U.S. patent application number 11/661524 was filed with the patent office on 2008-07-24 for purification of factor xlll polypeptides from biological materials.
This patent application is currently assigned to Novc Nordisk HealthCare A/G. Invention is credited to Susanne Bang.
Application Number | 20080176789 11/661524 |
Document ID | / |
Family ID | 35197792 |
Filed Date | 2008-07-24 |
United States Patent
Application |
20080176789 |
Kind Code |
A1 |
Bang; Susanne |
July 24, 2008 |
Purification of Factor Xlll Polypeptides From Biological
Materials
Abstract
The invention relates to a method for purifying a factor XIII
polypeptide from a biological material, the method comprising
subjecting the material to sequential chromatography on an
anion-exchange matrix and a hydrophobic interaction matrix.
Inventors: |
Bang; Susanne; (Bagsvaerd,
DK) |
Correspondence
Address: |
NOVO NORDISK, INC.;INTELLECTUAL PROPERTY DEPARTMENT
100 COLLEGE ROAD WEST
PRINCETON
NJ
08540
US
|
Assignee: |
Novc Nordisk HealthCare A/G
Zurich
CH
|
Family ID: |
35197792 |
Appl. No.: |
11/661524 |
Filed: |
August 26, 2005 |
PCT Filed: |
August 26, 2005 |
PCT NO: |
PCT/EP2005/054207 |
371 Date: |
September 20, 2007 |
Current U.S.
Class: |
514/14.2 ;
530/417 |
Current CPC
Class: |
A61P 25/00 20180101;
C07K 14/755 20130101; A61K 38/45 20130101; A61P 7/04 20180101; A61P
7/00 20180101; A61P 9/10 20180101 |
Class at
Publication: |
514/12 ;
530/417 |
International
Class: |
A61K 38/16 20060101
A61K038/16; C07K 1/22 20060101 C07K001/22; A61P 7/00 20060101
A61P007/00 |
Foreign Application Data
Date |
Code |
Application Number |
Aug 27, 2004 |
DK |
PA 2004 01301 |
Claims
1. A method for purifying a factor XIII polypeptide from a
biological material comprising subjecting the material to
sequential chromatography on an anion-exchange matrix and a
hydrophobic interaction matrix wherein no precipitation step to
produce a factor XIII polypeptide-containing precipitate is
performed.
2. A method according to claim 1, wherein the factor XIII
polypeptide is a recombinant factor XIII.
3. A method according to claim 2, wherein the factor XIII
polypeptide is human factor XIII.
4. A method according to claim 2, wherein the factor XIII
polypeptide is a dimer of A subunits.
5. A method according to claim 4, wherein the factor XIII
polypeptide is a dimer of human A subunits.
6. A method according to claim 1, wherein the biological material
is a biological fluid.
7. A method according to claim 6, wherein the biological fluid is
the supernatant of a cell lysate.
8. A method according to claim 7, wherein the biological fluid is
the supernatant of a yeast cell lysate.
9. A method for purifying a factor XIII polypeptide from a
biological material comprising: (a) subjecting a biological
material comprising a factor XIII polypeptide to chromatography on
a first anion-exchange chromatographic material, said
chromatography comprising: (i) applying said biological material to
said first anion-exchange chromatographic material; (ii) eluting
unbound material from the first anion-exchange chromatographic
material with a buffer A comprising one or more stabilizing agents
selected from sugars, alcohols, and alditols which are capable of
increasing the stability of the factor XIII polypeptide, which
buffer A is suitable for eluting material not bound to the first
anion-exchange chromatographic material; and (iii) eluting said
factor XIII polypeptide from the first anion-exchange
chromatographic material by gradient-elution with buffer A', which
buffer A' is suitable for eluting said factor XIII polypeptide from
said first anion-exchange chromatographic material; and (b)
subjecting the eluate from step (iii), or a fluid prepared by use
of the eluate from step (iii), to chromatography using a
hydrophobic interaction chromatographic material, said
chromatography comprising: (iv) applying the eluate from step
(iii), or a fluid prepared by use of the eluate from step (iii), to
said hydrophobic interaction chromatographic material; (v) 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 (vi) eluting
said factor XIII polypeptide from said chromatographic material by
gradient-elution with buffer B', which buffer B' is suitable for
eluting factor XIII from said hydrophobic interaction
chromatographic material.
10. (canceled)
11. (canceled)
12. A method according to claim 9, wherein the stabilizing agent is
a sugar, a C.sub.3-C.sub.8-alcohol, or an alditol.
13. A method according to claim 12, wherein the stabilizing agent
is a polyalcohol.
14. A method according to claim 12, wherein the stabilizing agent
is selected from the group consisting of glycerol, propylene
glycol, propan-1,3-diol, propyl alcohol, and isopropyl alcohol.
15. A method according to claim 14, wherein the stabilizing agent
is selected from the group consisting of glycerol, propylene
glycol, and propan-1,3-diol.
16. A method according to claim 15, wherein said stabilizing agent
is present in a concentration of from about 5% (v/v) to about 50%
(v/v).
17. A method according to claim 16, wherein said stabilizing agent
is present in a concentration of from about 10% (v/v) to about 50%
(v/v).
18. A method according to claim 17, wherein said stabilizing agent
is present in a concentration of from about 10% (v/v) to about 20%
(v/v).
19. A method according to claim 18, wherein said stabilizing agent
is present in a concentration of about 10% (v/v).
20. A method according to claim 18, wherein said stabilizing agent
is present in a concentration of about 20% (v/v).
21. A method according to claim 9, wherein the pH of buffer A is
between about 6.5 and about 9.
22. A method according to claim 21, wherein the pH of buffer A is
between about 7 and about 9.
23. A method according to claim 22, wherein the pH of buffer A is
about 8.
24. A method according to claim 9, wherein buffer A has a
conductivity of less than about 2 mS/cm.
25. A method according to claim 9, wherein no precipitation to form
a crystalline precipitate of the factor XIII polypeptide is
performed between step (iii) and step (iv).
26. A method according to claim 9, wherein no precipitation step
using sodium acetate to form a crystalline precipitate of the
factor XIII polypeptide is performed between step (iii) and step
(iv).
27. A method according to claim 9, wherein the hydrophobic
interaction chromatographic material uses phenyl as the ligand.
28. A method according to claim 27, wherein the hydrophobic
interaction chromatographic material is Source.TM. 15Phe.
29. A method according to claim 27, wherein the hydrophobic
interaction chromatographic material is Phenyl Sepharose.TM. 6 Fast
Flow High Substitution.
30. A method according to claim 27, wherein the hydrophobic
interaction chromatographic material is Phenyl Sepharose.TM. High
Performance High Substitution.
31. A method according to claim 9, wherein the pH of buffer B is
from about 6 to about 8.
32. A method according to claim 31, wherein the pH of buffer B is
about 7.5.
33. A method according to claim 9, wherein buffer B has a
conductivity of more than 25 mS/cm.
34. A method according to claim 33, wherein buffer B has a
conductivity of at most 50 mS/cm.
35. A method according to claim 9, wherein the eluate from stage
(vi), or a fluid prepared by use of the eluate from stage (vi), 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 factor XIII polypeptide in an amount effective to
significantly improve the stability thereof, and/or (2) adjusting
the pH of the eluate from stage (vi), or a fluid prepared by use of
the eluate from stage (vi), to a pH between about 7 and about
8.
36. A method according to claim 35, wherein the stabilizing agent
used in step (1) is a sugar, an alcohol, or an alditol.
37. A method according to claim 36, wherein the stabilizing agent
used in step (1) is a sugar, a C.sub.3-C.sub.8-alcohol, or an
alditol.
38. A method according to claim 37, wherein the stabilizing agent
used in step (1) is a polyalcohol.
39. A method according to claim 37, wherein the stabilizing agent
used in step (1) is selected from the group consisting of glycerol,
propylene glycol, propan-1,3-diol, propyl alcohol, and isopropyl
alcohol.
40. A method according to claim 39, wherein the stabilizing agent
used in step (1) is selected from the group consisting of glycerol,
propylene glycol, and propan-1,3-diol.
41. A method according to claim 40, wherein the stabilizing agent
used in step (1) is added to a concentration of from about 5% (v/v)
to about 50% (v/v).
42. A method according to claim 41, wherein the stabilizing agent
used in step (1) is added to a concentration of from about 10%
(v/v) to about 50% (v/v).
43. A method according to claim 42, wherein the stabilizing agent
used in step (1) is added to a concentration of from about 10%
(v/v) to about 20% (v/v).
44. A method according to claim 35 wherein the pH of the eluate
from stage (vi), or of a fluid prepared by use of the eluate from
stage (vi), is adjusted in step (2) to a pH between about 7 and
about 8.
45. A method according to claim 44, wherein the pH of the eluate
from stage (vi), or of a fluid prepared by use of the eluate from
stage (vi), is adjusted in step (2) to a pH of about 7.5.
46. A method according to claim 9, wherein no precipitation step to
produce a factor XIII polypeptide-containing precipitate is
performed after step (vi).
47. A method according to claim 46, wherein the method further
comprises a step of subjecting the eluate from the hydrophobic
interaction chromatography material, or a material prepared by use
of the eluate from the hydrophobic interaction chromatography
material, to chromatography on a second anion-exchange
chromatography matrix.
