Novas estratégias de purificação dos fatores de coagulação Fator VIII e Proteína C a partir de plasma humano empregando cromatografia líquida.

Abstract

IWASHITA, C. New strategies of purification of coagulation Factor VIII and Protein C from human plasma using liquid chromatography. 2012. 142p. [Masters thesis (Biotechnology)], Sao Paulo: Instituto de Ciencias Biomedicas, Universidade de Sao Paulo, Sao Paulo, 2012. In this work purification methods for the coagulation factor VIII (FVIII) and Protein C (PC) by chromatography was studied. Coagulation FVIII concentrate is used for Hemophilia A treatment, while PC concentrates is used in patients at risk of deep vein thrombosis. Activated PC concentrates can be used for the treatment of sepsis. The use of the anion exchange column ANX Sepharose FF as the first purification step allows the elution of FVIII and PC with good purification factors. In this context, we proposed the separation of FVIII and PC using immobilized metal ion affinity chromatography (IMAC). A systematic study was carried out using IMAC-Cu, Ni, Zn, Co and Fe columns and the desorption methods employing imidazole, ammonium chloride and pH variation. In the columns containing Fe and Ni metal ions, proteins from the loading samples did not adsorb to the resin. In general, PC interacted much weakly with IMAC columns than FVIII. In IMAC-Co, PC was not adsorbed to the resin, while FVIII could be eluted with 100 mM imidazole. In IMACCu PC could be eluted with 10 mM imidazole and FVIII with 200 mM. Proteins were much more strongly attached to Cu than to the other studied metals. It was not possible to elute the proteins from IMAC-Cu column with either 1M NH4Cl or when pH was descreased to 4,0. In IMAC-Zn, PC was not adsorbed and FVIII could be eluted with 200 mM imidazole or 1 M NH4Cl. By decreasing the pH, proteins could be eluted, but the recovery of the FVIII was very low, when the decreasing of the pH started at pH 7,0, or none, when the decreasing of the pH started at pH 6,0. We conclude that FVIII and PC could be well separated using IMAC-Cu, IMAC-Zn and IMAC-Co, and that the Co column gave the best yield and purification factors for the 2 proteins.