Ethanol Fractionation of Human Plasma: An Overview

Abstract

Human plasma is an extraordinarily complex aqueous solution of proteins, hormones, lipoids, carbohydrates etc., from which certain proteins are of interest in therapy. At the moment a small but increasing number of the hundreds of plasma proteins are used in clinical medicine and have to be prepared by fractionation. The methods applied in fractionation are more or less the same as for purification of proteins, but the aim is quite different; fractionation is meant to make the most out of plasma in terms of the clinical efficacy of the wanted protein(s). The safety of the preparations must be guaranteed by avoiding possible changes ill the native structure of the protein(s) or contamination that causes side effects. The economics of fractionation also have to be considered. To reach this aim, good recovery and efficacy of the protein(s) are necessary and are mainly dependent on controlled methods and technology. The safety is dependent mainly on a correct interpretation of "Good Manufacturing Practices", which primarily is to be found in well instructed and well educated personnel, adequate housing, sanitation and equipment. Continuous attention is needed to control the costs.During the last forty years a number of methods have been tried and performed in the fractionation of human plasma based on: differential solubility differential interaction with solid media differential interaction with physical fields. For the preparation of the bulk plasma proteins, i.e. fibrinogen, immunoglobulin and albumin, the cold ethanol method, based on different solubility is generally practised although other methods based on different solubility are or have been used like fractionation with ammonium sulphate (1) and ether(2).The separation between the proteins is performed under conditions where either the solubility of the desired protein is maximized and the solubility of the other proteins is minimized or reversed. The precipitate is separated from the supernatant by semi-continuous centrifugation and if needed, clarification.Variables determining the precipitation of the protein(s) are: ethanol concentration pH temperature ionic strength protein concentration from which the last two are less critical than the other variables and therefore mostly left out of the fractionation schemes. The first large scale batch wise fractionation of human plasma using acetate buffers was described by Cohn and coworkers who developed a number of methods from which the method 5 and 6 (3) and the immunoglobulin method 9 according to Oncley et aI. (4) are the best known.In the flowsheet ( fig. 1) other products like plasminogen, prothrombin complex etc., which can be prepared from fraction II+III and a further purification of fraction II has been left out. By this combined method five preparations can be derived from human plasma: C. T. Smit Sibinga et al. (eds.), Plasma Fractionation and Blood Transfusion

Factor Xa and Prothrombin: Mechanism of Action of FEIBA

“…Purification of prothrombin and factor Xa (FXa) was started from a prothrombin complex preparation made as described by Brummelhuis et al [13] and heat-treated for virus inactivation by treatment with hot vapour [14]. Detergent treatment was used as a second independent virus inactivation step.…”