Mammalian system, Chinese hamster ovary cells

Some drugs with low intrinsic permeability achieve acceptable oral bioavailability because they are substrates for uptake transporters, which normally function in nutrient uptake. The most prominent example is the peptide transporter, PepTl, which is active toward peptidomimetic antibiotics such as cephalexin, the antiviral agent valacyclovir [24] and other drugs. PepTl is natively expressed in Caco-2 cells, and adenovirus transduction has been used to increase PepTl expression levels [25]. However, the expression of PepTl was not polarized in this system and this expressed system appears to be of limited value as an improved screening model. PepTl has also been expressed in Chinese hamster ovary cells and a variety of other mammalian systems [26, 27]. 

Chinese Hamster CHO/Hgprt System. Chinese hamster ovary (CHO) cells have 21 or 22 chromosomes with one intact X chromosome and a large acrocentric marker chromosome (Natarajan and Obe, 1982). The use of these cells in mammalian mutation experiments was first reported by Hsie et al. (1975), and was refined into a quantitative assay for mutagenicity testing by O Neill. The performance of this system has been reviewed by the USA EPA Gene-Tox Program. The experimental procedure for this assay is similar to the V79/Hgprt system already described, and for more detailed descriptions the reader is referred to Li et al. (1987). 

The genotoxic data are largely negative, although 1,1,1-trichloroethane was mutagenic in some Salrmnella assays and induced chromosomal aberrations in Chinese hamster ovary cells and cell transformation in mammalian systems. ... 

Phenol induced mutations at the hprt locus of Chinese hamster V79 cells in the presence of an exogenous metabolic system from the livers of phenobarbital-induced mice and tk locus mutations in mouse lymphoma L5178Y cells in the presence or the absence of an exogenous metabolic activation system. Micronuclei were induced by phenol in Chinese hamster ovary cells in one study and sister chromatid exchanges in mammalian cells were increased in several studies, including three with human lymphocytes. 

For eukaryotic genes, expression in insect cells (Baculovirus system) or mammalian cells (Chinese hamster ovary cells, cos7 monkey cells) is possible and is often more likely to lead to active protein than in bacteria or yeast owing to a more efficient posttranslational machinery. However, expression rates are very low and not feasible for production of biocatalysts. 

O Neill, J.P., Brimer, P.A., Machanoff, R., Hirsch, G.P., Hsie, A.W. 1977. A quantitative assay of mutation induction at the hypoxanthine-quanine phosphoribosyl transferase locus in Chinese hamster ovary cells (CHO/HGPRT System) Development and definition of the system. Mutat. Res. 45 91-101. Huberman, E. 1976. Cell-mediated mutagenicity of different genetic loci in mammalian cells by carcinogenic polycyclic hydrocarbons. In Screening Tests in Chemical Carcinogenesis, eds. R. Montesano, H. Bartsch,... 

Mammalian cell suspension cultures are the preferred choice for large-scale recombinant protein production in stirred-tank bioreactors. The most widely used systems are Chinese hamster ovary (CHO) cells and the murine myeloma fines NSO and SP2/0. In half of the biological license approvals from 1996-2000, CHO cells were used for the production of monoclonal antibodies and other recombinant glycosylated proteins, including tPA (tissue plasminogen activator) and an IgGl fusion with the tumor necrosis factor (TNF) receptor, the latter marketed as Enbrel [7]. 

Etanercept is produced by recombinant technology in a Chinese hamster ovary mammalian cell expression system. The WCB is grown in a proprietary media system. The cells are cultured initially in flasks and then inoculated into the bioreactor vessel. The product is purified in a number of chromatographic steps, followed by viral inactivation and viral filtration steps. 

Mammalian cells in vitro are exposed to the test chemical with and without an exogenous mammalian metaboUc activation system and cultured for two rounds of replication in bromodeox3Uiridine (BrdU) containing medium. After treatment with a spindle inhibitor (e.g., colchicine) to accumulate cells in a metaphase-Uke stage of mitosis (c-metaphase), cells are harvested, stained, and metaphase cells analyzed for SCEs. Primary cultures (e.g., human lymphocytes) or established cell lines (e.g., Chinese hamster ovary or lung cells) may be used in the assay. At least three adequately spaced concentrations of the test substance should be used. 

The simplest and most sensitive assays for detecting clastogenic (i.e. chromosomal breaking) effects involve the use of mammalian cells. Cultures of established cell lines (e.g. Chinese hamster ovary) as well as primary cell cultures (e.g. human l)nnphocyte) may be used. After exposure to a range of chemical concentrations in the presence and absence of an appropriate metabolic activation system, the cell cultures are treated with a spindle inhibitor (e.g. vinblastine) to accumulate cells in a metaphaselike stage of mitosis. Cells are harvested at appropriate times and chromosome preparations are made, stained with DNA-specific dye and the metaphase cells are analysed under the microscope for chromosome abnormalities. 

Most IFNs have now been produced in a variety of expression systems including E. coli, fungi, yeast and also some mammalian cell lines such as Chinese hamster ovary (CHO) cell lines and monkey kidney cell lines. Most IFNs currently in medical use are recombinant human (rh) products produced in E. coli. The inability of E. coli to carry out post-translational modifications is in most instances irrelevant, as the majority of human IFN-as, as well as IFN-jS, are not normally glycosylated. While IFN-y is glycosylated, the E. co/i-derived unglycosylated form displays a biological activity identical to the native human protein. 

Although numerous cell lines have been screened for their efficiency as a host system for recombinant protein production, only a few have shown favorable properties for the expression of biopharmaceuticals (Hauser, 1997). Regulatory and economic issues for large-scale production and the intended application of the recombinant protein (diagnosis, therapy, etc.) have to be carefully considered (Makrides and Prentice, 2003). Three mammalian cell lines are now commonly used by the pharmaceutical industry Chinese hamster ovary (CHO) cells, the murine myeloma SP2/0 and the NS0 cell line (see Table 3.1). These cell lines have been used to produce 11 of 21 therapeutic products approved from 1996 to 2000 (Chu and Robinson, 2001). 

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