Purification, formulation and preclinical testing of recombinant FVIII produced in human cells.

Abstract

Hemophilia is a genetic disorder linked to the X chromosome which results in deficiency or abnormality of factor VIII protein (FVIII). The replacement therapy using protein derived from human plasma or recombinant produced in hamster cell lines (CHO and / or BHK) are the only treatment options available. However, one of the largest replacement therapy problems is the formation of inhibitory antibodies against recombinant factors, which is the most significant complication related to the treatment of hemophilia patients. The use of non-human strains introduces immunogenic epitopes (a-Gal and Neu5Gc) in FVIII affecting the efficacy and half-life of the protein. Over the past 10 years, it was developed at the Blood Center of Ribeirão Preto producing strains of human FVIII. Our research group succeeded in increasing our FVIII production scale at the beginning came a few milliliters to about 40 liters of broth. Currently, we are able to produce in an anaerobic mode perfusion about 50,000 IU of FVIII. Our FVIIIr is produced in human cells which contains glyco structures similar or identical to the plasma FVIII, resulting in more efficient product less immunogenic and safe for long term use. The purpose of this proposal is to purify FVIII produced in our laboratory to determine the glycosylation profile (in colanoration with Manitoba University), to formulate FVIII with nanoparticles and to performe the first pre-trial in the country. This work can bring as a benefit to Brazilian society the possibility of obtaining a more efficient medication, less immunogenic and with lower production costs. In addition to the technology that will be generated, this project contributes to development of human resources with multidisciplinary features. (AU)