Recombinant animal cells

Although capable of glycosylating heterologous human proteins, the glycosylation pattern usually varies from the pattern observed on the native glycoprotein (when isolated from its natural source, or when expressed in recombinant animal cell culture systems). 

The functional effects of glycosylation take on added significance in the context of producing gonadotrophins by recombinant means. As subsequently discussed, several are now produced for clinical application in recombinant (animal cell line) systems. While the glycosylation patterns observed on the recombinant molecules can vary somewhat in composition from those associated with the native hormone, these slight differences bear no negative influence upon their clinical applicability. 

Wagner R Lehmann J (1988) The growth and productivity of recombinant animal cells in a bubble-free aeration system. Trends in Biotechnology 6 101-104. 

Large-Scale Production of Proteins from Recombinant DNA Using Suspensions of Animal Cells... 

When sufficiently high levels of expression and protein accumulation are achieved, efficient downstream processing protocols must be developed to insure product quality and the economic feasibility of production. As the demand for safe, recombinant pharmaceutical proteins continues to expand, the market potential of plant-produced recombinant proteins is considerable. Molecular farming can produce recombinant proteins at a lower cost than traditional expression systems based on microbial or animal cell culture, and without the risk of contamination with human pathogens. 

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. 

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. 

The desired gene/cDNA is normally amplified, sequenced and then introduced into an expression vector that facilitates its introduction and expression (transcription and translation) in an appropriate producer cell type. All recombinant therapeutic proteins approved to date are produced in E. coli, S. cerevisiae or in animal cell lines (mainly CHO or BHK cells). The general... 

Expression of recombinant proteins in animal cell culture systems... 

In addition to recombinant biopharmaceuticals, animal cell culture is used to produce various other biologically based pharmaceuticals. Chief amongst these are a variety of vaccines and hy-bridoma cell-produced monoclonal antibodies (Chapter 13). Earlier interferon preparations were also produced in culture by a particular lymphoblastoid cell line (the Namalwa cell line), which was found to synthesize high levels of several IFN-a s naturally (Chapter 8). 

Over half of all biopharmaceuticals thus far approved are produced in recombinant E. coli or S. cerevisiae. Industrial-scale bacterial and yeast fermentation systems share many common features, an overview of which is provided below. Most remaining biopharmaceuticals are produced using animal cell culture, mainly by recombinant BHK or CHO cells (or hybridoma cells in... 

After its purification, sterile filtration and aseptic filling, human urokinase is normally freeze-dried. Because of its heat stability, the final product may also be heated to 60 °C for up to 10 h in an effort to inactivate any undetected viral particles present. The product utilized clinically contains both molecular mass forms, with the higher molecular mass moiety predominating. Urokinase can also be produced by techniques of animal cell culture utilizing human kidney cells or by recombinant DNA technology. 

Better methods for preparing clotting factors from blood and the development of recombinant clotting factors provided the solutions. Methods of detecting, inactivating, and removing viruses were improved, and none of the hemophilia replacement products— conventional or recombinant—has transmitted either HIV or hepatitis since 1987. As an alternative, recombinant clotting factors 8 and 9, produced in animal cells, were approved in 1992 and 1997, without the risk associated with human blood products. 

Over half of all biopharmacuticals thus far approved are produced in recombinant E. coli or S. cerevisiae. Industrial-scale bacterial and yeast fermentation systems share many common features, an overview of which is provided below. Most remaining biopharmaceuticals are produced using animal cell culture, mainly by recombinant BFIK or CFiO cells (or hybridoma cells in the case of some monoclonal antibodies Appendix 1). While industrial-scale animal cell culture shares many common principles with microbial fermentation systems, it also differs in several respects, as subsequently described. Microbial fermentation/animal cell culture is a vast speciality area in its own right. As such, only a summary overview can be provided below and the interested reader is referred to the Further Reading section. 

Recombinate (rhFactor VIII produced in an animal cell line) Baxter Healthcare/ Haemophilia A Genetics Institute 1992 (USA)... 

Epoxybutane induced morphological transformation, sister chromatid exchanges, chromosomal aberrations and mutation in cultured animal cells however, in a single study, it did not induce unscheduled DNA synthesis in rat primary hepatocytes. It induced sex-linked recessive lethal mutations and translocations in Drosophila melanogaster, mitotic recombination in yeast, and mutations in yeast and fungi. 1,2-Epoxybutane induced DNA damage and mutations in bacteria. 

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