Bìol. Tvarin, 2016, Volume 18, Issue 2, pp. 152–159

http://dx.doi.org/10.15407/animbiol18.02.152

DEVELOPMENT OF TECHNOLOGY OF BLOOD COAGULATION FACTOR VIII PURIFICATION BY THE METHOD OF AFFINITY CHROMATOGRAPHY ON SILICA SORBENTS

N. O. Shurko, T. V. Danysh

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SI «Institute of Blood Pathology and Transfusion Medicine NAMS of Ukraine»,
45 gen. Chuprynky str., Lviv 79044, Ukraine,
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The manufacturing processes of chromatographic purification are used increasingly for plasma fractionation in the last few years. Implementation and combination of ion-exchange, affinity and size-exclusion chromatography have allowed the development of a new generation of therapeutic plasma derivatives, especially coagulation factors with improved purity and virus safety.

The purpose of this work was to investigate the possibility of using the method of chromatographic purification of factor VIII on silica sorbents with triazine dyes as ligands.

The paper presents the results of studies of purification blood coagulation factor VIII which is a combination of the method of fresh frozen plasma fractionation with the next step of affinity chromatography on macroporous silica sorbents. The main advantages and disadvantages of using triazine dyes as ligands are described.

The suggested method contains a combination of previous precipitation of plasma proteins with barium citrate, adsorption with aluminium hydroxide, separation of precipitated proteins by centrifugation and using the method of negative affinity chromatography on the silica sorbents: Diasorb Active Bright-blue K, Diasorb Active-scarlet damask 4GT, Diasoorb-Procion Blue HB, Diasorb-Procion Gelb MXR.

We have shown that the combination of previous fractionation of plasma with method of negative affinity chromatography on macroporous silica sorbents with synthetic dyes as ligands can achieve high purity factor VIII (about 100 times). For example, using sorbent Diasorb Active-scarlet damask 4GT increases the specific activity of factor VIII from 0.017±0.004 IU/mg to 1.940±0.023 IU/mg protein. This technology can be used along with other types of chromatography in the scheme of preparative receiving of factor VIII.

Keywords: AFFINITY CHROMATOGRAPY, SILICA SORBENTS, TRIAZINE DYES, FRACTIONATION, PLASMA OF BLOOD, BLOOD COAGULATION FACTOR VIII, HAEMOPHILIA A

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