48. A method according to claim 9, wherein the method further
comprises a step of: subjecting the eluate from stage (vi), or a
fluid prepared by use of the eluate from stage (vi), to
chromatography on a second anion-exchange chromatographic material,
said chromatography comprising: (vii) applying the eluate from
stage (vi), or a fluid prepared by use of the eluate from stage
(vi), to said second anion-exchange chromatographic material;
(viii) eluting unbound material from the second anion-exchange
chromatographic material with buffer C, which buffer C is suitable
for eluting material not bound to the second anion-exchange
chromatographic material; and (ix) eluting said factor XIII
polypeptide from the second anion-exchange chromatographic material
with buffer C', wherein buffer C' is suitable for eluting factor
XIII polypeptides which bind to the second anion-exchange
chromatographic material in step (viii).
49. A method according to claim 48, wherein buffer C and/or buffer
C' comprises one or more stabilizing agents which are capable of
increasing the stability of the factor XIII polypeptide.
50. A method according to claim 49, wherein buffer C and/or buffer
C' comprises a stabilizing agent, which stabilizing agent is a
sugar, an alcohol, or an alditol.
51. A method according to claim 50, wherein buffer C and/or buffer
C' comprises a stabilizing agent, which stabilizing agent is a
sugar, a C.sub.3-C.sub.8-alcohol, or an alditol.
52. A method according to claim 51, wherein buffer C and/or buffer
C' comprises a stabilizing agent, which stabilizing agent is a
polyalcohol.
53. A method according to claim 51, wherein buffer C and/or buffer
C' comprises a stabilizing agent selected from the group consisting
of glycerol, propylene glycol, propan-1,3-diol, propyl alcohol, and
isopropyl alcohol.
54. A method according to claim 53, wherein buffer C and/or buffer
C' comprises a stabilizing agent selected from the group consisting
of glycerol, propylene glycol, and propan-1,3-diol.
55. A method according to claim 54, wherein said stabilizing agent
is present in a concentration of from about 5% (v/v) to about 50%
(v/v).
56. A method according to claim 55, wherein said stabilizing agent
is present in a concentration of from about 10% (v/v) to about 50%
(v/v).
57. A method according to claim 56, wherein said stabilizing agent
is present in a concentration of from about 10% (v/v) to about 20%
(v/v).
58. A method according to claim 57, wherein said stabilizing agent
is present in a concentration of about 10% (v/v).
59. A method according to claim 57, wherein said stabilizing agent
is present in a concentration of about 20% (v/v).
60. A method according to claim 48, wherein buffer C and/or buffer
C' has a pH of about 7.5.
61. A method according to claim 48, wherein the conductivity of the
eluate from step (ix) containing the factor XIII polypeptide is
adjusted to about 10 mS/cm.
62. A method according to claim 48, wherein the pH of the eluate
from step (ix) containing the factor XIII polypeptide is adjusted
to about 7.5.
63. A method according to claim 54, wherein no precipitation step
to produce a factor XIII polypeptide-containing precipitate is
performed after step (ix).
64. A method according to claim 12, wherein no precipitation step
to produce a factor XIII polypeptide-containing precipitate is
performed.
65-71. (canceled)
72. A pharmaceutical composition comprising a factor XIII
polypeptide prepared by use a method according to claim 9.
73. A pharmaceutical composition according to claim 72, wherein the
pharmaceutical composition is an aqueous composition and comprises
20% (v/v) propylene glycol.
74. A pharmaceutical composition according to claim 72, wherein the
pharmaceutical composition is an aqueous composition and comprises
1.5% (w/v) sucrose and 3.5% (w/v) mannitol.
75. A method of reducing blood loss in a patient comprising
administering to the patient an amount of a composition according
to claim 72 that is effective to reduce blood loss.
76. A method of reducing the incidence of intraventricular
hemorrhage in a premature infant comprising administering to the
infant an amount of a composition according to claim 72 that is
effective to prevent hemorrhage.
77. A method of reducing surgery-related blood loss in a patient
during or after surgery comprising administering to the patient an
amount of a composition according to claim 72 that is effective to
reduce blood loss during or after surgery.
78. A method of treating hemophilia A comprising administering to a
hemophilia A patient an amount of a composition according to claim
72 that is effective for treating hemophilia A.
79. A method of treating hemophilia B comprising administering to a
hemophilia B patient an amount of a composition according to claim
72 that is effective for treating hemophilia B.
80. A method of treating a platelet disorder comprising
administering to a platelet disorder patient an amount of a
composition according to claim 72 that is effective for treating
platelet disorders.
81-88. (canceled)
89. A method according to claim 35, wherein no precipitation step
to produce a factor XIII polypeptide-containing precipitate is
performed after step (vi).
90. A method according to claim 15, wherein no precipitation step
to produce a factor XIII polypeptide-containing precipitate is
performed after step (vi).
Description
FIELD OF THE INVENTION
[0001] The present invention relates to methods of protein
purification from biological materials, especially the purification
of factor XIII and factor XIII polypeptides from biological
fluids.
BACKGROUND OF THE INVENTION
[0002] The blood coagulation factor XIII (FXIII), the fibrin
stabilising factor, is a transglutaminase that binds to and
cross-links fibrin monomers in the haemostatic plug thereby
providing a fibrin structure with increased mechanical strength and
resistance against fibrinolysis (see Ariens et al, Blood 100(3),
743-754 (2002)). Factor XIII is also known as "fibrinoligase" and
"fibrin stabilizing factor". When activated, factor XIIIa is able
to form intermolecular gamma-glutamyl-.epsilon.-lysine cross-links
between side chains of fibrin molecules and other substrates.
Factor XIII is found in plasma and in platelets. The enzyme exists
in plasma as a tetrameric zymogen consisting of two A-subunits
(also referred to as "a") and two B subunits (also referred to as
"b") (this tetrameric zymogen is designated A.sub.2B.sub.2 (also
referred to as "a.sub.2b.sub.2")) and in platelets as a zymogen
consisting of two A-subunits (this dimeric zymogen is designated
A.sub.2-dimer (also referred to as "a.sub.2-dimer")).
[0003] It has been described that factor XIII may be used for
treating bleeding episodes, in patients having a congenital factor
XIII deficiency as well as in patients not having a congenital
factor XIII deficiency, see for instance U.S. Pat. No. 5,114,916,
U.S. Pat. No. 5,607,917, WO 2002038167, WO 2002036155, WO
200267981, and WO 200267980.
[0004] U.S. Pat. No. 5,612,456 concerns the preparation of factor
XIII from biological fluids using an acetate precipitation step in
combination with anion-exchange chromatography and hydrophobic
interaction chromatography.
SUMMARY OF THE INVENTION
[0005] The present invention concerns a method for purifying a
factor XIII polypeptide from a biological material, the method
comprising subjecting the material to sequential chromatography on
an anion-exchange matrix and a hydrophobic interaction matrix. In
one embodiment, the method for purifying a factor XIII polypeptide
from a biological material does not comprise a precipitation step
to produce a factor XIII-containing precipitate.
[0006] The present invention also concerns a method for purifying a
factor XIII polypeptide from a biological material, the method
comprising the step of subjecting the biological material to
chromatography using a hydrophobic interaction chromatographic
material, wherein said hydrophobic interaction chromatographic
material is Phenyl Sepharose.TM. High Performance High Substitution
from Amersham.
[0007] The present invention also concerns the use of polypeptides
prepared by the above method for reducing blood loss.
DETAILED DESCRIPTION OF THE INVENTION
[0008] The present invention concerns a method for purifying a
factor XIII polypeptide from a biological material, the method
comprising subjecting the material to sequential chromatography on
an anion-exchange matrix and a hydrophobic interaction matrix.
[0009] In one embodiment, the present invention concerns a method
for purifying a factor XIII polypeptide from a biological material,
the method comprising the steps of:
(a) subjecting a biological material comprising a factor XIII
polypeptide to chromatography on a first anion-exchange
chromatographic material, said chromatography comprising: [0010]
(i) applying said biological material to said first anion-exchange
chromatographic material; [0011] (ii) eluting unbound material from
the first anion-exchange chromatographic material with a buffer A,
which buffer A is suitable for eluting material not bound to the
first anion-exchange chromatographic material; and [0012] (iii)
eluting said factor XIII polypeptide from the first anion-exchange
chromatographic material by gradient-elution with buffer A', which
buffer A' is suitable for eluting said factor XIII polypeptide from
said first anion-exchange chromatographic material; (b) subjecting
the eluate from step (iii) or a fluid prepared by use of the eluate
from step (iii) to chromatography using a hydrophobic interaction
chromatographic material, said chromatography comprising: [0013]
(iv) applying the eluate from step (iii) or a fluid prepared by use
of the eluate from step (iii) to said hydrophobic interaction
chromatographic material; [0014] (v) 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 [0015] (vi) eluting said
factor XIII polypeptide from said chromatographic material by
gradient-elution with buffer B', which buffer B' is suitable for
eluting factor XIII polypeptide from said hydrophobic interaction
chromatographic material.
[0016] Purification of a factor XIII polypeptide is the process of
increasing the concentration of the factor XIII polypeptide in a
sample in relation to other components of said sample, resulting in
an increase in the purity of the factor XIII polypeptide. It should
be understood that the concentration of a factor XIII polypeptide
in a sample in relation to other components of said sample is not
equivalent to the concentration of factor XIII polypeptide in the
sample. The increase in the purity of the factor XIII 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).
