Chemistry: analytical and immunological testing – Including sample preparation – Liberation or purification of sample or separation of...
Reexamination Certificate
1999-10-21
2003-06-17
Le, Long V. (Department: 1641)
Chemistry: analytical and immunological testing
Including sample preparation
Liberation or purification of sample or separation of...
C436S018000, C436S161000, C436S162000, C436S174000, C436S175000, C436S176000, C436S177000, C436S178000, C436S824000, C436S069000, C435S007100, C435S007200, C435S272000, C435S288600, C435S803000, C530S380000, C530S381000, C530S383000, C530S412000, C530S413000, C530S418000, C530S427000, C422S051000, C422S051000, C422S070000, C422S073000, C422S105000
Reexamination Certificate
active
06579723
ABSTRACT:
FIELD OF THE INVENTION
The invention relates to a method of recovering a highly purified vWF or a factor VIII/vWF-complex, respectively, from a biological starting material by means of immunoaffinity chromatography, as well as to a stable preparation containing highly purified vWF or factor VIII/vWF-complex.
BACKGROUND
Blood coagulation is a complex process including the sequential interaction of a series of components, in particular of fibrinogen, factor II, factor V, factor VII, factor VIII, factor IX, factor X, factor XI and factor XII. The loss of one of these components or the inhibition of its functionality causes an increased tendency of blood coagulation which may be life-threatening in some patients.
Von Willebrand factor (vWF) circulates in plasma complexed with factor VIII, factor VIII aiding blood coagulation and vWF in the complex with factor VIII stabilising the latter and protecting it from proteolytic degradation. On account of its function in the platelet aggregation, vWF also directly interferes in blood coagulation. vWF exists in plasma in a series of multimer forms of a molecular weight of from 1×10
6
to 20×10
6
Dalton. vWF is a glycoprotein primarily formed in the endothelial cells of mammals and subsequently liberated into circulation. In this connection, starting from a polypeptide chain having a molecular weight of approximately 220 kD, a vWF dimer having a molecular weight of 550 kD is formed in the cells by the formation of several sulfur bonds. Thereupon, further polymers of the vWF with ever increasing molecular weights, up to 20 million Dalton, are formed from the vWF dimers by linking. It is presumed that particularly the high-molecular vWF multimers have an essential importance in blood coagulation.
In hemophilia, blood coagulation is disturbed by a deficiency of certain plasmatic blood coagulation factors. In hemophilia A, the bleeding inclination is caused by a deficiency of factor VIII or a deficiency of vWF, respectively, which constitutes an essential component of factor VIII. Treatment of hemophilia A is effected primarily by substituting the lacking coagulation factor with factor concentrates, e.g. by infusion of factor VIII, factor VIII-complex or vWF.
vWF syndrome has several clinical pictures caused by an underproduction or an overproduction of vWF. Thus, an overproduction of vWF causes an increased thrombosis tendency. An undersupply caused by the absence or reduction of high-molecular forms of vWF is manifested in an increased bleeding tendency and an increased bleeding time on account of the inhibition of platelet aggregation and of wound closure.
A vWF deficiency may also cause a phenotypic hemophilia A, since vWF is an essential component of functional factor VIII. In these instances, the half-life of factor VIII is reduced to such an extent that its function in the blood coagulation cascade is impaired. Patients suffering from von Willebrand syndrome (vWD) thus frequently exhibit a factor VIII deficiency. In these patients, the reduced factor VIII activity is not the consequence of a defect of the X chromosomal gene, but an indirect consequence of the quantitative and qualitative change of vWF in plasma. The differentiation between hemophilia A and vWD may normally be effected by measuring the vWF antigen or by determining the ristocetin-cofactor activity. Both the vWF antigen content and the ristocetin cofactor activity are lowered in most vWD patients, whereas they are normal in hemophilia A patients.
Conventional methods for a therapy of von Willebrand syndrome use vWF recovered from plasma, and there are a number of attempts to treat vWD patients with purified vWF or factor VIII/vWF-complex. The development of monoclonal and polyclonal antibodies to blood factors, in particular to vWF, has enabled an improved isolation and purification of vWF or of factor VIII/vWF-complex from plasma or other biological sources.
