The dissertation is devoted to development of a new method to obtain purified preparation of the blood coagulation factor VIII using macropore silica matrix with active dyes as ligands, to synthesis and investigation of properties of a dye-ligand macropore silica suitable for purification of the coagulation factor VIII, to use of various chemicalsas virus-inactivating agents in the process of purification, and to development of a scheme for obtaining a highly active virus-safe preparation of the factor VIII and studying its biochemical properties.
Research results demonstrated that the process of purification of the factor VIII is due to the phenomenon of the negative affinity adsorption. Factor VIII is not absorbed by any of the synthesized sorbents. However, its specific activity increases in the supernatant. This is due to its sorption from the investigated solution of additional proteins. A group of studied sorbents is isolated, which shows the best binding of additional proteins: Diasorb-Active purple 4GT, Diasorb-Procion Gelb M4R, Diasorb-Procion Blue HB, Diasorb-Procion Blue MXR and Diasorb-Active bright blue КН. It was determined that the pH of the solution affects the sorption of protein (the highest level of specific activity of factor VIII was at pH 7.4).
It has been established that in choosing a chromatographic sorbent, not only the choice of the ligands is important, but also the properties of the matrix. It was demonstrated that the best sorption of additional proteins was achieved on a sorbent with a pore size of 750 Å (the highest degree of the factor VIII purification).
To create a scheme for obtaining a highly purified preparation of the blood coagulation factor VIII from blood plasma, the research was conducted on combinations of the pre-fractionation method with the method of negative affinity sorption. It was determined that the pre-fractionation with barium citrate, aluminum hydroxide (III), and PEG-4000, provides removal of factors of prothrombin complex, fibrinogen, fibronectin, lipoproteins, denatured proteins, albumin etc. At this stage, we have reached hundredfold purification of the factor VIII preparation (for example, from 0.017±0.001 to 1.88±0.11 IU/mg protein for sorbent Diasorb-Active purple 4GT).
It has also been demonstrated that the use of ion-exchange chromatography on DEAE-Sepharose and affinity chromatography on selected sorbents allows obtaining factor VIII from Cryoprecipitate with a degree of purification of 129 to 242 times and preservation of up to 73–76 % of the initial procoagulant activity. The main losses from the initial activity of factor VIII and the von Willebrand’s factor (up to 27 %) occurred at the stage of ion-exchange chromatography.The phenomenon of negative affinity sorption provides almost 100.0 % (96.34 %) yield of the product. This is another significant advantage of this method in the technology of obtaining a purified preparation of the coagulation factor VIII.
It has been found that the combination of pre-fractionation, ion-exchange and affinity chromatography stages provides a purification rate of 239–700 times, depending on the type of selected sorbents. The best result was achieved when using Diasorb-Active purple 4GT.
To get the virus-safe preparation, the possibility of using known effective methods of antiviral treatment: solvent-detergent and ammonium thiocyanate, was investigated. Stages of inclusion of each of these methods in the scheme of purification of the coagulation factor VIII and methods of removal of virus-inactivating agents are proposed. The optimal application of these methods is prior to the cryopreservation stage, because most of the factors remain in the supernatant.