[0017] Factor XIII polypeptides include the complete factor XIII
zymogen tetramer, the A.sub.2B.sub.2 intermediate and factor XIIIa
as well as subunits thereof, including the A subunit and A.sub.2
dimers, as well as naturally occurring allelic variants of factor
XIII and genetically engineered variants of factor XIII that retain
at least part of the characteristic cross-linking activity of
factor XIII; an example of such a variant is the Val34Leu variant
of wild-type human factor XIII (i.e. a variant in which the Val
residue at position 34 in the amino acid sequence of wild-type
human factor XIII is replaced by a Leu residue). Derivatives and
fragments of such polypeptides, where the derivatives and fragments
have retained at least a significant part of the characteristic
cross-linking activity of factor XIII, are also encompassed. The
wild-type sequence of human factor XIII can be found in EP 268772
and EP 236978.
[0018] In one embodiment of the present invention, the factor XIII
polypeptide is a recombinant factor XIII. In one embodiment of the
present invention, the factor XIII polypeptide is human factor
XIII. In one embodiment of the present invention, the factor XIII
polypeptide is a dimer of A subunits. In one embodiment of the
present invention, the factor XIII polypeptide is a dimer of human
A subunits.
[0019] Biological material may be any material derived from or
containing cells, cell components or cell products. A biological
material may be a biological fluid.
[0020] 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.
[0021] The factor XIII polypeptides may be purified from a wide
variety of biological materials, including lysates, homogenizates
or extracts of cells which naturally produce a factor XIII
polypeptide, but also of cells which have been genetically modified
to produce a factor XIII polypeptide, such as yeast cells (for
instance Saccharomyces cerevisiae cells) transformed with DNA
coding for a factor XIII polypeptide.
[0022] The biological material may be treated by use of a number of
methods prior to application on the first anion-exchange
chromatographic material. Such methods include, but a not limited
to, centrifugation, lysis, homogenization, and high pressure
disruption. 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.
[0023] In one embodiment of the present invention, the factor XIII
polypeptide is purified from a cell culture, such as a yeast cell
culture, as described above. Prior to the chromatography in step
(a), the yeast cells may be lysed in a lysis buffer, and the
resulting supernatant may be subjected to disruption by high
pressure, such as for instance a pressure of about 2.0 kBar or
below, such as a pressure of from about 1.0 kBar to about 2.0 kBar,
such as at a pressure of about 1.5 kBar, optionally following a
homogenisation step. In one embodiment of the present invention,
the buffer used for lysing the cells is buffer A. The lysis buffer
may comprise protease inhibitors such as EDTA (ethylenediamine
tetraacetic acid), phenanthroline, pepstatin and particularly PMSF
(phenyl methyl sulfonyl fluoride), but other commercially available
protease inhibitors may also be used.
[0024] 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.
[0025] Prior to the chromatography in step (a), buffer A may be
added to the biological material, for instance in the amount of
about one volume, in the amount of about two volumes, in the amount
of about three volumes, or in an amount of more than about three
volumes. Buffer A may also be used to prepare the biological fluid,
for instance by resuspending a pellet of cells in buffer A. In one
embodiment, buffer A is used for lysing the cells.
[0026] The biological fluid is centrifuged, and may optionally also
be filtered, prior to the chromatography in step (a).
[0027] 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 HCl or 1 M NaOH or by other means known in the art.
[0028] The conductivity of the biological fluid may be adjusted to
the conductivity of buffer A prior the chromatography in step (a),
for instance by adding buffer A to the biological fluid or by other
means known in the art.
[0029] The first anion-exchange chromatographic material may be any
anion-exchange chromatographic material known in the art which is
capable of binding a factor XIII polypeptide under one set of
conditions and releasing it under a different set of conditions,
such as an anion-exchange chromatographic material comprising a
quaternary ammonium ion. Further non-limiting examples of
anion-exchange chromatographic materials include derivatised
dextrans, agarose, cellulose, polyacrylamide, and specialty
silicas, such as PEI, DEAE, QAE and Q derivatives. Suitable
anion-exchange chromatographic material may be identified by
subjecting a biological fluid comprising factor XIII polypeptide to
chromatography on the anion-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) 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
anion-exchange chromatographic materials include, but are not
limited to Source.TM. 30Q (Amersham Biosciences), Toyopearl.RTM.
SuperQ-650M (Tosoh Bioscience), Q-sepharose.TM. (Amersham
Biosciences) and DEAE Fast-Flow Sepharose (Amersham Biosciences).
The first anion-exchange chromatographic material may be
pre-equilibrated with buffer A prior to application of the
biological material.
[0030] In one embodiment of the present invention, buffer A and/or
buffer A' comprises one or more stabilizing agents which are
capable of increasing the physical and/or chemical stability of the
factor XIII polypeptide.
[0031] The term "physical stability" of the factor XIII polypeptide
as used herein refers to the potential tendency of the protein to
form biologically inactive and/or insoluble aggregates 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 factor XIII 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
formulations may be performed in a sharp focused light with a dark
background. The turbidity of the formulation may be characterized
by a visual score ranking the degree of turbidity, for instance on
a scale from 0 to 3 (a formulation showing no turbidity then
corresponding to a visual score 0, and a formulation showing visual
turbidity in daylight corresponding to visual score 3). A
formulation is classified as physically unstable with respect to
protein aggregation when it shows visual turbidity in daylight.
Alternatively, the turbidity of the formulation 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 (OD.sub.405). Physical stability of the
aqueous protein formulations 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.
[0032] 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, 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.
[0033] The term "chemical stability" of the factor XIII polypeptide
when present in buffer A as 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 formulation, 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 factor XIII
polypeptide when present in buffer A 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).
[0034] Any agent which is capable of significantly improving the
physical and/or chemical stability of factor XIII polypeptide when
present in buffer A (e.g. as determined by measuring turbidity at
OD.sub.405 over a period of time) may be used as a stabilizing
agent in buffer A or buffer A'.
[0035] An agent suitable for use as stabilizing agent in buffer A
may, for instance, be a salt (e.g. sodium chloride), a sugar, an
alcohol (such as an C.sub.3-C.sub.8 alcohol), an alditol, an amino
acid (e.g. glycine, L-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
HOCH.sub.2--[CH(OH)], --CH.sub.2OH, where n is 0, 1, 2, 3 . . .
etc. 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 and improves the physical stability of a factor
XIII polypeptide in solution. In this respect, reference is made to
Remington: The Science and Practice of Pharmacy, 19th edition,
1995.
[0036] In one embodiment of the present invention, buffer A
comprises one or more stabilizing agents of the polyalcohol
type.
[0037] In one embodiment of the present invention, buffer A
comprises 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). In one embodiment of the present
invention, buffer A comprises one or more stabilizing agents
selected from the group consisting of glycerol, propylene glycol
and propan-1,3-diol. In one embodiment of the present invention,
buffer A comprises propylene glycol.
[0038] In a further embodiment of the present invention, when the
stabilizing agent in buffer A 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
in buffer A 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 in buffer A 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 in buffer A 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
in buffer A is present in a concentration of about 20% (v/v).
[0039] Buffer A may comprise protease inhibitors such as EDTA
(ethylenediamine tetraacetic acid), phenanthroline, pepstatin and
particularly PMSF (phenyl methyl sulfonyl fluoride), but other
commercially available protease inhibitors may also be used.
[0040] 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.
[0041] In one embodiment of the present invention, the conductivity
of buffer A is less than about 2 mS/cm.
[0042] Buffer A' is used for the elution of the factor XIII
polypeptide by gradient-elution. Gradient elution means that the
composition of buffer A' 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 (ii), in this case buffer 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 is then increased during the course of elution. 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 anion-exchange chromatographic material, it is customary to add
a salt, for instance NaCl, to buffer A, creating buffer A', and
then increase the concentration of the salt until at least the
majority of the bound factor XIII polypeptide is eluted. The
determination of which fractions containing factor XIII polypeptide
to pool for further processing, for instance to exclude undesired
impurities eluting at the beginning or the end of the factor XIII
polypeptide elution, is within the knowledge of a person skilled in
the art. Likewise, the general art of performing an anion-exchange
chromatography with regard to for instance pre-equilibration,
elution time, washing, reconstitution of the anion-exchange
chromatographic material etc is well-known.
[0043] After eluting the factor XIII polypeptide in step (iii), the
eluate containing the factor XIII polypeptides is taken to step
(iv) without an intervening step involving precipitation of factor
XIII polypeptide. It may be that some factor XIII polypeptide
precipitates depending on the handling and treatment of the eluate,
however, no steps to intentionally precipitate a factor XIII
polypeptide should be taken. Other intervening steps may be
contemplated. The eluate may also be kept at, for instance,
4.degree. C. for 24 hours or longer, or at, for instance,
-80.degree. C.
[0044] 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 factor
XIII 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 Amberchrom.TM.
CG 71 (Tosoh Bioscience), Phenyl Sepharose.TM. High Performance
(Amersham, cat no 17-1082), Phenyl Sepharose.TM. 6 Fast Flow High
Substitution (Amersham, cat no 17-0973), Toyopearl.RTM. Butyl 650
(Tosoh Bioscience), Toyopearl.RTM. Phenyl (Tosoh Bioscience),
Source.TM. 15Phe (Amersham, cat no 17-0147), Butyl Sepharose.TM.
