Pb2450 Dye‐ligand Chromatography as an Alternative Method for Obtaining the Factors Viii/Von Willebrand Complex

Abstract

Background:A new dye‐ligand chromatographic procedure has been developed to produce a highly purified factors VIII/von Willebrand complex (FVIII/vWF) concentrate from plasma cryoprecipitate.Factor VIII (FVIII) and von Willebrand factor (vWF) circulate in the plasma as a non‐covalent protein complex FVIII/vWF. The levels of factor VIII in vivo are coordinately regulated with levels of vWF.Willebrand's disease together with hemophilia A are the most common heredity diseases of the hemostasis system. The concentrate of FVIII/vWF complex is used for the treatment of these diseases.The main method of obtaining of plasma concentrates of FVIII/vWF complex remains the method of ion‐exchange chromatography. With the use of ion exchange chromatography it is not possible to maintain the initial ratio because the elution of these two factors from the ion exchanger occurs at different ionic strength values. That is, the ion‐exchange chromatography method does not allow for the absolute yield of vWF together with FVIII.Aims:investigate the ratio of FVIII/vWF complex at various stages of the technology opurification of antihemophilic concentrate.Methods:Cryoprecipitation, ion‐exchange chromatography on DEAE‐Sepharose FAST FLOW; dye‐ligand affinity chromatography.Results:We development of a new method to obtain purified preparation of the complex FVIII/vWF, that inclused cryoprecipitation, ion‐exchange chromatography on DEAE‐Sepharose FAST FLOW and dye‐ligand affinity chromatography.The degree purification of FVIII was 95.3 times on the stage of ion‐exchange chromatography. The ratio FVIII/vWF has changed approximately twice (from 0.78 to 1.75) (Table 1). We used a buffer NaCl with an ionic strength of 0.3 M for co‐elution of FVIII and vWF. However, part of the vWF was eluted with the first fractions (0.15 M NaCl).The research demonstrated that the process of purification of the FVIII and vWF with dye‐ligand is due to the phenomenon of the negative affinity adsorption. This property we used in the technology of obtaining of FVIII/vWF complex. This property provides the output level of the relationship this provides the output level of the relationship (Table 1).Summary/Conclusion: It was established that at the stage of dye‐ligand chromatography, the loss from the initial activity of FVIII is practically absent and the original ratio of FVIII/vWF is saved.image