Milking rabbits' protein potential

Using transgenic animals to produce therapeutically useful recombinant proteins offer benefits compared with traditional methods. Alexandre Fouassier, business development manager at BioProtein Technologies, looks at using transgenic rabbits to produce proteins in milk

Over the past few years, biotechnology has generated new opportunities for the development of human pharmaceuticals. One particular success story is the use of recombinant technologies to produce therapeutically useful proteins, such as hormones, antibodies, growth factors and antigens for vaccines. Producing these proteins in the quantities required to meet clinical needs presents a challenge, however, and manufacturers are constantly looking for new solutions. Transgenic animals provide one answer, and can offer benefits compared with traditional methods of recombinant protein manufacture. The transgenic rabbit, in particular, gives a fast, cost-effective and efficient means of producing therapeutic protein in milk.

Through advances in biotechnological techniques, scientists are able to isolate sequences of bases coding for specific proteins (genes), and insert them into the DNA of other living cells. These 'hosts' are usually rapidly reproducing cells such as bacteria, yeast or cultured cell lines, which can be grown in large volumes to express the protein of interest on an industrial scale for use in therapeutics. Naturally occurring erythropoietin, for example, is a very rare glycoprotein hormone responsible for the regulation of red blood cell production. The erythropoietin gene was cloned in 1985 and today it is routinely expressed in cultured cells. The recombinant erythropoietin harvested from these cells is used to increase the production of red blood cells in patients with anaemia caused by a variety of conditions, such as cancer chemotherapy or in association with HIV.

several hurdles

The manufacture of proteins using recombination techniques is not always straightforward and must overcome a number of hurdles. To be successful the gene must be expressed at high levels and this depends on regulatory sequences. Vectors introducing the gene into the cell must be highly efficient, and the resulting proteins must be easy to extract from the protein-producing cells. Furthermore, most proteins are generally large molecular weight complex molecules, which require post-translational modifications such as glycosylation to be efficient. Moreover, these molecules are highly sensitive to both the environmental conditions, and the expression systems used. The efficacy and safety of the final therapeutic depends on the ability of the manufacturing process to consistently produce molecules that are structurally and functionally accurate.

There are several methods of manufacturing recombinant proteins, including microbial fermentation systems and the large-scale culture of mammalian cells. Some of these processes can be expensive, costing several thousand dollars per gram of protein.

Bacterial systems have the capacity to produce virtually unlimited amounts of pharmaceutical protein in large-scale fermentation plants. Production cycles are very short - in some cases just a matter of hours - and consequently production by this method is relatively cost-effective. Bacterial systems are, however, limited to the production of simple molecules because bacterial cells are unable to carry out the post-translational modifications necessary for the correct function and pharmacokinetic behaviour of many of the more complex proteins required for therapeutics. These modifications include changes such as cleavage, folding, subunit assembly, glycosylation, carboxylation, amidation, acetylation and phosphorylation.

cellular enzymes

These important alterations, which are necessary for the in vivo activity of the proteins, are carried out by cellular enzymes. Different cell types have different enzymatic capabilities. Glycosylation is one of the most important events for therapeutics as it is essential for the stability of many proteins in blood circulation. In eukaryotic cells, glycosylation takes place in the endoplasmic reticulum and Golgi body - two organelles absent in bacteria.

Fungi, plants and yeasts, which can all be grown on an industrial scale to produce recombinant proteins, share the problem of not being able to carry out post-translational modifications to varying extents. Yeasts, for example, while they are able to carry out glycosylation, do so inefficiently, and provide molecules that are often incompletely modified.

Certain mammalian animal cell lines such as Chinese hamster ovary (CHO) cells, can be programmed to express foreign genes and have been widely used to synthesise proteins that closely resemble native proteins. The costs of cell culture media, however, make this technique expensive and it is also difficult to grow animal cells in fermentors on the scale of those used for bacterial and yeast systems. The production process also takes longer - days or weeks rather than hours.

The use of transgenic animals and plants for the production of human therapeutic proteins is a relatively new manufacturing development. Ever since the birth of 'Tracey', in 1991, the first transgenic sheep expressing the human blood clotting factor, human α-1-antitypsin, the idea of transgenic animals as protein fermentors has taken off.

Since then, more than 100 foreign proteins have been produced experimentally from different organs in several animal species. Transgenic animals have the potential to make a significant contribution to the production of biopharmaceuticals because they can produce complex proteins at high volume and with low cost.

Transgenic animals can be created though two principal methods - microinjection and nuclear transfer.

foreign DNA

In the microinjection method, freshly fertilised oocytes are harvested and DNA constructs are injected into the male pronucleus (a vacuole that contains the male DNA) using a thin glass needle. The male and female pronuclei fuse to form the nucleus, which now contains foreign DNA. The cell then divides to form a two-cell embryo and is transferred into a recipient female.

The main disadvantage of this technique is that transgene integration within the male pronucleus genome is random. The expression of the transgene can, therefore, be affected by its position in the genome, meaning that the subsequent selection of efficient protein-producing animals is required.

Nuclear transfer involves transferring the nucleus from a somatic cell into an enucleated oocyte. Generating a transgenic animal in this way involves creating a genetic construct with the gene of interest in suitable cultured cells such as foetal fibroblasts. Nuclei from these somatic cells are transferred into enucleated oocytes and these are triggered to develop in vitro.