High Performance High Substitution (Amersham, cat no 17-3100),
Octyl-Sepharose.TM. (Amersham, cat no 17-0946) and Phenyl
Sepharose.TM. High Performance High Substitution (Amersham), and
the like. In one embodiment of the present invention, the
hydrophobic interaction chromatographic material uses phenyl as a
ligand.
[0045] Buffer B may be added to the eluate from stage (iii) or a
fluid prepared by use of the eluate from stage (iii) prior to the
chromatography in step (b) in an amount of about one to three
volumes or more, or a concentrated version of buffer B, comprising
the same ingredients as buffer B, but in, e.g., four times the
concentration, is added to the eluate from stage (iii) or a fluid
prepared by use of the eluate from stage (iii) in an amount
corresponding to the strength of the concentrated buffer (a
twice-concentrated buffer is added in the amount of one volume).
The pH is then adjusted to the pH of buffer B.
[0046] Buffer B may comprise protease inhibitors such as EDTA
(ethylenediamine tetraacetic acid), phenanthroline, pepstatin and
particularly PMSF (phenyl methyl sulfonyl fluoride), but other
commercially available protease inhibitors may also be used.
[0047] Buffer B may have a pH from about 6 to about 8, for instance
about 7.5. In one embodiment of the present invention, buffer B has
a conductivity of more than 25 mS/cm. In another embodiment of the
invention, buffer B has a conductivity of at most 50 mS/cm. This
may be achieved, for example, by use of a phosphate buffer or by
other means known in the art. In one embodiment of the present
invention, the conductivity of the eluate from step (iii), or of a
fluid prepared by use of the eluate from step (iii), is adjusted to
a conductivity of at least about 25 mS/cm.
[0048] Buffer B' is used for the elution of the factor XIII
polypeptide by gradient elution. In gradient 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 (v), 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 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 factor XIII polypeptide is eluted. The
determination of which fractions containing factor XIII polypeptide
to pool for further processing, e.g. in order to exclude undesired
impurities eluting at the beginning or the end of the factor XIII
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.
[0049] In one embodiment of the present invention, the eluate from
stage (vi) or a fluid prepared by use of the eluate from stage (vi)
is treated by use of a method comprising a step of [0050] (1)
addition of one or more stabilizing agents which are capable of
increasing the stability of the factor XIII polypeptide in an
amount effective to significantly improve the stability thereof,
and/or [0051] (2) adjusting the pH of the eluate from stage (vi),
or a fluid prepared by use of the eluate from stage (vi), to a pH
between about 7 and about 8.
[0052] These steps, 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 critical. The
person skilled in the art will be able to determine how and when to
perform these steps.
[0053] The stabilizing agent mentioned in step (1) should be
capable of increasing the physical and/or chemical stability, as
described above, of the factor XIII polypeptide. Any agent which is
capable of significantly improving the physical and/or chemical
stability of factor XIII polypeptide (e.g. as determined by
measuring turbidity at OD.sub.405 over a period of time) may be
used as a stabilizing agent in step (1).
[0054] An agent suitable for use as stabilizing agent in step (1)
may, for instance, be a salt (e.g. sodium chloride), a sugar, an
alcohol (such as an C.sub.3-C.sub.8 alcohol), an alditol, an amino
acid (e.g. glycine, L-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,
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 factor
XIII polypeptide in solution. In this respect, reference is made to
Remington: The Science and Practice of Pharmacy, 19th edition,
1995.
[0055] In one embodiment of the present invention, the stabilizing
agent used in step (1) is a polyalcohol.
[0056] In one embodiment of the present invention, the stabilizing
agent used in step (1) is 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). In one embodiment of the present invention, the
stabilizing agent used in step (1) is selected from the group
consisting of glycerol, propylene glycol and propan-1,3-diol. In
one embodiment of the present invention, the stabilizing agent used
in step (1) is propylene glycol.
[0057] In a further embodiment of the present invention, when the
stabilizing agent used in step (1) is a liquid alcohol or liquid
polyalcohol [such as, e.g., glycerol, propylene glycol,
propan-1,3-diol, propyl alcohol or isopropyl alcohol], it is added
to 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 used in step (1) is added to 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 used in step (1) is added to 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 used in step (1) is added to a
concentration of about 10% (v/v). In a still further embodiment, a
stabilizing agent of the liquid alcohol or liquid polyalcohol type
used in step (1) is added to a concentration of about 20%
(v/v).
[0058] In one embodiment of the present invention, the stabilizing
agent(s) used in step (1) is/are the same as the stabilizing
agent(s) used in buffer A. In a further embodiment of the present
invention, the stabilizing agent(s) is/are added to a concentration
similar to the concentration of the stabilizing agent(s) used in
buffer A.
[0059] In one embodiment of the present invention, the pH in step
(2) is adjusted to about 7.5.
[0060] After eluting the factor XIII polypeptide in step (vi), the
eluate containing the factor XIII polypeptide should not be
subjected to a precipitation step. It is possible that some factor
XIII polypeptide precipitates, depending on the handling and
further treatment of the eluate, but no steps to intentionally
precipitate a factor XIII polypeptide should be taken. Other
intervening steps may be contemplated. The eluate may also be kept
at, e.g., 4.degree. C. for 24 hours or longer, or at, e.g.,
-80.degree. C.
[0061] In one embodiment of the present invention, the method
further comprises a step of subjecting the eluate from stage (vi),
or a fluid prepared by use of the eluate from stage (vi), to
chromatography on a second anion-exchange chromatographic material,
said chromatography comprising: [0062] (vii) applying the eluate
from stage (vi), or a fluid prepared by use of the eluate from
stage (vi), to said second anion-exchange chromatographic material;
[0063] (viii) eluting unbound material from the second
anion-exchange chromatographic material with buffer C, which buffer
C is suitable for eluting material not bound to the second
anion-exchange chromatographic material; and [0064] (ix) eluting
said factor XIII polypeptide from the second anion-exchange
chromatographic material with buffer C', which buffer C' is
suitable for eluting factor XIII polypeptides which bind to the
second anion-exchange chromatographic material in step (viii).
[0065] A fluid prepared by use of the eluate from stage (vi) may,
for instance, be prepared by use of a method comprising one or both
of steps (1) and (2) as described above.
[0066] In one embodiment of the present invention, the conductivity
of the eluate from stage (vi), or a fluid prepared by use of the
eluate from stage (vi), is adjusted to less than about 2 mS/cm.
[0067] The components of buffer C and buffer C' may be chosen with
a view to the desired final pharmaceutical composition of the
factor XIII polypeptide. Such considerations are within the
knowledge of a person skilled in the art.
[0068] 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 factor XIII polypeptide. Any agent which
is capable of significantly improving the physical and/or chemical
stability of factor XIII polypeptide when present in buffer C (e.g.
as determined by measuring turbidity at OD.sub.405 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 C.sub.3-C.sub.8 alcohol), an alditol, an amino acid
(e.g. glycine, L-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, 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 factor
XIII polypeptide in solution. In this respect, reference is made to
Remington: The Science and Practice of Pharmacy, 19th edition,
1995.
[0069] In one embodiment of the present invention, buffer C
comprises one or more stabilizing agents of the polyalcohol
type.
[0070] In one embodiment of the present invention, buffer C
comprises 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). In one embodiment of the present
invention, buffer C comprises one or more stabilizing agents
selected from the group consisting of glycerol, propylene glycol
and propan-1,3-diol. In one embodiment of the present invention,
buffer C comprises propylene glycol.
[0071] In a further embodiment of the present invention, when the
stabilizing agent in buffer C is a liquid alcohol or liquid
polyalcohol [such as, e.g., glycerol, propylene glycol,
propan-1,3-diol, propyl alcohol or isopropyl alcohol], 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 in buffer C 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 used in buffer C 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 in buffer C 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 in buffer C
is present in a concentration of about 20% (v/v).
[0072] In one embodiment of the present invention, buffer C
comprises the same stabilizing agent(s) as used in buffer A. In a
further embodiment of the present invention, buffer C comprises the
stabilizing agent(s) in a concentration similar to the
concentration of the stabilizing agent(s) in buffer A.
[0073] If a lyophilized formulation of a factor XIII polypeptide is
considered, then buffer C and/or buffer C' should comprise, for
example, sucrose or mannitol or one or more other ingredients
suitable for lyophilization instead of, for example, propylene
glycol.
[0074] Buffer C' is used for the elution of the factor XIII
polypeptide by gradient elution, wherein the composition of buffer
C' is changed during the course of elution. Buffer C' is typically
derived from the buffer used for washing in step (viii), in this
case buffer C, in that the concentration of one or more of the
components of the latter buffer is increased or decreased during
the course of elution, or a new component is added to the latter
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 an anion-exchange
chromatographic material, buffer C' is typically derived from
buffer C by addition thereto of a salt, e.g. NaCl, the
concentration of the salt then being increased until at least a
major portion of the bound factor XIII polypeptide is eluted. The
determination of which fractions containing factor XIII polypeptide
to pool for further processing, e.g. in order to exclude undesired
impurities eluting at the beginning or the end of the factor XIII
polypeptide elution, is within the knowledge of a person skilled in
the art. Likewise, the general art of performing an anion-exchange
chromatography with regard to, e.g., pre-equilibration, elution
time, washing, reconstitution of the anion-exchange chromatographic
material, etc., is well known.
[0075] The conductivity and pH, as well as other parameters of the
eluate comprising factor XIII polypeptide, may be adjusted after
elution by means known in the art.
