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CA 02540986 2006-03-31
WO 2005/049070 PCT/DK2004/000810
THERAPEUTIC USE OF FACTOR XI
FIELD OF THE INVENTION
The present invention relates to the therapeutic use of human Factor XI for
the
prevention and/or treatment of bleeding episodes, methods for the purification
of factor XI
and factor XI polypeptides from biological fluids, as well as pharmaceutical
formulations.
BACKGROUND OF THE INVENTION
Human Factor XI is a serine protease consisting of two identical subunits,
each
having a molecular mass of about 80 kDa. FXI circulates in plasma as a
disulfide-linked
homodimer having a molecular mass of ~160KDa. FXI is activated by cleavage of
each
monomer between Argass and Ile3~o to form an amino-terminal heavy chain of 50
kDa and a
carboxy-terminal light chain of 35 kDa, which are disulfide-linked. The
protein is encoded by
a 23 kb gene located on chromosome 4 (4q35) 15 exons and 14 introns coding for
a mRNA
consisting of 2,097 nucleotides, which in turn encodes an amino-terminal
signal (leader)
peptide of 18 amino acids and the 607 amino acids present in each monomer of
the mature
protein. Exons III-X encode four tandem repeats sequences (Apple domains)
homologous to
similar domains found in human plasma PK (58% identity). Exons XI-XV encode
the typical
trypsin-like catalytic domain, which is activated by proteolytic cleavage of
the zymogen at an
internal Arg 369-Ile 370 bond to yield a heavy chain containing four Apple
domains (369
amino acids) and the light chain or catalytic domain (238 amino acids).
One mechanism for initiation of coagulation is via exposure to the circulation
of
tissue factor (TF) at sites of injury, followed in succession by (i) binding
of plasma Factor VII
(FVII) to TF and its proteolytic conversion to activated Factor VII (FVlla);
(ii) binding of Factor
X to the TF-FVlla complex and its proteolytic conversion to activated Factor X
(FXa); (iii)
proteolytic conversion by FXa of prothrombin to thrombin; and (iv) the
generation of a
complex between tissue factor pathway inhibitor (TFPI) and FXa, followed by
binding of the
TFPI:FXa complex to TF-FVlla, which attenuates FXa activation of thrombin and
limits the
flux of thrombin generated via the TF pathway. The relatively small amount of
thrombin
produced during this phase results in the activation of FXI to FXIa (which
activates Factor IX
to FIXa) and the activation of Factor V on the surface of platelets and the
further activation of
Factor X. These events further promote the formation of sufficient amounts of
thrombin (the
so-called "thrombin burst") to convert fibrinogen into fibrin, thereby
stabilizing an initial
platelet plug and resulting in appropriate hemostasis.
Dimeric FXI circulates in plasma as a zymogen in a non-covalent complex with
the
cofactor high molecular weight kininogen (HK) that promotes the binding of FXI
to negatively
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2
charged surfaces and its activation by its cognate proteases, FXlla, FXIa, and
thrombin. The
HK binding site to FXI involves multiple Apple domains (A1, A2, A4), with the
A2 domain
being the most important. Complex formation with HK in the presence of Zn2+
ions has been
shown to promote the binding of FXI to activated platelets. The interaction of
FXI with the
surface of activated platelets has been shown to be mediated via residues
Ser24$-Val2~~
within the A3 domain of FXI; residues Ser248, Arg2eo, Lyszss, Phe2so and
GIn~63 have also been
implicated in this interaction. The A3 domain of FXI also contains a heparin
binding site
within residues Thr249-Phe2so and residues Lys2s2 and Lys2sa have been
implicated in the
binding to platelets. Although FXI and HK circulate in plasma in a non-
covalent complex, and
HK has been shown to bind to the surface of activated platelets, the
interaction of FXI with
the platelet surface apparently does not require binding of HK-FXI complex.
Instead, it
appears that the FXI dimer binds directly to a high-affinity, specific site on
activated platelets
(approx. 1500 sites/platelet; Kd at approx 10 nM). The isolated recombinant A3
domain of FXI
binds to the same number of sites on activated platelets and with the same
affinity as the FXI
dimer.
The activated enzyme, FXIa, has also been shown to bind to high-affinity,
saturable
sites on activated platelets (Kd at approx 800 pM; 500 sites/platelet) and can
activate FIX at
rates similar to those observed in solution. The substrate FIX binding site in
FXI involves
both a subdomain (A1a~34-Leu~~2) in the A2 domain and two subdomains (I1e~84-
Val~s2 and
Ser259-Ser2ss) within the A3 domain. Binding to the platelet surface is
mediated by the
glycoprotein 1 b-V-IX complex utilizing one polypeptide chain of the FXI
dimer, thereby
presenting the other monomer as a substrate binding site for FIX. It is likely
that FIXa
generation serves to localize FIXa-catalyzed FX activation to the platelet
surface which also
promotes prothrombin activation by FXa.
In addition to forming membrane associated complexes leading to the local
explosive generation of thrombin on the platelet surface, FXIa is also subject
to regulation by
a variety of plasma and platelet protease inhibitors whose functional activity
appears to
depend on whether FXIa is bound to the platelet surface or whether it is free
in solution.
Thus, a number of serine protease inhibitors including a-1-protease inhibitor,
antithrombin III,
C1 inhibitor, a-2-antiplasmin, plasminogen activator inhibitor 1 (PAI-1 ), and
protein C inhibitor
have all been shown to inactivate FXIa in the plasma compartment. However,
within the
environment of activated platelets, it seems likely that the most
physiologically relevant
inhibitor of FXIa is protease nexin II (PNII), which is found in very low
concentration in
plasma but is secreted from platelet a-granules (1-1.5 nM PNII released per
10$ platelets)
suggesting a plasma concentration at 3-5 nM under normal physiological
conditions. PNII is
a potent inhibitor of FXIa with a Ki of 300-500 pM that is significantly
enhanced in the
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3
presence of heparin. Binding of FXI to the platelet surface in the presence of
HK and Zn2+
ions or in the presence of prothrombin and Caz+ is protected from inactivation
by both PNII
and a-1-protease inhibitor showing that FXIa activity generated on the surface
of platelet is
localized to the haemostatic thrombus whereas the site of regulation of FXIa
by PNII and
other protease inhibitors occurs in solution. It is also possible that
endothelial cells, which
contain heparan sulphate glycosaminoglycans, might promote the assembly of
FXIa/PNII
complexes thereby potentiating the inhibition of FXIa on the endothelium.
The participation of FXI in thrombin generation on the surface of of the
activated
platelet is also thought to play a role in inhibiting fibrinolysis via
thrombin-activatable
fibrinolysis inhibitor (TAFI), which proteolytically removes the carboxy-
terminal lysine
residues from fibrin that play a role in plasminogen binding and activation.
An intact FXI
feedback loop is believed to be necessary to generate sufficient thrombin for
significant TAFI
activation.
Notably, platelets and megakaryocytes apparently synthesize a second form of
FXI,
designated platelet-derived FXI (pd-FXI), which differs from the circulating
form in lacking
Exon V, which is the first exon of the two exons encoding the second Apple
domain, and in
vitro studies have shown that the preferred substrate for platelet factor Xla
may be plasma
FXI and not FIX. Platelet FXI (Mr 220 KDa) has been found to be associated
with the platelet
plasma membrane. Platelets contain about 300 molecules of pd-FXI/cell.
FXI deficiency is an autosomal recessive syndrome characterized by a variable
tendency to bleed. Even if severe, the deficiency may be clinically
asymptomatic until the
patient is challenged by surgical trauma; however, in some cases bleeding can
occur
regardless of the severity of the deficiency. Optimal management of patients
with FXI
deficiency requires attention to a number of features in addition to the FXI
level. First, it is
important to evaluate the bleeding tendency in an individual with partial
deficiency and
whether additional factors are making a significant contribution. Such
assessment should
include measurement of FVIIIC and von Willebrand factor levels, the bleeding
time and
platelet aggregation. Fresh frozen plasma has been used to treat the first
known cases of
FXI deficiency and was the main treatment until the development of FXI
concentrate. The
main disadvantages of plasma are the large volumes required, allergic
reactions and the
potential for transmission of infectious agents. In addition, there have been
reported a rather
variable FXI content in this product. Two FXl concentrates are currently
available. The FXI
concentrate from Bio Products Laboratory (BPL) (England) is formulated with a
high
concentration of antithrombin (mean 102 iu/ml) and heparin (10u/ml) which is
thought to
protect against any residual FXIa. A second FXI concentrate is produced by
Hemoleven
(France) and the product is formulated with 3-5 u/ml heparin, 2-3 iulml of
antithrombin and
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4
C1 inhibitor. Furthermore, it has been reported that it is possible to
pasteurize pooled fresh
frozen plasma with preservation of 75-95% activity of FXI. Patients with mild
FXI deficiency
are generally treated with fresh frozen plasma, though patients with severe
FXI deficiency
may be treated with a FXI concentrate.
Patients (including those not suffering from congenital FXI deficiency) who
bleed
excessively in association with surgery or major trauma and need blood
transfusions develop
more complications than those who do not experience any bleeding. Even
moderate
bleedings requiring the administration of human blood or blood products (such
as, e.g.,
platelets, leukocytes, plasma-derived concentrates for the treatment of
coagulation defects,
etc.) may lead to complications associated with the risk of transferring human
viruses
(hepatitis, HIV, parvovirus, and other, presently unknown viruses). Extensive
bleedings
requiring massive blood transfusions may lead to the development of multiple
organ failure
including impaired lung and kidney function. Once a subject has developed
these serious
complications a cascade of events involving a number of cytokines and
inflammatory
reactions is started making any treatment extremely difficult or, often,
unsuccessful.
Therefore a major goal in surgery as well as in the treatment of major tissue
damage is to
avoid or minimise the bleeding. To avoid or minimise such bleeding, it is of
importance to
ensure the formation of stable and solid haemostatic plugs that are not easily
dissolved by
fibrinolytic enzymes. Furthermore, it is of importance to ensure quick and
effective formation
of such plugs or clots.
W02003007983 discloses the use of a combination of factor Vlla and FXI for
treatment of bleeding episodes.
Thus, there is a need in the art for improved hemostatic treatment modalities
that
result in the rapid, controlled formation of stable fibrin clots.
SUMMARY OF THE INVENTION
The present invention provides methods and compositions for treating bleeding
episodes. The methods are carried out by administering to a patient in need
thereof a
preparation comprising a factor XI (FXI) polypeptide, in an amount effective
for such
treatment. The methods of the invention result in one or more of: reduced
clotting time;
enhancement of hemostasis; increase in clot lysis time; increase in clot
strength; and/or
increase in overall clot quality (OCQ) in said patient. In some embodiments,
following
administration of a FXI polypeptide, the patient exhibits an effective FXI
plasma
concentration of at least about 5 nM, 10 nM, 30 nM, 60 nM, or 120 nM.
In some embodiments, the FXI polypeptide comprises the sequence of SEQ ID
N0:1, or a fragment thereof that retains at least one FXI-associated
biological activity. In
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WO 2005/049070 PCT/DK2004/000810
some embodiments, the FXI polypeptide comprises the sequence of SEQ ID N0:2,
or a
fragment thereof that retains at least one FXI-associated biological activity.
In some
embodiments, the FXI polypeptide comprises a chemically modified derivative of
SEQ ID
N0:1 or SEQ ID N0:2, or a variant of either SEQ ID N0:1 or SEQ ID N0:2
containing one or
5 more amino acid sequence alterations. In some embodiments, the FXI
polypeptide has the
sequence of SEQ ID N0:1. In some embodiments, the FXI polypeptide has the
sequence of
SEQ ID N0:2.
In some embodiments, the patient does not suffer from a congenital FXI
deficiency.
In some embodiments, the bleeding episodes are secondary to surgery, a dental
procedure,
trauma, or hemodilution. In some embodiments, the patient suffers from aquired
FXI
deficiency.
The invention also provides methods and compositions for preventing bleeding
episodes. The methods are carried out by administering to a patient in need
thereof a
preparation comprising a FXI polypeptide, in an amount effective to prevent
bleeding.
In some embodiments, the methods of the invention further comprise, prior to
administration of a FXI polypeptide: (a) obtaining a sample of blood from said
patient; (b)
determining at least one of: FXI concentration, ratio of FXIa:FXI, or amount
of exogenous FXI
necessary to restore coagulation; and (c) based on the results of step (b),
determining said
amount of FXI effective for treatment.
In one embodiment the methods of the invention does not comprise
administration
of a Factor VII/Factor Vlla coagulation agent.
As used herein, a Factor VII/Factor Vlla coagulation agent is a Factor Vll
polypeptide or a Factor VII-related polypeptide as described in WO2003007933.
The invention also provides methods and compositions for treating bleeding
episodes in which a patient is administered (i) a first amount of a
preparation comprising a
FXI polypeptide and (ii) a second amount of a preparation comprising a non-
Factor VIUFactor
Vlla coagulation agent, under conditions in which the first and second amounts
in
combination are effective for such treatment. Non-limiting examples of non-
Factor VII/Factor
Vlla coagulation agents include: Factor XII, phospholipids, Factor XIII;
tissue factor pathway
inhibitor (TFPI) inhibitor; Factor IX; thrombin activatable fibrinolysis
inhibitor (TAFI);
plasminogen activator inhibitor-1 (PAI-1 ); Factor V; protein C inhibitor;
protein S inhibitor;
tissue plasminogen activator (tPA) inhibitor; prothrombin, Factor VIII,
fibrinogen, and Factor
X.
The invention also provides pharmaceutical formulations comprising (i)
isolated
recombinant FXI polypeptide and (ii) a pharmaceutically acceptable carrier or
excipient.
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6
The invention further provides methods for purifying a factor XI polypeptide
from a
biological material, the method comprising subjecting the material to
sequential
chromatography on an cation-exchange chromatographic material, a hydrophobic
interaction
chromatographic material, and a Hydroxyapatite chromatographic material.
BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1 is a graphic representation of the effect of increasing amounts of
FXI on
overall clot quality in blood obtained from patients before and after cardiac
surgery.
Figure 2 is a graphic representation of the effect of increasing amounts of
FXI on
overall clot quality in blood obtained from normal subjects.
Figure 3 is a graphic representation of the biological activity of different
FXI
formulations after storage at 5°C for 96 days.
Figure 4 is the preparative chromatogram of factor XI polypeptide-containing
fractions from first cation-exchange chromatography using Obelix ST CIEX (cat
no 11-0010)
as described in example 7.
Figure 5 is the preparative chromatogram of factor XI polypeptide-containing
fractions from Hydrophobic interaction chromatography using Butyl Sepharose
High
Performance High Substitution (cat no 17-3100) as described in example 0.
Figure 6 is the preparative chromatogram of factor XI polypeptide-containing
fractions from Hydroxyapatite chromatography using CHT Hydroxyapatite Type I
BioRad cat
no 157-0020) as described in example 9.
DETAILED DESCRIPTION OF THE INVENTION
The present invention is based on the surprising finding that exogenously
administered FactorXl (FXI) can be effective as a general hemostatic agent in
human blood
without the administration of Factor VII/Factor Vlla coagulation agents. The
therapeutic use
of FXI according to the invention may provide one or more of: a shortened
clotting time, a
firmer clot, and an increased resistance of the formed clots to fibrinolysis
and a reduction of
bledding-associated complications.
The present invention provides methods and compositions useful in the
therapeutic
use of FXI in human patients for treating or preventing bleeding episodes, for
enhancing
hemostasis, for increasing clot lysis time, andlor for increasing clot
strength. The methods
are carried out by administering to the patient an effective amount of Factor
XI for achieving
one or more of these desired therapeutic goals. The compositions include
pharmaceutical
formulations for the therapeutic use of FXI that comprise FXI. In one
embodiments, the
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7
compositions include pharmaceutical formulations for the therapeutic use of
FXI that
comprise isolated FXI. In one embodiments, the compositions include
pharmaceutical
formulations for the therapeutic use of FXI that comprise recombinant FXI. In
one
embodiments, the compositions include pharmaceutical formulations for the
therapeutic use
of FXI that comprise isolated recombinant FXI.
In one series of embodiments, the present invention relates to administration
of FXI
to normal human patients. As used herein, a "normal" human is one who does not
suffer from
a congenital deficiency in Factor XI (i.e., Hemophilia C, see, Seligsohn
(1993), Thromb.
Haemost. 70:68-71 ); normal humans include, without limitation, patients
exhibiting
thrombocytopenia (lowered count or activity of platelets), patients
contemplating or
undergoing a surgical or dental procedure, and patients who have been
subjected to trauma
or organ damage and who, as a consequence, may exhibit lowered platelet counts
and/or
lowered levels of fibrinogen, FVIII, and/or other coagulation proteins. Normal
human patients,
for example, encompass patients experiencing a transient decrease in the
plasma levels of
FXI (or any other coagulation-related protein or factor) due to bleeding,
trauma,
chemotherapy, liver disease, hemodilution (such as, e.g., may result from the
infusion of
plasma expanders or salt solutions to maintain blood volume or prevent shock),
or any other
circumstances not directly related to a congenital defect in a FXI gene.
In another series of embodiments, the present invention relates to
administration of
isolated and/or recombinant FXI to human patients suffering from a congenital
FXI
deficiency.
In another series of embodiments, the present invention relates to
administration of
isolated and/or recombinant FXI to human patients suffering from aquired FXI
deficiency.
In practicing the present invention, any FXI polypeptide may be used that is
effective
in preventing or treating bleeding. This includes FXI polypeptides derived
from blood or
plasma or from platelets or those produced by recombinant means in any
suitable host
organism or cell. Also encompassed are FXI polypeptides in their uncleaved
(zymogen) form,
as well as those that have been proteolytically processed to yield their
respective bioactive
forms (designated FXIa).
As used herein, FXI polypeptides encompass, without limitation, FXI as well as
FXI-
related polypeptides. The term "FXI" is intended to encompass, without
limitation,
polypeptides having the amino acid sequence of wild-type human plasma FXI, as
described,
e.g., in Fujikawa et al., Biochem. 25:2417 (1986), as well as wild-type FXI
derived from other
species, such as, e.g., bovine, porcine, canine, murine, rabbit, and salmon
FXI. In general, it
is preferred to use FXI proteins syngeneic with the subject, in order to
reduce the risk of
inducing an immune response. Preparation and characterization of non-human FXI
has been
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8
described, e.g., by Gailani (1997), Blood 90:1055. The present invention also
encompasses
the use of such factor XI proteins within veterinary procedures.