This technique allows the targeted integration of the transgene into the host genome by homologous recombination, so significantly accelerating the production of an efficient protein-producing group of transgenic animals.

The choice of animal for commercial protein production depends on a variety of factors, including generation time, number of offspring, potential yield and susceptibility to disease. A variety of animals have been used successfully, including mice, rabbits, goats, sheep and cows.

convenient source

The source of the protein is also an important consideration. Milk, blood, urine and seminal plasma can be used, as well as the egg white from birds' eggs and the cocoons of some insects.

The most convenient source is milk, however. The secretory properties of the mammary gland make it the ideal protein producer, and the milk is easy to collect.

Furthermore, milk does not contain endogenous proteases, and the composition of milk is specific and simple, making the identification and purification of the recombinant protein relatively easy. Complex and difficult to produce proteins, such as human b-NGF, hGH, lysozyme, erythropoietin, thrombopoietin, and parathyroid hormone, have been successfully harvested from the milk of transgenic animals.

In general, the larger the animal, the greater the milk yield, but this must be balanced against longer gestation periods and the time it takes to produce a functional transgenic herd. A key factor in the use of transgenic species is the efficiency of expression of the desired protein. For commercial use, expression levels of at least 1g/l are required. The rabbit has emerged as a key model for protein production based on: the speed at which transgenic animal colonies can be established; good milk yields; high protein content; an ability to produce complex functional proteins; and ease of handling.

Rabbits produce significant quantities of milk, in the range of 100-250ml/day (approx 10-15l of milk per rabbit per year). Their prolific nature reduces the time required to establish a transgenic line and so accelerates time-to-market for the recombinant protein. Large numbers of transgenic rabbits can be generated in a relatively short time - it takes just 11 months to produce a fully transgenic protein-producing colony, compared with 57 months for cows and 29 months for goats (figure 1). A colony of 400 rabbits can produce up to 10kg of protein per year assuming a 40% recovery in the purification process.

microinjection method

Transgenic rabbits are generally produced using the microinjection method. A female rabbit is implanted with 20 viable embryos containing DNA constructs. The founder generation (F0) are born after one month and are partially composed of transgenic animals. In addition, a number of transgenic founders do not exhibit the transgene insertion in all the cells, a phenomenon called mosaic expression.

After only four months, the founder generation reaches sexual maturity and these animals are then bred with non-transgenic rabbits through artificial insemination to produce heterozygous first generation (F1) transgenic animals. Sperm from one well-characterised F1 transgenic male provides a Master Sperm Bank (MSB), which will be used to give birth to the next generation of transgenic males. The sperm from these F2 males will provide a Working Sperm Bank (WSB) to scale up the colony for larger scale milk production (figure 2).

ideal model

Faster time to production is possible if only small volumes of milk are required, for example, in pre-clinical studies. Here, F1 rabbits can be used and these can be produced in just 5-6 months. This makes the rabbit an ideal model for clinical development studies. The rabbit mammary gland naturally secretes proteins that are N- or O-glycosylated, which means that the rabbit is able to produce complex proteins that are in an active and functional form for human therapeutic use (see table 1). Transgenic rabbit milk can produce recombinant protein with yields in the region of 1-10g/l.

The production of proteins in rabbit milk meets regulatory requirements for the stable expression and inheritance of the transgene, reliability of the protein manufacturing process, and quality of the final product. It also meets virus clearance validation and there is no known instance of prion disease in rabbits that could cause transmission of Creutzfeldt Jakob disease.^1-3 Although many experiments were performed to try to artificially transmit prion disease to rabbits, no evidence of transmission has been shown. No prion clearance validation is therefore required.

Protein production in rabbits is within the time scale required for CHO cell fermentors, but operates at a fraction of the cost. Transgenic rabbits, it is estimated, can reduce the costs of protein production by 20-40% compared with CHO systems, and capital costs can be reduced by 40-70%, as there is no need for an expensive and validated infrastructure containing fermentation tanks, air filtration and temperature control systems.

important benefits

The benefits of the rabbit are particularly important in light of the today's uncertain manufacturing environments. Biotechnology-derived products represent about 25% of all new medicines and it is predicted that this will increase to about 50% within 10 years.

Current trends indicate that there is going to be a shortfall in manufacturing capacity for biopharmaceuticals. While many companies are investing in increasing their manufacturing capacity, it can take four to five years to build a manufacturing plant at a cost of US$100-500m (Euro 94.5-473m). The use of transgenics offers pharmaceutical and biotechnology companies an attractive option as a highly flexible, scaleable source of protein with a fast time to full production.

cloning unpredictable

While advances have been made in cloning technologies, cloning is still regarded as an unpredictable and inefficient process. Some animals are more amenable to cloning than others. Rabbits are one of the mammalian species that have always been considered difficult to clone from adult somatic cells, but recent research has shown that cloning the rabbit is possible. Chesne et al (from Dr. Jean-Paul Renard's group at INRA) in collaboration with BioProtein Technologies, have modified cloning protocols for use in the rabbit, and have succeeded in producing living, fertile rabbit clones (figure 3).⁴ Although the process is still in development, it offers new possibilities for the production of proteins in rabbits. Cloning would improve integration of the gene of interest, and therefore be a more effective means of generating a large number of transgenic animals.