Hyperglycosylatedhuman coagulation factor VIII fusion protein and preparation method and application thereof

Abstract

The invention discloses a hyperglycosylated human coagulation factor VIII (FVIII) fusion protein and a preparation method and application thereof. The fusion protein successively contains human FVIII, a soft peptide connector, at least one human chorionic gonadotropin betasubunitcarboxyl terminal peptide rigid unit and prolonged half-life period portion (preferably ahumanIgG Fc variant) from an end N to an end C. The fusion protein has biological activity similar to that of recombinant FVIII and prolonged in-vivo activity half-life period, thereby improving the pharmacokinetics and drug efficacy.

Description

technical field [0001] The present invention relates to the field of fusion proteins, more specifically, to a fusion protein of human blood coagulation factor VIII (FVIII) and its preparation method and application, especially the application for treating various blood coagulation-related diseases. Background technique [0002] Coagulation factor VIII (FVIII), also known as antihemophilic factor, plays a very important role in the endogenous coagulation system. According to a large number of research results of FVIII molecular genetics, it is shown that the lack of FVIII in the sex chromosome X-linked gene will cause hemophilia A. According to statistics, the prevalence of hemophilia A in the male population is 1 / 5000, accounting for more than 80% of the total number of hemophilias. The current commonly used treatment for hemophilia A is replacement therapy, which is to supplement the lack of coagulation factor VIII in hemophilia patients. [0003] FVIII is a multistructur...

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Problems solved by technology

According to reports, HEK-293 cells were transfected with the double expression vector of rFVIIIFc and Fc constructed by Biogen, and the fusion in the form of rFVIIIFc homodimer was not expected to be detected in the expressed product, and only the monomer-dimer was expressed. Polymeric hybrid rFVIIIFc fusion protein and Fc dimer, the company's researchers speculate that the possible reason is that the expression system used was too large in molecular weight for the homodimer type, and the host cells failed to secrete rFVIIIFc homolog with a molecular weight of about 400KDa The dimeric protein may fail to polymerize due to the steric hindrance effect between rFVIIIFc monomers (Peters RT et al., J Thromb Haemost, 2013, 11(1):132-41), it can be seen that homologous two Expression of polymeric FVIII fusion proteins is quite difficult

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