In some embodiments, the FXI polypeptide is wild-type human plasma FXI (SEQ ID
N0:1 ). In other embodiments, the FXI is platelet-derived FXI (pd-FXI) (SEQ ID
N0:2), as
described, e.g., in Hsu et al. (1998), J. Biol. Chem. 273:13787-93.
FXI polypeptides further encompass natural allelic variations of FXI that may
exist
and differ from one individual to another. Also, the degree and location of
glycosylation or
other post-translational modifications may vary in some circumstances,
depending on the
source of FXI-encoding nucleic acid, the host cells in which the FXI is
produced, and the
conditions in which the FXI-producing cells are maintained.
FXI-related polypeptides include, without limitation, FXI polypeptides that
have
either been chemically modified relative to human FXI (i.e., FXI derivatives)
and/or contain
one or more amino acid sequence alterations relative to human FXI (i.e., FXI
variants). Such
FXI-related polypeptides may exhibit an alteration in one or more aspects of
biological
activity relative to human FXI, including, without limitation, altered
stability, altered
phospholipid binding, altered specific enzymatic activity, altered
immunogenicity, altered
bioavailability, altered binding to one or more FXI binding partners, altered
binding to FXI
inhibitors, and the like. FXI-related polypeptides encompass such polypeptides
in their
uncleaved (zymogen) form, as well as those that have been proteolytically
processed to yield
their respective bioactive forms, which may be designated "FXIa-related
polypeptides" or
"activated FXI-related polypeptides".
Non-limiting examples of FXI derivatives include: wild-type FXI or FXI
variants that
have been modified by phosphorylation, sulfation, PEGylation, or by the action
of one or
more glycosyltransferases and/or glycosidases, whether in vivo or in vitro
(see, e.g., Ekdahl
et al. (1999), Thromb. Haemost. 82:1283-8).
Non-limiting examples of FXI variants include: FXI in which one or more N-
linked or
O-linked glycosylation consensus sites have been modified, single-chain FXI
(i.e., FXI in
which the monomer polypeptides are not subject to intrachain proteolytic
cleavage as in the
wild-type), and cysteine variants in which one or more cysteine residues are
eliminated or
relocated, including, but not limited to, alterations that change the
disulfide bonding pattern of
the monomer or dimer. In one embodiment, Cys~~ (which is not believed to
participate in
inter- or intramolecular disulfide bonding) is eliminated or substituted.
In one series of embodiments, the FXI variant has decreased half-life in
plasma
relative to wild-type human FXI. In one embodiment the FXI variant has a half-
life lower than
50 hours. In one embodiment the FXI variant has a half-life lower than 24
hours. In one
embodiment the FXI variant has a half-life lower than 12 hours. In one
embodiment the FXI
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9
variant has a half-life lower than 6 hours. In one embodiment the FXI variant
has a half-life
lower than 3 hours.
In one series of embodiments, FXI variants are polypeptides in which N-linked
glycosylation at one or more sites has been disrupted by modified of the
cognate N-linked
glycosylation consensus sites, such as, e.g., by independent substitution with
any amino acid
of N72, N108, N335, N432, N473, or combinations of any of the foregoing. Non-
limiting
examples of such variants include FXI-N72Q; FXI-N108Q; FXI-N335Q, FXI-N432Q,
FXI-
N473Q; FXI-N72Q/N108Q; FXI-N72Q/N108Q/N335Q; FXI-N72Q/N108Q1N335Q/N432Q;
FXI-N72Q/N108Q/N335Q/N432Q/N473Q; FXI-N72Q/N432Q; FXI-N72Q/N473Q; FXI-
N108Q/N432Q; FXI-N108Q/N473Q; and FXI-N432Q/N473Q. Disruption of N-linked
glycosylation at one or more of the sites may also be achieved, e.g., by: (i)
independent
deletion of any of residues 72-74, 108-110, 335-337, 432-434, and 473-475
(i.e., one or
more residues at each site may be deleted and not substituted with any another
amino acid)
(ii) independent substitution of the N+2 residue (such as, e.g., substituting
T74 to any residue
other than S, substitution of S110 to any residue other than T, substitution
of S337 to any
residue other than S, substitution of S434 to any residue other than T,
substitution of T475 to
any residue other than T; (iii) substitution of the N+1 residues with a
glycosylation-disrupting
amino acid (exemplified by, but not limited to, proline (P). It will be
understood that any
combination of the above means may be used to independently disrupt
glycosylation at
different sites within the FXI polypeptide.
Also encompassed by the invention are chimeric or fusion polypeptides between
all
or part of the FXI sequence and other heterologous peptide sequences. For
example, one or
more of the four Apple domains may be substituted by similar apple domains
from other
polypeptides (see, e.g., Gailani et a1.(1999) Blood 94:621 a) or one or more
of the Apple
domains may be deleted in its entirety. In another embodiment, a binding site
for LDL
Receptor-associated protein (LRP) (such as, e.g., a peptide comprising
residues Phes4z-
Asn3~6 of Factor IXa, which has been shown to contribute to the interaction
with LRP,
Rohlena et al. (2003), J. Biol. Chem. 278:9394) is attached to the sequence of
a FXI
polypeptide to modify its pharmacokinetic properties.
The dimeric nature of FXI in its active form (and the asymmetric function of
the two
monomers in, e.g., platelet binding and FIX activation) also enables
preparations for use in
the present invention that comprise FXI heterodimers, i.e., combinations of
two non-identical
FXI (or FXI-related) monomer polypeptides. The only requirement is that the
heterodimer
exhibit one or more beneficial aspects of FXI bioactivity.
FXI polypeptides for use in the present invention include, without limitation,
polypeptides exhibiting substantially the same or improved biological activity
relative to wild-
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WO 2005/049070 PCT/DK2004/000810
type human FXI, as well as polypeptides in which the FXI biological activity
has been
substantially modified or reduced relative to the activity of wild-type human
FXI.
In practicing the present invention, useful compositions comprising FXIa or
FXIa-
related polypeptides, including variants, encompass those that exhibit at
least about 10%, at
5 least about 20%, at least about 30%, at least about 40%, at least about 50%,
at least about
60%, at least about 70%, at least about 80%, at least about 90%, at least
about 100%, at
least about 110%, at least about 120%, or at least about 130%, of the specific
activity of
compositions comprising solely wild-type FXI, when the wild-type FXI
equivalent is one that
has been obtained from the same source or produced in the same cell type, and
when the
10 activity comparison is made by parallel testing in an identical FXI
activity assay. As used
herein, the terms "activity" and "specific activity" apply, individually or in
aggregate, to any
aspect or aspects of FXI bioactivity.
In some embodiments, the ratio between the specific proteolytic activity of a
FXI-
related polypeptide and the proteolytic activity of wild-type human FXI is at
least about 1.25
when tested a FXI amidolytic assay; in other embodiments, the ratio is at
least about 2.0; in
further embodiments, the ratio is at least about 4Ø
FXI Biological Activity
In practicing the present invention, one or more different aspects of FXI
bioactivity
may be quantified and used, e.g., in (i) selection of: appropriate FXI
compositions for
therapeutic administration, formulations, methods for FXI production or
purification, and the
like; and/or (ii) assessment of the efficacy of different therapeutic
modalities. It will be
understood that "specific activity" of FXIa for any of these aspects of
bioactivity is expressed
as units of activity per unit mass of FXIa polypeptides. These aspects include
the following:
I. Proteolytic activity:
(a) Amidolytic acfivity may be quantified in vitro using a suitable
chromogenic
substrate, such as, e.g., S2355 (Chromogenix), as described in Ekdahl et al.
(1999), Thromb.
Haemost. 82:1283-8. The measured activity is compared with a standard FXIa
preparation
having a defined specific activity (Enzyme Research Laboratories) and values
are expressed
as AU of FXIa activity.
(b) FXI activation activity may be quantified directly in vitro by measuring
the
proteolytic conversion of factor IX to IXa as described for example, in
Gailani et al. (2001 ),
Blood 97: 3117-3122.
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11
II. Bindinq activities and inhibitors
Wild-type human FXI has a number of binding partners, including prekallikrein
(PK),
high-molecular weight kininogen (HK), thrombin/prothrombin, Factor IX (FIX),
and the
platelet-associated FXI receptor designated GP1 b-V-IX. In practicing the
present invention,
any conventional binding assay may be used to quantify the affinity of FXI
polypeptides for
any of these (or other) binding partners. Such binding assays include, but are
not limited to,
competition binding assays in which either binding partner is labelled.
Also encompassed by FXI polypeptide binding partners are FXI active site
inhibitors,
including, without limitation, antithrombin III, C1 inhibitor, a2 antitrypsin,
PAI-1, protein C
inhibitor, and protease nexin II (PNII). The affinity of these compounds for
FXI polypeptides
may be quantified by use of conventional binding assays; alternatively, the
inhibitory activity
of such compounds for the proteolytic activity of particular FXI polypeptide
preparations may
be measuring using an amidolytic or FIX-activation assay.
III. Clotting parameters:
Clotting time, clot lysis time, and clot strength are clinical parameters used
for
assaying the status of patient's haemostatic system. Blood samples are drawn
from the
patient at suitable intervals after administration of a FXI polypeptide and
one or more of
these parameters are assayed. Alternatively, a FXI polypeptide or preparation
may be used
for in vifrolex vivo treatment of blood that has been drawn from a human
subject.
Clotting time may be assayed by means of standard PT or aPTT assays.
Clot lysis time and clot strength may be measured by thromboelastograpy as
described by, e.g., Vig et al. (2001 ) Blood coagulation & fibrinolysis, Vol.
12 (7) pp. 555-561.
and Sorensen (2003) Throm Haemost 1:551-558. Alternatively, clot strength may
be assayed
as described by Carr et al, (1991 ), Am. J. Med. Sci. 302: 13-8.
One parameter that reflects the clotting activity of FXI as measured by
thromboelastography is the "overall clot quality" (OCQ). Once clot formation
has been
initiated (t=0), measurement of the clot strength as a function of time
reveals a maximum
velocity (max vel) of clot formation as welt as the time required to reach the
maximum
velocity (tmaxvel)~ Subsequently, addition of tissue plasminogen activator
(tPA) allows
measurement of fibrinolysis and derivation of the time required to reach the
maximum
velocity offibrinolysis (tminvel)~ OCQ is calculated as:
(Max vel l t max vel) X (t min vel - t max vel).
IV. Pharmacokineticparameters
Wild-type human FXI is believed to have a half-life in plasma of approximately
50
hours, which is mediated at least in part to its interaction with HK. In
practicing the present
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invention, FXI polypeptides, which exhibit pharmacokinetic properties that
differ from native
FXI, may be used. Non-limiting examples include FXI polypeptides that have
been treated
with sialidase to remove one or more terminal sialic acid residues from FXI-
associated
oligosaccharides, FXI polypeptides that have been modified by PEGylation, and
FXI
polypeptides exhibiting an altered interaction with HK. In practicing the
present invention,
pharmacokinetic properties may be calculated using, e.g., WinNonlin
Professional Version
3.1 (Pharsight Inc., Mountain View, CA, USA). Calculations are performed using
mean
concentration values at each time point, if more than one value was present.
The following pharmacokinetic parameters may be calculated: AUC, AUC~/oE,~rap~
CmaX, tmaX, A~, t~~z, CL, and Vz using the following formulas:
AUC Area under the plasma concentration-time curve from time
0 to infinity. Calculated using the linear/log trapezoidal rule with
extrapolation to infinity.
The linear trapezoidal rule is used from time 0 to tmax~
~ :.~Y( + _ri'x ,~" 1~.~ <
~; ~ _z ~~.
r~~,.:> ~ f ' ~ ~ ~ ..t'. '~? - , ~ttl yk
b, u.. ri
The log trapezoidal rule is used from time tmax to the last
time point t:
'~~j .,T(~~;~~ .". ~!"~~tT ,~...r..~ _
-r(.-~,i..y''~;;.~ ;a
Extrapolation to infinity is performed using:
.r~ ~.:'i (' -~;:~a~ .= ~'~#,'''1
AUCo~oExtrap Percentage of AUC that is due to extrapolation from the last
concentration to infinity:
"'. ''~''~r ~'°
,~t ~, <: - .~
~.r. .~ ,.
~. z: ~..,..:, ~, ,~.~'i~;.:.:
f u.. ;.~~ iii:
~ trJ
CmaX Maximum plasma concentration back extrapolated to time
zero
CL Total body clearance
.,.y~ _.. :.~v~~'
f , . ...
.t~ ~.#f
tmax Time at which maximum plasma concentration is observed.
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i
t~/2 :.xl, w
Half-life:
AZ Terminal rate constant. Calculated by log-linear regression
of (mean) concentrations versus time
Vz Volume of distribution based on the terminal phase:
r~:
p: t~7 Y
~,; ~ Isf~..,:' r
..::a l.. t: ~..~ r' J'~;.
Production and purification of FXI:
FXI polypeptides for use in the present invention may be prepared from plasma
or
from recombinant sources using any suitable method known in the art. As used
herein, the
term "isolated" refers to FXI polypeptides that have been separated from the
cell in which
they were synthesized or the medium in which they are found in nature (e.g.,
plasma or
blood).
Separation of polypeptides from their cell of origin may be achieved by any
method
known in the art, including, without limitation, removal of cell culture
medium containing the
desired product from an adherent cell culture; centrifugation or filtration to
remove non-
adherent cells; and the like. Optionally, FXI polypeptides may be further
purified. Purification
may be achieved using any method known in the art, including, without
limitation, affinity
chromatography, such as, e.g., on an anti-FXI antibody column or a peptide
affinity column
(non-limiting examples of which include Heparin, Blue, Red, L-arginine,
Benzamidine
peptide, other dyes, or RP-chromatography); hydrophobic interaction
chromatography; ion-
exchange chromatography; size exclusion chromatography; electrophoretic
procedures (e.g.,
preparative isoelectric focusing (IEF), differential solubility (e.g., any
precipitation or
crystallization using, e.g., salt, pH, ammonium sulphate, or other additives),
or extraction and
the like, as described in more detail above. Following purification, the
preparation preferably
contains less than about 10% by weight, more preferably less than about 5% and
most
preferably less than about 1 %, of non-FXI polypeptides derived from the host
cell.
Purification of FXI from plasma may also be achieved by known methods,
including,
without limitation, those disclosed by Koide et al. (1977), Biochem. 16: 2279
and Bouma et
al. (1977), J.Biol.Chem. 252:6432, incorporated herein by reference. Methods
for preparing
recombinant FXI are known in the art. See, for example, Kemball-Cook et al.
(1994), Gene
139:275, Fujikawa et al. (1986), Biochem. 25:2417, and Meijers et a1.(1992),
Blood 79:1435,
which are incorporated herein by reference in their entirety. FXIa is also
commercially
available from Enzyme Research Laboratories, South Bend, IN.
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The present invention further concerns a method for purifying a FXI
polypeptide,
such as recombinant FXI, from other biological material, the method comprising
subjecting
the material to chromatography on a ration-exchange chromatographic material.
The present invention further concerns a method for purifying a FXI
polypeptide,
such as recombinant FXI, from other biological material, the method comprising
subjecting
the material to chromatography on a a hydrophobic interaction chromatographic
material.
The present invention further concerns a method for purifying a FXI
polypeptide,
such as recombinant FXI, from other biological material, the method comprising
subjecting
the material to chromatography on a hydroxyapatite chromatographic material.
The present invention further concerns a method for purifying a FXI
polypeptide,
such as recombinant FXI, from other biological material, the method comprising
subjecting
the material to sequential chromatography on a ration-exchange chromatographic
material,
a hydrophobic interaction chromatographic material and Hydroxyapatite
chromatographic
material. It is to be understood that a sequential chromatography is performed
in the order as
described.
The term "Hydroxyapatite chromatographic material" as used herein means any
Hydroxyapatite chromatographic material known in the art which is capable of
binding a FXI
polypeptides, such as a Hydroxyapatite matrix.
The term "ration-exchange chromatographic material" as used herein means any
ration-exchange chromatographic material known in the art which is capable of
binding a FXI
polypeptides, such as a ration-exchange matrix.
The term "hydrophobic interaction chromatographic material" as used herein
means
any hydrophobic interaction chromatographic material known in the art which is
capable of
binding a FXI polypeptides, such as a hydrophobic interaction matrix.
In one embodiment, the present invention concerns a method for purifying a FXI
polypeptide from a biological material, the method comprising the step of:
subjecting a biological material comprising a FXI polypeptide to
chromatography on a first
ration-exchange chromatographic material, said chromatography comprising:
(i) applying said biological material to said first ration-exchange
chromatographic
material;
(ii) eluting unbound material from the first ration-exchange chromatographic
material
with a buffer A, which buffer A is suitable for eluting material not bound to
the first
ration-exchange chromatographic material; and
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(iii) eluting unbound material from the first cation-exchange chromatographic
material with a buffer A', which buffer A' is suitable for eluting material
not bound to
the first cation-exchange chromatographic material; and
(iv) eluting said FXI polypeptide from the first cation-exchange
chromatographic
5 material by elution with buffer A", which buffer A" is suitable for eluting
said FXI
polypeptide from said first cation-exchange chromatographic material.
In one embodiment, the present invention concerns a method for purifying a FXI
polypeptide from a biological material, the method comprising the steps of:
10 subjecting the eluate from step (iv) or a fluid prepared by use of the
eluate from step (iv) to
chromatography using a hydrophobic interaction chromatographic material, said
chromatography comprising:
(v) applying the eluate from step (iv) or a fluid prepared by use of the
eluate from
step (iv) to said hydrophobic interaction chromatographic material;
15 (vi) eluting unbound material from said hydrophobic interaction
chromatographic
material with buffer B, which buffer B is suitable for eluting material not
bound to the
hydrophobic interaction chromatographic material; and
(vii) eluting said FXI polypeptide from said hydrophobic interaction
chromatographic
material by gradient-elution with buffer B', which buffer B' is suitable for
eluting FXI
polypeptide from said hydrophobic interaction chromatographic material.
