The first microbial bioreactors, in particular Escherichia coli and Saccharomyces cerevisiae, were found to be satisfactory for the production of simple polypeptides such as insulin and human growth hormone. However, microbial bioreactors were found to be unsuitable for proteins with complex post-translational modifications or intricate folding requirements, such as the coagulation factors, or monoclonal antibodies. This led to the development of large-scale mammalian cell culture, for example, the use of Chinese Hamster Ovary (CHO) cell bioreactors.
These technologies permitted the development of numerous monoclonal antibodies, cytokines, and other complex bioactive biomolecules. However, there are proteins that, due to a combination of complex structure and large therapeutic dosing, have until now eluded recombinant production using traditional bacterial and cell culture bioreactors. 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 only source is human plasma because of the high dose needed.
Even human serum albumin, the therapeutic protein used in largest amounts (>400 tons, worldwide), the use of the recombinant form, produced in Saccharomyces cerevisiae, is limited to excipient applications. These are within the practical production capacity of this system but far too small for high-volume therapeutic indication (volume replacement).
Capital investments in production plants represent a significant portion of the development cost of new recombinant drugs. 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. Milk-specific production offers a way to lessen the bite.
MILK AS A POSSIBLE PRODUCTION MEDIA
Here is the method to achieve milk-specific recombinant protein production. Fuse an expression vector, comprising a gene that is encoded for the human or humanized target protein with mammary gland-specific regulatory sequences, and then insert into the germline of the selected production species. When integrated, the milk-specific expression construct becomes a dominant genetic characteristic that is inherited by the progeny of the founder animal (Figure 1). This general strategy makes it possible to harness the ability of dairy animal mammary glands to produce large quantities of complex proteins.
Figure 1. Schematic representation of the transgenic production process, using the production of rhAT in the milk of transgenic goats as an example.
Figure 2. Schematic representation of the somatic cell nuclear nuclear transfer process employed for the production of transgenic animals used for the production of recombinant proteins.
SCREENING THE MAMMALS
Transgenic mice have mainly been used for the testing of expression constructs prior to or concomitant with the generation of larger founder transgenic animals. 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. The model allows the definition of regulatory sequences that efficiently target expression of heterologous genes to the mammary gland. Obviously, the very limited milk yield from transgenic mice restricts expression of recombinant proteins to small amounts. But this can be sufficient to obtain meaningful data on the protein of interest. As an example, it was possible to purify enough Malaria antigen MSP142 from transgenic mouse milk to test for immune protection in a primate model.
The generation of transgenic rabbits by pronuclear microinjection is straightforward and inexpensive. Relative to ruminants, rabbits have a short gestation interval that allows up to eight lactations per year. However, only 1.5 L of milk can be obtained per lactation, and this limits the value of this expression system to products with a commercial scale in the low-kilogram range; Labor-intensive milking and high husbandry costs could become prohibitive for larger quantities of purified proteins.
Recombinant protein production in the milk of transgenic sows has been reported for human Protein C, factor VIII, and factor IX. 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.
Transgenic ruminants are obvious candidates for targeting expression of recombinant proteins to the mammary gland. Thousands of years of patient genetic selection have yielded breeds of sheep, goats, and cattle that can produce prodigious quantities of milk. The first published report of production of therapeutic proteins in the milk of transgenic dairy farm animals was the targeting of factor IX and alpha1-antitrypsin to the milk of transgenic ewes.17 Other proteins such as fibrinogen and factor VIII have also been expressed in the mammary gland of transgenic sheep.
Transgenic dairy goats, with an average milk output per doe on the order of 600 to 800 L per natural lactation, have shown to be well adapted to the production of therapeutic proteins. The timeline from initiation of transgene transfer to natural lactation of resulting transgenic does is 16 to 18 months for goats (Figure 3). A large number of production females can be easily generated from a transgenic male using artificial insemination or embryo transfer techniques. Relatively small herds of a few hundred transgenic does can then easily yield several hundred kilograms of purified product per year. This level of production can meet the manufacturing needs of several factors traditionally derived from plasma fractionation and for a large number of recombinant antibodies currently in development.
Dairy cows have a yearly milk output in the range of 10,000 L. Consequently, with concentrations routinely achieved with most mammary gland-specific proteins, yields of tens of kilograms of recombinant proteins can be produced by one lactating transgenic cow. In addition, embryo culture and transfer technologies are well established for cattle breeds, allowing efficient generation of transgenic cows by somatic cell nuclear transfer. However, it takes almost three years from the onset of transgene transfer to obtain milk from a cow's natural lactation. The tremendous scale-up potential offered by transgenic cattle may compensate for this drawback, especially for indications that necessitate large quantities of protein.
ATRYN: THE FIRST TRANSGENICALLY PRODUCED BIOPHARMACEUTICAL
The recombinant production of AT presented numerous challenges. Antithrombin is a complex glycoprotein carrying 4 N-linked glycosylation sites and 3 disulfide bonds. These characteristics, which are crucial for the functions of AT, precluded the use of microbial bioreactors for its recombinant production. In addition, the therapeutic use of AT calls for large amounts, often grams, of purified protein per course of treatment. This ruled out the use of standard mammalian cell culture bioreactors, because production costs with this approach would be prohibitive.
Expression in the milk of transgenic dairy goats was employed. The promoter region of the goat beta-casein gene was linked to hAT cDNA. This transgene was introduced into the chromosomes of goat embryos, which were then transferred to surrogate mothers. The resulting goats carrying this transgene produce the gene product, rhAT, in their milk. Transgenic offspring from the line selected for commercial development consistently express rhAT in their milk at approximately 2 g/L.18 Expression levels of up to 10 g/L were observed in other lines that were not developed further because of timing issues.
The rhAT protein is isolated from the milk of the transgenic females and conventionally purified using tangential flow filtration, heparin affinity chromatography, nanofiltration, anion exchange chromatography, and hydrophobic interaction chromatography, with a yield of greater than 50% (Figure 4). The human AT purified from transgenic goat's milk is structurally indistinguishable from human plasma-derived AT (hpAT) with the exception of the carbohydrates. 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. 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.
Several independent laboratories have determined that differences in glycosylation of AT do not affect the intrinsic rate constant of the uncatalyzed or heparin catalyzed inhibition of thrombin, indicating that the carbohydrate chains solely affect heparin binding and not heparin activation or proteinase binding functions. Thus, glycosylation does not impact the major biological activity of AT, which is thrombin inhibition, but explains the differences in affinity for heparin and in pharmacokinetics.
The manufacturing process for rhAT has been validated for its viral and prion removal capacity. 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. 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. The rhAT purification process has been validated for its ability to reduce scrapie contamination over a 100 billion fold (>11.3 log10 scrapie removal).
Figure 4. Schematic representation of the process used to purify ATryn from the milk of transgenic goats.
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