of protein transcription, translation, processing, and secretion in mammalian cells, the stages at which antibody expression is affected are still poorly determined. The aim of this work was to compare the productivity of two cell lines developed in CHO DG44 cells, deficient in dihydrofolate reductase, transfected with vectors carrying either heavy (H) or light (L) chains of chimeric antibody under different combinations of selective elements. Both H and L chains were cloned either in pOptiVEC or pcDNA3.3 vectors and different combinations were used to produce HL and LH cell lines. We have shown that Ab production has been low and comparable between HL and LH cells until selection on methotrexate (MTX) when LH but not HL cells have responded with 3.5 times increased productivity. Flow cytometry analysis has demonstrated that intracellular concentration of full size Abs in LH cells was 5.6 times higher than in HL ones due to higher amount of H chain synthesis. No differences in viability between HL and LH cells have been found. We have concluded that the expression of H chain in the pOptiVEC vector, which is responsible for MTX resistance, has led to the suppression of H chain synthesis and limitation in full Ab assembly.

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Fed-batch culture of recombinant NS0 myeloma cells with high monoclonal antibody production

Cited by 120 publications

References 28 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

Development of Animal-free, Protein-Free and Chemically-Defined Media for NS0 Cell Culture

Production of anti TNF-α antibodies in eukaryotic cells using different combinations of vectors carrying heavy and light chains

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