[0076] In one embodiment of the present invention, the method
further comprises a step of subjecting the eluate from stage (vi),
or a fluid prepared by use of the eluate from stage (vi), to
ultrafiltration in a manner known in the art.
[0077] In one embodiment of the present invention, the method
further comprises a step of subjecting the eluate from stage (vi),
or a fluid prepared by use of the eluate from stage (vi), to
diafiltration in a manner known in the art.
[0078] In one embodiment of a method according to the present
invention, the method does not comprise a precipitation step to
produce a factor XIII polypeptide-containing precipitate. It is
possible that some factor XIII polypeptide precipitates, depending
on the handling and treatment of the fluid containing the factor
XIII polypeptide, but no steps to intentionally precipitate a
factor XIII polypeptide should be taken.
[0079] The present invention provides a pharmaceutical composition
comprising a factor XIII polypeptide prepared by use of a method
according to the present invention. In one embodiment, such a
pharmaceutical composition has a pH from about 2.0 to about 10.0.
The pharmaceutical composition may further comprise a buffer
system, preservative(s), tonicity-adjusting agent(s), chelating
agent(s), stabilizer(s) and/or surfactant(s). In one embodiment of
the present invention the pharmaceutical composition is an aqueous
formulation, i.e. a formulation comprising liquid water. Such a
formulation is typically a solution or a suspension. In a further
embodiment of the present invention the pharmaceutical composition
is an aqueous solution. The term "aqueous formulation" as employed
in the context of the present invention refers to a formulation
comprising at least 50% by weight (w/w) of water. Likewise, the
term "aqueous solution" refers to a solution comprising at least
50% (w/w) water, and the term "aqueous suspension" refers to a
suspension comprising at least 50% (w/w) water.
[0080] In one embodiment, the factor XIII polypeptide in a
pharmaceutical composition of the invention in the form of an
aqueous formulation is present in a concentration from about 0.2
mg/ml to about 30 mg/ml, such as from about 0.2 mg/ml to about 10
mg/ml, e.g. from about 0.5 mg/ml to about 10 mg/ml, for instance
from about 1 mg/ml to about 10 mg/ml.
[0081] In one embodiment, the pharmaceutical composition is a dried
formulation (e.g. a freeze-dried or spray-dried formulation)
intended for reconstitution by the physician or the patient by
addition of solvents and/or diluents prior to use.
[0082] In one embodiment, the present invention relates to a
pharmaceutical composition comprising an aqueous solution of a
factor XIII polypeptide prepared by use of a method according to
the present invention, together with a buffer, wherein said
formulation has a pH from about 2.0 to about 10.0.
[0083] In one embodiment of the present invention the pH of the
formulation is selected from the list consisting of 2.0, 2.1, 2.2,
2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5,
3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8,
4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1,
6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4,
7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7,
8.8, 8.9, 9.0, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, and
10.0.
[0084] In a further embodiment of the present invention the buffer
is selected from the group consisting of 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.
[0085] In one embodiment of the present invention the formulation
further comprises a pharmaceutically acceptable preservative. In a
further embodiment of the present invention the preservative is
selected from the group consisting of 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) 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
well-known to the skilled person (see, e.g., Remington: The Science
and Practice of Pharmacy, 19th edition, 1995).
[0086] In one embodiment of the present invention the formulation
further comprises an tonicity-adjusting agent (normally
incorporated for the purpose of rendering the formulation
substantially isotonic). In a further embodiment of the present
invention the tonicity-adjusting agent is selected among salts
(e.g. sodium chloride), sugars, alcohols (such as C.sub.3-C.sub.8
alcohols), alditols, amino acids (e.g. glycine, L-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.
[0087] 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 another embodiment of the present invention, the
tonicity-adjusting agent is present in a concentration of from
about 1 mg/ml to about 7 mg/ml. In another further embodiment of
the present invention, the tonicity-adjusting agent is present in a
concentration of from about 8 mg/ml to about 24 mg/ml. In another
further embodiment of the present invention, the tonicity-adjusting
agent is present in a concentration of from about 25 mg/ml to about
50 mg/ml. The use of an tonicity-adjusting agent in pharmaceutical
compositions is well-known to the skilled person (see, e.g.,
Remington: The Science and Practice of Pharmacy, 19th edition,
1995).
[0088] In one embodiment of the present invention the formulation
further comprises a chelating agent. In a further embodiment of the
present invention the chelating agent is selected from salts of
EDTA, citric acid and aspartic acid, and mixtures thereof. In a
further embodiment of the present invention the chelating agent is
present in a concentration from 0.1 mg/ml to 5 mg/ml. In one
further embodiment of the present invention the chelating agent is
present in a concentration from 0.1 mg/ml to 2 mg/ml. In another
further embodiment of the present invention the chelating agent is
present in a concentration from 2 mg/ml to 5 mg/ml.
[0089] The use of a chelating agent in pharmaceutical compositions
is well-known to the skilled person (see, e.g., Remington: The
Science and Practice of Pharmacy, 19th edition, 1995).
[0090] The pharmaceutical compositions of the present invention
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 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 Spray-Drying
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 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, such as blockage of tubing, membranes or
pumps when the polypeptide-containing pharmaceutical composition is
administered using an infusion system.
[0091] 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 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 those carrying a charged side chain, such as
arginine, lysine, aspartic acid or glutamic acid. Any stereoisomer
(i.e., L or D) of a particular amino acid (methionine, histidine,
arginine, lysine, isoleucine, aspartic acid, tryptophan, threonine
and mixtures thereof), or combinations of these stereoisomers or
glycine or an organic base such as, but not limited to, imidazole,
may be present in the pharmaceutical compositions of the present
invention so long as the particular amino acid or organic base is
present either in its free base form or its salt form. In one
embodiment the L-stereoisomer of an amino acid 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
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.
[0092] In one embodiment of the present invention the amino acids
or amino acid analogues are used in a concentration which is
sufficient to prevent or delay aggregation of the protein.
[0093] In one embodiment, a pharmaceutical composition of the
present invention 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 XIII 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 or D 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
molar ratio of added methionine to methionine residues ranges from
about 1:1 to about 1000:1, such as 10:1 to about 100:1.
[0094] In one embodiment of the present invention the
pharmaceutical composition further comprises a stabilizer. The use
of a stabilizer in pharmaceutical compositions is well known to the
skilled person (see, e.g., Remington: The Science and Practice of
Pharmacy, 19th edition, 1995). In one embodiment of the present
invention the pharmaceutical composition comprises a stabilizer
selected from high-molecular-weight polymers or from
low-molecular-weight compounds. In a further embodiment, the
stabilizer is selected from 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).
[0095] A pharmaceutical composition of the present invention may
also comprise additional stabilizing agents which further enhance
stability of a therapeutically active polypeptide therein.
Stabilizing agents of particular interest in relation to the
present invention include, but are not limited to, methionine and
EDTA, which protect the polypeptide against methionine oxidation,
and a nonionic surfactant, which protects the polypeptide against
aggregation associated with freeze-thawing or mechanical
shearing.
[0096] In one embodiment of the present invention the
pharmaceutical composition or formulation further comprises a
surfactant. In a further embodiment the surfactant is selected from
detergents, ethoxylated castor oil, polyglycolyzed glycerides,
acetylated monoglycerides, sorbitan fatty acid esters,
polyoxypropylene-polyoxyethylene block polymers (e.g. poloxamers
such as Pluronic.RTM. 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, 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. C.sub.6-C.sub.12 fatty acids (such as
oleic acid or caprylic acid)] and salts thereof, acylcarnitines and
derivatives thereof, N.sup..alpha.-acylated derivatives of lysine,
arginine and histidine, side-chain acylated derivatives of lysine
and arginine, N.sup..alpha.-acylated derivatives of dipeptides
comprising any combination of lysine, arginine and histidine and a
neutral or acidic amino acid, N.sup..alpha.-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 [749]-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 .beta.-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.
[0097] The use of a surfactant in pharmaceutical compositions is
well-known to the skilled person (see, e.g., Remington: The Science
and Practice of Pharmacy, 19th edition, 1995).
[0098] Other ingredients may also be incorporated in a
pharmaceutical composition of the present invention. Such
additional ingredients may include wetting agents, emulsifiers,
antioxidants, bulking agents, tonicity modifiers
(tonicity-adjusting agents), chelating agents, metal ions,
oleaginous vehicles, proteins (e.g. human serum albumin, gelatin or
other proteins) and zwitterionic substances (e.g. betaine, taurine
or an amino acid such as arginine, glycine, lysine or histidine).
Such additional ingredients should not, of course, adversely affect
the overall stability of the pharmaceutical composition of the
present invention.
[0099] Pharmaceutical compositions containing a factor XIII
polypeptide prepared by use of a method according to the present
invention according to the present invention may be administered to
a patient in need of such treatment by several different routes,
e.g. topically (such as by application to the skin or to a mucous
membrane), by routes which bypass absorption (such as
administration in an artery, in a vein or in the heart), and by
routes which involve absorption (such as by administration in the
skin, beneath the skin, in muscle or in the abdomen).
[0100] Administration of pharmaceutical compositions according to
the present invention to patients in need thereof may be via
various routes of administration, e.g. lingual, sublingual, buccal,
oral, in the stomach or intestine, nasal, pulmonary (e.g. via the
bronchioles and alveoli or both), epidermal, dermal, transdermal,
vaginal, rectal, ocular (e.g. via the conjunctiva), urethral or
parenteral.
