Drug – bio-affecting and body treating compositions – Enzyme or coenzyme containing – Hydrolases
Reexamination Certificate
2000-03-13
2001-08-28
Carlson, Karen Cochrane (Department: 1653)
Drug, bio-affecting and body treating compositions
Enzyme or coenzyme containing
Hydrolases
C514S008100
Reexamination Certificate
active
06280729
ABSTRACT:
FIELD OF THE INVENTION
This invention relates to purifying and stabilizing factor IX, one of the proteins essential to the cascade of reactions which accomplishes blood coagulation at the site of a wound.
Factor IX is a globular protein which has a molecular weight of about 70,000 daltons and which, in a normal individual, is constantly produced in the liver and circulates at a normal blood plasma concentration of approximately 5 &mgr;g/ml.
Hemophilia B (also known as Christmas disease) is a very serious illness which results in decreased in vivo and in vitro clotting activity and requires extensive medical monitoring throughout the life of the affected person. Such persons show normal clotting times only when provided with exogenous factor IX which is extracted from the blood plasma of normal individuals. Except for such treatment, the afflicted person can suffer from spontaneous bleeds in joints which produce severe pain and debilitating immobility, bleeds into muscles resulting in large volumes of blood accumulating in the tissue, spontaneous bleeds in the throat and neck which may cause asphyxiation if not immediately treated, bleeding into the urine, and severe bleeding following surgery or minor accidental injuries or dental extractions.
Functional factor IX deficiencies can arise in different ways. The gene coding for factor IX is located on the X chromosome. This explains why hemophilia B is much more common in males than females. Some of the afflicted persons are known to have inherited an X chromosome with a complete deletion of the factor IX gene. These severely affected persons may even produce antibodies to therapeutically injected factor IX. Many hemophilia B patients are known to produce a factor IX molecule with an altered amino acid sequence which results in molecules of partial or no coagulation activity. Some hemophilia B patients produce normal factor IX, but in insufficient quantities to effect clotting within a normal time after injury.
As mentioned above, Factor IX activity can be restored in the patient by injection of normal human plasma. However, at minimum, several liters would have to be administered to raise the patients's circulating factor IX levels to an effective range. Accordingly, the emphasis in therapy for hemophilia B patients has been to provide injections of a plasma concentrate highly enriched in factor IX. The provision of such a concentrate is no easy task, as will become apparent from the discussion which follows.
The mechanisms whereby circulating blood is generally prevented from clotting, yet directed to clot at the site of a wound are very complex and involve numerous proteins, other macromolecules, cells and structures. This hemostatic mechanism also utilizes numerous feedback or amplification pathways to further regulate coagulation. Owing to the large number of individual protein species which make up the clotting pathway and the large number of other macromolecules in blood plasma, it is generally difficult to isolate useable quantities of any one component, including factor IX, in highly pure form. In addition, blood contains numerous proteases (enzymes which digest or damage other proteins) which can affect adversely the protein selected for isolation, such as factor IX, before it can be separated from other blood components.
Since the clotting ability of the blood is held in a controlled balance, factor IX and other components associated with coagulation must be held inactive most of the time to avoid unnecessary clotting. Yet, the proteins must always be present throughout the circulatory system—ready to react immediately when needed.
The blood therefore contains by necessity a very complicated mechanism to prevent clotting from taking place where it is not needed, to clear unwanted clotting, and to rapidly stop the loss of blood at an injured site. The elucidation of this complicated mechanism of regulation makes clear why it is so difficult to isolate therapeutic factor IX free of clinically dangerous contaminants.
The formation of an effective clot involves the complex interaction of many vascular system components, including platelet blood cells, collagen and microfibrils exposed by damage to the vascular epithelium, phospholipids, and circulating proteins. Proteins which circulate in the blood as inert proenzymes and which are involved in coagulation are typically referred to as “coagulation factors”. Upon activation, they generally function as highly specific enzymes which make specific alterations in other coagulation factor proenzymes. Thus, in turn, each sequential factor is activated. Some proenzymes, such as factor XII, may also be activated by contact with a damaged surface or by complexing with other macromolecules.
The mechanism of the clotting process is known in considerable detail. The active form of a coagulation factor is denoted by the subscript “a” and is typically produced from the inactive proenzyme by the action of another of the factor-specific proteases. In theory, administration of activated coagulation factors to hemophilia patients carries a risk of clot formation at many locations besides the site of injury.
The hemostatic mechanism may be characterized as a very delicate balance between those materials or processes which inhibit coagulation and those which enhance it. Oversupply of one or more substances, particularly activated coagulation factors, may lead to unwanted coagulation. Activated coagulation factors can therefore be dangerous contaminants in therapeutic preparations of coagulation proenzymes, such as factor IX preparations.
With respect to hemophilia B patients, the state of the art, however, involves their being typically treated with “prothrombin complex concentrate”, which is a plasma extract concentrated in factor IX, but containing also significant amounts of other plasma proteins, including factors II, VII, X, active forms thereof, and numerous other contaminating proteases. Such preparations can also routinely be contaminated with factor IX
a
.
There are numerous reports in the literature on the adverse clinical consequences of administering prothrombin complex concentrate (or other factor IX concentrates) contaminated with factor IX
a
and/or with active or degraded forms of other clotting factors. The most serious risk is the inadvertent activation of the clotting cascade. Deaths have been documented.
Solutions to the problems associated with the use of impure factor IX concentrates have been hampered by lack of understanding of exactly how and why such concentrates induce unwanted clotting. It has been proposed that factor IX concentrates may induce coagulation not only because amounts of factors IX
a
are present, but also because they are significantly contaminated with other clotting factors, thus overloading the blood with high circulating levels of one or more clotting factors, or activated forms thereof.
Based on determinations using sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE), factor IX
a
has an approximate molecular weight of 54,000. However, other peptide species which result from proteolytic degradation of factor IX have very similar molecular weights for example, from approximately 40,000 to approximately 65,000. It is not currently known whether factor IX activation products or degradation products are primarily responsible for adverse clinical consequences which have been observed on administration of prothrombin complex concentrate or other impure factor IX-containing concentrates. Consequently, the development of factor IX therapeutics which avoid the hazards of impure preparations is of great pharmaceutical interest.
The prior art discloses various strategies for the purification of factor IX, including the use of monoclonal antibody affinity technology to separate factor IX from other coagulation factors. However, it is important that such factor IX purifications include every effort to minimize the formation of factor IX
a
or degraded factor IX peptides. Since factor IX (the proenzyme) and activated factor IX (IX
a
) are
Enkoji Takashi
Feldman Fred
Ho Laura
Huang Chin C.
Kleszynski Richard R.
Aventis Behring LLC
Carlson Karen Cochrane
Synnestvedt & Lechner LLP
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