A Highly Purified Factor VIII:c Concentrate Prepared from Cryoprecipitate by Ion‐Exchange Chromatography

Abstract

A new ion-exchange chromatographic procedure has been developed to produce a highly purified factor VIII (FVIII) concentrate from plasma cryoprecipitate. Solubilized cryoprecipitate, after adsorption on aluminium hydroxide and cold precipitation, was treated with 0.3% tri(n-butyl)phosphate and 1% Tween 80 at 25 degrees C for at least 8 h to inactivate lipid-enveloped viruses. The fraction was then loaded onto a column packed with DEAE-Fractogel TSK 650 M and chromatographed. Most proteins and TnBP-Tween 80 flowed through the gel unretarded. FVIII:c, which bound to the gel, was eluted by increasing the ionic strength, then was directly filter-sterilized without ultrafiltration or addition of a protein stabilizer. Chromatographic recovery of FVIII:c was 80-90%. After freeze-drying, FVIII:c was at a concentration of 42.5 +/- 9.5 IU/ml and had a specific activity of 175.4 +/- 37.8 IU/mg (n = 40), corresponding to a purification factor of over 12,000 from plasma. The typical yield of the freeze-dried FVIII:c from cryoprecipitate was 55-65%. FVIII:c was stable for over 24 h at room temperature in the liquid state. The mean content of fibrinogen and immunoglobulin G was only 65 and 100 mg/l, respectively, corresponding to 1.4 and 2.3 mg/1,000 IU FVIII:c. This concentrate, which is much purer than traditional FVIII concentrates, has been found to be well tolerated and effective in clinical treatment of hemophilia A patients.

Influence of the type of factor VIII concentrate on the incidence of factor VIII inhibitors in previously untreated patients with severe hemophilia A

“…It was prepared by ion-exchange chromatography and was solvent-detergent treated. 29 FVIII:C-specific activity was more than 150 IU/mg protein. Von Willebrand factor (VWF) was present at a concentration of 200 to 400 IU VWF:Ag per 1000 IU FVIII:C, naturally stabilizing FVIII:C. There was no albumin in the formulation.…”

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.…”

“…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).…”

Development of Technology of Blood Coagulation Factor Viii Purification by the Method of Affinity Chromatography on Silica Sorbents

“…Кріопреципітат, який було доволі легко отримати з окремих доз плазми у місцевих центрах служби крові, а та-кож з великих пулів плазми на промислових підприємствах, був основним препаратом для лікування хворих на гемофілію до середини 90-х рр. (а в Україні -донедавна) [7].…”

“…Оскільки FVIII зв'язується у плазмі з VWF, який виступає природнім ста-білізатором його активності, в процесі хромато-графічного виділення важливо дотримувати-ся умов, що забезпечують збереження цього комплексу. При виборі афінних лігандів перед-бачають їх можливу специфічність до будь-якого з білків цього комплексу [7].…”