The targeting of heterologous proteins to the mammary gland of transgenic mice was independently reported by several groups during the late 1980s.1,2,3 These initial successes were followed by reports relating the generation of transgenic sheep, goats, cows, and pigs with milk-specific transgenes with the ultimate objective of producing recombinant proteins for clinical use (reviews by Clark,4 Meade et al.,5 Pollock et al.6 ). Transgenic Res 1997; 6:271-278. 23.

21.

The resulting goats carrying this transgene produce the gene product, rhAT, in their milk.

Maga EA and Murray JD. The targeting of a recombinant protein to the milk of a transgenic animal (Figure 1) is achieved by first generating an expression vector containing the gene encoding the protein of interest fused to milk-specific regulatory elements. The aim was to target recombinant proteins to the mammary gland of transgenic farm animals to solve problems associated with either microbial or animal cell expression systems. Clark, AJ.The mammary gland as a bioreactor: expression, processing, and production of recombinant proteins. Transgenically produced human antithrombin: Structural and functional comparison to human plasma-derived antithrombin. 19.

Yann Echelard, PhD, is vice president of research and development at GTC Biotherapeutics, Inc. 5 the Mountain Rd, Framingham, MA 01701, 508.370.5420, fax 508.370.5104, yann.echelard@gtc-bio.com, Carol A. Ziomek, PhD, is vice president for development at GTC Biotherapeutics, Inc. 175 Crossing Blvd., Framingham MA 01702, 508.370.5421, fax 508.370.5266, carol.ziomek@gtc-bio.com, Harry M. Meade, PhD, is senior vice president of research and development at GTC Biotherapeutics, Inc., 175 Crossing Blvd., Framingham MA 01702, 508.370.5256, fax 508.370.5266, harry.meade@gtc-bio.com. Transgenic pigs as bio-reactors: a comparison of gamma-carboxylation of glutamic acid in recombinant human protein C and factor IX by the mammary gland. Expression levels are dependent on the mammary-specific regulatory sequences employed, the gene expressed, and the integration site of the transgene. ATryn may be given in association with heparin or low molecular weight heparin in these situations. This is also a challenge found with mammalian cell culture, microbial expression systems, or transgenic plants. This article traces the development of protein technology from the microbe to the mammary glands. Recombinant protein production in the milk of transgenic sows has been reported for human Protein C,13 factor VIII,14 and factor IX.15 Lactating sows can yield a surprising amount of milk (100–200 L) and it has been reported that the porcine mammary gland cells can carry out the complex post-translational modifications (γ-carboxylation, proteolytic processing) on factor IX and Protein C at rates higher than those encountered with mammalian cell and transgenic mouse milk systems.16. Following germline integration, mammary gland-specific transgenes are predictably inherited by the offspring of the founder animal. However, it takes almost three years from the onset of transgene transfer to obtain milk from a cow's natural lactation. Another consideration may be a species-specific ability to perform specialized post-translational modifications more efficiently. Stinnackre, MG, Massoud M, Viglietta C, Houdebine LM.The preparation of recombinant proteins from mouse and rabbit milk for biomedical and pharmaceutical studies. The tremendous scale-up potential offered by transgenic cattle may compensate for this drawback, especially for indications that necessitate large quantities of protein. The use of recombinant proteins has steadily increased during the last two decades. A large number of production females can be easily generated from a transgenic male using artificial insemination or embryo transfer techniques. Expression in the milk of transgenic dairy goats was employed. Thromb Haemost 1997; 78:532-536. 17. Concentrations surpassing 1 g/L are attained routinely and levels of up to 20 g/L have also been achieved. Expression of bovine beta-lactoglobulin/human erythropoietin fusion protein in the milk of transgenic mice and rabbits. Schematic representation of the somatic cell nuclear nuclear transfer process employed for the production of transgenic animals used for the production of recombinant proteins.
to the mammary gland of the animal. All studies demonstrated that rhAT was well tolerated in these patient populations. 14. Bacteria often improperly fold complex proteins, leading to involved and expensive refolding processes, and both bacteria and yeast lack adequate post-translational modification machinery for mammalian-specific N- and O-linked glycosylation, γ-carboxylation, and proteolytic processing. For example, commercial recombinant production of complex molecules, such as antithrombin and alpha1-antitrypsin, has not yet been achieved in microbial or mammalian cell derived bioreactors. The development of a recombinant option for antithrombin will provide a safe and reliable supply of this important factor and will facilitate the resumption of clinical trials aimed at acquired deficiencies of antithrombin, such as cardiovascular surgery, severe burns, and severe sepsis. Eur J Biochem 1997; 245:482-489. On the other hand, transgenic livestock can be maintained and scaled-up in relatively inexpensive facilities, use animal feed as raw material, and can achieve impressive yields of recombinant proteins. 5. The main glycosylation differences observed for rhAT were the presence of fucose and GalNAc, a higher level of mannose, and a lower level of galactose and sialic acid. The development of very efficient expression systems has been the key to the full exploitation of the recombinant technology. Production of human recombinant protein pharmaceuticals in the milk of transgenic farm animals solves many of the problems associated with either microbial or animal cell expression systems. It will close with specific information on rhAT production and the status of clinical tests. Also, the inherent risk associated with the regulatory approval process is a stimulus for the development of flexible and inexpensive approaches for the manufacture of therapeutic proteins. © 2020 MJH Life Sciences™ and BioPharm International.

