Host cell lines

The identities and roles of many of the drug transporters are discussed in other chapters in this volume, and are not extensively reintroduced here. A goal is to develop a comprehensive panel of cells expressing individual, functional transporters as research reagents. To simplify data interpretation, the set of transporters should be expressed in the same host cell line and the abundance of functional proteins in the cell line should be known relative to the corresponding in vivo values. However, useful mechanistic data can be obtained from less comprehensive systems. 

Emphasis is placed not only upon ensuring the absence of contaminant proteins, but also other potential contaminants, particularly DNA. (Host cell-line-derived DNA could harbour oncogenes Chapter 7.)... 

Figure 5.1 Comparison of IgM complexed with DNA versus IgM decomplexed by pretreatment with 1.0 M NaCl and 0.05 M EDTA. Sample purified IgM plus partially degraded DNA extracted from the host cell line. Dotted trace marks the salt gradient. Arrow marks introduction of 5 mM EDTA to the salt strip. Mono-S HR5/5, pH 5.5. See text for explanation. (Data from P. Gagnon, 1996, Special weapons and tactics for removal of product-bound DNA, oral presentation, BioEast 96, Washington, D.C.)...

Introduction of a gene of interest into the host cell line by viral infection is a convenient method since a large number of cells can be infected simultaneously. Systems employing Semliki Forest Virus, Vaccinia Virus, and Retoviral vectors are used. However, drawbacks include the requirement for special precautions when engineering and preparing the viral... 

Yamane-Ohnuki N, Kinoshita S, Inoue-Urakubo M, Kusunoki M, Lida S, Nakano R, Wakitani M, Niwa R, Sakurada M, Uchida K, Shitara K, Satoh M. Establishment of FUT knockout Chinese Hamster Ovary cells an ideal host cell line for producing completely defucosylated antibodies with enhanced antibody-dependent cellular cytotoxicity. Biotech Bioeng 2004 87 614—622. 

Bacterial, yeast, and mammalian cells are all commonly used in recombinant DNA work. The choice of the type of cell, and the particular strain of that cell type, will depend on the type of product sought, the quantity of product desired, and the economics of production. For example, the cell type that produces the highest level of a particular protein may not be the best objective choice for production, if that cell type does not properly fold and modify the protein for full activity. Usually, each new product must be evaluated on its own when choosing a host cell line. 

Glycosylation in animal cells the effect of the host cell line... 

Transfection of the Vector into the Host Cell Line... 

One week after the third or fourth boost, antisera may be collected and tested by flow cytometry. Immunoreactivity of the antisera with receptor transfectants, however, does not correlate with immunoreactivity with the target receptor. This is because mice produce antibodies to many other cell surface determinants even when receptors are expressed in syngeneic cell lines. Depending on the transfectant host cell line, antisera may be better assessed by flow cytometry using transformed cell lines or leukocytes known to express the receptor or receptor transfectants made in a different cell line. 

A major breakthrough in FVIII replacement therapy safety was achieved with the development of recombinant FVIII concentrates [7]. The development of rFVIII was also a major accomplishment in biotechnology that required cloning, identification and transfection of the rFVIII gene into suitable host cell lines, and subsequent characterization of the expressed proteins. An equally remarkable feat of manufacturing expertise was required to... 

The manufacturers of recombinant proteins for chnical applications and regulatory agencies were in agreement that it was extremely important to minimize eventual risks associated with the use of recombinant mammalian cell hosts. Risks were seen in tumour principles, carried by the DNA of the host and in adventitious agents (vimses, mycoplasma, etc.) that could infect the host cell lines and thus eventually be transmitted to patients receiving products from those hosts. Also, the consistency and quality of the recombinant proteins were discussed in the con-... 

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