Large Scale Production of RH-Albumin Expressed in the Milk of Transgenic Cattle - an Economic and Technical Challenge

Albumin from plasma fractionation is generally considered to be a safe and effective product. Nevertheless, a growing number of factors makes recombinant albumin manufacture increasingly attractive, i.e. the perceived risk of viral contamination in serum albumin, likelihood of reduced availability of recovered plasma in the future, and price. As a result of production technology evaluation, the expression of human albumin in the milk of transgenic cows has been identified as the most promising approach. A major challenge for this project is the fact that the production of pharmaceutical proteins on the scale of many tons, essentially free of any impurities at the price level of a commodity has not been achieved in recombinant biotechnology so far. In collaboration with Genzyme Transgenics Corp. (Framingham) project activities were initiated in February 1997.

The naturally occuring plasma colloid albumin is the most abundant soluble protein in the body of vertebrates and human serum albumin (hSA) is probably the most studied of all proteins. Development of plasma fractionation technology during 1942 was the basis for the production of tons of highly purified albumin for battlefield use. Albumin is used as a plasma volume expander and in albumin deficiency situations, such as trauma, surgery, shock or burns. The function of albumin is not limited to the maintenance of isooncotic pressure (blood vs. tissue). It also serves as a carrier of smaller molecules of many types, such as fatty acids, hormones, and ions as well as an antioxidant [1].

Nowadays the annual worldwide albumin market is approx. 440 t and this market is expected to climb to 550 t/yr. by the year 2000. However, it is believed that a number of different factors will contribute to limited hSA availability in the future:

General shortages of human blood as a result of the reduced willingness of people to donate blood. Changes in transfusion practices due to the availability of new oxygen therapeutics in the future, accompanied by general safety concerns with products derived from blood and/or plasma sources. An increasing number of blood factors available from recombinant manufacture.

The last point is particularly important for plasma fractionating companies, because it is expected that the availability of recombinant blood factors will result in significant losses of revenue from plasma products. Production of hSA by means of recombinant technology requires extremely low production and downstream processing costs in order to be price-competitive with the plasma product. Recombinant human albumin was first cloned, expressed (in yeasts) and patented by Genentech in 1981 [2]. Human albumin derived from recombinant production will differ from the plasma counterpart in terms of quality (essentially no risk/TSE transmission) and purity (no trace amounts of impurities such as prekallikrein activator). Today, at least two companies (Centeon/Delta Biotechnology Ltd., UK and Green Cross Corp., Japan) have successfully developed large-scale production processes for recombinant hSA from yeasts. These products are in advanced clinical trials. Market launch of the Green Cross product (Albrec) is expected in the year 2000.

Protein-expression in the milk of transgenic animals offers another attractive approach to producing recombinant human albumin at very low costs. Due to their high annual milk yield, cows are considered to be the most suitable ‘producers‘. An individual dairy cow will produce approximately 8000 liters of milk per annum, or an estimated 80 Kilograms of albumin per year. Using transgenic cows as naturally occuring ‘bioreactors‘ would enable the production of hSA in the required range of many tons. At Genzyme Transgenics (GTC, Framingham, USA) the general feasibility of transgenic albumin expression was successfully evaluated in mice. Expression levels in mice are known to be predictive of the expression level in larger animals. Nevertheless, production of proteins using the mammary gland carries several risks. The most obvious technical problem is to efficiently remove the endogenous (bovine) serum albumin, which is fairly similar to the human counterpart (76 % homology). During a purification feasibility program initiated by GTC, a proprietary process for the purification of hSA at competitive costs, essentially free of bSA, was successfully developed. However, public acceptance, regulatory requirements and economic aspects (e. g. price development) are considered to be other potential risk factors for transgenic albumin production.

Since the sheep ‘Dolly‘ [3] last year, cloning as a specific aspect of transgenic technology has received considerable public attention. Compared to ‘standard‘ microinjection techniques, this technology opens up new possibilities for more sophisticated genetic manipulation. Therefore, it has been decided to incorporate this technology into the albumin program. A general agreement for the production of human therapeutic proteins in the milk of cloned transgenic animals between GTC and Advanced Cell Technology, Inc. (ACT) was reached in October, 1997. In January 1998, the birth of two calves (George/Charlie), created by combining cloning techniques with genetic engineering was announced by ACT. Birth of cloned bovines transgenic for human serum albumin is expected by late 1998.

Despite these rapid advances, the large scale production of recombinant human albumin in transgenic cattle still lies somewhat in the future.

Literatur

• 1 Peters. All about Albumin. Academic Press Ltd. 1996: 0-12552110-3
• 2 Jones. Assoc. J. Americ. Blood Res. 1996: 4-108-110
• 3 Wilmut. et al. .Nature 1996: 64-66