When selecting a transgenic manufacturing platform, one of the most important considerations for a biopharmaceutical company is SPEED, particularly if the product is already in clinical trials. Due to the high “cost of entry” inherent in both traditional biologic manufacturing and first generation transgenic technologies, many companies, not wanting to commit large sums of money to an unproven product, wait until they have some efficacy data before investing in manufacturing capacity for the product. Unfortunately, the lead time to acquire/build manufacturing capacity often delays the product market launch date.
TranXenoGen has addressed this critical issue by selecting what it considers to be the optimum production species: chickens. Chickens have the great advantage of short development cycles (21 days to hatch) and short maturation cycles (5 to 6 months). This means that from the receipt of the partner’s gene sequences, TranXenoGen anticipates generating first generation chimeric transgenic chickens in 6 to 9 months and full germline birds in 12 to 18 months. These timelines are highly competitive in meeting the needs of companies with products as advanced in development as Phase III clinical trials.
Establishing “founders”
The first “cost of entry” to a transgenic production system that a biopharmaceutical company is faced with is the establishment of a small number of “founder” transgenic animals, expressing the specific product of interest. Since these founder animals may ultimately be used to generate the animals that produce the therapeutic protein administered as a drug to human patients, the animals used to generate “transgenic founders” must meet significant health criteria. In milk-producing ruminants, such as cows, BSE (often known as mad cow disease) is of critical concern. In sheep and goats, “scrapie” (the ovine and caprine equivalents of BSE) are of equal concern. In addition, the technology most commonly used to generate transgenic mammals is pronuclear microinjecting. This approach involves injecting the transgene individually into hundreds of single cell embryos, which is both costly and inefficient.
Pathogen-free founders
TranXenoGen has the significant advantage of being able to source SPF (specific pathogen free) eggs and chickens, generated primarily for the “egg-derived” vaccine industry, from U.S. vendors. Consequently, TranXenoGen is assured of the health status of its founder birds. In addition to their availability, chickens are inherently faster and much less expensive to develop than large ruminant mammals. As a result, TranXenoGen believes that it will reduce the cost of entry of transgenic production.
CAPITAL COSTS
The “make” or “buy” decision
Capital costs are always a significant issue for any company bringing a biopharmaceutical product to market, particularly a large volume protein. The company will be faced sooner or later with the “make” or “buy” decision. If the company decides on the “make” route, it may have to invest in a dedicated manufacturing facility (typically a cell-culture plant). The typical construction /validation period may take 4 to 5 years and the cost can range from $250 million to $400 million. For this reason, many biopharmaceutical companies often produce their first products through a “contract manufacturer". By avoiding significant capital investment in manufacturing facilities before the product completes clinical trials, the company avoids the high risk of significant capital loss if the product fails. However, the company may be at risk for significant "take or pay" capacity commitments.
Yield up to 500kg or more per year
Avian transgenics provides a very attractive alternative. TranXenoGen estimates that the cost to develop an avian manufacturing facility which will produce 500kg or more of raw product per year is approximately two million dollars. In comparison, the estimated cost to set up a similar facility for mammals is between five and twenty million dollars. Purification facilities are normally similar in scope and cost, regardless of the production platform.
COST OF GOODS
Labor intensive
Another major disadvantage of establishing a mammalian cell culture plant is the high “cost of goods” due to the expensive cultured media, the manufacturing process, and significant labor requirements. Alternatively, transgenic expression platforms provide a reduced “cost of goods”; however, the intensive labor and expense associated with the animal husbandry and milking of transgenic mammals or collection, maceration, and separation of proteins from transgenic plants can reduce the cost savings.
Fully automated
Transgenic chickens, in a fully automated, contained and controlled facility, should be able to produce these complex proteins for a fraction of the cost of mammalian cell processes. The fully automated nature of the chicken husbandry, which has been developed by the food industry, minimizes the human labor costs, the benefits of which are reflected in the final cost of goods. Proteins produced by transgenic chickens are expressed at commercial levels and delivered in a convenient, sterile package – an egg.
