The ability to clone and produce a wide range of proteins from diverse sources became feasible with the advent of recombinant technology. The selection of expression hosts for commercial biotechnology proteins is based on the economics of fermentation and purification as well as the ability of the host to accomplish the post-translational modifications needed for full biological activity of the recombinant protein. Some of these post-translational modifications include: signal peptide processing, pro-peptide processing, protein folding, disulfide bond formation, glycosylation, gamma carboxylation and beta-hydroxylation. Some of the economic factors influencing the choice of an expression host include: rates of biomass production, equipment costs, medium composition and expense, processes for protein recovery and purification, product yields, and the potential for contamination.
Much of the early work in biotechnology was directed toward the expression of recombinant or "heterologous" proteins in prokaryotes, like Escherichia coli and Bacillus subtilis. Such work in procaryotes provided ease of genetic manipulation, growth of the organisms in batch culture and the possibility of large-scale fermentation.
E. coli can perform signal peptide processing, protein folding, and disulfide bond formation. However, it cannot secrete proteins extracellularly glycosylate, gamma carboxylate, beta hydroxylate or process propeptides. B. subtilis suffers from the same limitations E. coli except that it is capable of extracellular secretion.
Total production costs from bacteria are also high because of problems with product recovery, purification, and the inability of bacteria to perform many of the post-translational modifications mentioned above. Furthermore, E. coil and other bacteria are pathogens and contaminants, such as, pyrogens and endotoxins, must be removed from the recombinantly produced protein. In addition, extensive post-purification chemical and enzymatic treatments (e.g., to refold the protein into an active form) can be required to obtain biologically active protein.
Because proteins are not secreted from prokaryotes, like E. coli, such cells must be disrupted for product recovery. The subsequent release of bacterial contaminants and other proteins make product purification more difficult and expensive. Because purification accounts for up to 90% of the total cost of producing recombinant proteins in bacteria, proteins, like tissue Plasminogen Activator (tPA), can cost several thousand dollars per gram to produce from E. coli.
Because of the many inadequacies associated with prokaryotic hosts, the biotechnology industry has looked to eukaryotic hosts like mammalian cell tissue culture, yeast, fungi, insect cells, and transgenic animals, to properly and efficiently express recombinant proteins. However, these hosts can suffer from any or all of the following disadvantages: expensive fermentation, low yields, secretion problems, inappropriate modifications in protein processing, high operating costs, difficulties in scaling up to large volumes, and/or contamination that either kills the host culture or makes product purification more expensive. For these reasons, existing eukaryotic hosts are unable to provide high-volume, low-cost protein production of recombinant proteins.
For most of those proteins requiring extensive post-translational modifications for therapeutic and/or functional activity, mammalian cell culture is the most common alternative to E. coli. Although mammalian cells are capable of correctly folding and glycosylating bioactive proteins, the quality and extent of glycosylation can vary with different culture conditions among the same host cells. Furthermore, mammalian culture has extremely high fermentation costs (60-80% of total production expense), requires expensive media, and poses safety concerns from potential contamination by viruses and other pathogens. Yields are generally low, for example, in the range of 0.5-1.5% of cellular protein, or up to about 300-400 milligrams per liter.
Yeast, fungi, insect cells and transgenic animals are currently being used as alternatives to mammalian cell culture. Yeast, however, produces incorrectly glycosylated proteins that have excessive mannose residues and generally limited eukaryotic processing. Further, although the baculovirus insect cell system can produce high levels of glycosylated proteins, these are not secreted--making purification complex and expensive. Fungi represent the best current system for high-volume, low-cost production, but they are not capable of expressing many target proteins. Transgenic animals are subject to lengthy lead times to develop herds with stable genetics, high operating costs, and contamination by animal viruses.
The biochemical, technical and economic limitations on existing prokaryotic and eukaryotic expression systems has created substantial interest in developing new expression systems for recombinant proteins. Plants represent the most likely alternative to existing systems because of the advantageous economics of field-grown crops, the ability to synthesize proteins in storage organs like tubers, seeds, fruits and leaves and the ability of plants to perform many of the post-translational modifications previously described. However, existing plant expression systems suffer from low yield (&lt;1.5% of total cellular protein).
Furthermore, expression of the target protein occurs in the open field (in roots, stems, leaves, fruits and seeds), thus making it difficult to prevent the recombinant protein from entering the food and feed chain. This is an issue of much concern to government regulatory agencies.
Although the use of plant cell culture to express proteins has been discussed, the lack of knowledge about the genetics and biochemistry of plant gene expression and secretion has precluded this system from being developed into a commercially feasible one.
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