Note: Descriptions are shown in the official language in which they were submitted.
CA 02251558 1998-10-09
Highly Purified Factor VIII complex
The present invention relates to a highly purified
complex consisting of the components factor VIII
(factor VIII:C or FVIII) and von Willbrand factor
(vWF), a stable pharmaceutical preparation containing
the highly purified complex as well as a method of
preparing a highly purified factor VIII:C/vWF-complex.
Von Willebrand factor is a multimeric glycoprotein
encoded by a gene on chromosome 12 and circulating in
plasma at concentrations of from 5 to 10 ~g/ml freely
and as non-covalent complex with coagulation factor
VIII, the protein which is encoded by a gene on
chromosome 10 and which is impaired or is missing,
respectively, in hemophilia A.
Among the two most important functions of vWF in
haemostasis are:
the adhesion of the thrombocytes to an injured
endothelium, vWF binding to the injured sub-endothelium
and enabling a bridge between this surface and the
platelets as well as an aggregation of the platelets
among themselves. The first interaction between
thrombocytes and sub-endothelium is effected via the
glycoprotein Ib of the thrombocyte membrane and the
collagen fibres of the injured endothelium. vWF binds
to these two proteins and thus mediates the formation
of a first layer of thrombocytes. Further cross-linking
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CA 02251558 1998-10-09
of the platelets among themselves is mediated by vWF by
its binding to the glycoprotein complex IIb/IIIa. For
these tasks of primary haemostasis, mainly the large
multimers are responsible (Eller; Lab. Med. (1994); 18:
168-176).
by its binding site for FVIII, vWF also
influences plasmatic coagulation. FVIII is present in
plasma almost exclusively in a non-covalent complex
with vWF, approximately every tenth vWF molecule
carrying an FVIII molecule. Primarily dimers and small
multimers are used as carriers. By this complexing with
vWF, FVIII is prptected against an increased
proteolytic inactivation (e.g. by activated protein C).
Furthermore, FVIII is potentiated by complex
formation in respect of its cofactor activity in the
intrinsic coagulation (Eller; Lab. Med. (1994); 18:
168-176).
vWF is formed in the vascular endothelial cells,
which are the main source of this plasma protein, by
constitutive or stimulated liberation, but it is also
synthetized in a smaller portion by the megakariocytes.
(PNAS 92 (1995), 2428-2432).
The primary product of translation is comprised of
2813 amino acids. After splitting off the signal
peptide (22 amino acids), dimerisation takes place.
Further processing is effected in the Golgi apparatus,
the dimers polymerizing under splitting off of the
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CA 02251558 1998-10-09
propeptide (741 amino acids). The propeptide plays an
important role in the further linking of the dimers,
where it catalyses the formation of disulfide bridges
at the amino-terminal end. Thus, differently sized
oligomers ranging in size from a dimer of 500,000
daltons to large multimers of up to 20 million daltons
are developed. In addition to the proteolytic
procedures, vWF is subject to other post-translational
modifications, including glycosylation and sulfatizing.
(Mancuso et al.; Hamostaseologie (1989); 9: 122-129).
Thus, due to the complexity of the biosynthesis,
there is a large number of the most varying vWF
molecules having the most varying tasks and properties.
As a consequence, von Willebrand factor may exhibit
quite different binding activities to its natural
binding partners. In particular it has been shown that
the bindings of various vWF molecules to glycoprotein
Ib, to collagen, to heparin, to glycoprotein IIb/IIIa-
complex, to factor VIII and to the sub-endothelium may
differ in strength.
This means, however, that each vWF preparation is
composed of a mixture of these different vWF proteins
or vWF aggregates, respectively, and thus will be
heterogenous in terms of its properties, such as its
essential binding strength to factor VIII.