While in theory any transgenic manufacturing system should be capable of producing large volumes of product, from several kilograms up to several tons, there are logistical issues with some transgenic technologies. For example, large ruminant animals breed very slowly (cows produce one offspring per year; sheep and goats, two to four) and this makes the generation of a large herd (anything from tens to thousands of animals) a very slow process. Transgenic plants require large areas for planting and containment. Small volumes of product can be produced from plants grown in greenhouses where these genetically modified organisms can be contained. However, when the volumes of product required begin to escalate up into the tens of kilograms, reaching perhaps 100 kilograms or more, these plants will most likely have to be grown out in the open on huge multiple acre sites.
More is definitely better
Avian transgenics, as an alternative, has the distinct advantage that chickens lay 21 eggs a month (on average). If all of these eggs are fertilized, one hen can generate a flock of 120 chickens in six months of which approximately 30 would be transgenic hens used for production. As a result, the rapid breeding process generates large flocks capable of producing large volumes of product in a short time. An additional factor to be considered is that large ruminant animals require a considerable amount of space for grazing and exercise. A herd of 100 cows, for example, could produce a large volume of protein and would require multiple acres of space. TranXenoGen believes that a chicken facility of 15,000 square feet could yield over one ton of product per year, and ten chicken facilities could be located on a single 50-acre campus.
Scalability
When a new product is launched on the market, it is often difficult to predict the total volume of product that will be required, as demand often exceeds expectation. This means that a company may have to increase production dramatically in a short period of time. If the company has built a cell-culture facility and scaled it accordingly, the time to build additional capacity (often two years or more) will result in loss of revenues. Likewise, the significant expansion of a herd of ruminant animals can take a long time, due to the low numbers of offspring and long breeding cycles.
Rapid Ramp-up
A flock of transgenic chickens can be multiplied up by a factor of ten or more in a twelve-month period. New chicken facilities can also be constructed to accommodate the increased flock within this time frame. This is particularly important if the new product is being launched for a primary indication and production has been scaled accordingly, but secondary indications may follow. Of particular advantage is the fact that a small flock of transgenic chickens can be maintained to generate product for clinical trials, and this flock can be ramped up very rapidly, immediately prior to the market launch, and thus avoid the unnecessary expense of keeping large numbers of animals before the product is needed. This again helps to defer capital investment until product success is assured.
The issue of correct processing of the therapeutic protein, often ignored in the initial decision-making process by companies selecting a transgenic manufacturing technology, is now becoming recognized as being of critical importance. This realization is the result of various companies experimenting with transgenic expression systems. Companies have typically focused on high-level expression when selecting a transgenic technology, but the vast majority of therapeutic proteins are not simple linear protein sequences. Most are folded into secondary and tertiary structures and over half have post-translational modifications, including glycosylation structures, disulfide bonds, gamma carboxylation and amidation. These modifications usually have to be correctly completed to confer the required activity and/or plasma circulating half-life of the protein. Endogenous plasma proteins in particular tend to need a full complement of sialic acid to complete their glycosylation structures; otherwise, the half-life of the protein in plasma is often greatly reduced.
Glycosylation
Currently, only limited information and data are available regarding correct processing of transgenically produced proteins. Reported data has included comparisons of glycosylation structures on circulating IgG in multiple species; including human, pig, rat, mouse, chicken, goat, sheep and cow. One study showed that only human and chicken IgG contained a simple type of terminal sialic acid structure, NANA. The study suggests that the addition of sialic acid to carbohydrate groups in chickens is similar to that observed in humans. Human and chicken IgG structures contained a full complement of sialic acid. This evidence is encouraging and suggests that the avian transgenic system may confer post-translational glycosylation processing that is closer to humans than any other species currently used for transgenic production of proteins.
Transgenic chickens have the advantage over other transgenic animals that they can be housed in a contained/barrier facility from the moment they hatch from their shell. Chickens have been reared and housed this way for the egg production industry for decades. TranXenoGen can add the element of controlled, filtered air flow to its chicken production facility to provide a barrier to adventitious agents which could contaminate the production flock. The result is a totally enclosed, contained and controlled environment.
Containment
The egg white is contained in the sterile environment of the eggshell, and the therapeutic protein is maintained in a sterile state until the shell is broken as it enters the purification process.
Chicken eggs have been used for many years in the production of certain vaccines. As a result, the manufacturing infrastructure has been well established (including clean facilities, SPF birds and fully automated equipment), and the regulatory authorities are experienced in examining products derived from eggs and approving the facilities used to produce such products. The endogenous proteins contained in eggs are well characterized and extensively studied and purification technologies have been established to remove them.
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