[0101] 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, 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 situ precipitating or in
situ crystallizing), infusion solution, or as implants.
[0102] 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 XIII 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), poly(vinyl
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.
[0103] Pharmaceutical compositions comprising a factor XIII
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.
[0104] Pharmaceutical compositions comprising a factor XIII
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 XIII 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,
[0105] Methods for producing controlled release systems useful for
pharmaceutical compositions comprising a factor XIII 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 Drugs and the
Pharmaceutical Sciences vol. 99: Protein Formulation and Delivery
(MacNally, E. J., ed. Marcel Dekker, New York, 2000).
[0106] 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 XIII 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 XIII 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.
[0107] In one embodiment of the present invention, a pharmaceutical
composition comprising a factor XIII 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.
[0108] In another embodiment of the present invention, a
pharmaceutical composition comprising a factor XIII 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.
[0109] In a further embodiment of the present invention, a
pharmaceutical composition comprising a factor XIII 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.
[0110] In an still further embodiment of the present invention, a
pharmaceutical composition comprising a factor XIII 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.
[0111] The present invention also relates to a method for treating
bleeding episodes, which method comprises administration of a
pharmaceutical composition comprising an effective amount of a
factor XIII polypeptide prepared by use of a method according to
the present invention to a subject in need thereof.
[0112] In another aspect, the present invention relates to a method
for reducing clofting time in a subject, which method comprises
administration of a pharmaceutical composition comprising an
effective amount of a factor XIII polypeptide prepared by use of a
method according to the present invention to a subject in need
thereof.
[0113] In a further aspect, the present invention relates to a
method for prolonging the clot lysis time in mammalian plasma,
which method comprises administration of a pharmaceutical
composition comprising an effective amount of a factor XIII
polypeptide prepared by use of a method according to the present
invention to a subject in need thereof.
[0114] In another aspect, the present invention relates to a method
for increasing clot strength in mammalian plasma, which method
comprises administration of a pharmaceutical composition comprising
an effective amount of a factor XIII polypeptide prepared by use of
a method according to the present invention to a subject in need
thereof. In a further aspect, the present invention relates to a
method for enhancing fibrin clot formation in mammalian plasma,
which method comprises administration of a pharmaceutical
composition comprising an effective amount of a factor XIII
polypeptide prepared by use of a method according to the present
invention to a subject in need thereof.
[0115] In another aspect, the present invention relates to a method
for prevention of intraventricular haemorrhage in premature
infants, which method comprises administration of a pharmaceutical
composition comprising an effective amount of a factor XIII
polypeptide prepared by use of a method according to the present
invention to a subject in need thereof.
[0116] In a further aspect, the present invention relates to a
method for reducing surgery-related blood loss in a subject during
or after surgery, which method comprises administration of a
pharmaceutical composition comprising an effective amount of a
factor XIII polypeptide prepared by use of a method according to
the present invention to a subject in need thereof.
[0117] In another aspect, the present invention relates to a method
for treating hemophilia A, which method comprises administration of
a pharmaceutical composition comprising an effective amount of a
factor XIII polypeptide prepared by use of a method according to
the present invention to a subject in need thereof.
[0118] In a further aspect, the present invention relates to a
method for treating hemophilia B, which method comprises
administration of a pharmaceutical composition comprising an
effective amount of a factor XIII polypeptide prepared by use of a
method according to the present invention to a subject in need
thereof.
[0119] In another aspect, the present invention relates to a method
for treating platelet disorders, which method comprises
administration of a pharmaceutical composition comprising an
effective amount of a factor XIII polypeptide prepared by use of a
method according to the present invention to a subject in need
thereof.
[0120] In a further aspect, the present invention relates to a
method for treating von Willebrand's disease, which method
comprises administration of a pharmaceutical composition comprising
an effective amount of a factor XIII polypeptide prepared by use of
a method according to the present invention to a subject in need
thereof.
[0121] In a further embodiment of these methods, a factor XIII
polypeptide prepared by use of a method according to the present
invention is administered in combination with an effective amount
of a factor VIIa polypeptide as described in WO200185198. In a
further embodiment, the factor VIIa polypeptide and the factor XIII
polypeptide prepared by use of a method according to the present
invention are the sole active agents administered to the subject
for the indicated treatment. In one embodiment, the factor XIII
polypeptide prepared by use of a method according to the present
invention and the factor VIIa polypeptide are administered
simultaneously and in one-dosage form. In another embodiment, the
factor XIII polypeptide prepared by use of a method according to
the present invention and the factor VIIa polypeptide are
administered sequentially. In a further embodiment, the factor XIII
polypeptide prepared by use of a method according to the present
invention and the factor VIIa polypeptide are administered within
about 1-2 hours of each other, for example within 30 minutes of
each other, for instance within 10 minutes of each other. The
factor VIIa polypeptide and the factor XIII polypeptide may be
provided, e.g., in the form of a kit comprising a factor XIII
polypeptide prepared by use of a method according to the present
invention in a first unit-dosage form and a factor VIIa polypeptide
in a second unit-dosage form. In one embodiment, the effective
amount of factor XIII polypeptide is from 0.05 mg/day to 500 mg/day
(for a subject weighing 70 kg). In one embodiment, the effective
amount of factor VIIa polypeptide is from 0.05 mg/day to 500 mg/day
(70-kg subject).
[0122] In one embodiment, the subject to be treated is a human; in
another embodiment, the subject has an impaired thrombin
production; in one embodiment, the subject has a lowered plasma
concentration of fibrinogen (e.g., in the case of a
multi-transfused subject).
[0123] In the context of the present invention, an "effective
amount" of a factor VIIa polypeptide and an "effective amount" of a
factor XIII polypeptide are defined as the amount of factor VIIa
polypeptide and factor XIII polypeptide, respectively, that is
sufficient to prevent or reduce bleeding or blood loss, so as to
cure, alleviate or partially arrest the disease and its
complications alone or in combination with other administered
therapeutic agents.
[0124] The present invention also relates to the use of a factor
XIII polypeptide prepared by use of a method according to the
present invention for treating bleeding episodes.
[0125] In another aspect, the present invention relates to the use
of a factor XIII polypeptide prepared by use of a method according
to the present invention for reducing clotting time in a
subject.
[0126] In a further aspect, the present invention relates to the
use of a factor XIII polypeptide prepared by use of a method
according to the present invention for prolonging the clot lysis
time in mammalian plasma.
[0127] In another aspect, the present invention relates to the use
of a factor XIII polypeptide prepared by use of a method according
to the present invention for increasing clot strength in mammalian
plasma.
[0128] In a further aspect, the present invention relates to the
use of a factor XIII polypeptide prepared by use of a method
according to the present invention for enhancing fibrin clot
formation in mammalian plasma.
[0129] In another aspect, the present invention relates to the use
of a factor XIII polypeptide prepared by use of a method according
to the present invention for prevention of intraventricular
haemorrhage in premature infants.
[0130] In a further aspect, the present invention relates to the
use of a factor XIII polypeptide prepared by use of a method
according to the present invention for reducing surgery-related
blood loss in a patient during or after surgery.
[0131] In another aspect, the present invention relates to the use
of a factor XIII polypeptide prepared by use of a method according
to the present invention for treating hemophilia A.
[0132] In a further aspect, the present invention relates to the
use of a factor XIII polypeptide prepared by use of a method
according to the present invention for treating hemophilia B.
[0133] In another aspect, the present invention relates to the use
of a factor XIII polypeptide prepared by use of a method according
to the present invention for treating platelet disorders.
[0134] In a further aspect, the present invention relates to the
use of a factor XIII polypeptide prepared by use of a method
according to the present invention for treating von Willebrand's
disease.
[0135] The present invention also relates to the use of a factor
XIII polypeptide prepared by use of a method according to the
present invention for the preparation of a medicament for treating
bleeding episodes.
[0136] In another aspect, the present invention relates to the use
of a factor XIII polypeptide prepared by use of a method according
to the present invention for the preparation of a medicament for
reducing clotting time in a subject.
[0137] In a further aspect, the present invention relates to the
use of a factor XIII polypeptide prepared by use of a method
according to the present invention for the preparation of a
medicament for prolonging the clot lysis time in mammalian
plasma.
[0138] In another aspect, the present invention relates to the use
of a factor XIII polypeptide prepared by use of a method according
to the present invention for the preparation of a medicament for
increasing clot strength in mammalian plasma.
[0139] In a further aspect, the present invention relates to the
use of a factor XIII polypeptide prepared by use of a method
according to the present invention for the preparation of a
medicament for enhancing fibrin clot formation in mammalian
plasma.
[0140] In another aspect, the present invention relates to the use
of a factor XIII polypeptide prepared by use of a method according
to the present invention for the preparation of a medicament for
prevention of intraventricular haemorrhage in premature
infants.
[0141] In a further aspect, the present invention relates to the
use of a factor XIII polypeptide prepared by use of a method
according to the present invention for the preparation of a
medicament for reducing surgery-related blood loss in a patient
during or after surgery.
[0142] In another aspect, the present invention relates to the use
of a factor XIII polypeptide prepared by use of a method according
to the present invention for the preparation of a medicament for
treating hemophilia A.
[0143] In a further aspect, the present invention relates to the
use of a factor XIII polypeptide prepared by use of a method
according to the present invention for the preparation of a
medicament for treating hemophilia B.
