Transgenic Goats Are Key To Antithrombin Production

With their high milk production and short generational time, these bovids are ideal bioreactors able to dramatically increase the protein yield

Sanofi-Aventis CEO Chris Viehbacher told the Financial Times that his company had “missed the boat” when it comes to diversifying into biologics. He expressed his realization that biologic drugs have revolutionized the pharmaceutical industry. But the biotech industry has just had a development that may be even more revolutionary.

The purest definition of a biologic drug is one that is derived from a living organism or cell. Their manufacture requires a large cell culture, the extraction of the active ingredient from the media or cell, and its purification in a facility called a bioreactor.

Three different protein production techniques have been the mainstay of manufacturing in the United States. These methods require large capital outlays to build the facility to grow the tissue cultures — think brewery-size vats — and result in relatively low yields.

Transgenic animals

Transgenic animals were first developed in 1985 and were useful in the production of biopharmaceuticals shortly thereafter. The ideal transgenic animal produces plenty of milk, and has relatively short generation times. Typically, dairy animals are used because of their high volume of milk production. Goats have been selected for a number of reasons, the chief of which is the short generational time of 18 months.

Goats produce about 800 liters of milk per year. Using goats has been shown to dramatically increase the yield of the active protein by more than 10 times that from the cell culture model.

An initial “founder” transgenic animal is created by first injecting an engineered segment of DNA into the pronucleus of a one-cell embryo. This DNA segment contains a mammary-specific promoter sequence that codes for the protein of interest and the regulatory DNA that tells the cell how to manufacture the protein. The embryo is then transferred into a surrogate female animal, resulting in a transgenic offspring. If, after maturing and being induced to produce milk, the animal expresses the desired protein in the milk in sufficient quantity and quality, it can be included in the producing pool of animals.

The key to this method of drug development is the use of the mammary gland. The major function of the mammary gland is to produce a solution that is loaded with a variety of proteins. Some of the proteins that can be created in milk are difficult or impossible to create in tissue culture-based bioreactors. Also, the mammary gland is capable of processing completed proteins through glycosylation and gamma carboxylation, functions that occur after translation of the DNA into proteins.

Transgenically produced proteins are isolated from milk in a multistep process that includes methods normally used by cell culture bioreactors as well as processes adapted from the dairy industry. The resulting proteins are held to the same standards as any other recombinant protein. Obviously safety concerns have been addressed: All goats are certified to be free of scrapie by the United States Department of Agriculture, the farm is inspected by the FDA, and the goats receive superb medical care.

Hereditary antithrombin deficiency

On Feb. 9, the FDA approved ATryn, recombinant antithrombin manufactured by GTC Biotherapeutics in Massachusetts. While the approval of this agent may not be particularly revolutionary (human-derived antithrombin has been available in the United States for many years), the approval of ATryn may signal the start of a disruptive technologic breakthrough, as this is the first approved transgenic therapeutic protein in the United States.

ATryn is indicated for the prevention of peri-operative and peripartum thromboembolic events in patients with hereditary antithrombin deficiency. HD is a genetic disorder leading to excessive clotting because of a lack of normal antithrombin activity. Antithrombin (AT) is a 432 amino acid protein that regulates thrombin and the downstream clotting processes. There are two distinct types of HD — Type I and Type II. Type I is a quantitative deficiency, meaning there are low absolute levels of AT. Type II is a functional deficiency, meaning the existing AT is not able to act on the clotting system because of a defect in the structure of the AT. Both types of HD are the result of mutations or point deletions in the DNA that regulates the manufacture or structure of the protein.

HD is diagnosed based on two tests, one measuring the quantity and the other measuring the function of AT. It is a diagnosis of exclusion — other causes of reduced AT such as liver disease or nephritic syndrome must be absent.

Treatment of HD depends on the risk of a thromboembolic event. If a patient has a venous clot, acute anticoagulation agents are used. If an HD patient has had multiple episodes of thrombosis or embolism, long term anticoagulation may be desired. AT becomes the treatment of choice as a prophylactic agent during periods of high risk of thrombosis.

There are two forms of AT available — the plasma products taken from pooled whole blood and the new ATryn, transgenic recombinant antithrombin. The amino acid sequences of ATryn and human antithrombin are indistinguishable.

Dose is dependent on pretreatment functional AT activity level, weight, and the patient’s reason for therapy (pregnancy dosing is different from surgical dosing). The goal of treatment is to restore and maintain AT activity levels between 80 percent and 120 percent of normal. Treatment is initiated approximately 24 hours before surgery or before delivery. Frequent monitoring is needed after surgery and once or twice daily thereafter. ATryn is expected to be available in the second quarter of 2009.

The future

Although the mammary epithelial cell does not typically express antibodies, GTC Biopharmaceuticals has found that the intracellular machinery needed to properly fold and assemble the heavy and light chains of antibodies are functional within these cells.

Transgenic goats can be used to produce a wide variety of biologically active recombinant proteins and antibodies. They include both small and large proteins as well as complex glycosylated proteins containing specific sugars attached at precise sites in the mature protein molecule. The types of antibodies that can be created using transgenic animals include mouse antibodies, partially humanized antibodies, and fully human antibodies. Of intense interest is the creation of proteins that can be fused to the targeting sequence of an anti-tumor antibody. This next generation of biotechnology is certain to be a future topic of Tomorrow’s Medicine!

Thomas Morrow, MD, is the immediate past president of the National Association of Managed Care Physicians. He has 24 years of managed care experience at the payer or health plan level.
The author is a director in the value-based health department at Genentech Inc. During the three years before taking the Genentech position, he received honoraria or other financial benefits from: Amgen, Amylin Pharmaceuticals, AstraZeneca, Biogen Idec, Centocor, Galderma, GlaxoSmithKline, Johnson & Johnson, Merck, Novartis, Novo Nordisk, Pfizer, Procter & Gamble, Q-Med, Sanofi-Aventis, Teva Pharmaceuticals Industries, UCB, and Wyeth. The views expressed in Tomorrow’s Medicine are the author’s alone.