Purificação de fatores de coagulação VIII e VII recombinantes para o tratamento das hemofilias A e B produzidos a partir de células humanas

Abstract

In this work, several chromatographic methods were studied for the purification of recombinant clotting factors VII (FVIIr) and VIII (FVIIIr) derived from human SK-Hep cell lines. The FVIIIr is used for the treatment of Hemophilia A, while the FVIIr is used for the treatment of Hemophilia B and Hemophilia A. Producing these factors in human cell lines results in glycosylation, sulphation and folding patterns similar to the endogenous factors produced in the human organism. Purification of FVIIIr by multimodal chromatography techniques using CaptoMMC resin, affinity using FVIIISelect resin and ion exchange (SP-Sepharose) yielded a fairly homogeneous and well-defined band profile (by SDS-PAGE) which demonstrated the expected presence of the light and heavy chains, Westen-Blott indicated that commercial antibodies recognized the heavy chain of the studied molecule. The techniques allowed a high reproducibility of the process where purification sequences indicated the same behavior of chromatographic profiles and the process eliminated 99.5% ± 0.5% nonspecific proteins and recovering up to 64% FVIIIr. FVIIr was purified with only a single chromatographic technique using the FVIISelect resin which isolated the protein by removing about 99% impurities and recovering virtually the entire product. The affinity chromatography eluate was dialyzed on 5 kDa membranes which resulted in the autoactivation process of the FVIIr molecule resulting in a signal increase of up to 5 fold over the initial amount. The SDS-PAGE gel and Westen-Blott demonstrated the auto-activation process where a migration of 50 kDa to 30 kDa band was observed and the commercial antibodies against FVII were able to detect the band. The purification method was also quite reproducible and the band profile very similar compared to the commercial products. Thus, it was possible to obtain purification platforms for the FVIIr and FVIIIr proteins.

Tratamento de Hemofílicos A Grave Atendidos no Hospital de Hematologia da Fundação HEMOPE, com Inibidor de Fator VIII Através da Imunotolerância

“…O Fator Recombinante VII ativado (FVIIr) ou complexo protrombínico parcialmente ativado (CPPa) conhecido como agente by passing é utilizado em eventos hemorrágicos agudos em que o FVIII não tem resposta diante dos inibidores contra a proteína do FVIII, contudo, ainda há discussões sobre o manejo, pois são produtos caros e não apresentam uma hemostasia eficiente em todos os pacientes que o utilizam (PEPE et al, 2017;GRANOVSKI, 2018;SILVA et al, 2019).…”