The occurrence of various forms of vWF is also the
cause of the complex and different phenotypes in the
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CA 02251558 1998-10-09
pathophysiology of von Willebrand Disease which, in
certain cases, is due to an underproduction and in
other cases to an overproduction of von Willebrand
factor. Thus, e.g., an overproduction of vWF leads to
an increased thrombosis tendency, whereas an
undersupply of vWF results in an increased bleeding
tendency or in increased bleeding times; this, however,
is not always so, for it is decisive in which form von
Willbrand factor is over- or underproduced.
To differentiate and characterize the properties of
vWF and of vWF syndrome, a number of analytical methods
are used.
Thus, the ristocetin cofactor activity
determination is essential for diagnostical purposes.
In doing so, the thrombocyte aggregation in the
presence of the antibiotic ristocetin is assayed which
is reduced or not present at all in patients afflicted
with vWF syndrome. (Macfarlane et al.; Thrombosis et
Diathesis Haemorrhagica 1775; 34: 306-308).
Moreover, the collagen binding activity of vWF can
be used for differentiating the vWF syndrome (Thomas et
al.; Hamastaseologie (1994); 14: 133-139).
The binding dissociation constant between vWF and
FVIII can be determined according to the method of Vlot
et al. (Blood 83 (11) (1995); 3150-3157).
The molecular structure of vWF is determined by
analysis of the multimer structure by means of SDS
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CA 02251558 1998-10-09
electrophoresis in 1.2% agarose gels (Ruggeri et al.;
Blood (1981); 57: 1140-1143).
To determine the total amount of vWF antigen,
various commercially available ELISA test kits are
used.
The preparation of an optimal FVIII/vWF complex
should aim at providing a stable product which, above
all, is free from undesired accompanying proteins,
since any unnecessary protein load harbors the risk of
undesired side effects.
Thus, any concentration of vWF which is not
necessary for stabilizing FVIII is a load on the
hemophiliac.
In the course of methods employed for producing von
Willebrand preparations, in particular from plasma
pools, hitherto it has not been possible to eliminate
the risk of a heterogenous composition due to the
various forms of von Willebrand factor present; in the
prior art it has not been possible so far to obtain a
vWF preparation having uniform properties, e.g. with
regard to its binding activity to a certain ligand.
It had been known to purify factor VIII complex by
various anti-vWF-monoclonal antibodies both from plasma
and also from cryoprecipitate (Thromb. Haemostas. 57
(1987), 102-105). In doing so, also the stability of
the FVIII/vWF complex in various buffers at pH 6.5 was
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CA 02251558 1998-10-09
tested, among them buffers containing glycols, amino
acids, chaotropic substances, amines, other salts or
organic solvents and/or detergents. The addition of
lysin to buffer solutions containing chaotropic
substances at high concentrations, such as, e.g., 3M
urea, resulted in a protection of factor VIII:C/vWF:Ag
relative to denaturing effects. The activities of
factor VIII:C and of vWF R.cof. after incubation with
20% v/v ethylene glycol + 1 M KJ amounted, e.g., to 72%
and 48%, respectively, after treatment with 3M urea to
88% and 77%, respectively.
The specific acitivity of factor VIII:C in an end
product, eluted with 1 M KJ + 1 M lysin + 20 mM
imidazole + 5 mM CaCl2, pH 6.5, was 45 I.U./mg total
protein, that of vWF was 60 I.U./mg.
It has also been reported to chromatographically
purify factor VIII/vWF-complex from cryoprecipitate
after adsorption on A1(OH)3 and carrying out a virus
inactivation measure by using anti-vWF monoclonal
antibodies (Biotechnol. Blood Prot.~227 (1993), 109-
114). Elution of the adsorbed complex was effected at
pH 6.5 by the addition of the chaotropic agent KJ (1
M). The ratio factor VIII:C/vWF:AG was 0.8, the
specific activity of factor VIII:C was 38 I.U./mg.
Finally, it has been known from EP-0 295 645-A2 to
purify factor VIII complex from heterogenous biological
liquids by means of affinity chromatography, using
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CA 02251558 1998-10-09
specific peptides directed against vWF. In doing so,
the complex was eluted using pH gradients or buffers of
high ionic strength (cf. Example 5 of EP-0 295 645-A2).