In one embodiment, the present invention concerns a method for purifying a FXI
polypeptide from a biological material, the method comprising the steps of:
subjecting the eluate from step (vii) or a fluid prepared by use of the eluate
from step (vii) to
chromatography using a Hydroxyapatite chromatographic material, said
chromatography
comprising:
(viii) applying the eluate from step (vii) or a fluid prepared by use of the
eluate from
step (vii) to said hydroxyapatite chromatographic material;
(ix) eluting unbound material from the hydroxyapatite chromatographic material
with
buffer C, which buffer C is suitable for eluting material not bound to the
hydroxyapatite chromatographic material; and
(x) eluting said FXI polypeptide from said hydroxyapatite chromatographic
material
by gradient-elution with buffer C', which buffer C' is suitable for eluting
FXI
polypeptide from said hydroxyapatite chromatographic material.
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In one embodiment, the present invention concerns a method for purifying a FXI
polypeptide from a biological material, the method comprising the steps of:
(a) subjecting a biological material comprising a FXI polypeptide to
chromatography on a first
cation-exchange chromatographic material, said chromatography comprising:
(i) applying said biological material to said first cation-exchange
chromatographic
material;
(ii) eluting unbound material from the first cation-exchange chromatographic
material
with a buffer A, which buffer A is suitable for eluting material not bound to
the first
cation-exchange chromatographic material; and
(iii) eluting unbound material from the first cation-exchange chromatographic
material with a buffer A', which buffer A' is suitable for eluting material
not bound to
the first cation-exchange chromatographic material; and
(iv) eluting said FXI polypeptide from the first cation-exchange
chromatographic
material by elution with buffer A", which buffer A" is suitable for eluting
said FXI
polypeptide from said first cation-exchange chromatographic material;
(b) subjecting the eluate from step (iv) or a fluid prepared by use of the
eluate from step (iv)
to chromatography using a hydrophobic interaction chromatographic material,
said
chromatography comprising:
(v) applying the eluate from step (iv) or a fluid prepared by use of the
eluate from
step (iv) to said hydrophobic interaction chromatographic material;
(vi) eluting unbound material from said hydrophobic interaction
chromatographic
material with buffer B, which buffer B is suitable for eluting material not
bound to the
hydrophobic interaction chromatographic material; and
(vii) eluting said FXI polypeptide from said hydrophobic interaction
chromatographic
material by gradient-elution with buffer B', which buffer B' is suitable for
eluting FXI
polypeptide from said hydrophobic interaction chromatographic material;
(c) subjecting the eluate from step (vii) or a fluid prepared by use of the
eluate from step (vii)
to chromatography using a Hydroxyapatite chromatographic material, said
chromatography
comprising:
(viii) applying the eluate from step (vii) or a fluid prepared by use of the
eluate from
step (vii) to said hydroxyapatite chromatographic material;
(ix) eluting unbound material from the hydroxyapatite chromatographic material
with
buffer C, which buffer C is suitable for eluting material not bound to the
hydroxyapatite chromatographic material; and
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(x) eluting said FXI polypeptide from said hydroxyapatite chromatographic
material
by gradient-elution with buffer C', which buffer C' is suitable for eluting
FXI
polypeptide from said hydroxyapatite chromatographic material.
Purification of a FXI polypeptide is the process of increasing the
concentration of the
FXI polypeptide in a sample in relation to other components of said sample,
resulting in an
increase of the purity of the FXI polypeptide. It should be understood that
the concentration
of a FXI polypeptide in a sample in relation to other components of said
sample is not
equivalent to the concentration of FXI polypeptide in the sample. The increase
in the purity of
the FXI polypeptide may be followed measured by use of methods known in the
art, such as
for instance by use of SDS-PAGE (Sodium Dodecyl Sulfate Polyacrylamide Gel
Electrophoresis), HPLC (High Performance Liquid Chromatography) or Berichrome
assays
(Dade Behring Diagnostics), or Clot activity assay.
Biological material may be any material derived from or containing cells, cell
components or cell products. A biological material may be a biological fluid.
A biological fluid may be any fluid derived from or containing cells, cell
components
or cell products. Biological fluids include, but are not limited to cell
cultures, cell culture
supernatants, cell lysates, cleared cell lysates, cell extracts, tissue
extracts, blood, plasma,
serum, all of which may also be homogenizates and filtrates, and fractions
thereof, for
instance collected by chromatography of unfractionated biological fluids.
The FXI polypeptides may be purified from a wide variety of biological
materials,
including cell culture supernatants, which naturally produce a FXI
polypeptide, but also of
cells which have been genetically modified to produce a FXI polypeptide, such
as
mammalian cells (for instance CHO cells) transformed with DNA coding for a FXI
polypeptide.
The biological material may be treated by use of a number of methods prior to
application on the first cation-exchange chromatographic material. Such
methods include,
but a not limited to, centrifugation, filtration. In one embodiment, the
biological material is a
biological fluid. In one embodiment of the present invention, the biological
fluid is the
supernatant of a cell lysate. In one embodiment of the present invention, the
biological fluid is
the supernatant of a yeast cell lysate.
In one embodiment of the present invention, the FXI polypeptide is purified
from a
cell culture, such as a mammalian cell culture, as described above. Prior to
the
chromatography in step (a), the mammalian cells may be separated from cell
culture
supernatant by centrifugation and / or filtration. Inhibitors such as EDTA
(ethylenediamine
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tetraacetic acid) and benzamidineHCl may be included before being subjected to
chromatographic step (a).
A buffer is a solution comprising a substance, which substance is capable of
preventing significant changes in the pH of solutions to which small amounts
of acids or
bases are added and thereby of maintaining largely the original acidity or
basicity of the
solution. A buffer usually comprises a weak acid or weak base together with a
salt thereof.
The pH of the biological fluid may be adjusted to the pH of buffer A prior the
chromatography in step (a), for instance by using 1 M HCI or 1 M NaOH or by
other means
known in the art.
The first cation-exchange chromatographic material may be any cation-exchange
chromatographic material known in the art which is capable of binding a FXI
polypeptide
under one set of conditions and releasing it under a different set of
conditions, such as an
cation-exchange chromatographic material comprising a sulphopropyl group.
Further non-
limiting examples of cation-exchange chromatographic materials include
derivatised
dextrans, agarose, cellulose, polyacrylamide, and specialty silicas, such as
carboxymetyl.
Suitable cation-exchange chromatographic material may be identified by
subjecting a
biological fluid comprising FXI polypeptide to chromatography on the cation-
exchange
chromatographic material of choice, collecting fractions and determining the
purity and
content of the fractions, for instance by use of SDS-PAGE (Sodium Dodecyl
Sulfate
Polyacrylamide Gel Electrophoresis), HPLC (High Performance Liquid
Chromatography),
clotactivity or Berichrome assays (Dade Behring Diagnostics), monitoring the
absorbance of
the eluate at 280 nm and by use of other methods known in the art. Examples of
suitable
cation-exchange chromatographic materials include, but are not limited to
Streamline SP XL
(Amersham Biosciences cat no 17-5073), Obelix ST CIEX (Amersham Biosciences
cat no
11-0010), Streamline Direct CST (Amersham Biosciences 17-5266), S-Support
Unosphere,
BioRad cat no 156-0113 or Toyopearl SP-550C Toso Haas cat no 14028. In one
embodiement Obelix ST CIEX is used.
The first cation-exchange chromatographic material may be pre-equilibrated
with
buffer A prior to application of the biological material.
Buffer A may comprise protease inhibitors such as EDTA (ethylenediamine
tetraacetic acid) and benzamidineHCl, but other commercially available
protease inhibitors
may also be used.
In one embodiment of the present invention, the pH of buffer A is between 6.5
and
9. In a further embodiment, the pH of buffer A is between 7 and 9. In a
further embodiment,
the pH of buffer A is about 8.
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In one embodiment of the present invention, the conductivity of buffer A is
less than
about 40 mS/cm.
Buffer A" is used for the elution of the FXI . Typically, the concentration of
one or
more of the components of the buffer used for washing in step (ii), in this
case buffer A and
A', is increased or decreased during the course of elution or a new component
is added to
the buffer, and the concentration of this component . This increase or
decrease may take
place continuously or in discrete steps as it is known in the art. For elution
of material bound
to an cation-exchange chromatographic material, it is customary to add a salt,
for instance
NaCI, to buffer A. This specific cation exchanger can also be used as a
hydrophobic
interaction chromatographic resin, for this kind of resin it is customary to
add a propandiol /
glycerol to buffer A creating buffer A'. If both NaCI and Propandiol /
glycerol is added to
buffer A then FXI can be eluted. The determination of which fractions
containing FXI
polypeptide to pool for further processing, for instance to exclude undesired
impurities eluting
at the beginning or the end of the FXI polypeptide elution, is within the
knowledge of a
person skilled in the art. Likewise, the general art of performing an cation-
exchange
chromatography with regard to for instance pre-equilibration, elution time,
washing,
reconstitution of the cation-exchange chromatographic material etc is well-
known.
After eluting the FXI polypeptide in step (iv), the eluate containing the FXI
polypeptides protease inhibitors such as EDTA (ethylenediamine tetraacetic
acid) and
Benzamidine is added and then taken to step (v) . The eluate may also be kept
at, for
instance, 4°C for 24 hours or longer, or at, for instance, -
80°C.
The hydrophobic interaction chromatographic material for use in step (b) may
be
any hydrophobic interaction chromatographic material known in the art, which
is capable of
binding a FXI polypeptide under one set of conditions and releasing it under a
different set of
conditions, such as a hydrophobic interaction chromatographic material
derivatised with
phenyl, butyl or octyl groups, or polyacrylic resins. Non-limiting examples of
suitable
hydrophobic interaction chromatographic material are AmberchromTM CG 71 (Tosoh
Bioscience), Phenyl SepharoseTM High Performance (Amersham, cat no 17-1082),
Phenyl
SepharoseTM 6 Fast Flow High Substitution (Amersham, cat no 17-0973),
Toyopearl~ Butyl
650 (Tosoh Bioscience), Toyopearl~ Phenyl (Tosoh Bioscience), Source TM l5Phe
(Amersham, cat no 17-0147), Butyl SepharoseT"' High Performance High
Substitution
(Amersham, cat no 17-3100), Octyl-SepharoseT"~ (Amersham, cat no 17-0946) and
Phenyl
SepharoseTM High Performance High Substitution (Amersham), and the like. In
one
embodiment of the present invention, the hydrophobic interaction
chromatographic material
uses butyl as a ligand.
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Buffer B and NaCI may be added to the eluate from stage (iv) or a fluid
prepared by
use of the eluate from stage (iv) prior to the chromatography in step (b) in
an amount of
about one to two volumes or more, or a concentrated version of buffer B,
comprising the
same ingredients as buffer B, but in, e.g., twice the concentration, is added
to the eluate from
5 stage (iv) or a fluid prepared by use of the eluate from stage (iv) in an
amount corresponding
to the strength of the concentrated buffer (a twice-concentrated buffer is
added in the amount
of 1,5 volumes).
Buffer B may have a pH from about 5 to about 9, for instance about 8. In one
embodiment of the present invention, buffer B has a conductivity of more than
25 mS/cm, for
10 instance more than 70 mSlcm. This may be achieved, for example, by use of a
phosphate
buffer or by other means known in the art, e.g. NaCI. In one embodiment of the
present
invention, the conductivity of the eluate from step (iv) or a fluid prepared
by use of the eluate
from step (iv) is adjusted to a conductivity of at least about 60 mS/cm.
Buffer B' is used for the elution of the FXI polypeptide by gradient elution.
In gradient
15 elution, the composition of buffer B' is changed during the course of
elution. Typically, the
concentration of one or more of the components of the buffer used for washing
in step (vi), in
this case buffer B, is increased or decreased during the course of elution, or
a new
component is added to the buffer and the concentration of this component is
then increased
during the course of elution. This increase or decrease may take place
continuously or in
20 discrete steps, as is well known in the art. For elution of material bound
to a hydrophobic
interaction chromatographic material, it is customary to dilute the washing
buffer with water
until at least a major portion of the bound FXI polypeptide is eluted. The
determination of
which fractions containing FXI polypeptide to pool for further processing,
e.g. in order to
exclude undesired impurities eluting at the beginning or the end of the FXI
polypeptide
elution, is within the knowledge of a person skilled in the art. Likewise, the
general art of
performing a hydrophobic interaction chromatography with regard to, e.g., pre-
equilibration,
elution time, washing, reconstitution of the hydrophobic interaction
chromatographic material,
etc., is well known.
In one embodiment of the present invention, the eluate from stage (vii) or a
fluid
prepared by use of the eluate from stage (vii) is treated by use of a method
comprising a step
of
(1 ) addition of one or more stabilizing agents which are capable of
increasing the
stability of the FXI polypeptide in an amount effective to significantly
improve the
stability thereof, and/or
This step, and optionally other steps of post-processing known in the art, may
be
carried out alone or in combination, and the order in which the steps are
performed is not
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21
critical. The person skilled in the art will be able to determine how and when
to perform these
steps.
In one embodiment of the present invention, a stabilizing agents which are
capable
of increasing the physical and/or chemical stability of the FXI polypeptide is
added to the
fractions containing FXI.
The term "physical stability" of the FXI polypeptide as used herein refers to
the
potential tendency of the protein to form biologically inactive and/or
insoluble aggregates or
multimers of the protein as a result of exposure of the protein to thermo-
mechanical stresses
and/or interaction with interfaces and surfaces that are destabilizing, such
as hydrophobic
surfaces and interfaces. Physical stability of the FXI polypeptide when
present in buffer A
may be evaluated by means of visual inspection and/or turbidity measurements
after
exposing the formulation filled in suitable containers (e.g. cartridges or
vials) to
mechanical/physical stress (e.g. agitation) at different temperatures for
various time periods.
Visual inspection of the FXI polypeptide when present in buffer may be
performed in
a sharp focused light with a dark background. The turbidity of the composition
may be
characterized by a visual score ranking the degree of turbidity, for instance
on a scale from 0
to 3 (a composition showing no turbidity then corresponding to a visual score
0, and a
composition showing visual turbidity in daylight corresponding to visual score
3). A
composition is classified as physically unstable with respect to protein
aggregation when it
shows visual turbidity in daylight. Alternatively, the turbidity of the
composition may be
evaluated by simple turbidity measurements well-known to the skilled person,
for instance by
measuring the optical density of the solution at a wavelength of 405 nm
(OD4os). Physical
stability of the aqueous protein compositions may also be evaluated by using a
spectroscopic
agent or probe of the conformational status of the protein. The probe is
preferably a small
molecule that preferentially binds to a non-native conformer of the protein.
One example of a
small-molecule spectroscopic probe of protein structure is Thioflavin T.
Thioflavin T is a
fluorescent dye that has been widely used for the detection of amyloid
fibrils. In the presence
of fibrils, and perhaps other protein configurations as well, Thioflavin T
gives rise to a new
excitation maximum at about 450 nm and enhanced emission at about 482 nm when
bound
to a fibril protein form. Unbound Thioflavin T is essentially non-fluorescent
at the
wavelengths.
Other small molecules can be used as probes of the changes in protein
structure
from native to non-native states. For instance the "hydrophobic patch" probes
that bind
preferentially to exposed hydrophobic patches of a protein. The hydrophobic
patches are
generally buried within the tertiary structure of a protein in its native
state, but become
exposed as a protein begins to unfold or denature. Examples of these small
molecular,
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22
spectroscopic probes are aromatic, hydrophobic dyes, such as antrhacene,
acridine,
phenanthroline or the like. Other spectroscopic probes are metal-amino acid
complexes,
such as cobalt metal complexes of hydrophobic amino acids, such as
phenylalanine, leucine,
isoleucine, methionine, and valine, or the like.
The term "chemical stability" of the FXI polypeptide when used herein refers
to
chemical covalent changes in the protein structure leading to formation of
chemical
degradation products with potentially lower biological potency and/or
potentially increased
immunogenic properties compared to the native protein structure. Various
chemical
degradation products can be formed depending on the type and nature of the
native protein
and the environment to which the protein is exposed. Elimination of chemical
degradation
can most probably not be completely avoided, and an increase in amounts of
chemical
degradation products is often seen during storage and use of the protein
composition, as
well-known to a person skilled in the art. Most proteins are prone to
deamidation, a process
in which the side-chain amide group in glutaminyl or asparaginyl residues is
hydrolysed to
form a free carboxylic acid. Other degradation pathways involve formation of
high-molecular-
weight transformation products wherein two or more protein molecules are
covalently bound
to each other via transamidation and/or disulfide interactions, leading to
formation of
covalently bound dimer, oligomer and polymer degradation products (Stability
of Protein
Pharmaceuticals, Ahern. T.J. & Manning M.C., Plenum Press, New York 1992).
Oxidation
(e.g. of methionine residues) can be mentioned as another variant of chemical
degradation.
The chemical stability of the FXI polypeptide when present in buffer B' can be
evaluated by
measuring the amounts of chemical degradation products at various times after
exposure to
different environmental conditions; the formation of degradation products can,
for example,
often be accelerated by increase in temperature. The amount of each individual
degradation
product is often determined by separation of the degradation products
depending on
molecule size and/or charge using various chromatographic techniques (e.g. SEC-
HPLC
and/or RP-HPLC).
Any agent which is capable of significantly improving the physical and/or
chemical
stability of FXI polypeptide when present in buffer B' (e.g. as determined by
measuring
turbidity at OD4os over a period of time) may be used as a stabilizing agent.
An agent suitable for use as stabilizing agent, for instance, be a salt (e.g.
sodium
chloride), a sugar, an alcohol (such as an C4-Ca alcohol), an alditol, an
amino acid (e.g.
glycine, histidine, arginine, lysine, isoleucine, aspartic acid, tryptophan or
threonine), a
polyethyleneglycol (e.g. PEG400), or a mixture of one or more thereof. Any
sugar, such as a
mono-, di-, or polysaccharide, or a water-soluble glucan, may be used. An
alditol is a
polyalcohol of structure HOCH2-[CH(OH)]~ CH20H, where n is 1, 2, 3....etc. Non-
limiting
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23
examples of substances which are sugars, alcohols or alditols are fructose,
glucose,
mannose, sorbose, xylose, maltose, lactose, sucrose, trehalose, dextran,
pullulan, dextrin,
cyclodextrin, soluble starch, hydroxyethyl starch, carboxymethylcellulose-Na,
mannitol,
sorbitol, inositol, galactitol, dulcitol, xylitol, arabitol, glycerol
(glycerine), propan-1,2-diol
(propylene glycol), propan-1,3-diol, and butan-1,3-diol. The sugars, alcohols
and alditols
mentioned above may be used individually or in combination. There is no fixed
limit to the
amount used, as long as the substance is soluble in the liquid preparation and
improves the
physical stability of a FXI polypeptide in solution. In this respect,
reference is made to
Remington: The Science and Practice of Pharmacy, 19th edition, 1995.
