Mammalian cells, recombinant

However, various assays utilizing nonstandardized sources of HDAC protein preparations and substrates appear to generate large variations in the resulting data. As an example. Table 6.1 summarizes the observed variations in the recently reported in vitro data for the effects of SAHA on individual H DAC isoforms [14,19-23]. HDAC isoforms used for in vitro screening assays have been expressed in Escherichia coli (HDACl, 3), Picchia, SF9 insect cells (HDACl-11) or mammalian cells (recombinant HDACl, 3) these sources may lead to differential enzyme activities. 

Farm animals produce recombinant proteins less expensively than bacteria or cells in culture because the farm animals produce large volumes of milk containing up to 5 g/L of recombinant protein. In addition, modifications to the proteins that can be performed only by mammalian cells are made by the cells of the mammary gland. Therefore, numerous pharmaceuticals that previously could only be made by cells in culture or extracted from human tissue or blood are being produced by lactating farm animals. 

Recombinant DNA technology has already provided several products of therapeutic interest from mammalian cells. Table 2 gives examples of products from mammalian cells, the use, and the technology used for production. Technology development for these products has centered around the differences in characteristics of mammalian versus microbial cells, notably, the shear sensitivity and susceptibiUty to contamination of the mammalian lines. 

Product formation kinetics in mammalian cells has been studied extensively for hybridomas. Most monoclonal antibodies are produced at an enhanced rate during the Gq phase of the cell cycle (8—10). A model for antibody production based on this cell cycle dependence and traditional Monod kinetics for cell growth has been proposed (11). However, it is not clear if this cell cycle dependence carries over to recombinant CHO cells. In fact it has been reported that dihydrofolate reductase, the gene for which is co-amplified with the gene for the recombinant protein in CHO cells, synthesis is associated with the S phase of the cell cycle (12). Hence it is possible that the product formation kinetics in recombinant CHO cells is different from that of hybridomas. 

Kost, T. A., and Condreay, J. P. (2002). Recombinant baculovirases as mammalian cell gene-delivery vectors. Trends Biotechnol. 20 173—180. 

Lazaris, A., Arcidiacono, S., Huang, Y., Zhou, J.F., Duguay, F., Chretien, N., Welsh, E.A., Soares, J.W., and Karatzas, C.N., Spider silk fibers spun from soluble recombinant silk produced in mammalian cells. Science, 295(5554), 472- 76, 2002. 

Mammalian cells Cultured human lymphocytes Mitotic recombination Aberrant metaphases No data - L Vova 1984... 

Factor IX Replacement Hemophilia B therapy may include recombinant (produced via transfection of mammalian cells with the human factor IX gene) or plasma-derived (concentrate from pooled plasma) factor IX (see Table 64-2). Guidelines for choosing the factor-concentrate formulation for hemophilia B are similar to the guidelines for hemophilia A. However, older-generation factor IX concentrates containing other vitamin K-dependent proteins (e.g., factors II, VII, and IX), called prothrombin complex concentrates (PCCs), have been associated with thrombogenic side effects. Consequently, these products are not first-line treatment for hemophilia B.11... 

This approach is not restricted to bacterial or viral cells. Mammalian cells under highly proliferating conditions can be cultured at increasing exposure to a compound in attempts to create resistant mutants. Alternatively, one can sometimes use a structural biology approach to predict amino acid changes that would abrogate inhibitor affinity from study of enzyme-inhibitor complex crystal structures. If the recombinant mutant enzyme displays the diminished inhibitor potency expected, one can then devise ways of expressing the mutant enzyme in a cell type of interest and look to see if the cellular phenotype is likewise abolished by the mutation. 

Geisse, S. and Henke, M. (2005) Large-scale transient transfection of mammalian cells a newly emerging attractive option for recombinant protein production. Journal of Structural and Functional Genomics, 6 (2-3), 165-170. 

Fang, X., et al. Functional characterization of a recombinant sodium-dependent nucleoside transporter with selectivity for pyrimidine nucleosides (cNTlrat) by transient expression in cultured mammalian cells. Biochem. 

Studies have shown that plants can make biologically active recombinant proteins through both transgenic and transient expression approaches. Although the plant post-translational machinery is similar to that of mammalian cells, there are some notable differences, e.g. differences in glycosylation, particularly the absence of sia-lation, which may impact the activity of certain proteins. The absence of mammalian enzymes may prevent complex maturation processes that are critical for the biological activity of proteins such as insulin. Fortunately these shortcomings affect the activity of only a limited number of proteins. 

One of the most obvious benefits of plants is the potential for production scale up, leading to the production of virtually limitless amounts of recombinant antibody at minimal cost Plants are easy to grow, and unlike bacteria or animal cells their cultivation is straightforward and does not require specialist media, equipment or toxic chemicals. It has been estimated that plantibodies could be produced at a yield of 10-20 kg per acre at a fraction of the cost associated with production in mammalian cells [2,18] The use of plants also avoids many of the potential safety issues associated with other expression systems, such as contaminating mammalian viruses or prions, as well as ethical considerations involving the use of animals. 

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]. 

The third time constraint depends on whether the product can be extracted from seeds or fruits. This uncouples protein expression and purification. Large batches of seeds containing the recombinant protein can be produced and stored at low costs. Provided the protein remains stable in the stored seeds, purification can be carried out on demand or shifted according to free capacities. The advantage of one large harvest, with seeds mixed to uniformity, is that this allows production on demand. In contrast, mammalian cell culture is prone to minor batch-to-batch variations in... 

Wurm, F. 2004. Production of recombinant protein therapeutics in cultivated mammalian cells. Nature Biotechnology 22, 1393-1398. 

Most interferons have now been produced in a variety of expression systems, including E. coli, fungi, yeast and some mammalian cell lines, such as CHO cell lines and monkey kidney cell lines. Most interferons currently in medical use are recombinant human (rh) products produced in E. coli. E. coli s inability to carry out post-translational modifications is irrelevant in most instances, as the majority of human IFN-as, as well as IFN- 3, are not normally glycosylated. Whereas IFN-y is glycosylated, the E. coli-derived unglycosylated form displays a biological activity similiar to the native human protein. 

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