Chemistry: natural resins or derivatives; peptides or proteins; – Proteins – i.e. – more than 100 amino acid residues – Separation or purification
Reissue Patent
2000-07-17
2003-07-22
Brumback, Brenda (Department: 1642)
Chemistry: natural resins or derivatives; peptides or proteins;
Proteins, i.e., more than 100 amino acid residues
Separation or purification
C530S388250, C530S389300
Reissue Patent
active
RE038202
ABSTRACT:
FIELD OF THE INVENTION
This 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 INVENTION
Inherited 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 resulting in major clinical signs of bleeding or thrombosis. One of the most common bleeding disorders is Haemophilia A, which is due to deficiency or dysfunction of the coagulation Factor VIII. Less frequently occurring, but equally severe bleeding disorders include deficiencies of the haemostatic proteins Factor IX (Haemophilia B), Factor VII, or Factor X. On the other hand, thrombosis may occur as the result of even partial (heterozygous or acquired) deficiency of Protein C or Protein S, which are major components of a system that acts as an antagonist of the coagulation pathway (for reviews on haemostatic disorders see A. L. Bloom and D. P. Thomas (Eds.), Haemostasis and Thrombosis, 2nd edition, Churchill-Livingstone, Edinburgh, 1987, pp 393-436 and 452-464). Replacement therapy is considered as a powerful and effective means to restore the haemostatic balance in vivo. For instance, concentrates containing Factor IX have proven highly valuable blood products which are life-saving when used to control bleeding in patients suffering from Factor IX deficiency.
Commercially available Factor IX concentrates (so-called prothrombin complex concentrates) usually are prepared with ion exchange resins to separate Factor IX from the other plasma proteins. This technique however, yields Factor IX preparations that also contain a number of other, closely related haemostatic proteins. These include Factor VII, Factor X, Factor II, Protein C and Protein S, which all belong to the class of the vitamin K-dependent proteins. The term “vitamin K-dependent” is referring to the fact that these proteins contain glutamic acid residues that are carboxylated during biosynthesis in a vitamin K-dependent process. Carboxylation provides these proteins with unique Ca
2+
-binding sites that are obligatory for the biological activity of these proteins within the Ca
2+
-dependent haemostatic process. Due to these and other structural similarities (see B. Furie and B. C. Furie, Cell vol 53, 1988, pp 505-518), the vitamin K-dependent proteins are readily co-purified. Thus, most Factor IX concentrates are also containing other haemostatic proteins such as Factor VII, Protein C and Protein S, and consequently the same concentrates have been used for the treatment of deficiencies of those proteins as well. However, treatment of bleeding disorders with compositions containing anticoagulant proteins such as Protein C and Protein S is in fact highly undesired, as is treatment of thromboembolic disease with compositions containing Factor IX, Factor VII or other procoagulant components as major contaminants. Therefore, the ideal therapeutic composition to correct a deficiency of one specific haemostatic protein should consist of solely that single component in an intact conformation and nothing else except solvent, and sometimes an inert carrier. As a consequence, the purification strategies needed to achieve the desired degree of purity have become increasingly complex.
Along with the introduction of advanced, more complex purification protocols a novel problem of undesired proteolysis of the target species was encountered. Limited proteolysis is a key mechanism in the regulation of a number of biological systems (see H. Neurath and K. A. Walsh, Proc. Natl. Acad. Sci. USA vol 73, 1976, pp 3825-3832). Typical examples of such biological systems include the complement system, the fibrinolytic system, and the blood coagulation system. These biological cascade systems involve the sequential conversion of intact, inactive precursor proteins into active enzymes or cofactors by proteolysis of one or more specific peptide bonds. On the other hand, feedback mechanisms exist to maintain these processes under local control and lead to proteolytic inactivation of the target proteins. Accordingly, the components of the coagulation cascade are present in blood plasma in a precursor form lacking biological activity. With regard to replacement therapy, the presence of coagulation proteins that are no longer intact is troublesome, since after having been subject to limited proteolysis, such cleaved species may bypass the natural, local control of haemostasis in vivo. Although natural mechanisms effectively control proteolysis under physiological conditions, these can no longer be maintained when haemostatic proteins are isolated from their natural source. As such protease-sensitive sequences are exposed within the tertiairy structure of these proteins, they provide easily accessible targets for proteolysis in a non-physiological environment lacking natural control mechanisms. Therefore, it is virtually impossible to completely prevent partial proteolysis during purification. Uncontrolled proteolysis of these vulnerable proteins is not limited to purification from a natural source as human plasma or fractions thereof, but may equally occur when the same proteins are obtained by recombinant DNA technology from transformed cell lines in vitro, or from biological fluids, including milk, of transgenic animals in vivo.
The presence of cleaved species in therapeutic products is clearly not desired, because the presence of activated proteins may trigger thrombogenic responses of the haemostatic system, whereas the presence of inactivated proteins leads to products with suboptimal biological activity that may competitively inhibit the reactions to be corrected. Prothrombin complex concentrates contain activated species of virtually all vitamin K-dependent coagulation factors, and this has been established as a causative agent for the occurrence of thromboembolic complications since the 1970s is (G. C. White et al., Blood vol 49, 1977, pp 159-170; J. M. Lusher, Seminars in Hematology vol 28, suppl 6, 1991, pp 3-5). Theoretically, in particular those species that participate in the initiation phase of the coagulation system, and thereby are the most amplified in the cascade mechanism, are to be considered as the most potent in disturbing the physiological haemostatic balance. Indeed, in vivo studies employing purified activated coagulation factors have identified activated Factor IX (S. Gitel et al., Proc. Natl. Acad. Sci. USA vol 74, 1977, pp 3028-3032) and activated Factor VII (K. Mertens et al., Thromb. Haemostasis vol 64, 1990, pp 138-144) as thrombogenic even in extremely low dosage. This may raise particular concern for activated forms that are relatively resistent to inhibition in vivo. Most activated vitamin K-dependent coagulation factors are subject to almost instantaneous inhibition by the abundance of protease inhibitors in blood plasma. However, Factor IXa is only slowly inhibited, whereas Factor VIIa and activated Protein C have in vivo half-lives up to 2 hours (K. Mertens et al., Thromb. Haemostasis vol 64, 1990, pp 138-144; P. C. Comp, Hematology, McGraw-Hill, New York, N.Y., 1990, pp 1290-1303). Thus, upon infusing vitamin K-dependent haemostatic proteins into patients, it should be noted that even traces of activated forms may remain in the patients circulation sufficiently long to bypass physiological control. Therefore, Protein C
Mertens Koenraad
van Mourik Jan Aart
Brumback Brenda
Hoffmann & Baron , LLP
Stichting Sanquin Bloedvoorziening
LandOfFree
Antibodies specific for a haemostatic protein, their use for... does not yet have a rating. At this time, there are no reviews or comments for this patent.
If you have personal experience with Antibodies specific for a haemostatic protein, their use for..., we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Antibodies specific for a haemostatic protein, their use for... will most certainly appreciate the feedback.
Profile ID: LFUS-PAI-O-3019818