In one embodiment of the present invention, one or more stabilizing agents of
the
polyalcohol type is added.
In one embodiment of the present invention, one or more stabilizing agents
selected
from the group consisting of glycerol (propan-1,2,3-triol), propylene glycol
(propan-1,2-diol),
propan-1,3-diol, propyl alcohol (1-propanol) and isopropyl alcohol (2-
propanol) is added. In
one embodiment of the present invention, one or more stabilizing agents
selected from the
group consisting of glycerol, propylene glycol and propan-1,3-diol is added.
In a further embodiment of the present invention, when the stabilizing is a
liquid
alcohol or liquid polyalcohol [such as, e.g., glycerol, propylene glycol,
propan-1,3-diol, propyl
alcohol or isopropyl alcohol], the stabilizing agent is present in a
concentration of from about
5% by volume (v/v) to about 50% (v/v). In a further embodiment, a stabilizing
agent of the
liquid alcohol or liquid polyalcohol type is present in a concentration of
from about 10% (v/v)
to about 50% (v/v). In a further embodiment, a stabilizing agent of the liquid
alcohol or liquid
polyalcohol type is present in a concentration of from about 10% (v/v) to
about 20% (v/v). In
a further embodiment, a stabilizing agent of the liquid alcohol or liquid
polyalcohol type is
present in a concentration of about 10% (v/v). In a still further embodiment,
a stabilizing
agent of the liquid alcohol or liquid polyalcohol type is present in a
concentration of about
20% (v/v).
The stabilizing agent mentioned should be capable of increasing the physical
and/or
chemical stability, as described above, of the FXI polypeptide. Any agent
which is capable of
significantly improving the physical and/or chemical stability of FXI
polypeptide (e.g. as
determined by measuring turbidity at OD4os over a period of time) may be used
as a
stabilizing agent.
The eluate from step (vii) may be used for the preparation of a pharmaceutical
composition. This may involve a change of buffer, and/or adjustment of the
conductivity
and/or pH to physiological values, and/or other actions to render the eluate
acceptable for
use in mammals, such as humans; means of rendering such an eluate acceptable
for use in
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24
this manner are well known in the art. The eluate may also be kept at, e.g.,
4°C for 24 hours
or longer, or at, e.g., -80°C.
In one embodiment of the present invention, the method further comprises a
step of
subjecting the eluate from stage (vii), or a fluid prepared by use of the
eluate from stage (vii),
to chromatography on a hydroxyapatite chromatographic material, said
chromatography
comprising:
(viii) applying the eluate from stage (vii), or a fluid prepared by use of the
eluate from
stage (vii), to said hydroxyapatite chromatographic material;
(ix) eluting unbound material from the hydroxyapatite chromatographic material
with
buffer C, which buffer C is suitable for eluting material not bound to the
hydroxyapatie chromatographic material; and
(x) eluting said FXI polypeptide from the hydroxyapatite chromatographic
material
with buffer C', which buffer C' is suitable for eluting FXI polypeptides which
bind to
the hydroxyapatite chromatographic material in step (ix).
A fluid prepared by use of the eluate from stage (vii) may, for instance, be
prepared
before application.
In one embodiment of the present invention, the conductivity of the eluate
from
stage (vii), or a fluid prepared by use of the eluate from stage (vi), is
adjusted to less than
about 20 mS/cm by adding water. pH is adjusted to 5,8 to 9. In one embodiment
pH is
adjusted to 6,0
The components of buffer C and buffer C' may be chosen with a view to the
desired
final pharmaceutical composition of the FXI polypeptide. Such considerations
are within the
knowledge of a person skilled in the art.
In one embodiment of the present invention, buffer C comprises one or more
stabilizing agents, which stabilizing agents are capable of increasing the
physical and/or
chemical stability, as described above, of the FXI polypeptide. Any agent
which is capable of
significantly improving the physical and/or chemical stability of FXI
polypeptide when present
in buffer C (e.g. as determined by measuring turbidity at OD4os over a period
of time) may be
used as a stabilizing agent in buffer C or buffer C'.
An agent suitable for use as stabilizing agent in buffer C may, for instance,
be a salt
(e.g. sodium chloride), a sugar, an alcohol (such as an C4-C$ alcohol), an
alditol, an amino
acid (e.g. glycine, histidine, arginine, lysine, isoleucine, aspartic acid,
tryptophan or
threonine), a polyethyleneglycol (e.g. PEG400), or a mixture of one or more
thereof. Any
sugar, such as a mono-, di-, or polysaccharide, or a water-soluble glucan, may
be used.
Non-limiting examples of substances which are sugars, alcohols or alditols are
fructose,
glucose, mannose, sorbose, xylose, maltose, lactose, sucrose, trehalose,
dextran, pullulan,
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dextrin, cyclodextrin, soluble starch, hydroxyethyl starch,
carboxymethylcellulose-Na,
mannitol, sorbitol, inositol, galactitol, dulcitol, xylitol, arabitol,
glycerol (glycerine), propan-1,2-
diol (propylene glycol), propan-1,3-diol, and butan-1,3-diol. The sugars,
alcohols and alditols
mentioned above may be used individually or in combination. There is no fixed
limit to the
5 amount used, as long as the substance is soluble in the liquid preparation
and improves the
physical stability of a FXI polypeptide in solution. In this respect,
reference is made to
Remington: The Science and Practice of Pharmacy, 19th edition, 1995.
In one embodiment of the present invention, buffer C comprises one or more
stabilizing agents of the polyalcohol type.
10 In one embodiment of the present invention, buffer C comprises NaCI.
One or more stabilizing agents selected from the group consisting of glycerol
(propan-1,2,3-triol), propylene glycol (propan-1,2-diol), propan-1,3-diol,
propyl alcohol
(1-propanol) and isopropyl alcohol (2-propanol) is added to the fluid from
(x).
In one embodiment of the present invention, one or more stabilizing agents
selected
15 from the group consisting of glycerol, propylene glycol and propan-1,3-diol
is added. In one
embodiment of the present invention, propylene glycol is added.
In a further embodiment of the present invention, when the stabilizing agent
it is
present in a concentration of from about 5% (v/v) to about 50% (v/v). In a
further
embodiment, a stabilizing agent of the liquid alcohol or liquid polyalcohol
type is present in a
20 concentration of from about 10% (vlv) to about 50% (vlv). In a further
embodiment, a
stabilizing agent of the liquid alcohol or liquid polyalcohol type used is
present in a
concentration of from about 10% (v/v) to about 20% (v/v). In a further
embodiment, a
stabilizing agent of the liquid alcohol or liquid polyalcohol type is present
in a concentration of
about 10% (v/v).
25 Buffer C' is used for the elution of the FXI polypeptide by gradient
elution, wherein
the composition of buffer C' is changed during the course of elution.
Typically, the
concentration of one or more of the components of the buffer used for washing
in step (ix), in
this case buffer C, is increased or decreased during the course of elution, or
a new
component is added to the buffer and the concentration of this component is
then increased
during the course of elution. This increase or decrease may take place
continuously or in
discrete steps, as is well known in the art. For elution of material bound to
hydroxyapatite
chromatographic material, it is customary to add a salt, e.g. K-P04 or NaCI,
to buffer C and
then increase the concentration of the salt until at least a major portion of
the bound FXI
polypeptide is eluted. The determination of which fractions containing FXI
polypeptide to pool
for further processing, e.g. in order to exclude undesired impurities eluting
at the beginning or
the end of the FXI polypeptide elution, is within the knowledge of a person
skilled in the art.
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26
Likewise, the general art of performing an hydroxyapatite chromatography with
regard to,
e.g., pre-equilibration, elution time, washing, reconstitution of the cation-
exchange
chromatographic material, etc., is well known.
In a series of embodiments, the use of one or more stabilizing agents in any
or all of
the solutions used in purification of FXI results in an increase in the
physical and/or chemical
stability of FXI by at least 10%, 25%, 50%, or 100% over the physical and/or
chemical
stability of a control (i.e., FXI subjected to the same treatment but in the
absence of the
stabilizing agent). In another series of embodiments, the use of one or more
stabilizing
agents in any or all of the solutions used in purification of FXI results in
an increase in the
physical and/or chemical stability of FXI by at least 2-fold, 5-fold, 10-fold,
or 20-fold over the
physical and/or chemical stability of a control (i.e., FXI subjected to the
same treatment but in
the absence of the stabilizing agent).
Activation of FXI
Wild-type human FXI is normally activated by proteolytic cleavage between
Arg3so
and Ile3~o, which may be catalyzed by FXIa, FXlla, or thrombin. If desired,
activation of FXI
for use in the present invention may be achieved using FXIa or FXlla (both
from Enzyme
Research Laboratories, South Bend, IN) or thrombin (Sigma). See, e.g., Sun et
al. (1999) J.
Biol Chem 51:36373-36373 and Baglia (2003) J. Biol Chem 24:21744-21750. It is
also within
the scope of the invention to utilize other proteases to activate FXI
polypeptides and in
particular, FXI-related polypeptides.
The present invention encompasses methods and compositions for the therapeutic
administration of FXI that utilize preparations having different FXI
activation levels. In some
embodiments, the methods and compositions employ FXI polypeptides that have
not been
subjected to any activation procedure. In some embodiments, the preparation of
FXI or FXI-
related polypeptide exhibits a ratio (by mass) of activated:zymogen FXI or FXI-
related
polypeptide of between about 1:99 to about 99:1, such as, e.g., between about
5:95 to about
95:5; about 10:90 to about 90:10; about 20:80 to about 80:20; about 30:70 to
about 70:30;
about 40:60 to about 60:40; and about 50:50. In some embodiments, the
preparation
contains not more than about 5% FXIa relative to the total FXI on a molar
basis; more
preferably, not more than about 2.5%, even more preferably, not more than
about 1 %, most
preferably not more than about 0.5% or 0.1 %. In some embodiments, the
preparation
contains not more than about 0.01-0.05% FXIa on a molar basis. In some
embodiments, the
preparation contains not more than about 0.01-0.04% FXIa on a molar basis. In
some
embodiments, the preparation contains not more than about 0.01-0.03% FXIa on a
molar
basis.
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27
The invention also relates to FXI-related polypeptides that exhibit a
differential
capacity to be activated relative to wild-type FXI, such as, e.g., FXI-related
polypeptides that
are more easily activated by FXlla than by thrombin, and vice versa;
polypeptides that are
constitutively activated, even in the absence of proteolytic cleavage;
heterodimers in which
one monomer (by virtue of mutation or chemical modification) cannot be
proteolytically
activated; and the like.
Furthermore, the invention also relates to FXI-related polypeptides that are
resistant
to autoactivation, i.e. variants where the ratio between rate of activation by
thrombin (and/or
FXlla) versus rate of activation by FXIa is higher than for wild-type FXI.
The methods and compositions of the invention may also employ treatment, pre-
treatment, storage, or co-administration of a FXI polypeptide with additional
agents that
inhibit and/or promote activation. Non-limiting examples of agents that
inhibit activation
include C1 esterase inhibitor (C1lnb), a-2 antiplasmin, (a2AP), a1-antitrypsin
(a1AT),
protease Nexin II, benzamidine, heparin, and antithrombin III; non-limiting
examples of
agents that promote activation include FXIa, FXlla, and thrombin.
Pharmaceutical formulations comprising FXI:
The present invention encompasses pharmaceutical compositions comprising a
preparation of FXI or FXI-related polypeptide for prophylactic and/or
therapeutic treatment.
Pharmaceutical compositions or formulations according to the invention
comprise a
a FXI polypeptide, such as, e.g., at concentrations between 0.001-100 mg/ml,
that is
preferably dissolved in a pharmaceutically acceptable carrier, preferably an
aqueous carrier
or diluent. Briefly, pharmaceutical compositions suitable for use according to
the present
invention are made by mixing a preparation comprising FXI and/or a FXI-related
polypeptide,
preferably in purified form, with suitable adjuvants and a suitable carrier or
diluent. A variety
of aqueous carriers may be used, such as water, buffered water, 0.4% saline,
0.3% glycine,
sugars, detergents, salts, buffers, glycerols, preservatives, protease
inhibitors, glycols, and
the like. The preparations of the invention can also be formulated using non-
aqueous
carriers, such as, e.g., in the form of a gel or as liposome preparations for
delivery or
targeting to the sites of injury. Liposome preparations are generally
described in, e.g., U.S.
Patents Nos. 4,837,028, 4,501,728, and 4,975,282. The compositions may be
sterilised by
conventional, well-known sterilisation techniques. The resulting aqueous
solutions may be
packaged for use or filtered under aseptic conditions and lyophilised, the
lyophilised
preparation being combined with a sterile aqueous solution prior to
administration.
The compositions may contain pharmaceutically acceptable auxiliary substances
or
adjuvants, including, without limitation, pH adjusting and buffering agents,
tonicity adjusting
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28
agents, preservatives, stabilizers, surfactants, chelating agents, and the
like. One skilled in
this art may formulate the compositions of the invention an appropriate
manner, and in
accordance with accepted practices, such as those disclosed in Remington's
Pharmaceutical
Sciences, Gennaro, ed., Mack Publishing Co., Easton, PA, 1990.
In one embodiment of the invention, the pharmaceutical compositions comprising
a
preparation of FXI or FXI-related polypeptide further comprises a pH adjusting
and buffering
agent. In one embodiment of the invention, the pharmaceutical compositions
comprising a
preparation of FXI or FXI-related polypeptide further comprises a tonicity
adjusting.agent. In
one embodiment of the invention, the pharmaceutical compositions comprising a
preparation
of FXI or FXI-related polypeptide further comprises a preservative. In one
embodiment of the
invention, the pharmaceutical compositions comprising a preparation of FXI or
FXI-related
polypeptide further comprises a stabilizer. In one embodiment of the
invention, the
pharmaceutical compositions comprising a preparation of FXI or FXI-related
polypeptide
further comprises a surfactant. In one embodiment of the invention, the
pharmaceutical
compositions comprising a preparation of FXI or FXI-related polypeptide
further comprises a
chelating agent.
Non-limiting examples of suitable buffers include acetate buffers, carbonate
buffers,
citrate buffers, glycylglycine buffers, histidine buffers, glycine buffers,
lysine buffers, arginine
buffers, phosphate buffers (containing, e.g.,sodium dihydrogen phosphate,
disodium
hydrogen phosphate or trisodium phosphate), TRIS
[tris(hydroxymethyl)aminomethane]
buffers, bicine buffers, tricine buffers, malate buffers, succinate buffers,
maleate buffers,
fumarate buffers, tartrate buffers, aspartate buffers, and mixtures thereof.
Non-limiting examples of pharmaceutically acceptible preservatives include
phenol,
o-cresol, m-cresol, p-cresol, chlorocresol, methyl p-hydroxybenzoate, ethyl p-
hydroxybenzoate, propyl p-hydroxybenzoate, butyl p-hydroxybenzoate, 2-
phenoxyethanol, 2-
phenylethanol, benzyl alcohol, chlorobutanol, thiomerosal, bronopol, benzoic
acid, imidurea,
chlorohexidine, sodium dehydroacetate, benzethonium chloride, chlorphenesine
(3-p-
chlorphenoxypropane-1,2-diol), benzamidine and mixtures thereof. In a further
embodiment
of the present invention the preservative is present in a concentration from
0.1 mg/ml to 20
mg/ml. In one further embodiment of the present invention the preservative is
present in a
concentration from 0.1 mg/ml to 5 mg/ml. In another further embodiment of the
present
invention the preservative is present in a concentration from 5 mg/ml to 10
mg/ml. In another
further embodiment of the present invention the preservative is present in a
concentration
from 10 mg/ml to 20 mg/ml. The use of a preservative in pharmaceutical
compositions is
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29
well-known to the skilled person (see, e.g., Remington: The Science and
Practice of
Pharmacy, 19th edition, 1995).
Non-limiting examples of tonicity-adjusting agents (which are normally
incorporated
for the purpose of rendering the formulation substantially isotonic include
salts (e.g. sodium
chloride), sugars, alcohols (such as C~-C$ alcohols), alditols, amino acids
(e.g. glycine,
histidine, arginine, lysine, isoleucine, aspartic acid, tryptophan or
threonine),
polyethyleneglycols (e.g. PEG400), and mixtures thereof. Any sugar, such as a
mono-, di-, or
polysaccharide, or a water-soluble glucan, may be used. Non-limiting examples
of
substances which are sugars, alcohols or alditols are fructose, glucose,
mannose, sorbose,
xylose, maltose, lactose, sucrose, trehalose, dextran, pullulan, dextrin,
cyclodextrin, soluble
starch, hydroxyethyl starch, carboxymethylcellulose-Na, mannitol, sorbitol,
inositol, galactitol,
dulcitol, xylitol, arabitol, glycerol (glycerine), propan-1,2-diol (propylene
glycol), propan-1,3-
diol, and butan-1,3-diol. The sugars, alcohols and alditols mentioned above
may be used
individually or in combination. There is no fixed limit to the amount used, as
long as the
substance is soluble in the liquid preparation.
In one embodiment, the tonicity-adjusting agent is present in a concentration
of from
about 1 mg/ml to about 150 mg/ml. In a further embodiment of the present
invention, the
tonicity-adjusting agent is present in a concentration of from about 1 mg/ml
to about 50
mg/ml. In one embodiment, the tonicity-adjusting agent is NaCI. In one
embodiment, the
tonicity-adjusting agent is NaCI present in a concentration of from about 1
mg/ml to about
150 mg/ml. In a further embodiment of the present invention, the tonicity-
adjusting agent is
NaCI present in a concentration of from about 1 mg/ml to about 50 mg/ml.
Non-limiting examples of chelating agents include salts of EDTA, citric acid
and
aspartic acid, and mixtures thereof. In some embodiments, a chelating agent is
present in a
concentration from 0.1 mg/ml to 5 mg/ml; from 0.1 mg/ml to 2 mg/ml; or from 2
mg/ml to 5
mg/ml.