In EP 0 416 983 A1, a preparation with factor
VIII/vWF-complex is described which was prepared by
anion exchange chromatography. According to WO 86/01718
A1, a factor VIII/vWF-complex preparation was obtained
by chromatography on a monoclonal antibody.
What is common to all these factor VIII/vWF-
complexes of the prior art is that they could not
provide a preparation homogenous in terms of vWF, as
regards its binding relative to factor VIII: C, despite
a high enrichment and purification of the preparations,
and thus there did not exist a native factor VIII/vWF-
complex of high specific activity.
Thus, the present invention has as its object to
provide a factor VIII/vWF-preparation comprising a
factor VIII/vWF-complex which has a uniform structure
as regards the binding properties of vWF relative to
factor VIII, and which thus is particularly well
tolerated or stable, respectively.
According to the invention, this object is achieved
by a preparation comprising a highly purified complex
comprised of the components factor VIII and von
Willebrand factor with a specific activity of at least
70, preferably 100 to 300 U factor VIII:C/mg, which is
obtainable by purifying a starting material comprising
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i ~ iinl, i I~ ~i~i~i il i i
CA 02251558 2004-09-17
24242-544
factor VIII:C and vWF by means of affinity chromatography,
the complex of factor VIII:C and vWF as well as the non-
complexed factor VIII:C and vWF, respectively, being eluted
fractionally. Non-complexed, i.e. excessive, factor VIII:C
and von Willebrand factor, respectively, are separated by
immunoaffinity chromatography, in particular by the
fractional elution, in which the complexed and the non-
complexed factor are obtained in separate fractions.
By the inventive separating step of non-complexed
factor VIII:C and von Willebrand factor, respectively,
particularly von Willebrand factor molecules having an
impaired affinity to factor VIII are purposefully separated,
and for the first time a homogenous, native factor VIII/vWF-
complex is obtained which contains a fraction of vWF that is
defined as regards its affinity to factor VIII.
According to one aspect of the present invention,
there is provided a method for purifying a starting material
comprising factor VIII:C and von Willebrand factor (vWF) by
means of immunoaffinity chromatography, characterised by the
following steps: contacting the starting materials with an
immunoaffinity chromatography material comprising
antibodies, eluting factor VIII/vWF-complex with a first
elution buffer and further elution of non-complexed factor
VIII:C and vWF, respectively, with a second elution buffer
having an increased concentration and conductivity,
respectively, as compared to the first elution buffer.
According to another aspect of the present
invention, there is provided a preparation containing a
highly purified complex comprising the components factor
VIII (factor VIII: C) and von Willebrand factor (vWF) having
a specific activity of at least 70 U of factor VIII:C/mg,
obtained by a method described herein.
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il,vl, i ~ li ~i:,~i..J i ~ i
CA 02251558 2004-09-17
24242-544
According to still another aspect of the present
invention, there is provided a preparation containing a
highly purified complex comprising the components factor
VIII (factor VIII: C) and von Willebrand factor (vWF) having
a specific activity of 100 to 300 U of factor VIII:C/mg,
obtained by a method described herein.
Surprisingly, the complex according to the
invention is stable in a chaotropic environment during
immunoaffinity chromatography. Even in a medium having a
conductivity of up to 30 mS, preferably up to 40 mS, a
dissociation of the complex could not be found. Thus, the
stable complex of factor VIII and vWF from the anti-vWF
column can be eluted and recovered even at a relatively high
ionic strength corresponding to a 2- to 3-fold isotonic
solution.
In the complex according to the invention, the vWF
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CA 02251558 1998-10-09
preferably has a collagen binding activity in the range
of from 0.2 to 0.6, based on the vWF antigen (plasma
unit/plasma unit).
The complex according to the invention preferably
is obtainable by purifying a starting material
containing the two components, the complex as well as
the separate factor VIII:C or vWF, respectively, being
eluted from the immunoaffinity carrier in fractions.
