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

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, This email address is being protected from spambots. You need JavaScript enabled to view it.

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

1. Orlova N. A., Kovnir S. V., Vorobiev I., Gabibov A. G., Vorobiev A. I. Blood clotting factor VIII: from evolution to therapy. Acta Naturae, 2013, vol. 5, no. 2, pp. 19–39.

2. Nichols T. C., Raymer R. A., Franck H. W., Merricks E. P., Bellinger D. A., De Friess N., Margaritis P., Arruda V. R., Kay M. A., High K. A. Prevention of spontaneous bleeding in dogs with haemophilia A and haemophilia B. Haemophilia, 2010, vol. 16, no. 3, pp. 19–23. https://doi.org/10.1111/j.1365-2516.2010.02255.x

3. Timothy C., Nichols M. D. Lessons learned from animal models of inherited bleeding disorders. Hematology Education, 2014, vol. 8, no. 1, pp. 39–46. https://doi.org/10.1016/j.tmrv.2006.11.001

4. De Waure C., Cadeddu C., Gualano M.-R., Niolotti N., Di Nardo F., Walter R. Gestione terapeutica dell’emofilia A. Italian Journal of Public Health, 2011, vol. 8, no. 2, pp. 17–30. (in Italian)

5. Burnouf T. Modern plasma fractionation. Transfusion Medicine Reviews, 2007, vol 21, no. 2, pp. 101–117. https://doi.org/10.1016/j.tmrv.2006.11.001

6. Burnouf T. Chromatography in plasma fractionation: benefits and future trends. Journal of Chromatography, 1995, vol. 664, pp. 3–15. https://doi.org/10.1016/0378-4347(94)00532-A

7. Burnouf T., Burnouf-Radosevich M., Huart J. J., Goudemand M. A highly purified factor VIII: concentrate prepared from cryoprecipitate by ion-exchange chromatography. Vox Sanguinis, 1991, vol. 60, pp. 8–15. https://doi.org/10.1111/j.1423-0410.1991.tb00864.x

8. Radosevich M., Burnouf T. Affinity chromatography — fractionated and DNA-engineered plasma proteins. Encyclopedia of Industrial Biotechnology: Bioprocess, Bioseparation, and Cell Technology, 2009, pp. 1–12.

9. Clonis Y. D. Affinity separation: dye-ligands. Encyclopedia of Separation Science. Academic Press Ltd, London, 2000, pp. 259–265. https://doi.org/10.1016/B0-12-226770-2/00531-7

10. Osterman L. A. The chromatography of proteins and nucleic acids, Moscow, Science, 1985, 536 p. (in Russian)

11. Muller-Berghaus G., Potzsch B., Schwinn H. Method for the affinity-chromatographic purification of factor VIII. Patent US, no. 09/202,800, 2000.

12. Ro Sen S., Casoni Chiaroi M. Determination of factor VIII activity in plasma and concentrates: comparison between methods. American Clinical Laboratory, 2002, pp. 32–37.

13. Larsson P.-O. High-performance liquid affinity chromatography. Methods in Enzymology, 1984, vol. 104, pp. 213–223. https://doi.org/10.1016/S0076-6879(84)04091-X

14. Read S. M., Northcote D. H. Minimization of variation in the response to different proteins of the Commssie Blue G Dye-binding assay for proteins. Analytical Biochemistry, 1981, vol. 116, no. 1, pp. 53–64. https://doi.org/10.1016/0003-2697(81)90321-3

15. Laemmly U. K. Cleavage of structural proteins during the assemble of the head of bacteriophage T4. Nature, 1970, vol. 227, no. 5259, pp. 680–685. https://doi.org/10.1038/227680a0

16. Burnouf T., Radosevich M. Affinity chromatography in the industrial purification of plasma proteins for therapeutic use. Journual of Biochemical and Biophysical Methods, 2001, vol. 49, pp. 575–586. https://doi.org/10.1016/S0165-022X(01)00221-4

17. Shurko N. O., Danysh T. V., Novak V. L. The method of purification of coagulation factor VIII. Patent UA, no. 2906990, 2011. (in Ukrainian)

18. Scoups K. P. Current protocols in protein Science. John Wiley and Sons Inc., 1995, 3800 p.

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