Purification of recombinant proteins for pharmaceutical use.

Estimates of the potential market size for genetically engineered pharmaceutical products have varied widely over the past several years. The 1988 market size has been estimated at about $800 million and this is forecast to reach about $4000 million by 1 Y9S [ I ] . It is difficult to be sure of the accuracy of any of these figures; however, with nine biotechnology-derived products currently on the market, and an apparently endless list of potential products in clinical trials or at earlier stages of development, the future appears very bright indeed [ 2 ) . There are many stages involved in bringing a biotechnology-derived pharmaceutical product to the market, these include: ( i ) identification of a potential product and its biological function; (ii) preparation o f recombinant plasmid and introduction into the host cell; (iii) development of fermentation (tissue culture) process; (iv) separation of the protein of interest from contaminating material; ( v ) preparation of product for clinical testing; (vi) scale up design, implementation and validation of the manufacturing processes. This paper reviews some of the issues which effect largescale purification of proteins for pharmaceutical use. The development and scale up of efficient methods for the separation and purification of proteins is a major goal for producers of biotechnology-derived products, including therapeutic drugs. Extensive recovery and purification processes are required to achieve a highly pure protein which guarantees the safety of the product in humans. The purification process can significantly effect the yield, stability, purity and cost of the final product. At Avondale (Brinny) Chemical Company, the Irish subsidiary of Schering-Plough Corporation, we have during the past 2 years set-up production-scale purification processes for two recombinant-DNA-derived pharmaceuticz’ products, a-2b interferon, currently in use worldwide for a variety of anti-cancer and anti-viral indications, and granulocyte macrophage colony stimulating factor (GMCSF) currently being used in clinical trials for a number of immune deficiency disorders. The purification processes for these products were developed and scaled up up by the Schering-Plough Research Division in the U.S.A. The annual requirements for recombinant-derived proteins are relatively small, often in the gram to kilogram range. While quantities are low, cost tends to be high, with many biotechnology-derived products costing thousands of dollars per gram to produce. A large proportion o f these costs is associated with purification. A production-scale purification system must meet the following requirements: ( i ) practicability at large scale; ( i i ) product stability; (iii) product purity; ( iv) final dosage form formulation; ( v ) economic feasibility. In addition, the purification process for a pharmaceutical product must result in protein which meets the following criteria: ( i ) > 95% bioactive; (ii) non-altered; (iii) homogeneous; (iv) freedom from impurities; ( V ) freedom from host pyrogens; (vi) < 10 pg/dose of nucleic acid [ 3 I. The steps involved in processing the product of fermentation/tissue culture to highly purified protein can be divided into three stages: ( a ) cell harvesting/disruption; ( h ) crude extractionlfractionation and (c) final purification.