The eluting agents may also contain relatively high
concentrations of chaotropic substances or of
chaotropically active salts, since, preferably, the
immunoaffinity chromatography is carried out on
antibodies which retain their affinity or avidity to
factor VIII or vWF even under stringent conditions, the
factor VIII/vWF-complex obtained remaining stable even
under these conditions.
Antibodies are chosen, e.g., which bind the antigen
without adversely affecting the binding properties up
to 1 M NaSCN or 0.5 M guanidinium hydrochloride, or
even up to a 100% saturation with ammonium sulfate. 50%
of the antigen are each still bound up to 1.5 M NaSCN
or 0.75 M guanidinium hydrochloride, respectively, 80%
ethylene glycol or 0.75 M urea. The antibodies used for
the immunoaffinity chromatography preferably are strong
antibodies which, in a binding test, bind to the
immobilized antigen even from diluted solutions of
30 ng/ml at the most, preferably 15 ng/ml at the most,
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CA 02251558 1998-10-09
corresponding to an antibody/antigen ratio of 1:5 to
1:20.
Due to the excellent properties of the complex
according to the invention in terms of its homogeneity,
in particular with a view to the factor VIII binding
properties of the vWF contained, it is particularly
well suited for the production of pharmaceutical
preparations for administering factor VIII and/or vWF.
It has been shown that the complex according to the
invention can be obtained in a concentration which is
at least 5,000-fold, preferably 7,100- to 21,400-fold,
enriched over plasma. A particular embodiment of the
present invention thus is a stable pharmaceutical
preparation comprising the highly purified complex of
the invention at a concentration which is at least
5,000-fold, preferably 7,100- to 21,400-fold, enriched
over plasma, the complex having a high complex binding
strength and being substantially free from non-
complexed vWF and factor VIII: C, respectively. In this
way it is ensured that no excessive vWF or other
proteins are a load on the patient, while the
physiological activity of the factor VIII/vWF-complex
is maintained. The preparation according to the
invention is also characterized by its excellent
physiological acceptance.
Its freedom from non-complexed vWF and/or factor
VIII:C, respectively, means that less than 5%,
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CA 02251558 1998-10-09
preferably less than lo, of free vWF or factor VIII: C,
based on the total protein content, can be found in the
pharmaceutical preparation. Particularly preferred are
preparations in which no non-complexed vWF:Ag or factor
VIII: C, respectively, can be detected.
The detection of non-complexed vWF or factor VIII,
respectively, is effected by re-chromatography of the
complex of the invention on immunoaffinity material,
this material being loaded with a certain amount of
free vWF or of factor VIII, respectively, the complex
being adsorbed again and eluted in the manner described
before. The material obtained after re-chromatography
contains the same ratio of factor VIII:C to vWF:Ag as
the starting material and thus does not exhibit any
relative losses. A further test for detecting non-
complex-bound factor VIII and vWF is the binding of the
complex to immobilized factor VIII:C antibodies or vWF
antibodies, no substantial portion, i.e. less than 5%,
of non-bound vWF or factor VIII: C, respectively, being
detectable after adsorption.
The pharmaceutical preparation according to the
invention is characterized by its high reliability as
regards its range of action, the risk of a degradation
or of activation of factor VIII, respectively, being
remarkably reduced by the small portion of free factor
VIII:C or of easily dissociable factor VIII: C,
respectively, as compared to known factor VIII:C/vWF-
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CA 02251558 1998-10-09
complex preparations.
It goes without saying that the pharmaceutical
preparation comprising the complex of the invention may
include suitable pharmaceutically active ingredients,
buffers, auxiliary agents or additives which are used
for factor VIII/vWF preparations. Because of the
excellent stability of the factor VIII/vWF-complex, the
latter may be processed into a stable pharmaceutical
preparation without any further use of common
stabilizers, such as albumin, sugar, in particular
trehalose or sucrose. ,
Also in solution at a neutral pH, the
pharmaceutical preparation according to the invention
is sufficiently stable to be provided as a liquid
preparation of a liquid-deep-frozen preparation. Upon
reconstitution of a respective lyophilized preparation,
also an about unchanged composition of the complex can
be shown.