Purification of factor VIII and of factor VIII/vWF-complex, respectively, from plasma is particularly rendered difficult by the fact that factor VIII occurs only in very small amounts in plasma, is extremely labile, and that the association of factor VIII with vWF is reversible under specific conditions.
By expressing factor VIII in recombinant cells (Wood et al., Nature 312: 330-337, 1984), it has been possible to prepare factor VIII by genetic engineering, yet it was only by the addition of or coexpression with vWF that a commercially usable yield of recombinant factor VIII could be achieved. Likewise, vWF has been prepared by genetical engineering methods and expressed in recombinant cells (Fischer et al., 1994, FEBS Letters 351: 345-348).
The recovery of purified factor VIII, vWF or factor VIII/vWF-complex from plasma or of factor VIII or vWF, respectively, from recombinant cells by means of various purification methods has also been described.
Zimmerman et al. (U.S. Pat. No. 4,361,509) describe a method of purifying factor VIII, wherein factor VIII/vWF complex is bound to a monoclonal anti-vWF antibody, and factor VIII is dissociated from the complex by means of CaCl
2
ions. The thus obtained factor VIII subsequently is recovered in pure state via a further chromatographic step. The immunoaffinity carrier to which vWF is still adsorbed is regenerated by means of a chaotropic agent, in particular NaSCN, a vWF/NaSCN solution being incurred as a by-product and being discarded.
U.S. Pat. No. 5,006,642 describes the recovery of vWF from a solution that comprises vWF and a chaotropic agent and is incurred as a by-product according to U.S. Pat. No. 4,361,509, by dialysis against a suitable buffer or by desalting the solution in a further chromatographic step.
EP 0 383 234 describes the preparation of a vWF concentrate by means of anion exchange chromatography, wherein a factor VIII/vWF-complex contained in a solution is dissociated by the addition of a calcium- and amino acid-containing buffer, and a vWF concentrate is recovered.
EP 0 469 985 describes a method of preparing vWF from plasma cryoprecipitate, which is largely free from factor VIII, in which vWF is separated from factor VIII in a first purification step, wherein factor VIII binds to an anion exchanger, whereas vWF is not bound. In a second step, the salt concentration of the vWF-containing eluate is reduced, whereby vWF can bind to a second anion exchanger. Thereafter vWF is eluted by increasing the ionic strength.
A purified vWF which optionally is complexed with factor VIII is desirable for use in the therapy of patients afflicted with von Willebrand syndrome, but also of patients suffering from phenotypic hemophilia A. Furthermore, due to the stabilizing effect of vWF, a better storage stability of factor VIII preparations is attained.
To purify factor VIII/vWF-complex, it has been suggested to precipitate contaminating proteins, such as, e.g., fibrinogen, by means of high concentrations of amino acids, in particular glycine (WO 82/04395, EP 0 383 234).
EP 0 600 480 describes the purification of factor VIII/vWF-complex from plasma by means of a combination of anion/cation exchange chromatography, wherein the factor VIII/vWF-complex is stabilized with heparin and optionally lysine is added as an antiprotease.
EP 0 295 645 describes the purification of factor VIII/vWF-complex on an affinity matrix, wherein specific peptides binding to vWF are used as the affinity carrier, the complex binding to these peptides and subsequently being eluted with a pH gradient.
WO 96/10584 describes a method of recovering highly-purified recombinant vWF by means of a combined anion exchange/heparin-affinity chromatography, and EP 0 705 846 describes the separation between high- and low-molecular fractions of vWF.
Mejan et al. (1988, Thromb. Haemost. 59: 364-371) have suggested to purify factor VIII/vWF-complex from plasma by means of immunoaffinity. By using a vWF-specific antibody coupled to a carrier, factor VIII/vWF-complex is bound to the carrier under physiological conditions, and the complex is elated under alkaline conditions at a pH of 10.2. To neutrali
Dorner Friedrich
Eibl Johann
Fiedler Christian
Fischer Bernhard
Mitterer Artur
Baxter Aktiengesellschaft
Gabel Gailene R.
Le Long V.
Townsend and Townsend / and Crew LLP
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