The pharmaceutical compositions of the present invention may include as a
therapeutically active component a polypeptide that possibly may exhibit
aggregate formation
during storage in liquid pharmaceutical compositions. The term "aggregate
formation" is
intended to indicate a physical interaction between the polypeptide molecules
that results in
formation of oligomers which may remain soluble, or of large visible
aggregates that
precipitate from the solution. The term "during storage" refers to a liquid
pharmaceutical
composition or formulation which, once prepared, is not immediately
administered to a
subject. Rather, following preparation, it is packaged for storage in a liquid
form, in a frozen
state, or in a dried form for later reconstitution into a liquid form or other
form suitable for
administration to a subject. The term "dried form" refers to a liquid
pharmaceutical
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composition or formulation dried by freeze-drying [i.e. lyophilization; see,
for example,
Williams and Polli (1984), J. Parenteral Sci. Technol. 38:48-59], by spray-
drying [see Masters
(1991 ) in Saray-Dryina Handbook (5th ed; Longman Scientific and Technical,
Essex, U.K.),
pp. 491-676; Broadhead et al. (1992) Drug Devel. Ind. Pharm. 18:1169-1206; and
5 Mumenthaler et al. (1994) Pharm. Res. 11:12-20] or by air-drying [Carpenter
and Crowe
(1988), Cryobiology 25:459-470; and Roser (1991 ) Biopharm. 4:47-53].
Aggregate formation
by a polypeptide during storage of a liquid pharmaceutical composition can
adversely affect
biological activity of that polypeptide, resulting in loss of therapeutic
efficacy of the
pharmaceutical composition. Furthermore, aggregate formation may cause other
problems,
10 such as blockage of tubing, membranes or pumps when the polypeptide-
containing
pharmaceutical composition is administered using an infusion system.
In one embodiment of the present invention, the pharmaceutical composition
comprises an amount of an amino acid base sufficient to decrease aggregate
formation by
the polypeptide during storage of the composition. The term "amino acid base"
indicates an
15 amino acid or a combination of amino acids where any given amino acid is
present either in
its free base form or in its salt form. When a combination of amino acids is
used, all of the
amino acids may be present in their free base forms, all may be present in
their salt forms, or
some may be present in their free base forms while others are present in their
salt forms. In
one embodiment, amino acids for use in preparing compositions of the present
invention are
20 those carrying a charged side chain, such as arginine, lysine, aspartic
acid or glutamic acid.
Any stereoisomer of a particular amino acid (e.g. glycine, methionine,
histidine, arginine,
lysine, isoleucine, aspartic acid, tryptophan, threonine or a mixture of one
or more thereof),
or combinations of these stereoisomers, may be present in pharmaceutical
compositions of
the present invention so long as the particular amino acid is present either
in its free base
25 form or its salt form. In one embodiment the L-stereoisomer is used.
Compositions of the
present invention may also be formulated with analogues of these amino acids.
By "amino
acid analogue" is intended a derivative of the naturally occurring amino acid
that brings about
the desired effect of decreasing aggregate formation by the polypeptide during
storage of the
liquid pharmaceutical compositions of the present invention. Suitable arginine
analogues
30 include, for example, aminoguanidine, ornithine and N-monoethyl L-arginine,
suitable
methionine analogues include ethionine and buthionine and suitable cysteine
analogues
include S-methyl-L cysteine. As with the other amino acids, the amino acid
analogues are
incorporated into the compositions in either their free base form or their
salt form. The
compound imidazole is also to be regarded as an amino acid analogue in the
context of the
present invention. Typcally, the amino acids or amino acid analogues are used
in a
concentration which is sufficient to prevent or delay aggregation of the
protein.
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31
In one embodiment, the pharmaceutical formulation comprises methionine (or
another sulfur-containing amino acid or amino acid analogue) to inhibit
oxidation of
methionine residues to their sulfoxide form when the factor XI polypeptide is
a polypeptide
comprising at least one methionine residue susceptible to such oxidation. The
term "inhibit
oxidation" is intended to indicate minimization of accumulation of oxidized
species (of
methionine) with time. Inhibition of methionine oxidation results in greater
retention of the
polypeptide in its proper molecular form. Any stereoisomer of methionine (L, D
or DL isomer)
or combinations thereof can be used. The amount to be added should be an
amount
sufficient to inhibit oxidation of the methionine residues such that the
amount of sulfoxide
form of methionine is acceptable to regulatory agencies. Typically, this means
that the
composition contains no more than from about 10% to about 30% methionine
sulfoxide form.
This can in general be achieved by adding methionine in an amount such that
the ratio of
added methionine to methionine residues ranges from about 1:1 to about 1000:1,
such as
10:1 to about 100:1.
Non-limiting examples of stabilizers include high-molecular-weight polymers or
low-
molecular-weight compounds, such as, e.g., polyethylene-glycols (e.g. PEG
3350), polyvinyl
alcohol (PVA), polyvinylpyrrolidone, carboxy-/hydroxycellulose and derivatives
thereof
(including HPC, HPC-SL, HPC-L and HPMC), cyclodextrins, sulfur-containing
substances as
monothioglycerol, thioglycolic acid and 2-methylthioethanol, various salts
(e.g. sodium
chloride), glycerol, propylene glycol, propan-1,3-diol, propyl alcohol (1-
propanol) and
isopropyl alcohol (2-propanol).
Non-limiting examples of surfactants include detergents, ethoxylated castor
oil,
polyglycolyzed glycerides, acetylated monoglycerides, sorbitan fatty acid
esters,
polyoxypropylene-polyoxyethylene block polymers (e.g. poloxamers such as
Pluronic~ F68,
poloxamer 188 and 407, Triton X-100 ), polyoxyethylene sorbitan fatty acid
esters,
polyoxyethylene and polyethylene derivatives such as alkylated and alkoxylated
derivatives
("Tweens", e.g. Tween-20, Tween-40, Tween-80 and Brij-35), monoglycerides and
ethoxylated derivatives thereof, diglycerides and polyoxyethylene derivatives
thereof,
alcohols, glycerol, lectins and phospholipids (eg. phosphatidyl-serine,
phosphatidyl-choline,
phosphatidyl-ethanolamine, phosphatidyl-inositol, diphosphatidyl-glycerol and
sphingomyelin), derivatives of phospholipids (e.g. dipalmitoyl-phosphatidic
acid) and
lysophospholipids (e.g. palmitoyl lysophosphatidyl-L-serine and 1-acyl-sn-
glycero-3-
phosphate esters of ethanolamine, choline, serine or threonine), and alkyl-,
alkoxyl- (alkyl
ester) and alkoxy- (alkyl ether) derivatives of lysophosphatidyl and
phosphatidylcholines, e.g.
lauroyl and myristoyl derivatives of lysophosphatidylcholine,
dipalmitoylphosphatidylcholine,
and modifications of the polar head group, i.e. cholines, ethanolamines,
phosphatidic acid,
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32
serines, threonines, glycerol, inositol, and the positively charged DODAC,
DOTMA, DCP,
BISHOP, lysophosphatidylserine and lysophosphatidylthreonine, and
glycerophospholipids
(e.g. cephalins), glyceroglycolipids (e.g. galactopyranoside),
sphingoglycolipids (e.g.
ceramides, gangliosides), dodecylphosphocholine, hen egg lysolecithin, fusidic
acid
derivatives (e.g. sodium tauro-dihydrofusidate etc.), long-chain fatty acids
[e.g. C6-C~2 fatty
acids (such as oleic acid or caprylic acid)] and salts thereof, acylcarnitines
and derivatives
thereof, Na-acylated derivatives of lysine, arginine and histidine, side-chain
acylated
derivatives of lysine and arginine, N"-acylated derivatives of dipeptides
comprising any
combination of lysine, arginine and histidine and a neutral or acidic amino
acid, N"-acylated
derivatives of a tripeptide comprising any combination of a neutral amino acid
and two
charged amino acids, DSS (docusate sodium, CAS registry no [577-11-7]),
docusate
calcium, CAS registry no [128-49-4]), docusate potassium, CAS registry no
[7491-09-0]),
SDS (sodium dodecyl sulfate or sodium lauryl sulfate), sodium caprylate,
cholic acid and
derivatives thereof, bile acids and salts thereof, and glycine or taurine
conjugates,
ursodeoxycholic acid, sodium cholate, sodium deoxycholate, sodium
taurocholate, sodium
glycocholate, N-hexadecyl-N,N-dimethyl-3-ammonio-1-propanesulfonate, anionic
(alkyl-aryl-
sulfonates) monovalent surfactants, zwitterionic surfactants (e.g. N-alkyl-N,N-
dimethylammonio-1-propanesulfonates, 3-cholamido-1-propyldimethylammonio-1-
propanesulfonate), cationic surfactants (quaternary ammonium bases; e.g. cetyl-
trimethylammonium bromide, cetylpyridinium chloride), non-ionic surfactants
(eg. Dodecyl [i-
D-glucopyranoside), and poloxamines (eg. Tetronic's), i.e. tetrafunctional
block copolymers
derived from sequential addition of propylene oxide and ethylene oxide to
ethylenediamine;
or the surfactant may be selected from the group of imidazoline derivatives,
or mixtures
thereof. In one embodiment, the pharmaceutical formulation comprises a
surfactant in a
concentration of about 0.01 mglml to about 50 mg/ml. In one embodiment, the
pharmaceutical formulation comprises Tween-80. In one embodiment, the
pharmaceutical
formulation comprises poloxamer 188.
In one embodiment, the pharmaceutical formulation comprises an electrolyte. In
one
embodiment, the pharmaceutical formulation comprises an electrolyte, such as
NaCI. In one
embodiment, the pharmaceutical formulation comprises an electrolyte, such as
KCI.
In one embodiment an electrolyte, such as NaCI, such as in a concentration of
150
mM, is employed when Tween 80 is employed as stabiliser. In one embodiment
aggregation
following storage is avoided.
In a series of embodiments, the use of one or more stabilizing agents used in
a
pharmaceutical formulation comprising a preparation of FXI or FXI-related
polypeptide
results in an increase in the physical andlor chemical stability of FXI by at
least 10%, 25%,
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33
50%, or 100% over the physical and/or chemical stability of a control (i.e.,
FXI subjected to
the same treatment but in the absence of the stabilizing agent). In another
series of
embodiments, the use of one or more stabilizing agents in a pharmaceutical
formulation
comprising a preparation of FXI or FXI-related polypeptide results in an
increase in the
physical and/or chemical stability of FXI by at least 2-fold, 5-fold, 10-fold,
or 20-fold over the
physical and/or chemical stability of a control (i.e., FXI subjected to the
same treatment but in
the absence of the stabilizing agent).
The following table provides non-limiting examples of suitable formulations.
No
aggregation was observed after at least a month at ambient temperature when
such
formulations contained 1.7 mg/ml FXI.
Antimicrobial
ID pH Buffer Isotonic Stabiliserpreservative
agent
50 mM
TRIS(HYDROXYMETHYL)AMINOMETHANE
A1 8.5 (TRIS), pH 8.5 na na na
50 mM
TRIS(HYDROXYMETHYL)AMINOMETHANE
A2 8.5 (TRIS), pH 8.5 150 mM NaCIna na
50 mM
TRIS(HYDROXYMETHYL)AMINOMETHANE 0.001
% w/v
A3 8.5 (TRIS), pH 8.5 na Tween na
80
50 mM
TRIS(HYDROXYMETHYL)AMINOMETHANE 0.1 %
w/v
A4 8.5 (TRIS), pH 8.5 na Tween na
80
50 mM
TRIS(HYDROXYMETHYL)AMINOMETHANE 0.1 %
Tween
A5 8.5 (TRIS), pH 8.5 150 mM NaCI80 na
50 mM
TRIS(HYDROXYMETHYL)AMINOMETHANE16.0 mg/ml
A6 8.5 (TRIS), pH 8.5 glycerol na 0.5 w/v
% phenol
5 w/v
50 mM hydroxypropyl-
TRIS(HYDROXYMETHYL)AMINOMETHANE beta-
A7 8.5 (TRIS), pH 8.5 na cyclodextrinna
50 mM 0.1 w/v
TRIS(HYDROXYMETHYL)AMINOMETHANE human
serum
A8 8.5 (TRIS), pH 8.5 na albumin na
'
50 mM
TRIS(HYDROXYMETHYL)AMINOMETHANE 0.5 M
A9 8.5 (TRIS), pH 8.5 na Sucrose na
~A10 8.5 50 mM ~ na 0.3 w/v na
~ ~ %
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34
TRIS(HYDROXYMETHYL)AMINOMETHANE Poloxamer
188
(TRIS), pH 8.5
50 mM
TRIS(HYDROXYMETHYL)AMINOMETHANE 18.6 mg/ml
A11 8.5 (TRIS), pH 8.5 na EDTA
50 mM
TRIS(HYDROXYMETHYL)AMINOMETHANE
B1 8.0 (TRIS), pH 8.0 na na na
50 mM
TRIS(HYDROXYMETHYL)AMINOMETHANE
B2 8.0 (TRIS), pH 8.0 150 mM NaCIna na
50 mM
TRIS(HYDROXYMETHYL)AMINOMETHANE 0.001
% w/v
B3 8.0 (TRIS), pH 8.0 na Tween na
80
50 mM
TRIS(HYDROXYMETHYL)AMINOMETHANE 0.1 %
w/v
B4 8.0 (TRIS), pH 8.0 na Tween na
80
50 mM
TRIS(HYDROXYMETHYL)AMINOMETHANE 0.1 %
Tween
B5 8.0 (TRIS), pH 8.0 150 mM NaCI80 na
50 mM
TRIS(HYDROXYMETHYL)AMINOMETHANE16.0 mg/ml
B6 8.0 (TRIS), pH 8.0 glycerol na 0.5 w/v
% phenol
5 w/v
50 mM hydroxypropyl-
TRIS(HYDROXYMETHYL)AMINOMETHANE beta-
B7 8.0 (TRIS), pH 8.0 na cyclodextrinna
50 mM 0.1 w/v
TRIS(NYDROXYMETHYL)AMINOMETHANE human
serum
B8 8.0 (TRIS), pH 8.0 na albumin na
50 mM
TRIS(HYDROXYMETHYL)AMINOMETHANE 0.5 M
89 8.0 (TRIS), pH 8.0 na Sucrose na
50 mM
TRIS(HYDROXYMETHYL)AMINOMETHANE 0.3 w/v
l
B10 8.0 (TRIS), pH 8.0 na Poloxamerna
188
50 mM
TRIS(HYDROXYMETHYL)AMINOMETHANE. 18.6 mg/ml
811 8.0 (TRIS), pH 8.0 na EDTA
0.3 w/v
B12 6.1 Histidine 1.36 mg/ml, 40 mg/ml Poloxamer6 mg/ml
pH 6.1 mannitol 188 phenol
C1 7.4 50 mM phosphate, pH na na na
7.4
C2 7.4 50 mM phosphate, pH 150 mM NaCIna na
7.4
0.001
% w/v
C3 7.4 50 mM phosphate, pH na Tween na
7.4 80
0.1 %
w/v
C4 7.4 50 mM phosphate, pH na Tween na
7.4 80
C5 7.4 50 mM phosphate, pH 150 mM NaCI0.1 % na
7.4 I Tween
I
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so
16.0 mg/ml
C6 7.4 50 mM phosphate, pH glycerol na 0.5 w/v
7.4 % phenol
5 w/v
hydroxypropyl-
beta-
C7 7.4 50 mM phosphate, pH na cyclodextrinna
7.4
- 0.1 w/v
human
serum
C8 7.4 50 mM phosphate, pH na albumin na
7.4
0.5 M
C9 7.4 50 mM phosphate, pH na Sucrose na
7.4
0.3 w/v
C10 7.4 50 mM phosphate, pH na Poloxamerna
7.4 188
18.6 mg/ml
C11 7.4 50 mM phosphate, pH na EDTA
7.4
14 mg/ml
C12 7.7 50 mM phosphate, pH propylene na 5.5 mg/ml
7.7 glycol phenol
D1 6.0 50 mM citrate, pH 6.0 na na na
D2 6.0 50 mM citrate, pH 6.0 150 mM NaCIna na
0.001
% w/v
D3 6.0 50 mM citrate, pH 6.0 na Tween na
80
0.1 %
Tween
D5 6.0 50 mM citrate, pH 6.0 150 mM NaCI80 na
16.0 mg/ml
D6 6.0 50 mM citrate, pH 6.0 glycerol na 0.5 w/v
% phenol
5 w/v
hydroxypropyl-
beta-
D7 6.0 50 mM citrate, pH 6.0 na cyclodextrinna
0.1 w/v
human
serum
D8 6.0 50 mM citrate, pH 6.0 na albumin na
0.5 M
D9 6.0 50 mM citrate, pH 6.0 na Sucrose na
0.3 w/v
D10 6.0 50 mM citrate, pH 6.0 na Poloxamerna
188
18.6 mg/ml
D11 6.0 50 mM citrate, pH 6.0 na EDTA
E2 10.050 mM glycine, pH 10.0 150 mM NaCIna na
'
0.001
% w/v
E3 10.050 mM glycine, pH 10.0 na Tween na
80
0.1 %
w/v
E4 10.050 mM glycine, pH 10.0 na Tween na
80
0.1 %
Tween
E5 10.050 mM glycine, pH 10.0 150 mM NaCI80 na
16.0 mg/ml
E6 10.050 mM glycine, pH 10.0 glycerol na 0.5 w/v
% phenol
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36
5wlv%
hydroxypropyl-
beta-
E7 10.050 mM glycine, pH 10.0 na cyclodextrinna
0.1 w/v
human
serum
E8 10.050 mM glycine, pH 10.0 na albumin na
0.5 M
E9 10.050 mM glycine, pH 10.0 na Sucrose na
0.3 w/v
E10 10.050 mM glycine, pH 10.0 na Poloxamerna
188
F1 7.0 50 mM phosphate, pH na na na
7.0
F2 7.0 50 mM phosphate, pH 150 mM NaCIna na
7.0
0.001
l w/v
F3 7.0 50 mM phosphate, pH na Tween na
7.0 80
0.1 %
, Tween
F5 7.0 50 mM phosphate, pH 150 mM NaCI80 na
7.0
16.0 mg/ml
F6 7.0 50 mM phosphate, pH glycerol na 0.5 w/v
7.0 % phenol
5 w/v
hydroxypropyl-
beta-
F7 7.0 50 mM phosphate, pH na cyclodextrinna
7.0
0.1 w/v
human
serum
F8 7.0 50 mM phosphate, pH na albumin na
7.0
0.5 M
F9 7.0 50 mM phosphate, pH na Sucrose na
7.0
0.3 w/v
F10 7.0 50 mM phosphate, pH na Poloxamerna
7.0 188
G2 5.0 50 mM citrate, pH 5.0 150 mM NaCIna na
0.1 %
Tween
G5 5.0 50 mM citrate, pH 5.0 150 mM NaCI80 na
16.0 mg/ml
G6 5.0 50 mM citrate, pH 5.0 glycerol na 0.5 w/v
% phenol
5 w/v
hydroxypropyl-
beta-
G7 5.0 50 mM citrate, pH 5.0 na cyclodextrinna
0.1 w/v
human
serum
G8 5.0 50 mM citrate, pH 5.0 na albumin na
0.5 M
G9 5.0 50 mM citrate, pH 5.0 na Sucrose na
0.3 w/v
G10 5.0 50 mM citrate, pH 5.0 na Poloxamerna
188
0.05 mg/ml0.21 mg/ml
G12 7.6 50 mM glycylglycine, na Tween phenol
pH 7.6 20
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37
H2 3.0 50 mM citrate, pH 3.0 150 mM NaCIna na
0.1 %
w/v
H4 3.0 50 mM citrate, pH 3.0 na Tween na
80
0.1 %
Tween
H5 3.0 50 mM citrate, pH 3.0 150 mM NaCI80 na
16.0 mg/ml
H6 3.0 50 mM citrate, pH 3.0 glycerol na 0.5 w/v
% phenol
5 w/v
hydroxypropyl-
beta-
H7 3.0 50 mM citrate, pH 3.0 na cyclodextrinna
0.1 w/v
human
serum
H8 3.0 50 mM citrate, pH 3.0 na albumin na
0.5 M
H9 3.0 50 mM citrate, pH 3.0 na Sucrose na
0.3 w/v
H10 3.0 50 mM citrate, pH 3.0 na Poloxamerna
188
H12 7.3 5.7 mM phosphate, pH 137 mM NaCI5,4 mM
7.3 I I KCI
In a non-limiting embodiment, a suitable formulation that allows recovery of
active
FXI after freeze-drying contains:
FXI cone: 0.2 mg/ml
Buffer: 20 mM buffer (Histidine or TRIS) (pH 5.5, 6.5 of 7.4), 25 mg/ml
Mannitol
(bulking agent), 2.5 mg/ml NaCI (bulking agent), with 0.01 % Tween 80
Preferably, the pharmaceutical compositions are administered parenterally,
i.e.,
intravenously, subcutaneously, or intramuscularly; intravenously being most
preferred. They
may also be administered by continuous or pulsatile infusion. It will be
understood that any
effective method for administering a FXI polypeptide may be used, including,
e.g., using
mucosal or inhalation methods of administration.
