Efficient methods now exist to produce a wide variety of biologically active recombinant proteins -- both large and small, simple and complex.

GTC has successfully produced over 20 monoclonal antibodies or Ig fusions in the milk of transgenic animals. These are among the most complex recombinant proteins to be expressed transgenically. They require stable integration of two gene constructs, simultaneous synthesis of two proteins in the same cell in roughly equal proportions, and proper assembly into tetrameric protein complexes.

Unlike other types of biotherapeutics such as growth factors or hormones, antibody therapeutics may require repeated treatments with relatively large doses in large numbers of patients. This translates into total annual requirements of hundreds of kilograms of any given monoclonal antibody. As the need for large quantities of monoclonal antibodies grows, transgenic animals will provide a cost-competitive large-scale production alternative for these complex proteins.

A transgenic animal carries heterologous DNA stably integrated into its genome. In most cases, for GTC Biotherapeutics, the heterologous DNA or transgene directs expression of therapeutic proteins into the milk of the animal. The transgene is inherited in a Mendelian fashion which permits the generation of large scale production herds using a conventional breeding program.

The major function of the mammary gland is to produce proteins. The mammary gland is capable of producing milk that carries over 40g/L of protein. We take advantage of the unique properties of this "natural protein secretion organ". By utilizing molecular biology technology, we can design DNA constructs that reliably express high levels of therapeutic proteins in the milk of the animals that carry the transgene. We take advantage of the normal mammalian protein processing mechanisms to synthesize properly folded and assembled complex proteins. Although the epithelial cells in the mammary gland do not usually express antibodies, we have found that the machinery needed to properly fold and assemble the heavy and light chains of antibodies are well represented in these cells. By utilizing the milk specific promoters to express the heavy and light chains, the cellular machinery is capable of secreting high levels of properly folded antibody.

There are four other means of commercial protein production. E. coli production, which was the first commercialized, is very efficient, but limited to simple non-glycosylated proteins. Although the cost of production is low, the cost of processing and refolding the proteins is significant. Fungal systems, such as Pichia or filamentous fungi allow efficient production of some secreted proteins, but the glycosylation is usually high mannose which can effect the pharmokinetics of the protein. There is also the baculovirus production system, which can produce a wide range of proteins in small scale, but has yet to be scaled up to commercial levels. The standard method for producing complex glycosylated proteins, (i.e. Monoclonal Antibodies) is with cell tissue culture. The protein may be properly folded and modified, but the low yields per cost of production facility limit the number of proteins that can be developed.

As a dairy production animal goats are utilized all over the world. In choosing a species of animal it is optimal to have animals that have been bred for significant milk production and also have a relative short generation time. Formally, choices range from mice, our model system with a generation time of 3 months and a milk yield of 1 ml, to rabbits with an 8 month generation time and 4 liter yield, to the largest commercial species, the cow. Cows have a generation time of 3 years, with an annual milk yield of 8000 liters.

The amount of recombinant protein that a goat can produce is a product of the level of recombinant protein in the milk and the yield per lactation of the animal. The dairy breeds of goats that we use yield an average of 2.5 liters of milk per day. A typical goat can produce 800 or more liters of milk over a 10-month lactation period. Our recent imports of high producing New Zealand goats have given us the potential to increase the lactation yield of our herds by conventional herd improvement. The level of recombinant protein found in the milk of our transgenic goat lines vary from low to over 14g/L. In all of our commercial programs we have produced animals with a recombinant protein level of greater than 2g/L. At 2g/L and a 800L yearly production, this results in over 1KG of recombinant protein per lactation per animal.

A transgene construct is introduced into very early stage embryos which are then transferred to surrogate mothers. After a period of gestation, first-generation offspring are born. These offspring are analyzed to identify animals that have integrated the transgene.

A piece of DNA is assembled specifically for each application. It includes several components: the gene sequence encoding the target human protein; regulatory regions from the gene of a milk-specific protein, other DNA regions that help assure high-level protein production; and a piece of backbone DNA that allows the construct to be handled, replicated and microinjected into embryos.

There are half a dozen proteins that occur only in milk, and these fall into two biochemical classes: caseins and whey proteins. The genes for these proteins have been cloned from a variety of species. These mammary-specific regulators are combined with cloned gene sequences that encode a specific target protein and used to create transgenic animals. In these animals, the recombinant protein is expressed only in the lactating mammary gland. Additional regulatory sequences such as enhancers or insulators can help control the level of recombinant gene or protein expression.

Transgenically produced proteins are expressed in the milk of females during lactation. Milking typically begins upon the birth of offspring, however, initial milk containing recombinant protein may be obtained by hormonal induction of lactation in either females or males, before they reach sexual maturity -- in goats, at approximately 2 months of age (7 months after microinjection of the embryos). Often, enough milk may be obtained to confirm successful recombinant protein expression, to begin characterizing the protein, to start purification process development and even to begin preclinical studies.

Proteins are isolated in a multi-step process that combines methods used to recover proteins from cell culture supernatant with processes from the dairy industry. GTC has developed proprietary steps designed for milk as a starting material, as well as specific capture steps designed for each protein. Proteins may be recovered in extremely pure form suitable for use as therapeutics.

After purification, transgenically produced recombinant proteins are analyzed to characterize their structure, purity and activity. They are held to the same standards as any other recombinant protein. Assays are selected and/or developed as appropriate for each individual protein. Complex proteins that require post-translational modification are further analyzed to confirm that they have been properly processed. This would include analyzing the structure and configuration of sugars that are added to glycoproteins, or verifying chemical cleavage of immature propeptide forms of the protein.