Products and Partnering





QUESTIONS BY POTENTIAL CUSTOMERS

1. What is the business of transgenic production?

The business of transgenic production is the manufacturing of potentially unlimited quantities of biologically active proteins in the milk of transgenic animals, at production levels that meet market demands, and at costs that make the product feasible for its intended applications.

2. What is the advantage of transgenic production?

Transgenic production provides high level expression of recombinant proteins in the milk of transgenic animals. It is an enabling technology: it permits large-scale production of valuable therapeutic proteins, with flexible scale-up and significantly lower capital costs and risks. It enables the manufacture of complex or unique molecules that cannot be produced efficiently by any other method. It provides a safe and renewable source of product.

3. What is GTC's core competency in transgenics?

 GTC's capabilities encompass the development, production, recovery, characterization and testing of recombinant protein products derived from the milk of primarily transgenic goats and mice. Our expertise spans the entire production process, from transgenic and genetic manipulation methods, through animal production and protein isolation to the production of finished product.

4. Why produce proteins in transgenic animals?

 Transgenic production takes advantage of normal mammalian protein processing mechanisms to synthesize properly folded and assembled complex proteins -- all within the cells of the mammary gland. This method permits flexible scale-up of protein manufacturing to meet increasing production needs throughout the product development process. Scale-up is as simple as breeding more transgenic animals. This is easier and less expensive than building and validating a larger biopharmaceutical fermentation or mammalian cell culture facility, therefore reducing overall capital costs.

5. How does transgenic expression compare with other production methods?

 Recombinant protein concentrations in the milk of transgenic animals are substantially higher than levels attained in cultures of yeast, bacteria, insect cells or mammalian cells. Expression levels of 2 to 10 grams of recombinant protein per liter of milk are readily achievable in transgenic livestock. In comparison, highly optimized cell cultures can typically generate 0.2 to 1 gram per liter of culture medium.

6. Why not just use cell culture or fermentation?

Because transgenic production provides natural biological efficiency and resulting favorable production economics. The mammary gland is an ideal bioreactor for producing recombinant proteins. It has a cell density up to 1,000 times greater than a mammalian cell culture bioreactor, and it can achieve sustainable protein yields of 10 or more grams of recombinant protein per liter of milk per day. Protein production is continuous throughout the lactation cycle, which lasts up to ten months in each animal. Although goats are seasonal breeders, appropriate animal care and breeding methods enable uninterrupted year-round milk production.

7. How well do transgenic animals glycosylate proteins?

 The mammary gland is designed to produce glycoproteins to nourish suckling newborns. Recombinant proteins expressed in the milk of transgenic animals have mammalian patterns of glycosylation. These glycosylation patterns may differ slightly from native human proteins. Nonetheless, a large number of different complex recombinant proteins have been expressed successfully in transgenic animals with proper protein folding, correct assembly of multiple protein chains and full bioactivity.

8. What kinds of proteins can be produced transgenically in milk?

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

GTC Biotherapeutics has successfully produced several different classes of proteins in the milk of transgenic animals. Some of these are complex glycosylated proteins, which contain specific sugars attached at precise sites in the mature protein molecule. We have produced various types of monoclonal antibodies, which are made up of two pairs of different protein chains that must be assembled correctly in vivo. In addition, we have produced fusion proteins that combine the active sites from two different proteins into one molecule, such as a biologically active enzyme fused to targeting sequences from an anti-tumor antibody. We also have expressed various proteins in milk that ordinarily are not secreted from the cell.

9. What is the transgenic advantage for monoclonal antibodies?

 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.

10. What types of monoclonal antibodies has GTC Biotherapeutics produced?

 We have expressed a broad range of monoclonal antibodies in transgenic animals, including mouse antibodies, both partially humanized and fully human antibodies, fusion proteins containing antibody molecules linked to enzymes for in vivo targeting, antibodies comprised of a single light chain, and hybrid molecules containing ligands attached to an immunoglobulin-like scaffold.

GTC Biotherapeutics 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 cells in roughly equal proportions, and proper assembly into tetrameric protein complexes.

11. Why use goats?

Goats are an ideal dairy species. They have reasonably short gestation and maturation times, produce large volumes of milk with a high protein content, and are generally accepted as a source of dietary milk. They are relatively easy to breed and maintain. Goat milk has been extensively characterized biochemically, and this makes it more straightforward to develop protein purification procedures.

12. How much recombinant protein can a goat produce?

 The breeds of goats that we use are prodigious milk producers, yielding 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. This can provide up to 0.8 kilograms (kg) of raw protein per year per goat for a protein expressed at a concentration of 1 gram per liter of milk, or 8 kg at expression levels of 10 grams per liter of milk.

13. How are transgenic animals produced?

 The gene for the target protein is linked with a milk-specific promoter and introduced into very early stage embryos which are then transferred to surrogate mothers. After a period of gestation, first-generation offspring are born. Blood samples and skin biopsies are collected from them and tested to identify animals that have integrated the transgene.

14. What is a transgenic founder animal?

 A genetic founder, denoted "F0 ," is a first-generation transgenic animal that develops directly from a microinjected embryo and carries the transgene stably integrated in its genome.

15. How many founders are needed?

 GTC Biotherapeutics typically produces, breeds and evaluates approximately 5 founders for each transgene construct. This provides enough animals to select for optimal genetics, protein expression, milk production and reproductive characteristics.

16. How is a founder production line chosen?

 Transgene sequences are analyzed by biochemical and molecular methods to confirm that they are complete and intact. If they are, then the animals are raised to maturity and bred to non-transgenic mates. Their offspring are tested to confirm that the transgene is inherited at the expected frequency.

Milk is collected from these transgenic animals and analyzed to measure the expression levels and biochemical characteristics of the recombinant protein. Milk may be collected directly from female founders, or from the daughters of male founders.

In order for a transgenic animal to be designated as the founder of a production line, it must meet several minimal criteria: stable transgene integration, suitable transmission of the transgene to its progeny; commercially feasible protein levels in milk; and good overall health.

17. How soon is recombinant protein available from transgenic animals?

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.

In parallel, other female founders are allowed to reach sexual maturity, give birth to offspring and commence full-scale milk production. The total time from transgene introduction to first natural lactation is about 18 months for female goats. Those founders that are males must produce daughters and these daughters must have offspring of their own before full-scale milk collection may begin. The time to first lactation is about 28 months for transgenic lines derived from male founders.

18. How are recombinant proteins purified from milk?

 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 Biotherapeutics 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 parenteral therapeutics.

19. How are transgenically produced recombinant proteins regulated?

A series of regulatory documents has been issued by the FDA (Food and Drug Administration), CPMP (Committee for Proprietary Medical Procedures) and ICH (International Conference on Harmonization of Technical Requirements for Registration of Pharmaceuticals for Human Use) to address important issues relevant to the manufacture, development and licensing of biological products. These cover gene therapy, recombinant protein therapeutics, monoclonal antibodies and transgenically produced therapeutic proteins. For example, both the FDA and EMEA (European Agency for the Evaluation of Medicinal Products) have issued guidance documents for the manufacture of human therapeutic products in transgenic animals. Together, these documents deal with important aspects of transgenic production and purification, with a special focus on generating the founders, establishing and maintaining production herds and assuring consistent production among lots, lactations and lines.


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