Engineered cell lines

Information concerning the function of NT-4/5 in the nervous system in vivo comes either from targeted deletions of the NT-4/5 gene, or from the admininstration of the protein or the grafting of cell lines engineered to secrete high levels of NT-4/5. 

The blastocyst is an early embryonic stage in mammalian development. Murine blastocysts can be harvested at day 3.5 p.c. Their inner cell mass contains embryonic stem cells. Multiple murine embryonic stem cell lines have been established. Embryonic stem cells carrying genetically engineered mutations are injected into blastocysts, which are subsequently implanted into pseudopregnant foster mothers. 

Glatt, H., Engst, W., and Hagen, M. et al. (1997). The use of cell lines genetically engineered for human xenohiotic metahohsing enzymes. In L.F.M. van Zutphen and M. Balls (Eds.) Animal Alternatives, Welfare and Ethics. AmsX rdwu Elsevier 81-94. 

Monteiro-Riviere, N.A., Inman, A.O. and Zhang, L.W. (2009) Limitations and relative utility of screening assays to assess engineered nanoparticle toxicity in a human cell line. Toxicology and Applied Pharmacology, 234 (2), 222-235. 

Many of the initial biopharmaceuticals approved were simple replacement proteins (e.g. blood factors and human insulin). The ability to alter the amino acid sequence of a protein logically coupled to an increased understanding of the relationship between protein structure and function (Chapters 2 and 3) has facilitated the more recent introduction of several engineered therapeutic proteins (Table 1.3). Thus far, the vast majority of approved recombinant proteins have been produced in the bacterium E. coli, the yeast S. cerevisiae or in animal cell lines (most notably Chinese hamster ovary (CHO) cells or baby hamster kidney (BHK) cells. These production systems are discussed in Chapter 5. 

The biopharmaceutical sector is largely based upon the application of techniques of molecular biology and genetic engineering for the manipulation and production of therapeutic macromolecules. The majority of approved biopharmaceuticals (described from Chapter 8 onwards) are proteins produced in engineered cell lines by recombinant means. Examples include the production of insulin in recombinant E. coli and recombinant S. cerevisiae, as well as the production of EPO in an engineered (Chinese hamster ovary) animal cell line. 

In order to overcome environmental concerns in particular, some companies are investigating the use of engineered plant cell lines as opposed to intact transgenic plants in the context of biopharmaceutical production. One company (DowAgroSciences) gained approval in 2006 for a veterinary subunit vaccine against Newcastle disease in poultry produced by such means. 

Enbrel is a product now approved for medical use that is based upon this strategy. The product is an engineered hybrid protein consisting of the extracellular domain of the TNF p75 receptor fused directly to the Fc (constant) region of human IgG (see Box 13.2 for a discussion of antibody structure) The product is expressed in a CHO cell line from which it is excreted as a dimeric soluble protein of approximately 150 kDa. After purification and excipient addition (mannitol, sucrose and trometamol), the product is freeze-dried. It is indicated for the treatment of rheumatoid arthritis and is usually administered as a twice-weekly s.c. injection of 25 mg product reconstituted in WFI. Enbrel functions as a competitive inhibitor of TNF, a major pro-inflammatory cytokine. Binding of TNF to Enbrel prevents it from binding to its true cell surface receptors. The antibody Fc component of the hybrid protein confers an extended serum half-life on the product, increasing it by fivefold relative to the soluble TNF receptor portion alone. 

In chlorate production the EMOS system has also been used to determine the formation of deposits on the electrodes, either the anode or cathode and combined with the information on process and electrolyte composition the system determines the need for cell cleaning or acid rinsing. The close monitoring of individual cell voltages has allowed plant engineers to establish the most appropriate current density for production lines dependent upon the state of the anode coatings. This allows for the same overall production capacity while permitting the operation of two different cell lines in the cell room at different current densities based upon the state of the anodes and cathodes in the cell. 

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