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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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post-translational modifications. Patterns of glycosylation achieved can, however, differ from typical patterns obtained when a glycoprotein is expressed in a mammalian cell line.
Most fungal host strains also naturally produce significant quantities of extracellular proteases, which can potentially degrade the recombinant product. This difficulty can be partially overcome by using mutant fungal strains secreting greatly reduced levels of proteases. Although researchers have produced a number of potential therapeutic proteins in recombinant fungal systems, no biopharmaceutical produced by such means has thus far sought or gained marketing approval.
Transgenic animals
The production of heterologous proteins in transgenic animals has gained much attention in the recent past. The generation of transgenic animals is most often undertaken by directly microinjecting exogenous DNA into an egg cell. In some instances, this DNA will be stably integrated into the genetic complement of the cell. After fertilization, the ova may be implanted into a surrogate mother. Each cell of the resultant transgenic animal will harbour a copy of the transferred DNA. As this includes the animal’s germ cells, the novel genetic information introduced can be passed on from one generation to the next.
A transgenic animal harbouring a gene coding for a pharmaceutically useful protein could become a live bioreactor, producing the protein of interest on an ongoing basis. In order to render such a system practically useful, the recombinant protein must be easily removable from the animal, in a manner which would not be injurious to the animal (or the protein). A simple way of achieving this is to target protein production to the mammary gland. Harvesting of the protein thus simply requires the animal to be milked.
Mammary-specific expression can be achieved by fusing the gene of interest with the promoter-containing regulatory sequence of a gene coding for a milk-specific protein. Regulatory sequences of the whey acid protein (WAP), b-casein and a- and b-lactoglobulin genes have all been used to date to promote production of various pharmaceutical proteins in the milk of transgenic animals (Table 3.13).
One of the earliest successes in this regard entailed the production of human tissue plasminogen activator (tPA) in the milk of transgenic mice. The tPA gene was fused to the upstream regulatory sequence of the mouse whey acidic protein — the most abundant protein found in mouse milk. More practical from a production point of view was the subsequent production of tPA in the milk of transgenic goats, again using the murine WAP gene regulatory sequence to drive expression (Figure 3.8). Goats and sheep have proved to be the most attractive host systems, as they exhibit a combination of attractive characteristics. These include:
• high milk production capacities (Table 3.14);
• ease of handling and breeding, coupled to well-established animal husbandry techniques.
A number of additional general characteristics may be cited which render attractive the production of pharmaceutical proteins in the milk of transgenic farm animals. These include:
• ease of harvesting of crude product—which simply requires the animal to be milked;
• pre-availability of commercial milking systems, already designed with maximum process hygiene in mind;
• low capital investment (i.e. relatively low-cost animals replace high-cost traditional fermentation equipment) and low running costs;
Table 3.13. Proteins of actual/potential therapeutic use that have been produced in the milk of transgenic animals
Protein Animal species Expression levels in milk
tPA Goat 6 g/l
Interleukin-2 Rabbit 0.5mg/l
Factor VIII Pig 3mg/l
Factor IX Sheep 1 g/l
a1-Antitrypsin Goat 20g/l
Fibrinogen Sheep 5 g/l
Erythropoietin Rabbit 50 mg/l
Antithrombin III Goat 14 g/l
Human a-lactalbumin Cow 2.5 g/l
Insulin-like growth factor I Rabbit 1 g/l
Protein C Pig 1 g/l
Growth hormone Rabbit 50 mg/l
• high expression levels of proteins are potentially attained. In many instances, the level of expression exceeds 1 g protein/litre milk. In one case, initial expression levels of 60 g/l were observed, which stabilized at 35 g/l as lactation continued (the expression of the a1-antitrypsin gene, under the influence of the ovine b-lactoglobulin promoter, in a transgenic sheep). Even at expression levels of 1 g/l, one transgenic goat would produce a similar quantity of product in 1 day as would likely be recoverable from a 50-1001 bioreactor system;
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