Chemistry: natural resins or derivatives; peptides or proteins; – Proteins – i.e. – more than 100 amino acid residues – Blood proteins or globulins – e.g. – proteoglycans – platelet...
Reexamination Certificate
1992-08-18
2001-07-10
Low, Christopher S. F. (Department: 1653)
Chemistry: natural resins or derivatives; peptides or proteins;
Proteins, i.e., more than 100 amino acid residues
Blood proteins or globulins, e.g., proteoglycans, platelet...
C530S383000, C530S384000, C530S413000
Reexamination Certificate
active
06258938
ABSTRACT:
The present invention is concerned generally with methods for the isolation of proteins, e.g., blood clotting proteins, from a mixture of proteins in a fluid. Proteins are commonly purified by immunoaffinity chromatography, in which a protein-containing mixture is contacted with an immobilized antibody to the protein, and the protein is then eluted using non-specific, harsh conditions to disrupt the protein-antibody complex.
One class of proteins for which immunoaffinity chromatography has been attempted are the proteins involved in the blood clotting-process. The general overall process of blood clotting involves two stages: an activation stage in which the proenzyme prothrombin, through the action of many Factors and calcium ions, is converted into its active enzyme form, thrombin, and a conversion stage in which the proteolytic enzyme thrombin acts upon fibrinogen to form fibrin, which forms a three dimensional network mesh that holds the formed elements of blood.
The requisite Factors for blood clotting are all proteins, several of which share some similarities in structure and function, while others are distinct moieties unlike any other. For example, six blood proteins (the “vitamin K-dependent proteins”) require vitamin K for their complete synthesis: Factor IX, Factor X, Factor VII, prothrombin, protein S, and protein C. As a group, these proteins share marked homology in amino acid sequence, are activated by limited proteolysis from the zymogen to active enzyme form, and contain the novel metal binding amino acid &ggr;-carboxyglutamic acid. These blood clotting proteins are representative of a unique class of metal-ion binding proteins which are able to bind a large number of bivalent and trivalent cations. Upon combination with metal-ion, such as calcium, magnesium, manganese and gadolinium ions, these proteins undergo a structural conformational transition involving changes in the peptide backbone and changes in exposure of specific amino acid residues, which can be monitored by fluorescence, circular dichroism, or immunochemical techniques.
Other blood clotting proteins also share this ability to bind with metal-ions. Factor V, proaccelerin, is essential in the conversion of prothrombin to thrombin and is a very labile protein which rapidly disappears from stored plasma. Factor VIII, antihemophilic factor, is essential for the making of thrombin and is deficient in the plasma of persons with classical hemophilia. Hemophilia is congenital and the blood of hemophiliacs appears normal relative to the coagulation mechanism except for the deficiency of Factor VIII.
The vitamin K dependent proteins are deficient, on an acquired basis, in liver disease, in vitamin K deficiencies and in the presence of vitamin K antagonist drugs such as sodium warfarin (Coumadin). Hemophilia B is a disorder characterized as a hereditary deficiency of Factor IX; of the 25,000 persons in the United States with hemophilia, approximately 10-12% are afflicted with Hemophilia B.
The treatment of persons whose disorders comprise acquired or congenital deficiencies of blood clotting proteins continues to be a high risk and costly therapy. For example, Hemophilia B is presently treated in two ways: use of fresh frozen plasma or use of a commercial preparation of Factor-IX. This latter material is a concentrate obtained by partial fractionation of normal human plasma and is, at best, only of intermediate purity. Both therapies, the frozen plasma and the impure Factor IX concentrate, present a significant risk of hepatitis to the patient, but the Factor IX concentrate presents a far greater risk of infection since it is prepared from pooled human plasma. Essentially all hemophiliacs receiving multiple transfusions of either of these plasma products have been exposed to hepatitis and show seriological evidence of such exposure. Clinically, most have some form of abnormal liver function. However, the impure Factor IX concentrate adds risk to major complications, such as disseminated intravascular coagulation, thrombosis, and hepatitis, among others, believed to be directly caused or aggravated by the impurities in the preparations. More recently, an increased risk for the development of the highly fatal Acquired Immune Deficiency Syndrome (AIDS) has been reported in patients with hemophilia who received plasma concentrates. Although plasma protein infusion therapy is still the treatment of choice in these disorders, it is clear the complications of such infusion therapy, caused directly by the impurities in the prepared product, diminish its use and effectiveness. For this reason, any method which would provide blood clotting plasma protein in concentrated form of substantial purity would eliminate or significantly reduce the undesirable medical complications of current infusion therapy. Such an advance would satisfy a long recognized need and provide additional advantages such that infusion therapy may be used regularly and prophylactically by hemophiliacs to reduce or eliminate the protein deficiencies associated with such disorders.
As mentioned above, general procedures are known for purifying blood clotting factors in plasma by passing the plasma through an affinity chromatographic column comprising inert matrix support, usually in the form of beads, such as Sepharose to which is bound the antibody to the factor it is desired to isolate. The factor specifically complexes with the fixed antibody and thereafter the factor (antigen) is eluted from the column. However, prior to this invention, it has been very difficult to obtain therapeutically useful purifications of the desired blood clotting factors by this process since the blood clotting factors are very difficult to elute successfully. This is because the chemical or physical conditions necessary to separate the antibody from the protein can destroy the function of the protein.
Accordingly, it would be highly desirable to provide a means for isolating proteins including individual blood clotting factors whereby both the structural and functional integrity of the protein can be retained and whereby the proteins can be recovered in quantity.
SUMMARY OF THE INVENTION
In general, the invention features highly effective methods for isolating proteins which undergo conformational changes (i.e., a change in tertiary structure) when complexed with ligands. The methods employ antibodies (either polyclonal or monoclonal) which either specifically react with protein-ligand complexes, and substantially fail to react with the protein in the absence of the ligand, or specifically react with ligand-free protein, and substantially fail to react with the protein complexed with the ligand. (Since the protein is generally stabilized by the ligand, the protein, complexed with the ligand, is sometimes referred to herein as a “ligand-stabilized” conformer.)
Where the antibody used in the method is specific for the ligand-stabilized protein to be isolated, the method involves immobilizing the antibody on a solid support and then contacting a mixture containing the protein with the immobilized antibody, in the presence of the ligand, to bind the ligand-stabilized protein to the immobilized antibody. To release the protein, the protein-antibody complex is contacted with a compound having a binding affinity for the ligand higher than the affinity of the protein for the ligand; this higher affinity compound removes the ligand from the protein, changing the protein's conformation so that the antibody no longer binds to it, and the protein is thus released. This releasing step is specific and is carried out under mild conditions, and thus provides a high degree of purification without the risk of denaturation and loss of function associated with the non-specific, harsh conditions under which proteins are conventionally eluted from immunoaffinity columns.
When, in the above method, the protein containing mixture is citrated plasma, the method preferably involves removing the calcium from the plasma, e.g., by dialysis against a buffer, and then adding to the plasma a n
Furie Barbara C.
Furie Bruce E.
Lewis Richard M.
Liebman Howard A.
Fish & Richardson P.C.
Low Christopher S. F.
NE Medical Center Hospital, Inc.
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