The dose to be administered or concentration,
respectively, of the complex in the preparation can
also be determined easily by the skilled artisan on the
basis of prior art administering regimens for factor
VIII/vWF preparations.
Factor VIII and/or vWF may be contained in the
preparation according to the invention as native
protein, or as derivative thereof, e.g. as a protein
mutated by deletion, substitution or insertion, or as a
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CA 02251558 1998-10-09
chemical derivative or fragment, respectively, insofar
as the high binding affinity of factor VIII to vWF is
retained in the complex.
Subject matter of the present invention is also a
method by which the complex according to the invention
can be prepared, which comprises purifying the starting
material containing factor VIII:C and vWF by means of
immunoaffinity chromatography, the complex of factor
VIII:C and vWF as well as the non-complexed factor
VIII:C and vWF, respectively, being eluted
fractionally. By the purposeful fractional elution of
the non-complexed factors from the complex it has for
the first time been possible to obtain a von Willebrand
factor preparation or a factor VIII/vWF-complex,
respectively, that is uniform in terms of a specific
binding strength.
Preferably, antibodies directed against vWF are
used for immunoaffinity chromatography. In this manner,
the purification method which is substantially directed
at fractionating different vWF molecules or vWF units
can be even better directed at the different nature of
the von Willebrand molecule.
According to a particular embodiment of the method
according to the invention, monoclonal antibodies are
used as the antibodies. In terms of homogeneity, they
are to be preferred to a polyclonal antiserum.
A preferred elution buffer for the inventive
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CA 02251558 1998-10-09
complex during affinity chromatography is a buffer
comprising a thiocyanate and/or an ammonium salt,
preferably at a concentration not exceeding 2 M, most
preferred in the range of from 0.05 M to 1.5 M. This
first elution buffer may preferably contain ethylene
glycol, glycerol or a polyalkylene glycol.
Non-complexed factor VIII:C or vWF, respectively,
preferably are eluted with a buffer containing a
chaotropic agent, in particular thiocyanate, and/or
ammonium salt, and/or alkaline salts and/or ethylene
glycol, glycerol or a polyalkylene glycol and also are
recovered as homogenous preparations. In doing so, the
buffer has a concentration or conductivity,
respectively, which is increased over the first elution
buffer, in particular a concentration or conductivity,
respectively, which is more than 20%, preferably more
than 50% to 100%, higher.
A further subject matter of the present invention
thus consists in a von Willebrand preparation having a
reduced binding or complexing capacity relative to
factor VIII:C and which can be recovered by the present
method.
The highly purified complex, but also the inventive
non-complexed factor VIII:C or vWF preparations,
respectively, may optionally be further purified by
further chromatographic steps, preferably ion exchange
chromatography, gel filtration, hydrophobic
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CA 02251558 1998-10-09
chromatography, affinity chromatography, in particular
on immobilized heparin, or metal ion chelate
chromatography, and be processed in a known and
suitable manner into pharmaceutical and also diagnostic
preparations.
For producing pharmaceutical preparations as a rule
it is necessary to provide a measure for inactivating
or depleting viruses, preferably a heat treatment
and/or a physical or chemical treatment. Suitable
methods have been described in EP-0 159 311,
EP 0 519 901 as well as WO 94/13329. Since the complex
of the invention has a high stability, this virus
inactivation measure preferably is performed on the
highly purified complex.
According to a preferred embodiment of the method
of the invention, the starting material is selected
from the group consisting of plasma, a plasma fraction,
such as, e.g., cryoprecipitate or an alcohol
precipitate, a cell culture supernatant and a
pharmaceutical preparation.
The invention will be explained in more detail by
way of the following examples to which, however, it
shall not be restricted.