Nat Biotechnol 1998; 16:647-651. Pronuclear microinjection of one-cell embryos (Figure 1) or, alternatively, transfection into a primary cell population suitable for somatic cell nuclear transfer (Figure 2) have both been used to generate transgenic founders. The recombinant production of AT presented numerous challenges. These are within the practical production capacity of this system but far too small for high-volume therapeutic indication (volume replacement). In addition, embryo culture and transfer technologies are well established for cattle breeds, allowing efficient generation of transgenic cows by somatic cell nuclear transfer. The final section describes the process by which goats express rhAT in their milk at approximately 2 g/L. Blood 1998; 91, 4561-4571. The expression level of the protein(s) of interest is variable. A large number of human proteins and potential therapeutic targets and their development for therapeutic uses have been identified. Clark AJ, Bessos H, Bishop JO, Brown P, Harris S, Lathe R, McClenaghan M, Prowse C, Simons JP, Whitelaw CBA, Wilmut I.

A phase III, double-blind, placebo-controlled, multicenter study on the efficacy of recombinant human antithrombin in heparin-resistant patients scheduled to undergo cardiac surgery necessitating cardiopulmonary bypass. Genet Anal Biomol Eng 1999; 15:155-160. 7. Bio/Technology 1989; 7:487-492. 22. Approximately 50% are fatal, representing a major unmet medical need of significant interest in critical care. Thousands of years of patient genetic selection have yielded breeds of sheep, goats, and cattle that can produce prodigious quantities of milk. Avidan MS, Levy JH, van Aken H, Feneck RO, Latimer RD, Ott E, Martin E, Birnbaum DE, Bonfiglio LJ, Kajdasz DK, Despotis GJ. Ann Hematol 2003; 82:S84. 11. 12. Two clinical indications were pursued: In all the human studies completed, rhAT has not generated significant adverse events and has met the primary clinical endpoints. The generation of transgenic rabbits by pronuclear microinjection is straightforward and inexpensive. The rhAT purification process has been validated for its ability to reduce scrapie contamination over a 100 billion fold (>11.3 log10 scrapie removal). Transgenic milk as a method for the production of recombinant antibodies. Lubon H and Paleyanda RK.

This transgene is then introduced in the germline of the chosen production species. Although transgenic expression systems are able to perform complex post-translational modifications, such as γ-carboxylation, β-hydroxylation or N- and O-linked glycosylation, there are species- and tissue-specific characteristics for these modifications that may affect the appropriateness of a given system for the expression of specific proteins. Tait RC, Morfini M, Walker ID, Makris M, Dolan G, Menon R, Noonan PK, Frieling J, Bonfiglio J. Pharmacokinetics of recombinant human antithrombin in hereditary antithrombin deficient patients. This model allows the relatively inexpensive and rapid evaluation and optimization of transgene constructs and has proven crucial to the development of milk expression technology. Amsterdam: Harwood Academic Publishers; 1997. p. 461-463. Press Release. Production of biologically active salmon calcitonin in the milk of transgenic rabbits. Tait RC, Konkle BA, Bauer KA, Bonfiglio J, Dolan G, Frieling J, Greist A, Holmes HE, Makris M, Morfini M, Noonan B, Streisand JB, Weinstein R, Walker ID, Echelard Y. Here is the method to achieve milk-specific recombinant protein production. J Mammary Gland Biol Neopl 1998; 3:337-350. Methods 1999; 137:147-157. There was also substitution of 40-50% of the N-acetyl neuraminic acid with N-glycolyl-neuraminic acid.18 The terminal sialic acid in the rhAT contained the same 2-6 linkage found in hpAT. Pollock DP, Kutzko JP, Birck-Wilson E, Williams JL, Echelard Y, Meade HM. This general strategy makes it possible to harness the ability of dairy animal mammary glands to produce large quantities of complex proteins. Korhonen VP, Tolvanen M, Hyttinen JM, Uusi-Oukari M, Sinervirta R, Alhonen L, Jauhiainen M, Janne OA, Janne J. The timeline from initiation of transgene transfer to natural lactation of resulting transgenic does is 16 to 18 months for goats (Figure 3). San Diego: Academic Press; 1998 p. 399-427. 15. 2006 June 2. GTC Biotherapeutics and other have generated transgenic animal herds that yield large amounts of proteins as diverse as: human antithrombin (AT), alpha1-antitrypsin (AAT), C1 esterase inhibitor, fibrinogen, albumin, and monoclonal antibodies (Table 1). Now let us show this applied to a special product. The rhAT viral validation studies demonstrated that a significant virus reduction of >8.5 to >25.3 log10 (roughly 300 million fold to septillion fold) was accomplished across the distinctly different modes of the rhAT process.19 All GTC goats are certified free of scrapie in the United States Department of Agriculture (USDA) Scrapie Flock Certification Program and various risk minimization measures have been instituted to protect this highly controlled closed donor goat population. Milk-specific production offers a way to lessen the bite. 6. 2. This was the first positive opinion by a regulatory agency for a transgenically produced biopharmaceutical (from either plant or animal sources). Edmunds T, Van Patten SM, Pollock J, Hanson E, Bernasconi R, Higgins E, Manavalan P, Ziomek C, Meade H, McPherson JM, Cole ES. Schematic representation of the process used to purify ATryn from the milk of transgenic goats. 3.

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