Local delivery of the preparations of the present invention, such as, for
example,
topical application, may be carried out, e.g., by means of a spray, perfusion,
double balloon
catheters, stent, incorporated into vascular grafts or stents, hydrogels used
to coat balloon
catheters, incorporation into gauze or other bandage materials, or other well
established
methods.
Pharmaceutical compositions of the present invention may be administered in
various dosage forms, e.g. as solutions, suspensions, emulsions,
microemulsions, multiple
emulsion, foams, salves, pastes, plasters, ointments, tablets, coated tablets,
rinses, capsules
(e.g. hard gelatin capsules or soft gelatin capsules), suppositories, rectal
capsules, drops,
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38
gels, sprays, powder, aerosols, inhalants, eye drops, ophthalmic ointments,
ophthalmic
rinses, vaginal pessaries, vaginal rings, vaginal ointments, injection
solutions, in situ
transforming solutions (e.g. in situ gelling, in situ setting, in sifu
precipitating or in situ
crystallizing), infusion solution, or as implants.
Pharmaceutical compositions of the present invention may further be compounded
in, or bound or conjugated to (e.g. via covalent, hydrophobic or electrostatic
interactions), a
drug carrier, drug delivery system or advanced drug delivery system in order
to further
enhance stability of the factor XI polypeptide, to increase bioavailability,
to increase solubility,
to decrease adverse effects, to achieve chronotherapy well known to those
skilled in the art,
and/or to increase patient compliance. Examples of carriers, drug delivery
systems and
advanced drug delivery systems include, but are not limited to, polymers, e.g.
cellulose and
derivatives thereof, other polysaccharides (e.g. dextran and derivatives
thereof, starch and
derivatives thereof), polyvinyl alcohol), acrylate and methacrylate polymers,
polylactic acid
and polyglycolic acid and block co-polymers thereof, polyethyleneglycols,
carrier proteins
(e.g. albumin), gels (e.g. thermogelling systems, such as block co-polymeric
systems well
known to those skilled in the art), micelles, liposomes, microspheres,
nanoparticulates, liquid
crystals and dispersions thereof, L2 phase and dispersions thereof well known
to those
skilled in the art of phase behaviour in lipid-water systems, polymeric
micelles, multiple
emulsions (self-emulsifying and self-microemulsifying), cyclodextrins and
derivatives thereof,
and dendrimers.
Pharmaceutical compositions comprising a factor XI polypeptide prepared by use
of
a method according to the present invention are suitable for use in the
formulation of solids,
semisolids, powders and solutions for pulmonary administration using, for
example, a
metered dose inhaler, dry powder inhaler or a nebulizer, all of which are
devices well known
to those skilled in the art.
Pharmaceutical compositions comprising a factor XI polypeptide prepared by use
of
a method according to the present invention are suitable for use in the
formulation of
controlled-release, sustained-release, protracted-release, retarded-release or
slow-release
drug delivery systems. Pharmaceutical compositions comprising a factor XI
polypeptide
prepared by use of a method according to the present invention are, for
instance, useful in
formulation of parenteral controlled-release and sustained-release systems
(both systems
leading to a many-fold reduction in number of administrations) of types well
known to those
skilled in the art, such as controlled-release and sustained-release systems
for subcutaneous
administration. Without limiting the scope of the present invention, examples
of useful
controlled-release systems and compositions are hydrogels, oleaginous gels,
liquid crystals,
polymeric micelles, microspheres and nanoparticles,
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39
Methods for producing controlled release systems useful for pharmaceutical
compositions comprising a factor XI polypeptide prepared by use of a method
according to
the present invention include, but are not limited to, crystallization,
condensation, co-
crystallization, precipitation, co-precipitation, emulsification, dispersion,
high-pressure
homogenisation, encapsulation, spray-drying, microencapsulation, coacervation,
phase
separation, solvent evaporation to produce microspheres, extrusion and
supercritical fluid
processes. General reference is made to Handbook of Pharmaceutical Controlled
Release
(Wise, D.L., ed., Marcel Dekker, New York, 2000) and to Druas and the
Pharmaceutical
Sciences vol. 99: Protein Formulation and Delivery (MacNally, E.J., ed. Marcel
Dekker, New
York, 2000).
Parenteral administration may be performed by subcutaneous, intramuscular,
intraperitoneal or intravenous injection by means of a syringe, for example a
syringe in a
device of the pen type. Alternatively, parenteral administration can be
performed by means of
an infusion pump. A further option for administration of a composition in the
form of a solution
or suspension containing a factor XI polypeptide prepared by use of a method
according to
the present invention is administration as a nasal or pulmonary spray. As
another option,
pharmaceutical compositions containing a factor XI polypeptide prepared by use
of a method
according to the present invention may be adapted to transdermal
administration, e.g. by
needleless injection, by application of a patch (such as an iontophoretic
patch) or by
transmucosal (e.g. buccal) administration.
In one embodiment of the present invention, a pharmaceutical composition
comprising a factor XI polypeptide prepared by use of a method according to
the present
invention is stable for more than 6 weeks of usage and for more than 3 years
of storage.
In another embodiment of the present invention, a pharmaceutical composition
comprising a factor XI polypeptide prepared by use of a method according to
the present
invention is stable for more than 4 weeks of usage and for more than 3 years
of storage.
In a further embodiment of the present invention, a pharmaceutical composition
comprising a factor XI polypeptide prepared by use of a method according to
the present
invention is stable for more than 4 weeks of usage and for more than 2 years
of storage.
In an still further embodiment of the present invention, a pharmaceutical
composition
comprising a factor XI polypeptide prepared by use of a method according to
the present
invention is stable for more than 2 weeks of usage and for more than 2 years
of storage.
In some embodiments, FXI polypeptide formulations have a pH from about 4.0 to
about 10Ø In some embodiments, FXI polypeptide formulations have a pH from
about 4.0 to
about 8Ø In some embodiments, FXI polypeptide formulations have a pH from
about 4.0 to
about 7Ø In some embodiments, FXI polypeptide formulations have a pH from
about 4.0 to
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about 6.5. In some embodiments, FXI polypeptide formulations have a pH from
about 4.0 to
about 6Ø In some embodiments, FXI polypeptide formulations have a pH of
about 6.5 or
below, such as, e.g., between about pH 5.0 and about 6.5; such as between
about 5.5 and
6.5.
5 Therapeutic administration of Factor XI:
The present invention provides for the prevention and treatment of bleeding
using
FXI.
Bleeding refers to extravasation of blood from any component of the
circulatory
system. A bleeding episode encompasses unwanted, uncontrolled and often
excessive
10 bleeding in connection with surgery, trauma, or other forms of tissue
damage, as well as
unwanted bleedings in subjects having bleeding disorders. Bleedings may occur
as a
spontaneous events, such as intra-cerebral hemorrhage (ICH). Bleeding episodes
may occur
in subjects having a basically normal coagulation system but experiencing a
(temporary)
coagulophathy, as well as in subjects having congenital or acquired
coagulation or bleeding
15 disorders. In subjects having a defective platelet function, the bleedings
may be likened to
bleedings caused by haemophilia because the haemostatic system, as in
haemophilia, lacks
or has abnormal essential clotting "compounds" (e.g., platelets or von
Willebrand factor
protein). In subjects who experience extensive tissue damage, for example in
association
with surgery or vast trauma, the normal haemostatic mechanism may be
overwhelmed by the
20 demand of immediate haemostasis and they may develop excessive bleeding in
spite of a
basically (pre-trauma or pre-surgery) normal haemostatic mechanism. Such
subjects, who
further often are multi transfused, develop a (temporary) coagulopathy as a
result of the
bleeding and/or transfusions (i.e., a dilution of coagulation proteins,
increased fibrinolysis and
lowered number of platelets due to the bleeding and/or transfusions).
Bleedings may also
25 occur in organs such as the brain, inner ear region and eyes; these are
areas with limited
possibilities for surgical haemostasis and thus problems with achieving
satisfactory
haemostasis.
Similar problems may arise in the process of taking biopsies from various
organs
(liver, lung, tumour tissue, gastrointestinal tract) as well as in
laparoscopic surgery and
30 radical retropubic prostatectomy. Common for all these situations is the
difficulty in providing
haemostasis by surgical techniques (sutures, clips, etc.) which also is the
case when
bleeding is diffuse (e.g., haemorrhagic gastritis and profuse uterine
bleeding). Bleedings may
also occur in subjects on anticoagulant therapy in whom a defective
haemostasis has been
induced by the therapy given; these bleedings are often acute and profuse.
Anticoagulant
35 therapy is often given to prevent thromboembolic disease. Such therapy may
include
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41
heparin, other forms of proteoglycans, warfarin or other forms of vitamin K-
antagonists,
inhibitors of coagulation proteins, as well as aspirin and other platelet
aggregation inhibitors,
such as, e.g., antibodies or other inhibitors of GP Ilb/II la activity. The
bleeding may also be
due to so-called thrombolytic therapy which comprises combined treatment with
an
antiplatelet agent (e.g., acetylsalicylic acid), an anticoagulant (e.g.,
heparin), and a fibrinolytic
agent (e.g., tissue plasminogen activator, tPA). Bleeding episodes are also
meant to include,
without limitation, uncontrolled and excessive bleeding in connection with
surgery or trauma
in subjects having acute haemarthroses (bleedings in joints), chronic
haemophilic
arthropathy, haematomas, (e.g., muscular, retroperitoneal, sublingual and
retropharyngeal),
bleedings in other tissue, haematuria (bleeding from the renal tract),
cerebral haemorrhage,
surgery (e.g., hepatectomy), dental extraction, and gastrointestinal bleedings
(e.g., UGI
bleeds). The bleeding episodes may be associated with inhibitors against
factor VIII;
haemophilia A; haemophilia A with inhibitors; haemophilia B; deficiency of
factor VII;
deficiency of factor XI; thrombocytopenia; deficiency of von Willebrand factor
(von
Willebrand's disease); severe tissue damage; severe trauma; surgery;
laparoscopic surgery;
acidosis, hemodilution, consumption coagulopathies, hyperFibrinolysis,
hyopthermia,
haemorrhagic gastritis; taking biopsies; anticoagulant therapy; upper
gastroentestinal
bleedings (UGI); or stem cell transplantation. The bleeding episodes may be
profuse uterine
bleeding; occurring in organs with a limited possibility for mechanical
haemostasis; occurring
in the brain; occurring in the inner ear region; or occurring in the eyes.
A lowered count or activity of platelets refers to the number of platelets
(thrombocytes) present in the subject's plasma and to the biological,
coagulation-related
activity of such platelets. Lowered counts may be due, e.g., to increased
platelet destruction,
decreased platelet production, and pooling of a larger than normal fraction of
platelets in the
spleen. Thrombocytopenia, for example, is defined as a platelet count less
than 150,000
platelets per microliter; the upper limit of the normal platelet count is
generally considered to
be between 150,000 and 450,000 platelets per microliter. Platelet count may be
measured by
automated platelet counters; this is a well known method to the skilled
worker. Syndromes
due to lowered platelet count include, without limitation, thrombocytopenia,
coagulophathy.
Aspects of platelet activity include, without limitation, aggregation,
adhesion, and coagulant
activity of the platelets. Decreased activity may be due, e.g., to
glycoprotein abnormalities,
abnormal membrane-cytoskeleton interaction, abnormalities of platelet
granules,
abnormalities of platelet coagulant activity, abnormalities of signal
transduction and
secretion. Platelet activity, including aggregation, adhesion, and coagulant
activity, are
measured by standard methods known to the skilled worker, see e.g.,Platelets.
A Practical
Approach, Ed. S. P. Watson ~ K.S. Authi: Clinical Aspects of Platelet
Disorders (K.J.
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42
Clemetson) 15:299-318, 1996, Oxford University Press; Williams Hematology,
Sixth Edition,
Eds. Beutler, Lichtman, Coller, Kipps & Seligsohn, 2001, McGraw-Hill.
Syndromes due to
lowered platelet activity include, without limitation, Glanzmann
thrombathenis, Bernard-
Soulier syndrome, storage poll disease, anticoagulant treatment and
thrombolytic treatment.
In the context of the present invention, treatment encompasses both prevention
of
bleeding, including, without limitation, prevention of an expected bleeding,
such as, for
example, might be expected to occur during or consequent to a surgical
procedure, as well
as regulation of an already occurring bleeding, such as, for example, in
trauma, with the
purpose of inhibiting or minimizing the bleeding. The bleeding may be at an
identified site or
may be at an undetermined site. Prophylactic administration of a preparation
comprising a
FXI polypeptide is thus included in treatment.
In some embodiments, a normal human patient, i.e., one not suffering from a
congenital deficiency of FXI, may be administered FXI and/or a FXI-related
polypeptide at a
dosage that corresponds to about 0.05 mg to about 500 mg of wild-type FXI per
day or per
bleeding episode, e.g., from about 1 mg to about 200 mg, or, e.g., from about
1 mg to about
175 mg per day or per bleeding episode for a 70-kg subject as loading and
maintenance
doses, depending on the weight of the subject, the condition and the severity
of the
condition.
In some embodiments, blood is drawn from a patient in need of treatment with a
FXI
polypeptide and an assay is performed (prior to FXI polypeptide
administration) to assess
one or more of: (i) the plasma level of FXI; (ii) the ratio of
activated:zymogen FXI; and/or (iii)
the concentration of FXI needed to be added exogenously in order to restore
effective
coagulation; based on the results of the assay, an appropriate amount of FXI
polypeptide is
administered using a predetermined regimen. Any suitable assay may be used for
these
determinations, including, e.g., an ELISA or a gel-based method. Appropriate
calibration
standards are used in order to allow the comparison of the measured level with
the usual
level of FXI in human plasma (about 30 nM). Typically, it will be desired to
replenish FXI
levels to at least about 5 nM, such as about 10 nM, such as about 15 nM, such
as about 20
nM, and such as at least about 30 nM FXI, such as 60 nM, such as 120 nM.
When a FXI-related polypeptide is being used to replenish FXI activity in a
patient,
the FXI-related polypeptide will exhibit a particular level of at least one
FXI bioactivity and the
goal of the treatment is to provide an amount of that bioactivity that
corresponds to a
predetermined amount of wild-type FXI (i.e., an "effective FXI plasma
concentration").
In some embodiments, the present invention encompasses therapeutic
administration of FXI polypeptide to patients whose plasma level of FXI is
below about 3 nM;
5 nM; or 10 nM.
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Combination Treatments
The present invention also encompasses methods and compositions that provide
combination therapies in which FXI polypeptide is administered with a non-
Factor VII/Factor
Vlla coagulation agent. Suitable non-Factor VII/Factor VIIA coagulation agents
include,
without limitation, Factor XIII (see, e.g., WO 01/85198); inhibitors of tissue
factor pathway
inhibitor (TFPI inhibitors) (see, e.g., WO 01/85199); Factor IX (see, e.g., WO
02/062376);
thrombin activatable fibrinolysis inhibitor (TAFI) (see, e.g., PCT/DK02/00734;
PAI-1 (see,
e.g., PCT/DK02/00735; Factor V (see, e.g., PCT/DK02/00736); protein C
inhibitors (see, e.g.,
PCT/DK02/00737); thrombomodulin (see, e.g., PCT/DK02/00738); protein S
inhibitors (see,
e.g., PCT/DK02/00739); tissue plasminogen activator inhibitors (see, e.g.,
PCT/DK02/00740); a2-antiplasmin (see, e.g., PCT/DK02/00741 ); aprotinin (see,
e.g.,
PCT/DK02/00742); tranexamic acid (see, e.g., PCT/DK02/00751); ~-aminocaproic
acid (see,
e.g., PCT/DK02/00752); prothrombin, thrombin, Factor VII, Factor X, and
fibrinogen.