E x a m p 1 a 1 . (at present considered by
applicant to be the best mode of carrying out the
claimed invention)
500 g of cryoprecipitate are dissolved 1 + 4 in
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CA 02251558 1998-10-09
dissolution buffer at 30°C. Subsequently, the solution
is clarified via a 0.45 ~,m filter (e. g. Millipore
Durapore). A column filled with 0.5 1 anti-vWF gel
(IMMUNOTECH, France) is prepared with approximately 10
column volumes (CV) equibuffer, and the clarified
cryoprecipitate solution is applied thereto at 0.5
cm/min. Thereafter, adversely affecting impurities are
washed out with 5 to 10 CV of equibuffer (possibly with
an increased NaCl concentration >250 mM) at a flow rate
of 1 cm/min. Factor VIII/vWF-complex is then eluted
with elution buffer 1 at an approximate ratio of 1:1.
Elution with elution buffer 2 yields vWF which is
almost completely free from factor VIII. The second
eluting step simultaneously serves for -purifying the
column to allow for an immediate equilibration
thereafter.
Dissolution buffer: 7.5 mM Tris
60 mM NaCl
100 mM lysin
100 mM Na acetate pH 6.8
25 U/ml heparin
Equibuffer: 10 mM Tris
100 mM NaCl
100 mM lysin
3.25 mM CaClz pH 6.8
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CA 02251558 1998-10-09
Elution buffer 1: 10 mM Tris
100 mM glycin
250 mM NaCl
300 mM AMS
3 mM CaClz
40 % ethylene glycol pH 6.5
Elution buffer 2: 10 mM Tris
100 mM glycin
1.25 M NaCl
1.25 mM NaSCN
3 mM CaClz
40 % ethylene glycol pH 7.0
Results:
Sample U/ml 7 Yield U/mg U/ml 7 Yield U/mgFVIII:C Collag.B.
FVIII: C FVIII:FVIII:vWF vWF vWF tr~ vWF t. m
C vWF
Start 11.0 100.07 0.4 16.3 100.07 0.6 1:1.48 0.7
FVIII/vWF12.7 50.97 133.714.7 39.87 1:1.16 0.3
vWF 0.2 0.5~ 23.4 37.27 236.40.7
E x a m p 1 a 2 .
1 1 of fresh frozen plasma is thawed at T >25°C and
subsequently diluted 1 + 2 with equibuffer. The
solution is clarified through a 0.45 ~m filter. A
column filled with 50 ml anti-vWF gel (IMMUNOTECH) is
prepared with approximately 5 CV of equibuffer, and
then the clarified plasma solultion is applied at a
flow rate of 1 cm/min. The undesired impurities are
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CA 02251558 1998-10-09
washed out with 10 to 20 CV of equibuffer + 250 mM
NaCl. The first elution yields factor VIII/vWF-complex;
the second elution vWF free from factor VIII activity.
Then the column may immediately be equilibrated again.
Equibuffer: 5 g/1 Na citrate
2 U/ml heparin pH 7.0
Elution buffer l: 5 g/1 Na citrate
30 g/1 AMS
20 g/1 NaSCN
75 g/1 histidine
g/1 NaCl
0.4 g/1 CaCl2 pH 7.0
Elution buffer 2: 5 g/1 Na citrate
50 g/1 AMS
50 g/1 NaSCN
75 g/1 histidine
25 g/1 NaCl
0.4 g/1 CaCl2 pH 7.0
Results:
Sample U/ml 7 YieldU/mg U/ml 7 YieldU/mg FVIII:C Collag.B.
FVIII:CFVIII:CFVIIIvWF vWF vWF to vWF 1
relat.tn
vWF
Start 0.3 100.0' 0.01 0.3 100.07 0..011:-1 I 1.0
FVIII/vWF2.4 33.37 42.861.8 24.37 1:0.75 0.5
vWF 0.0 0.2' 4.5 76.07 112.50 0.8
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