Direct Capture of Plasma Factor VIII:C by Ion Exchange Chromatography

Abstract

An ion exchange medium, DEAE-Fractogel 650M, can be used to capture factor VIII (FVIII) directly from plasma. Previous reports have focussed on the use of this medium to capture FVIII from cryoprecipitate. In this report, citrate phosphate dextrose plasma was batch-stirred with DEAE-Sephadex A50, filtered, diluted, loaded onto a column packed with DEAE-Fractogel TSK 650M, and chromatographed. Most of the unwanted proteins flowed through the gel unadsorbed. Bound FVIII was eluted by increasing the ionic strength of the buffer. A citrate- based buffer gave an overall FVIII:C yield of 670 IU/kg plasma with a specific activity of 0.68 IU FVIII:C/mg protein. This process gave a higher yield of FVIIEC but significantly more prothrombin complex factors than cryoprecip- itation. This method is a promising alternative to cryoprecipitation of FVIII.

Separation of antihemophilic factor VII from human plasma by column chromatography

“…TMAE-Fractogel provides the highest binding capacity independent of the sample pH as compared to DEAE-Fractogel. DEAE-Fractogel is a tentacle type weak anion exchanger which has previously been used in the purification of FVIII and vWF (6,8,14). However, in two cases the investigators used cryoprecipitate as a source material (6,14) Tech etal.…”

“…However, in two cases the investigators used cryoprecipitate as a source material (6,14) Tech etal. (8) used DEAE-Fractogel ion exchange chromatography for purification of FVIII from plasma, but they have not studied the effect of Virus Inactivation and lyophilisation on the activity of FVIII. This work is a modification of the method of Tech etal.…”

“…Although the production of cryoprecipitate is a simple procedure, it has two major disadvantages, the recovery of FVIII is low about 40% of that in normal plasma, and the purity is poor (2,3). To overcome these disadvantages, the cryoprecipitate is further purified using various techniques such as adsorption (4), gel filtration (5) and ion exchange chromatography (6,7,8). The application of ion exchange chromatography for the isolation of FVIII from cryoprecipitate has been demonstrated in a number of reports using DEAEFractogel (Dimethylaminoethyl) and Q Sepharose Fast Flow resin (6,7,8).…”