[0144] In another aspect, the present invention relates to the use
of a factor XIII polypeptide prepared by use of a method according
to the present invention for the preparation of a medicament for
treating platelet disorders.
[0145] In a further aspect, the present invention relates to the
use of a factor XIII polypeptide prepared by use of a method
according to the present invention for the preparation of a
medicament for treating von Willebrand's disease.
[0146] In one embodiment, the mammalian plasma referred to in
certain aspects of the invention as described above is human
plasma. In another embodiment, the mammalian plasma is normal
plasma; in a further embodiment, the plasma is normal human plasma;
in another embodiment, the mammalian plasma is plasma from a
subject (e.g. a human subject) having an impaired thrombin
generation. In a still further embodiment, the mammalian plasma is
from a subject (e.g. a human subject) having a lowered
concentration of fibrinogen.
[0147] In a further aspect of the invention, the factor XIII
polypeptide in question prolongs the in vitro clot lysis time in
normal human plasma.
[0148] A list of embodiments of the present invention is given
below:
EMBODIMENT 1
[0149] A method for purifying a factor XIII polypeptide from a
biological material, the method comprising subjecting the material
to sequential chromatography on an anion-exchange matrix and a
hydrophobic interaction matrix.
EMBODIMENT 2
[0150] A method according to embodiment 1, wherein the factor XIII
polypeptide is a recombinant factor XIII.
EMBODIMENT 3
[0151] A method according to embodiment 1 or embodiment 2, wherein
the factor XIII polypeptide is human factor XIII.
EMBODIMENT 4
[0152] A method according to embodiment 1 or embodiment 2, wherein
the factor XIII polypeptide is a dimer of A subunits.
EMBODIMENT 5
[0153] A method according to embodiment 4, wherein the factor XIII
polypeptide is a dimer of human A subunits.
EMBODIMENT 6
[0154] A method according to any of embodiments 1 to 5, wherein the
biological material is a biological fluid.
EMBODIMENT 7
[0155] A method according to embodiment 6, wherein the biological
fluid is the supernatant of a cell lysate.
EMBODIMENT 8
[0156] A method according to embodiment 7, wherein the biological
fluid is the supernatant of a yeast cell lysate.
EMBODIMENT 9
[0157] A method according to any of embodiments 1 to 8, wherein the
method comprises the steps of:
(a) subjecting a biological material comprising a factor XIII
polypeptide to chromatography on a first anion-exchange
chromatographic material, said chromatography comprising: [0158]
(i) applying said biological material to said first anion-exchange
chromatographic material; [0159] (ii) eluting unbound material from
the first anion-exchange chromatographic material with a buffer A,
which buffer A is suitable for eluting material not bound to the
first anion-exchange chromatographic material; and [0160] (iii)
eluting said factor XIII polypeptide from the first anion-exchange
chromatographic material by gradient-elution with buffer A', which
buffer A' is suitable for eluting said factor XIII polypeptide from
said first anion-exchange chromatographic material; (b) subjecting
the eluate from step (iii), or a fluid prepared by use of the
eluate from step (iii), to chromatography using a hydrophobic
interaction chromatographic material, said chromatography
comprising: [0161] (iv) applying the eluate from step (iii), or a
fluid prepared by use of the eluate from step (iii), to said
hydrophobic interaction chromatographic material; [0162] (v)
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 [0163]
(vi) eluting said factor XIII polypeptide from said chromatographic
material by gradient-elution with buffer B', which buffer B' is
suitable for eluting factor XIII from said hydrophobic interaction
chromatographic material.
EMBODIMENT 10
[0164] A method according to embodiment 9, wherein buffer A
comprises one or more stabilizing agents which are capable of
increasing the stability of the factor XIII polypeptide.
EMBODIMENT 11
[0165] A method according to embodiment 10, wherein buffer A
comprises a stabilizing agent, which stabilizing agent is a sugar,
an alcohol or an alditol.
EMBODIMENT 12
[0166] A method according to embodiment 11, wherein buffer A
comprises a stabilizing agent, which stabilizing agent is a sugar,
a C.sub.3-C.sub.8-alcohol or an alditol.
EMBODIMENT 13
[0167] A method according to embodiment 12, wherein buffer A
comprises a stabilizing agent, which stabilizing agent is a
polyalcohol.
EMBODIMENT 14
[0168] A method according to embodiment 12, 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 15
[0169] A method according to embodiment 14, wherein buffer A
comprises a stabilizing agent selected from the group consisting of
glycerol, propylene glycol and propan-1,3-diol.
EMBODIMENT 16
[0170] A method according to any of embodiments 13 to 15, wherein
said stabilizing agent is present in a concentration of from about
5% (v/v) to about 50% (v/v).
EMBODIMENT 17
[0171] A method according to embodiment 16, wherein said
stabilizing agent is present in a concentration of from about 10%
(v/v) to about 50% (v/v).
EMBODIMENT 18
[0172] A method according to embodiment 17, wherein said
stabilizing agent is present in a concentration of from about 10%
(v/v) to about 20% (v/v).
EMBODIMENT 19
[0173] A method according to embodiment 18, wherein said
stabilizing agent is present in a concentration of about 10%
(v/v).
EMBODIMENT 20
[0174] A method according to embodiment 18, wherein said
stabilizing agent is present in a concentration of about 20%
(v/v).
EMBODIMENT 21
[0175] A method according to any of embodiments 9 to 20, wherein
the pH of buffer A is between about 6.5 and about 9.
EMBODIMENT 22
[0176] A method according to embodiment 21, wherein the pH of
buffer A is between about 7 and about 9.
EMBODIMENT 23
[0177] A method according to embodiment 22, wherein the pH of
buffer A is about 8.
EMBODIMENT 24
[0178] A method according to any of embodiments 9 to 23, wherein
buffer A has a conductivity of less than about 2 mS/cm.
EMBODIMENT 25
[0179] A method according to any one of embodiments 9 to 24,
wherein no precipitation to form a crystalline precipitate of the
factor XIII polypeptide is performed between step (iii) and step
(iv).
EMBODIMENT 26
[0180] A method according to embodiment 25, wherein no
precipitation step using sodium acetate to form a crystalline
precipitate of the factor XIII polypeptide is performed between
step (iii) and step (iv).
EMBODIMENT 27
[0181] A method according to any of embodiments 1 to 26, wherein
the hydrophobic interaction chromatographic material uses phenyl as
the ligand.
EMBODIMENT 28
[0182] A method according to embodiment 27, wherein the hydrophobic
interaction chromatographic material is Source.TM. 15Phe.
EMBODIMENT 29
[0183] A method according to embodiment 27, wherein the hydrophobic
interaction chromatographic material is Phenyl Sepharose.TM. 6 Fast
Flow High Substitution.
EMBODIMENT 30
[0184] A method according to embodiment 27, wherein the hydrophobic
interaction chromatographic material is Phenyl Sepharose.TM. High
Performance High Substitution.
EMBODIMENT 31
[0185] A method according to any of embodiments 9 to 30, wherein
the pH of buffer B is from about 6 to about 8.
EMBODIMENT 32
[0186] A method according to embodiment 31, wherein the pH of
buffer B is about 7.5.
EMBODIMENT 33
[0187] A method according to any of embodiments 9 to 32, wherein
buffer B has a conductivity of more than 25 mS/cm.
EMBODIMENT 34
[0188] A method according to embodiment 33, wherein buffer B has a
conductivity of at most 50 mS/cm.
EMBODIMENT 35
[0189] A method according to any of embodiments 9 to 34, wherein
the eluate from stage (vi), or a fluid prepared by use of the
eluate from stage (vi), 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 factor XIII polypeptide in an
amount effective to significantly improve the stability thereof,
and/or (2) adjusting the pH of the eluate from stage (vi), or of a
fluid prepared by use of the eluate from stage (vi), to a pH
between about 7 and about 8.
EMBODIMENT 36
[0190] A method according to embodiment 35, wherein the stabilizing
agent used in step (1) is a sugar, an alcohol or an alditol.
EMBODIMENT 37
[0191] A method according to embodiment 36, wherein the stabilizing
agent used in step (1) is a sugar, a C.sub.3-C.sub.8-alcohol or an
alditol.
EMBODIMENT 38
[0192] A method according to embodiment 37, wherein the stabilizing
agent used in step (1) is a polyalcohol.
EMBODIMENT 39
[0193] A method according to embodiment 37, wherein the stabilizing
agent used in step (1) is selected from the group consisting of
glycerol, propylene glycol, propan-1,3-diol, propyl alcohol and
isopropyl alcohol.
EMBODIMENT 40
[0194] A method according to embodiment 39, wherein the stabilizing
agent used in step (1) is selected from the group consisting of
glycerol, propylene glycol and propan-1,3-diol.
EMBODIMENT 41
[0195] A method according to any of embodiments 38 to 40, wherein
the stabilizing agent used in step (1) is added to a concentration
of from about 5% (v/v) to about 50% (v/v).
EMBODIMENT 42
[0196] A method according to embodiment 41, 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 43
[0197] A method according to embodiment 42, 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 44
[0198] A method according to any of embodiments 35 to 43, wherein
the pH of the eluate from stage (vi), or of a fluid prepared by use
of the eluate from stage (vi), is adjusted in step (2) to a pH
between about 7 and about 8.
EMBODIMENT 45
[0199] A method according to embodiment 44, wherein the pH of the
eluate from stage (vi), or of a fluid prepared by use of the eluate
from stage (vi), is adjusted in step (2) to a pH of about 7.5.
