Development of Large-Scale Fractionation Methods

Abstract

A modification of the method of Johnson for the preparation of clinical factor VIII concentrates is described by which a concentrate may consistently be obtained from 100-liter plasma volumes at a potency of 10 factor VIII units/ml or greater and a specific activity of 0.3 factor VIII units/mg protein (18 to 20-fold purification over plasma) with a final recovery close to 40% of the actual factor VIII activity of the starting plasma; these potency and purity values exceed the minimum requirements of the Division of Biologies Standards of the National Institutes of Health. The major changes that were introduced were: (a) an accelerated cryoprecipitation step to minimize the risk of pyrogen formation; (b) the elimination of ethanol to avoid coprecipitation of nonspecific proteins, thereby improving the purity of the final concentrate; (c) the reduction of the tris extraction volume to obtain a higher final factor VIII potency and smaller volumes for lyophilization; (d) the control of pH during the Al (0H)3 adsorption step to improve the purity and filtrability of the concentrate; (e) the removal of the Al (OH)(3) precipitate by a foamless centrifugation technique to minimize dénaturation of the factor VIII in the supernatant ; and (f ) the use of new clarification media to facilitate the final sterile filtration step.

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“…Advances in our understanding of the physical chemistry of proteins provided insight into factors that governed solubility. In particular, our understanding of the effect of pH, ionic strength and temperature lead to significant improvements in the Cohn fractionation process [44–b50]. Whereas some of these advances resulted in the development of Factor VIII concentrates with somewhat improved potency [51–b53], solubility and yield issues persisted.…”