The following is a list of embodiments of the present invention:
Embodiment 1: A method for treating bleeding episodes, said method comprising
administering to a patient in need thereof a preparation comprising Factor XI
(FXI) or FXI-
related polypeptide, in an amount effective for such treatment.
Embodiment 2: A method as defined in embodiment 1, wherein said administering
results in a reduced clotting time in said patient.
Embodiment 3: A method as defined in embodiment 1 or embodiment 2, wherein
said administering results in an enhancement of hemostasis in said patient.
Embodiment 4: A method as defined in any of embodiments 1 to 3, wherein said
administering results in an increase in clot lysis time in said patient.
Embodiment 5: A method as defined in any of embodiments 1 to 4, wherein said
administering results in an increase in clot strength in said patient.
Embodiment 6: A method as defined in any of embodiments 1 to 5, wherein said
administering results in an increase in overall clot quality (OCQ) in said
patient.
Embodiment 7: A method as defined in any of embodiments 1 to 6, wherein,
following said administration, said patient exhibits an effective FXI plasma
concentration of at
least about 5 nM.
Embodiment 8: A method as defined in embodiment 7, wherein said effective FXI
plasma concentration is at least about 10 nM.
Embodiment 9: A method as defined in embodiment 8, wherein said effective FXI
plasma concentration is at least about 30 nM, such as at least about 60 nM,
such as at least
about 120 nM.
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Embodiment 10: A method as defined in any of embodiments 1 to 9, wherein said
FXI or FXI-related polypeptide comprises the sequence of SEQ ID N0:1, or a
fragment
thereof that retains at least one FXI-associated biological activity.
Embodiment 11: A method as defined in any of embodiments 1 to 9, wherein said
FXI or FXI-related polypeptide comprises the sequence of SEQ ID N0:2, or a
fragment
thereof that retains at least one FXI-associated biological activity.
Embodiment 12: A method as defined in any of embodiments 1 to 11, wherein said
patient does not suffer from a congenital FXI deficiency.
Embodiment 13: A method as defined in any of embodiments 1 to 12, wherein said
bleeding episodes are secondary to a condition selected from the group
consisting of:
surgery, a dental procedure, trauma, or hemodilution.
Embodiment 14: A method as defined in any of embodiments 1 to 13, further
comprising, prior to said administering:
(a) obtaining a sample of blood from said patient; (b) determining at least
one of: FXI
concentration, ratio of FXIa:FXI, or amount of exogenous FXI necessary to
restore
coagulation; and (c) based on the results of step (b), determining said amount
of FXI
effective for treatment.
Embodiment 15: A method for treating bleeding episodes, said method comprising
administering to said patient (i) a first amount of a preparation comprising a
FXI polypeptide
and (ii) a second amount of a preparation comprising a non-Factor VIUFactor
Vlla
coagulation agent, wherein said first and second amounts in combination are
effective for
such treatment.
Embodiment 16: A method as defined in embodiment 15, wherein said non-Factor
VII/Factor Vlla coagulation agent is selected from the group consisting of:
Factor XIII; tissue
factor pathway inhibitor (TFPI) inhibitor; Factor IX; thrombin activatable
fibrinolysis inhibitor
(TAFI); plasminogen activator inhibitor-1 (PAI-1 ); Factor V; protein C
inhibitor; protein S
inhibitor; and tissue plasminogen activator (tPA) inhibitor.
Embodiment 17: A method as defined in embodiment 15 or embodiment 16, wherein
said administering results in a reduced clotting time in said patient.
Embodiment 18: A method as defined in any of embodiments 15 to 17, wherein
said
administering results in an enhancement of hemostasis in said patient.
Embodiment 19: A method as defined in any of embodiments 15 to 18, wherein
said
administering results in an increase in clot lysis time in said patient.
Embodiment 20: A method as defined in any of embodiments 15 to 19, wherein
said
administering results in an increase in clot strength in said patient.
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Embodiment 21: A method as defined in any of embodiments 15 to 20, wherein
said
administering results in an increase in overall clot quality (OCQ) in said
patient.
Embodiment 22: A method as defined in any of embodiments 15 to 21, wherein,
following said administration, said patient exhibits an effective FXI plasma
concentration of at
5 least about 5 nM.
Embodiment 23: A method as defined in embodiment 22, wherein said effective
FXI
plasma concentration is at least about 10 nM.
Embodiment 24: A method as defined in embodiment 23, wherein said effective
FXI
plasma concentration is at least about 30 nM, such as at least about 60 nM,
such as at least
10 about 120 nM.
Embodiment 25: A method as defined in any of embodiments 15 to 14, wherein
said
FXI or FXI-related polypeptide comprises the sequence of SEQ ID NO:1, or a
fragment
thereof that retains at least one FXI-associated biological activity.
Embodiment 26: A method as defined in any of embodiments 15 to 24, wherein
said
15 FXI or FXI-related polypeptide comprises the sequence of SEQ ID N0:2, or a
fragment
thereof that retains at least one FXI-associated biological activity.
Embodiment 27: A method as defined in any of embodiments 15 to 26, wherein
said
patient does not suffer from a congenital FXI deficiency.
Embodiment 28: A method as defined in any of embodiments 15 to 27, wherein
said
20 bleeding episodes are secondary to a condition selected from the group
consisting of:
surgery, a dental procedure, trauma, or hemodilution.
Embodiment 29: A method as defined in any of embodiments 15 to 28, further
comprising, prior to said administering:
(a) obtaining a sample of blood from said patient; (b) determining at least
one of: FXI
25 concentration, ratio of FXIa:FXI, or amount of exogenous FXI necessary to
restore
coagulation; and (c) based on the results of step (b), determining said amount
of FXI
effective for treatment.
Embodiment 30: A method as defined in embodiment 1, wherein said method does
not comprise administration of a Factor VII/Factor Vlla coagulation agent.
30 Embodiment 31: A pharmaceutical formulation comprising (i) isolated
recombinant a
FXI polypeptide and (ii) a pharmaceutically acceptable carrier or excipient.
Embodiment 32: Use of a FXI polypeptide for treating bleeding episodes.
Embodiment 33: Use according to embodiment 32, wherein said bleeding episodes
are secondary to a condition selected from the group consisting of: surgery, a
dental
35 procedure, trauma, or hemodilution.
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Embodiment 34: Use according to embodiment 32 or embodiment 33, wherein said
bleeding episodes are not treated with a Factor VII/Factor Vlla coagulation
agent.
Embodiment 35: Use of a FXI polypeptide for enhancement of hemostasis in a
patient in need thereof .
Embodiment 36: Use of a FXI polypeptide for increasing clot lysis time in a
patient in
need thereof.
Embodiment 37: Use of a FXI polypeptide for increasing clot strength in a
patient in
need thereof.
Embodiment 38: Use of a FXI polypeptide for increasing overall clot quality
(OCQ) in
a patient in need thereof.
Embodiment 39: Use of a FXI polypeptide for reducing clotting time in a
patient in
need thereof.
Embodiment 40: Use according to any of embodiments 32 to 39, wherein the
effective FXI plasma concentration in the patient is increased to at least
about 5 nM.
Embodiment 41: Use according to embodiment 40, wherein the effective FXI
plasma
concentration is increased to at least about 10 nM.
Embodiment 42: Use according to embodiment 41, wherein the effective FXI
plasma
concentration is increased to at least about 30 nM, such as at least about 60
nM, such as at
least about 120 nM.
Embodiment 43: Use according to any of embodiments 32 to 42, wherein the
patient
to be treated is not treated with a Factor VII/Factor Vlla coagulation agent.
Embodiment 44: Use of a FXI polypeptide for preparation of a pharmaceutical
formulation for treating bleeding episodes.
Embodiment 45: Use according to embodiment 44, wherein said bleeding episodes
are secondary to a condition selected from the group consisting of: surgery, a
dental
procedure, trauma, or hemodilution.
Embodiment 46: Use according to embodiment 44 or embodiment 45, wherein said
bleeding episodes are not being treated with a Factor VII/Factor Vlla
coagulation agent.
Embodiment 47: Use of a FXI polypeptide for preparation of a pharmaceutical
formulation for enhancement of hemostasis in a patient in need thereof .
Embodiment 48: Use of a FXI polypeptide for preparation of a pharmaceutical
formulation for increasing clot lysis time in a patient in need thereof.
Embodiment 49: Use of a FXI polypeptide for preparation of a pharmaceutical
formulation for increasing clot strength in a patient in need thereof.
Embodiment 50: Use of a FXI polypeptide for preparation of a pharmaceutical
formulation for increasing overall clot quality (OCQ) in a patient in need
thereof.
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Embodiment 51: Use of a FXI polypeptide for preparation of a pharmaceutical
formulation for reducing clotting time in a patient in need thereof..
Embodiment 52: Use according to any of embodiments 47 to 51, wherein the
effective FXI plasma concentration in the patient is increased to at least
about 5 nM.
Embodiment 53: Use according to embodiment 52, wherein the effective FXI
plasma
concentration is increased to at least about 10 nM.
Embodiment 54: Use according to embodiment 53, wherein the effective FXI
plasma
concentration is increased to at least about 30 nM, such as at least about 60
nM, such as at
least about 120 nM.
Embodiment 55: Use according to any of embodiments 44 to 54, wherein the
patient
to be treated is not treated with a Factor VII/Factor Vlla coagulation agent.
Embodiment 56: Use according to any of embodiments 32 to 55, wherein the
patient
to be treated does not suffer from a congenital FXI deficiency.
Embodiment 57: Use according to any of embodiments 32 to 56, wherein said FXI
polypeptide comprises the sequence of SEQ ID N0:1, or a fragment thereof that
retains at
least one FXI-associated biological activity.
Embodiment 57: Use according to any of embodiments 32 to 56, wherein said FXI
polypeptide comprises the sequence of SEQ ID N0:2, or a fragment thereof that
retains at
least one FXI-associated biological activity.
Embodiment 58: Use according to any of embodiments 32 to 57, wherein said FXI
polypeptide is to be administered in combination with a non-Factor VII/Factor
Vlla
coagulation agent.
Embodiment 59: Use according to embodiment 58, wherein said non-Factor
VII/Factor Vlla coagulation agent is selected from the group consisting of:
Factor XIII; tissue
factor pathway inhibitor (TFPI) inhibitor; Factor IX; thrombin activatable
fibrinolysis inhibitor
(TAFI); plasminogen activator inhibitor-1 (PAI-1 ); Factor V; protein C
inhibitor; protein S
inhibitor; and tissue plasminogen activator (tPA) inhibitor.
Embodiment 60. A method for purifying a FXI polypeptide from a biological
material,
the method comprising subjecting the material to sequential chromatography on
an cation-
exchange chromatographic material, a hydrophobic interaction chromatographic
material and
a hydroxyapatite chromatographic material.
Embodiment 61. A method according to embodiment 60, wherein the FXI
polypeptide is a recombinant FXI.
Embodiment 62. A method according to embodiment 60 or embodiment 61,
wherein the FXI polypeptide is human FXI.
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Embodiment 63. A method according to embodiment 60 or embodiment 61,
wherein the FXI polypeptide is a dimer.
Embodiment 64. A method according to embodiment 63, wherein the FXI
polypeptide is a dimer of human subunits.
Embodiment 65. A method according to any of embodiments 60 to 64, wherein the
biological material is a biological fluid.
Embodiment 66. A method according to embodiment 65, wherein the biological
fluid
is the supernatant of a mammalian cell.
Embodiment 67. A method according to embodiment 66, wherein the biological
fluid
is the supernatant of a CHO culture.
Embodiment 68. A method according to any of embodiments 60 to 67, wherein the
method comprises the steps of:
(a) subjecting a biological material comprising a FXI polypeptide to
chromatography on a first
cation-exchange chromatographic material, said chromatography comprising:
(i) applying said biological material to said first cation-exchange
chromatographic
material;
(ii) eluting unbound material from the first cation-exchange chromatographic
material
with a buffer A', which buffer A is suitable for eluting material not bound to
the first
cation-exchange chromatographic material; and
(iii) eluting unbound material from the first cation-exchange chromatographic
material with a buffer A', which buffer 'A is suitable for eluting material
not bound to
the first cation-exchange chromatographic material; and
(iv) eluting said FXI polypeptide from the first cation-exchange
chromatographic
material by elution with buffer A", which buffer A" is suitable for eluting
said FXI
polypeptide from said first cation-exchange chromatographic material;
(b) subjecting the eluate from step (iv), or a fluid prepared by use of the
eluate from step (iv),
to chromatography using a hydrophobic interaction chromatographic material,
said
chromatography comprising:
(v) applying the eluate from step (iv), or a fluid prepared by use of the
eluate from
step (iv), to said hydrophobic interaction chromatographic material;
(vi) eluting unbound material from the chromatographic material with buffer B,
which
buffer B is suitable for eluting material not bound to the hydrophobic
interaction
chromatographic material; and
(vii) eluting said FXI polypeptide from said chromatographic material by
gradient-
elution with buffer B', which buffer B' is suitable for eluting FXI from said
hydrophobic interaction chromatographic material.
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Embodiment 69. A method according to embodiment 68, wherein buffer A
comprises one or more stabilizing agents which are capable of increasing the
stability of the
FXI polypeptide.
Embodiment 70. A method according to embodiment 69, wherein buffer A
comprises a stabilizing agent, which stabilizing agent is a sugar, an alcohol
or an alditol.
Embodiment 71. A method according to embodiment 70, wherein buffer A
comprises a stabilizing agent, which stabilizing agent is a sugar, a C4-C$-
alcohol or an alditol.
Embodiment 72. A method according to embodiment 71, wherein buffer A
comprises a stabilizing agent, which stabilizing agent is a polyalcohol.
Embodiment 73. A method according to embodiment 72, wherein buffer A
comprises a stabilizing agent selected from the group consisting of glycerol,
propylene
glycol, propan-1,3-diol, propyl alcohol and isopropyl alcohol.
Embodiment 74. A method according to embodiment 73, wherein buffer A
comprises a stabilizing agent selected from the group consisting of glycerol,
propylene glycol
and propan-1,3-diol.
Embodiment 75. A method according to any of embodiments 72 to 74, wherein said
stabilizing agent is present in a concentration of from about 5% (v/v) to
about 50% (v/v).
Embodiment 76. A method according to embodiment 75, wherein said stabilizing
agent is present in a concentration of from about 10% (v/v) to about 50%
(v/v).
Embodiment 77. A method according to embodiment 76, wherein said stabilizing
agent is present in a concentration of from about 10% (v/v) to about 20%
(v/v).
Embodiment 78. A method according to embodiment 77, wherein said stabilizing
agent is present in a concentration of about 10% (v/v).
Embodiment 79. A method according to embodiment 78, wherein said stabilizing
agent is present in a concentration of about 20% (v/v).
Embodiment 80. A method according to any of embodiments 68 to 79, wherein the
pH of buffer A is between about 6.5 and about 9.
Embodiment 81. A method according to embodiment 80, wherein the pH of buffer A
is between about 7 and about 9.
Embodiment 82. A method according to embodiment 81, wherein the pH of bufferA
is about 8.
Embodiment 83. A method according to any of embodiments 68 to 82, wherein
buffer A has a conductivity of less than about 50 mSlcm.
Embodiment 84. A method according to any of embodiments 60 to 83, wherein the
hydrophobic interaction chromatographic material uses butyl or phenyl as the
ligand.
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Embodiment 85. A method according to embodiment 84, wherein the hydrophobic
interaction chromatographic material is Phenyl Sepharose High Performance High
Substitution.
Embodiment 86. A method according to embodiment 84, wherein the hydrophobic
5 interaction chromatographic material is Butyl Sepharose High Performance
High
Substitution.
Embodiment 87. A method according to any of embodiments 68 to 86, wherein the
pH of buffer B is from about 6 to about 9.
Embodiment 88. A method according to embodiment 87, wherein the pH of buffer B
10 is about 8.
Embodiment 89. A method according to any of embodiments 68 to 88, wherein
buffer B has a conductivity of more than 50 mS/cm.
Embodiment 90. A method according to embodiment 89, wherein buffer B has a
conductivity of more than 70 mS/cm.
15 Embodiment 91. A method according to any of embodiments 68 to 90, wherein
the
eluate from stage (vii), or a fluid prepared by use of the eluate from stage
(vii), is treated by
use of a method comprising a step of
(1 ) addition of one or more stabilizing agents which are capable of
increasing the stability of
the FXI polypeptide in an amount effective to significantly improve the
stability thereof, and/or
20 (2) adjusting the pH of the eluate from stage (vii), or of a fluid prepared
by use of the eluate
from stage (vii), to a pH between about 7 and about 9.
Embodiment 92. A method according to embodiment 91, wherein the stabilizing
agent used in step (1 ) is a sugar, an alcohol or an alditol.
Embodiment 93. A method according to embodiment 92, wherein the stabilizing
25 agent used in step (1 ) is a sugar, a C~-C$-alcohol or an alditol.
Embodiment 94. A method according to embodiment 93, wherein the stabilizing
agent used in step (1 ) is a polyalcohol.
Embodiment 95. A method according to embodiment 94, wherein the stabilizing
agent used in step (1 ) is selected from the group consisting of glycerol,
propylene glycol,
30 propan-1,3-diol, propyl alcohol and isopropyl alcohol.
Embodiment 96. A method according to embodiment 95, wherein the stabilizing
agent used in step (1 ) is selected from the group consisting of glycerol,
propylene glycol and
propan-1,3-diol.
Embodiment 97. A method according to any of embodiments 94 to 96, wherein the
35 stabilizing agent used in step (1 ) is added to a concentration of from
about 5% (v/v) to about
50% (vlv).
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Embodiment 98. A method according to embodiment 97, wherein the stabilizing
agent used in step (1 ) is added to a concentration of from about 10% (v/v) to
about 50%
(v/v).
Embodiment 99. A method according to embodiment 98, wherein the stabilizing
agent used in step (1 ) is added to a concentration of from about 10% (v/v) to
about 20%
(v/v).
Embodiment 100. A method according to any of embodiments 60 to 99, wherein the
method further comprises a step of subjecting the eluate from the hydrophobic
interaction
chromatography, or a material prepared by use of the eluate from the
hydrophobic interaction
chromatography, to chromatography on a Hydroxyapatite chromatographic
material.
