Process development for the production of recombinant antibodies using the glutamine synthetase (GS) system

Brown, Martin C.; Renner, G.; Field, Ray; Hassell, Tom

doi:10.1007/bf02521750

Cited by 89 publications

(75 citation statements)

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“…This allowed convenient expression and purification of IL-27 as a functional monomeric cytokine as described previously (33). We produced scIL-27 with the GS expression system, which is considered to be one of the most powerful systems for the production of recombinant protein in mammalian cells (8). The final concentration of the scIL-27 was 50 mg/l in the supernatant of scIL-27-expressing CHO cells.…”

Section: Resultsmentioning

confidence: 99%

“…The sequence was confirmed, and the DNA fragment was directly subcloned into the pEE12.4 expression vector with Hind III and EcoR I sites to construct the scIL-27 expression vector. The GS expression vector pEE12.4 carries the GS gene, and its product catalyzes the formation of glutamine from glutamate and ammonia (8). CHOK1 cells were transfected with the scIL-27 expression vector and cultured in the glutamine-free CD-CHO medium in the presence of the GS inhibitor methionine sulfoximine (Sigma, St. Louis, MO), at a concentration of 50 M, followed by adaptations for suspension cultures in Erlenmeyer flasks on an orbital shaker at 37°C.…”

Section: Methodsmentioning

confidence: 99%

“…Several reports appeared recently suggesting the possible role of IL-27 in IBD (14,30); however, the physiological effect of exogenously treated IL-27 on IBD mice to suppress the intestinal inflammation has not been reported. Thus, to assess the therapeutic effect of IL-27 on intestinal inflammation, we prepared single-chain human IL-27 (scIL-27) protein generated by the flexible linking of the human EBI3 protein and human p28 polypeptide in CHO cells by using the glutamine synthetase (GS) expression system (8). scIL-27 clearly inhibited the differentiation of both human and mouse Th17 cells from naive CD4 ϩ T cells under Th17-polarizing conditions in vitro.…”

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confidence: 99%

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Treatment with IL-27 attenuates experimental colitis through the suppression of the development of IL-17-producing T helper cells

Sasaoka

Itô

Yamashita

et al. 2011

American Journal of Physiology-Gastrointestinal and Liver Physi

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Inflammatory bowel disease (IBD) represents a group of chronic inflammatory diseases characterized by inflammation and relapsing gastrointestinal disorders. Recent studies have shown that Th17 cells, which are well known as key mediators of chronic inflammation, have a pivotal role in onset and development of IBD in humans and mice, alike. In recent years, it has been reported that IL-27, which is an IL-12-related heterodimeric cytokine consisting of EBI3 and p28 subunits, act directly on naive T cells to suppress the differentiation of Th17 cells. However, effects of exogenous IL-27 on the IBD are not well elucidated. To clarify the suppressive effect of IL-27 treatment on IBD, we applied the flexible linking method to EBI3 and p28 subunits and generated a single-chain human IL-27 (scIL-27). scIL-27 inhibited xenogenic mouse Th17 cell differentiation in vitro, indicating that scIL-27 also acts in mouse immune systems. In a 2,4,6-trinitrobenzene sulfonic acid (TNBS)-induced mouse acute colitis model, subcutaneous scIL-27 treatment significantly improved the colon length, extent of necrosis, and ulceration and thickened epithelium and several pathological scores in a dose-dependent manner. scIL-27 clearly suppressed several inflammatory cytokines, including IL-17, in inflamed colon, except for anti-inflammatory cytokine IL-10. The mesenteric lymph node cells from scIL-27-treated mice also exhibited a reduced inflammatory response and, furthermore, a lower population of Th17 cells than those of PBS-treated mice. Finally, we showed the therapeutic efficacy of scIL-27 on TNBS-induced colitis even after active colitis was established. These results suggest new possible therapeutic approaches for IBD, including disorders such as Crohn's disease and ulcerative colitis.

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Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

mentioning

confidence: 99%

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Treatment with IL-27 attenuates experimental colitis through the suppression of the development of IL-17-producing T helper cells

Sasaoka

Itô

Yamashita

et al. 2011

American Journal of Physiology-Gastrointestinal and Liver Physi

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“…Some examples are the use of the GS as a selection and amplification system and also as an optimization approach for the cellular metabolism to get rid for the need for glutamine in the culture medium (Sanders et al 1987;Bebbington et al 1992;Brown et al 1992); or the overexpression of cytosolique pyruvate carboxylase in continuous cell lines for improving the connection between glycolysis and the Krebscycle (Irani et al 1999: BHK21;Elias et al 2003: HEK293); or the overexpression of anti-sense LDH-A and of cytoplasmic glycerol-3-phosphate dehydrogenase in CHO cells for increasing the oxidative phosphorylation, for decreasing cellular respiration and thus reducing sensitivity to reactive oxygen species and overall, reducing apoptosis (Jeong et al 2004). Cellular behaviour against environmental stress conditions is often characterized by an induction of apoptosis, thus leading to a precious loss of viable biomass and the stopping of a productive culture.…”

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confidence: 99%

Introduction to animal cell culture technology—past, present and future

Merten

2006

Cytotechnology

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“…However, since too many receptors are detected, its applicability will practically depend on how well one can determine and manipulate the physiologically important pathways in a given culture system. Using the above approaches, researchers have published several papers on the development of serum-free media for NS0 culture processes (Brown et al 1992;Buser et al 1994;Robinson et al, 1995;Dempsey et al 2003). Break-through has also been in using these media for cell freezing and preservation (Merten et al 1995).…”

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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Process development for the production of recombinant antibodies using the glutamine synthetase (GS) system

Cited by 89 publications

References 9 publications

Treatment with IL-27 attenuates experimental colitis through the suppression of the development of IL-17-producing T helper cells

Treatment with IL-27 attenuates experimental colitis through the suppression of the development of IL-17-producing T helper cells

Introduction to animal cell culture technology—past, present and future

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

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