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biopharmaceuticals biochemistry and biotecnology - Walsh G.

Walsh G. biopharmaceuticals biochemistry and biotecnology - John Wiley & Sons, 2003. - 572 p.
ISBN 0-470-84327-6
Download (direct link): biochemistryandbiotechnology2003.pdf
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At the time of writing, no therapeutic protein produced in the milk of transgenic animals had been approved for general medical use. A number of companies, however, continue to pursue this strategy. These include GTC Biotherapeutics USA (formerly Genzyme Transgenics) and PPL Therapeutics (Scotland). ^-Antitrypsin, antithrombin and a range of monoclonal antibody-based products produced via transgenic technology continue to be evaluated by these companies.
In addition to milk, a range of recombinant proteins have been expressed in various other targeted tissues/fluids of transgenic animals. Antibodies and other proteins have been produced in the blood of transgenic pigs and rabbits. This mode of production is, however, unlikely to be pursued industrially for a number of reasons:
only relatively low volumes of blood can be harvested from the animal at any given time point;
serum is a complex fluid, containing a variety of native proteins. This renders purification of the recombinant product more complex;
many proteins are poorly stable in serum;
the recombinant protein could have negative physiological side effects on the producer animal.
Therapeutic proteins have also been successfully expressed in the urine and seminal fluid of various transgenic animals. Again, issues of sample collection, volume of collected fluid and the appropriateness of these systems renders unlikely their industrial-scale adoption. One system, however, that does show industrial promise is the targeted production of recombinant proteins in the egg white of transgenic birds. Targeted production is achieved by choice of an appropriate egg white protein promoter sequence. Large quantities of recombinant product can potentially accumulate in the egg, which can then be collected and processed with relative ease.
Transgenic plants
The production of pharmaceutical proteins using transgenic plants has also gained some attention over the last decade. The introduction of foreign genes into plant species can be undertaken by a number of means of which Agrobacterium-based vector-mediated gene transfer is most commonly employed. Agrobacterium tumefaciens and A. rhizogenes are soil-based plant pathogens. Upon infection, a portion of Agrobacterium Ti plasmid is translocated to the plant cell and is integrated into the plant cell genome. Using such approaches, a whole range of therapeutic proteins have been expressed in plant tissue (Table 3.16). Depending upon the specific promoters used, expression can be achieved uniformaly throughout the whole plant or can be limited, e.g. to expression in plant seeds.
Plants are regarded as potentially attractive recombinant protein producers for a number of reasons, including:
cost of plant cultivation is low;
harvest equipment/methodologies are inexpensive and well established;
ease of scale-up;
Table 3.16. Some proteins of potential/actual therapeutic interest that have been expressed (at laboratory level) in transgenic plants
Protein Expressed in Production levels achieved
Erythropoietin Tobacco 0.003% of total soluble plant protein
Human serum albumin Potato 0.02% of soluble leaf protein
Glucocerebrosidase Tobacco 0.1% of leaf weight
Interferon-a Rice Not listed
Interferon-b Tobacco 0.00002% of fresh weight
GM-CSF Tobacco 250 ng/ml extract
Hirudin Canola 1.0% of seed weight
Hepatitis B surface antigen Tobacco 0.007% of soluble leaf protein
Antibodies/antibody fragment Tobacco Various
proteins expressed in seeds are generally stable in the seed for prolonged periods of time (often years);
plant-based systems are free of human pathogens (e.g. HIV).
However, a number of potential disadvantages are also associated with the use of plant-based expression systems, including:
variable/low expression levels sometimes achieved;
potential occurrence of post-translational gene silencing (a sequence-specific mRNA degradation mechanism);
glycosylation patterns achieved differ significantly from native human protein glycosylation patterns;
seasonal/geographical nature of plant growth.
For these reasons, as well as the fact that additional tried and tested expression systems are already available, production of recombinant therapeutic proteins in transgenic plant systems has not as yet impacted significantly on the industry.
The most likely focus of future industry interest in this area concerns the production of oral vaccines in edible plants or fruit, such as tomatoes and bananas. Animal studies have clearly shown that ingestion of transgenic plant tissue expressing recombinant sub-unit vaccines (see Chapter 10 for a discussion of sub-unit vaccines) induces the production of antigen-specific antibody responses, not only in mucosal secretions but also in the serum. The approach is elegant in that direct consumption of the plant material provides an inexpensive, efficient and technically straightforward mode of large-scale vaccine delivery, particularly in poorer world regions. However, several hurdles hindering the widespread application of this technology include:
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