Embodiment 101. A method according to any of embodiments 68 to 100, wherein
the method further comprises a step of:
subjecting the eluate from stage (vii), or a fluid prepared by use of the
eluate from stage (vii),
to chromatography on a hydroxyapatite chromatographic material, said
chromatography
comprising:
(viii) applying the eluate (diluted and pH adjusted) from stage (vii), or a
fluid
prepared by use of the eluate from stage (vii), to said hydroxyapatite
chromatographic material;
(ix) eluting unbound material from the hydroxyapatite chromatographic material
with
buffer C, which buffer C is suitable for eluting material not bound to the
hydroxyapatite chromatographic material; and
(x) eluting said FXI polypeptide from the hydroxyapatite chromatographic
material
with buffer C', wherein buffer C' is suitable for eluting FXI polypeptides
which bind to
the hydroxyapatite chromatographic material in step (viii).
Embodiment 102. A method according to embodiment 101, wherein buffer C and/or
buffer C' comprises one or more stabilizing agents which are capable of
increasing the
stability of the FXI polypeptide.
Embodiment 103. A method according to embodiment 101, wherein a stabilizing
agent is added to the fXl containing fractions, which stabilizing agent is a
sugar, an alcohol or
an alditol.
Embodiment 104. A method according to embodiment 103, wherein a stabilizing
agent is added, which stabilizing agent is a sugar, a C4-C$-alcohol or an
alditol.
Embodiment 105. A method according to embodiment 104, wherein a stabilizing
agent is added, which stabilizing agent is a polyalcohol.
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Embodiment 106. A method according to embodiment 105 wherein a stabilizing
agent selected from the group consisting of glycerol, propylene glycol, propan-
1,3-diol, propyl
alcohol and isopropyl alcohol is added.
Embodiment 107. A method according to embodiment 106, wherein a stabilizing
agent selected from the group consisting of glycerol, propylene glycol and
propan-1,3-diol is
added.
Embodiment 108. A method according to any of embodiments 105 to 107, wherein
said stabilizing agent is added to a concentration of from about 5% (v/v) to
about 50% (v/v).
Embodiment 109. A method according to embodiment 108, wherein said stabilizing
agent is adde to a concentration of from about 10% (v/v) to about 50% (v/v).
Embodiment 110. A method according to embodiment 109, wherein said stabilizing
agent is added to a concentration of from about 10% (v/v) to about 20% (v/v).
Embodiment 111. A method according to embodiment 110, wherein said stabilizing
agent is added to a concentration of about 10% (v/v).
Embodiment 112. A method according to any of embodiments 101 to 111, wherein
buffer C and/or buffer C' has a pH from about 5,8 to about 7,8.
Embodiment 113. A method according to any of embodiments 101 to 112, wherein
buffer C and/or buffer C' has a pH of about 6,0.
Embodiment 114. A pharmaceutical composition comprising a FXI polypeptide
prepared by use of a method according to any of embodiments 60 to 113.
The following are intended as non-limiting examples of the present invention.
EXAMP LES
Example 1: Effect of FXI on hemostasis in cardiac patients:
Blood was obtained before and after surgery from 5 patients undergoing cardiac
surgery with cardiopulmonary bypass. The effect of FXI on clot formation and
stability was
evaluated using roTEG (rotational thromboelastography), using the method of
Vig et al.
(2001 ), Blood Coagulation & Fibrinolysis 12:555. Briefly, coagulation was
initiated by adding
Innovin (final dilution: 1:50,0000) (Dade Behring) and CaCl2 (final
concentration: 15 nM), in
the presence or absence of FXI (2.5, 10, or 25 nM) (HTI/Enzyme Research
Laboratories,
Essen). Fibrinolysis was initiated by addition of 4 nM tPA (American
Diagnostica).
Measurements were made using a ROTEG-04 Whole Blood Haemostasis System
Rotation
Thrombelastography apparatus (Pentapharm GmBH). Overall Clot Quality (OCQ) is
calculated as:
Max vel l t max vel) X (t min vel - t max vel)
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OCQ is then normalized to the control sample (incubated in the absence of any
hemostatic agents.
The results are shown in Figure 1. FXI considerably improved the overall clot
formation and clots formed in the presence of FXI had an increased resistant
to fibrinolysis.
Example 2: Effect of FXI on hemostasis in normal blood
Blood was obtained from 4 normal subjects, and the effect of FXI on clot
formation
was evaluated by ROTEG as described in Example 1.
Figure 2 illustrates that FXI caused a dose-dependent increase is OCQ in
normal
blood.
Example 3: Activity of alycosylation-disrupted FXI polypeptides
FXI variant containing the following substitutions were constructed using
standard
methodologies and were expressed after transfection in HEK293 cells. Crude
cell culture
supernatants were collected from cells grown for 96 h at 37°C. FXI
activity was measured by
ROTEG as described in Example 1.
The results are shown in the following Table.
Protein FXI activity in % of
expected
glues
NHP (Normal human plasma)
(31nM
FXI
FXI N72Q- l,2nM 42
FXI N108Q- 1,3nM 62
FXI N335Q - 0,4nM 75
FXI N432Q - 1,2nM 33
FXI N473Q - 0,6nM 83
Example 4: Storage stability of FXI formulations
The following solutions of FXI were prepared and stored for 5 weeks at
5°C, after
which FXI activity was measured as described in Example 1.
1. 384 nM FXI in 4 mM acetate, 150 mM NaCI, pH 5.4
2. 190 nM FXI in 50 mM acetate buffer, 150 mM NaCI, pH 5.4
3. 190 nM FXI in 50 mM acetate buffer, 150 mM NaCI, pH 5.4, 1 mM CaCl2
4. 190 nM FXI in 50 mM acetate buffer, 75 mM NaCI, pH 5.4, 300 mg/ml sucrose
5. 190 nM FXI in 50 mM MES buffer, pH 6.5, 150 mM NaCI
6. 190 nM FXI in 50 mM MES buffer, pH 6.5, 150 mM NaCI, 1 mM CaCl2
7. 190 nM FXI in 50 mM MES buffer, pH 6.5, 75 mM NaCI, 300 mg/ml sucrose
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The results are shown in Figure 3.
Example 5: Binding peptides for FXI
The following experiments were performed to identify peptides that bind FXI.
1. Synthesis of peptide libraries:
The following libraries were synthesized using Fmoc solid phase peptide
synthesis
on Tentagel resin bead from Rapp Polymere (Germany). Three different peptide
bead
libraries were used in the screening. They are named BL121, BL122 and BL123.
The format of the library BL121 is:
O~-OZ-O3-O4-OS-O6-O7-O$-Og-O~p-O~~-O~2-O13-0~4-Tentagel resin, where On is a L-
amino
acid and n=1,2 and 11,12 can be any proteinogenic L-amino acid except
methionine and
cysteine and n=4,5 and 7,8 and 10,11 and 13,14 can be any proteinogenic L-
amino acid
except Methionine and cysteine and deletion,
and n=3,6,9,12 can be Phe, Trp, Tyr, Leu.
The format of the library BL122 is
O~-0~-Oa-04-Os-Os-OwOs-O9-O~o-0,~-0~2-Tentagel resin, where On is a L-amino
acid and
n=1 -12 can be any proteinogenic L-amino acid except methionine and cysteine.
The format of the library BL124 is:
O~-OZ-O3-O4-OS-O6-O7-ASp-Phe-Pro-Og-Og-O~0-0~1- Tentagel resin, where On is a
L-amino
acid and n=1 -11 can be any proteinogenic L-amino acid except methionine and
cysteine.
2. Screening the peptide bead libraries:
Recombinant factor XI from Heamatologic Technologies was purchased and
biotinylated according to standard laboratory protocols. Then 5 u1 of factor
XI (1,2 uM) and 1
u1 streptavidin-alkaline phosphatase (1 mg/ml, Sigma) were added to three
synthetic peptide
bead libraries, BL121, BL122 and BL124, respectively, and allowed to incubate
for about 2-3
hours. The incubation buffer was l5mM TRIS-HCI, pH=7,4, 0,15M NaCI, 0,5%
bovine
Serume Albumin (BSA) and 0,05% Tween20. After washing with washing buffer (M
TRIS-
HCI, pH=7,4, 0,15M NaCI, and 0,05% Tween20), BCIP and NBT were added in
colorbuffer
(50mM TRIS-HVI pH = 8,8, 0,15M NaCI, 0,05% Tween20 and 15 mM MgCl2) and
coloration
was allowed to proceed 30 min - 1,5 h.
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3. Sequence determination
Active blue beads were removed from the library and sequenced by the Edman
sequencer (Procise, Applied Biosystems).
RESULTS:
5 Specific factor XI and factor XI-like binding peptides found in library
BL121, BL122
andBL123 according to the invention include peptides comprising amino acid
sequences are
outlined below:
Sequences found in BL121
10 SEQ ID N0:03: SRWPWSVFPDFPD
SEQ ID N0:04: DVWDYVVFDDFPS
SEQ ID N0:05: QRWVPYDDFPSLRS
SEQ ID N0:06: RHFHVFPDFPFVH
SEQ ID N0:07: HHFPPFSHFPDLPQ
15 SEQ ID N0:08: RRLPLSRLPDFP
SEQ ID N0:09: HPFFRGYPDFPD
SEQ ID N0:10: HPWHLVYPDFPS
SEQ ID N0:11: HDWLVRWPDFPS
SEQ ID N0:12: SHFWRQWPDFSD
20 SEQ ID N0:13: PQLRWHDFPDFGS
SEQ ID N0:14: VVWRHWQDFDQFW
SEQ ID N0:15: VDWQWSRFDDFPS
SEQ ID N0:16: HPWFDDFPHLFQ
Sequences from libraryL122
B
25 SEQ ID N0:17: YKWIHHDDFPLV
SEQ ID N0:18: FDRKRVHPDFPH
SEQ ID N0:19: DVWDYVVFDDFPS
SEQ ID N0:20: QQPIQRFPDFP
SEQ ID N0:21: QAIFTRFPDFPN
30 SEQ ID N0:22: EWFPDFPEGSDG
SEQ ID N0:23: HTHAFPDFPPH
SEQ ID N0:24: LVKGFPDFPNHN
SEQ ID N0:25: GPFPYAYEDFPE
SEQ ID N0:26: FYLKTRYYDFPE
35 SEQ ID N0:27: FQARHTIGDFPA
SEQ ID N0:28: RIKDFPSDSNTV
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SEQ ID N0:29: IWESHKVIEDFP
SEQ ID N0:30: QWFSVSRYQDFD
SEQ ID N0:31: QKDFHWRILPDF
SEQ ID N0:32: KIVKFPHTFPDL
SEQ ID N0:33: HLYDFDLDNEY
SEQ ID N0:34: KTILGDVDFDI
SEQ ID NO:35: RQLHPFHHFHG
SEQ ID N0:36: RSWLRYGYGH
SEQ ID N0:37: FNWNNVDEYYDW
SEQ ID N0:38: DQWDWEDYDEAW
SEQ ID N0:39: YDIYDDYEIWA
Sequences found in BL124
SEQ ID N0:40: YPKHIYADFPSTRL
SEQ ID N0:41: YPRHIYPDFPTDTT
SEQ ID N0:42: YLKHAWPDFPKLQQ
SEQ ID N0:43: YVRHRFEDFPTALP
SEQ ID N0:44: FPWHKYEDFPSPRT
SEQ ID N0:45: QPAHRYPDFPRNNH
SEQ ID N0:46: LPKTRFLDFPHVSF
SEQ ID N0:47: LPPARYPDFPAAKK
SEQ ID N0:48: IPKNRFSDFPDAQG
SEQ ID NO:49: LPSFRFPDFPATKT
SEQ ID N0:50: RVLNRYPDFPTTNQ
SEQ ID N0:51: FFKKTYADFPTSQT
SEQ ID N0:52: IFKKTYEDFPRFVY
SEQ ID N0:53: VLHNKYDDFPRVKK
SEQ ID N0:54: KVKHRFNDFPVWGN
It is concluded that l FXI-binding peptides include those
usefu having a core amino
acid motif of Asp-Phe-Pro.
Example 6
The cells from mammalian cell culture was separated from the supernatant by
centrifugation or filtration. Benzamidine and EDTA was added to final
concentration 1 mM.
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Example 7
First cation-exchange chromatography using Obelix ST CIEX (cat no 11-0010)
A Obelix matrix was equilibrated with 5 column volumes (cv) of buffer A, and a
load
corresponding to 150m1 supernatant pr ml packed column was applied to the
column. The
column was washed with 4 cv of buffer A (30mM Tris pH 8,0) and then with 5 cv
of buffer A'
(50mM Tris 50%Glycerol $~oo pH 9,0) . Elution was then performed with 5 cv
buffer A"
(50mM Tris 50%Glycerol $~oa 1 M NaCI pH 9,0). Flowrate was 16 cv/h,
temperature was 0-
10°C. The column was regenerated with 1 M NaOH. Fractions were
collected from at about
50% of peak height, the first peak eluting is discarded but the next main peak
contains FXI.
Analysis of FXI polypeptide-containing fractions was performed by HPLC (vide
infra) using
C4 Jupiter Phenomonex cat no OOG-4167-EO, 4.6x250 mm and by SDS-PAGE on a
NUpage 4-12% Bis/Tris Gel (Invitrogen) with MOPS running buffer under
reductive
conditions. Benzamidine and EDTA were added (to 1 mM) to the fractions
containing FXI
polypeptide and kept at approx. 4°C in a refrigerator, or frozen at -
80°C, until further use.
Alternative to Obelix ST CIEX is Streamline Direct CST Amersham cat no 17-
5266-03. (Figure 4, preparative chromatograme)
Example 8
Hydrophobic interaction chromatography using Butyl Sepharose Hiah Performance
Hiah
Substitution (cat no 17-3100)
1,5 volume of a buffer containing 2M NaCI 40mM Tris pH 8 was added to the
combined fractions containing FXI polypeptide from Example 7, and the pH was
adjusted to
8,2 if not already between 8,0 and 8,4. A Butyl Sepharose High Performance
High
Substitution matrix was equilibrated with 3 cv of buffer B (1 M NaCI 20mM Tris
pH 8,0) and
a load corresponding to approximately 1 mg/ml was applied to the column. The
column was
then washed with 2 cv of buffer B and then subjected to gradient elution going
from buffer B
to 100% elution buffer B' (20mM Tris pH 8,0) over 20 cv followed by 2 cv of
100% elution
buffer B'. Flowrate was12 cv/h, temperature was 0-10°C. Fractions were
collected after
elution of approximately 10 cv and until 15 cv. Analysis of FXI polypeptide-
containing
fractions was performed by HPLC (vide infra) using C4 Jupiter Phenomonex cat
no OOG-
4167-EO, 4.6x250 mm and by SDS-PAGE on a NUpage 4-12% Bis/Tris Gel
(Invitrogen) with
MOPS running buffer under reductive conditions. A'/ volume of propylene glycol
was
immediately added to the pool of FXI polypeptide-containing fractions to a
final concentration
of 20% (v/v) propylene glycol, and the resulting pool was then kept at approx.
4°C, or frozen,
until further use
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Alternative to Butyl Sepharose High Performance High Substitution is Phenyl
Sepharose High Performanc High Substitution. This alternative matrix results
in later elution.
(Figure 5, preparative chromatograme)
Example 9
Hydroxyapatite chromatography using CHT Hydroxyapatite Type I BioRad cat no
157-0020)
The pH of the pool of FXI polypeptide-containing fractions from Example 8 was
adjusted to 6,0 and 1 volume water was added to a conductivity of below 20
mS/cm. A
Hydroxyapatite Type I 20pm matrix was equilibrated with 6 cv of buffer C, and
then a load
corresponding to 5 mg/ml gel was applied to the column. The column was then
washed with
15 cv of buffer C (20mM K-P04 pH 6,0), and a buffer containing 95% buffer C
and 5%
elution buffer C' (20mM K-P04 2M NaCI pH 6,0) was performed as a washing step.
A
gradient elution from 5%C' to 100%C' was performed and used to elute the FXI
polypeptide
in small fractions. The conductivity of the pool containing the FXI
polypeptide fractions was
about 60 mS/cm and the pH about 6,0. Analysis of FXI polypeptide-containing
fractions was
performed by HPLC (vide infra) using C4 Jupiter Phenomonex cat no OOG-4167-EO,
4.6x250 mm and by SDS-PAGE on a NUpage 4-12% Bis/Tris Gel (Invitrogen) with
MOPS
running buffer under reductive conditions. The HPLC purity is >97% and
concentration of FXI
is about 1,2mg/ml.
The FXI containing fraction of high purity was collected and Propylene Glycol
was
added to final 10% v/v and stored below -20°C. (Figure 6, preparative
chromatograme)
Example 10
HPLC Analysis Procedure
High-Performance Liquid Chromatography (HPLC; referred to in Examples 7-9,
above) was performed using C4 Jupiter Phenomonex cat no OOG-4167-EO, 4.6x250
mm
and employing buffers as follows:
Buffer I: 0,1 %TFA in H20
Buffer II: 0,07%TFA in CH3CN
Equilibration of the column was carried out using a mixture of 75% (v/v)
Buffer I with
25% (v/v) Buffer II for 5 minutes (flow rate 1 ml/min.).
Elution of the column took place using a gradient going from 75% Buffer I /
25%
Buffer II to 39% Buffer I / 61 % Buffer II over a period of 18 minutes (flow
rate 1 ml/min.).
Regeneration of the column was performed by washing with 100% Buffer II for 2
minutes. (flow rate 0.5 ml/min.). The detection wavelength employed was 214
nm.
Temperature was 50°C. Samples of from 2 to 50 ~,g were loaded onto the
column.
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59
All patents, patent applications, and literature references referred to herein
are
hereby incorporated by reference in their entirety.
Many variations of the present invention will suggest themselves to those
skilled in
the art in light of the above detailed description. Such obvious variations
are within the full
intended scope of the appended claims.
DEMANDES OU BREVETS VOLUMINEUX
LA PRESENTE PARTIE DE CETTE DEMANDE OU CE BREVETS
COMPRI~:ND PLUS D'UN TOME.
CECI EST L,E TOME 1 DE 2
NOTE: Pour les tomes additionels, veillez contacter 1e Bureau Canadien des
Brevets.
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