Antibodies specific for a haemostatic protein, their use for isolating protein, haemostatic compositions devoid of proteolytic cleavage products of the protein

Abstract

A method for the generation of Ca++ independent antibodies against blood coagulation factors is described wherein an antibody selection strategy based upon small peptides comprising target sequences for limited proteolysis is employed. These antibodies which distinguish between intact and cleaved species of haemostatic protein provide novel tools for the isolation of intact haemostatic proteins.

Description

FIELD OF THE INVENTIONThis invention relates to the preparation of therapeutic compositions consisting of purified blood coagulation factors for the treatment of haemostatic disorders. Said compositions are obtained by affinity chromatography employing antibodies, more in particular monoclonal antibodies, that distinguish between intact and cleaved molecular species. Methods are disclosed to obtain such antibodies, which allow the isolation of vitamin K-dependent blood coagulation proteins, including Factor IX, Factor VII, Protein C or Protein S, as intact polypeptides, devoid of cleavage products representing activated or degraded species.BACKGROUND OF THE INVENTIONInherited or acquired deficiencies of proteins of the blood coagulation system provide a major cause for the occurrence of haemostatic disorders. Even the lack or shortage of one single component of this system may be sufficient to disturb the delicate balance between procoagulant and anticoagulant pathways in a manner r...

AI Technical Summary

Benefits of technology

The process of the present invention is a major breakthrough in the field of immuno-affinity chromatography in that it achieves unique selectivity for intact, non-cleaved target proteins. This allows for methods to obtain intact haemostatic proteins, which can thereafter be formulated into improved therapeutic blood products. These products provide safer and more effective agents than are available thus far for the treatment of patients encountering critical bleeding or clotting episodes.

Problems solved by technology

However, in spite of their high selectivity, those procedures have not been able to distinguish between the intact and activated or degraded coagulation factors, and as a consequence cleaved products are co-purified with the desired intact, non-activated target product.

Moreover, antibodies that are Ca.sup.2+ -dependent have the disadvantage that Ca.sup.2+ -ions cannot be added to most source materials, including plasma or fractions thereof, without simultaneously triggering the Ca.sup.2+ -dependent coagulation system that leads to proteolytic cleavage of the vitamin K-dependent target proteins.

This represents a major drawback for the applicability of the final therapeutic product as it has been demonstrated that even small amounts of activated coagulation factors have been implicated as causative agents for disseminated intravascular coagulation and thromboembolism.