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.6
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
Now let us show this applied to a special product. 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.
Figure 4. Schematic representation of the process used to purify ATryn from the milk of transgenic goats.