EMBODIMENT 46
[0200] A method according to any of embodiments 9 to 45, wherein no
precipitation step to produce a factor XIII polypeptide-containing
precipitate is performed after step (vi).
EMBODIMENT 47
[0201] A method according to any of embodiments 1 to 46, 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 an anion-exchange matrix.
EMBODIMENT 48
[0202] A method according to any of embodiments 9 to 47, wherein
the method further comprises a step of: subjecting the eluate from
stage (vi), or a fluid prepared by use of the eluate from stage
(vi), to chromatography on a second anion-exchange chromatographic
material, said chromatography comprising: [0203] (vii) applying the
eluate from stage (vi), or a fluid prepared by use of the eluate
from stage (vi), to said second anion-exchange chromatographic
material; [0204] (viii) eluting unbound material from the second
anion-exchange chromatographic material with buffer C, which buffer
C is suitable for eluting material not bound to the second
anion-exchange chromatographic material; and [0205] (ix) eluting
said factor XIII polypeptide from the second anion-exchange
chromatographic material with buffer C', wherein buffer C' is
suitable for eluting factor XIII polypeptides which bind to the
second anion-exchange chromatographic material in step (viii).
EMBODIMENT 49
[0206] A method according to embodiment 48, wherein buffer C and/or
buffer C' comprises one or more stabilizing agents which are
capable of increasing the stability of the factor XIII
polypeptide.
EMBODIMENT 50
[0207] A method according to embodiment 49, wherein buffer C and/or
buffer C' comprises a stabilizing agent, which stabilizing agent is
a sugar, an alcohol or an alditol.
EMBODIMENT 51
[0208] A method according to embodiment 50, wherein buffer C and/or
buffer C' comprises a stabilizing agent, which stabilizing agent is
a sugar, a C.sub.3-C.sub.8-alcohol or an alditol.
EMBODIMENT 52
[0209] A method according to embodiment 51, wherein buffer C and/or
buffer C' comprises a stabilizing agent, which stabilizing agent is
a polyalcohol.
EMBODIMENT 53
[0210] A method according to embodiment 52, wherein buffer C and/or
buffer C' comprises a stabilizing agent selected from the group
consisting of glycerol, propylene glycol, propan-1,3-diol, propyl
alcohol and isopropyl alcohol.
EMBODIMENT 54
[0211] A method according to embodiment 53, wherein buffer C and/or
buffer C' comprises a stabilizing agent selected from the group
consisting of glycerol, propylene glycol and propan-1,3-diol.
EMBODIMENT 55
[0212] A method according to any of embodiments 52 to 54, wherein
said stabilizing agent is present in a concentration of from about
5% (v/v) to about 50% (v/v).
EMBODIMENT 56
[0213] A method according to embodiment 55, wherein said
stabilizing agent is present in a concentration of from about 10%
(v/v) to about 50% (v/v).
EMBODIMENT 57
[0214] A method according to embodiment 56, wherein said
stabilizing agent is present in a concentration of from about 10%
(v/v) to about 20% (v/v).
EMBODIMENT 58
[0215] A method according to embodiment 57, wherein said
stabilizing agent is present in a concentration of about 10%
(v/v).
EMBODIMENT 59
[0216] A method according to embodiment 57, wherein said
stabilizing agent is present in a concentration of about 20%
(v/v).
EMBODIMENT 60
[0217] A method according to any of embodiments 48 to 59, wherein
buffer C and/or buffer C' has a pH of about 7.5.
EMBODIMENT 61
[0218] A method according to any of embodiments 48 to 60, wherein
the conductivity of the eluate from step (ix) containing the factor
XIII polypeptide is adjusted to about 10 mS/cm.
EMBODIMENT 62
[0219] A method according to any of embodiments 48 to 61, wherein
the pH of the eluate from step (ix) containing the factor XIII
polypeptide is adjusted to about 7.5.
EMBODIMENT 63
[0220] A method according to any of embodiments 48 to 62, wherein
no precipitation step to produce a factor XIII-containing
precipitate is performed after step (ix).
EMBODIMENT 64
[0221] A method according to any of embodiments 1 to 62, wherein no
precipitation step to produce a factor XIII-containing precipitate
is performed.
EMBODIMENT 65
[0222] A method for purifying a factor XIII polypeptide from a
biological material, the method comprising the step of subjecting
the biological material to chromatography using a hydrophobic
interaction chromatographic material, wherein said hydrophobic
interaction chromatographic material is chosen among Phenyl
Sepharose.TM. High Performance High Substitution, Source.TM. 15 Phe
and Sepharose.TM. 6 Fast Flow High Substitution.
EMBODIMENT 66
[0223] A method according to embodiment 65, wherein the factor XIII
polypeptide is a recombinant factor XIII.
EMBODIMENT 67
[0224] A method according to embodiment 65 or embodiment 66,
wherein the factor XIII polypeptide is human factor XIII.
EMBODIMENT 68
[0225] A method according to embodiment 65 or embodiment 66,
wherein the factor XIII polypeptide is a dimer of A subunits.
EMBODIMENT 69
[0226] A method according to embodiment 68, wherein the factor XIII
polypeptide is a dimer of human A subunits.
EMBODIMENT 70
[0227] A method according to any of embodiments 65 to 69, wherein
the biological material is a biological fluid.
EMBODIMENT 71
[0228] A method according to any of embodiments 65 to 70, wherein
no precipitation step to produce a factor XIII-containing
precipitate is performed.
EMBODIMENT 72
[0229] A pharmaceutical composition comprising a factor XIII
polypeptide prepared by use of a method according to any of
embodiments 1 to 71.
EMBODIMENT 73
[0230] A pharmaceutical composition according to embodiment 72,
[0231] wherein the pharmaceutical composition is an aqueous
composition and comprises 20% (v/v) propylene glycol.
EMBODIMENT 74
[0232] A pharmaceutical composition according to embodiment 72,
[0233] wherein the pharmaceutical composition is an aqueous
composition and comprises 1.5% (w/v) sucrose and 3.5% (w/v)
mannitol.
EMBODIMENT 75
[0234] Use of a pharmaceutical composition according to any of
embodiments 72 to 74 for reducing blood loss.
EMBODIMENT 76
[0235] Use of a pharmaceutical composition according to any of
embodiments 72 to 74 for the prevention of intraventricular
haemorrhage in premature infants.
EMBODIMENT 77
[0236] Use of a pharmaceutical composition according to any of
embodiments 72 to 74 for reducing surgery-related blood loss in a
patient during or after surgery.
EMBODIMENT 78
[0237] Use of a pharmaceutical composition according to any of
embodiments 72 to 74 for treating hemophilia A.
EMBODIMENT 79
[0238] Use of a pharmaceutical composition according to any of
embodiments 72 to 74 for treating hemophilia B.
EMBODIMENT 80
[0239] Use of a pharmaceutical composition according to any of
embodiments 72 to 74 for treating platelet disorders.
EMBODIMENT 81
[0240] Use of a pharmaceutical composition according to any of
embodiments 72 to 74 for treating von Willebrand's disease.
EMBODIMENT 82
[0241] Use of a factor XIII polypeptide prepared by use of a method
according to any of embodiments 1 to 71 for preparation of a
pharmaceutical composition for use in reducing blood loss.
EMBODIMENT 83
[0242] Use of a factor XIII polypeptide prepared by use of a method
according to any of embodiments 1 to 71 for preparation of a
pharmaceutical composition for prevention of intraventricular
haemorrhage in premature infants.
EMBODIMENT 84
[0243] Use of a factor XIII polypeptide prepared by use of a method
according to any of embodiments 1 to 71 for preparation of a
pharmaceutical composition for reducing surgery-related blood loss
in a patient during or after surgery.
EMBODIMENT 85
[0244] Use of a factor XIII polypeptide prepared by use of a method
according to any of embodiments 1 to 71 for preparation of a
pharmaceutical composition for treating hemophilia A.
EMBODIMENT 86
[0245] Use of a factor XIII polypeptide prepared by use of a method
according to any of embodiments 1 to 71 for preparation of a
pharmaceutical composition for treating hemophilia B.
EMBODIMENT 87
[0246] Use of a factor XIII polypeptide prepared by use of a method
according to any of embodiments 1 to 71 for preparation of a
pharmaceutical composition for treating platelet disorders.
EMBODIMENT 88
[0247] Use of a factor XIII polypeptide prepared by use of a method
according to any of embodiments 1 to 71 for preparation of a
pharmaceutical composition for treating von Willebrand's
disease.
[0248] The present invention is illustrated by the followed
non-limiting examples.
EXAMPLES
Example 1
Disruption of Yeast Cells
[0249] A culture comprising cells of the yeast Saccharomyces
cerevisiae expressing the amino acid sequence 1-731 of human factor
XIII was centrifuged for 8 min. in a Sorvall SLA centrifuge at 6000
rpm at 4.degree. C., and the cells were resuspended in freshly made
buffer (20% by volume (v/v) propylene glycol, 20 mM Tris, 1 mM
PMSF, pH 8.0) in an amount of 3 ml of buffer per gram of cell
pellet. The suspension was then subjected to a pressure of 1.5 kBar
at a temperature below 15.degree. C. The resulting suspension was
centrifuged for 30 min. in a Sorvall SLA centrifuge at 9000 rpm at
4.degree. C., and the pH of the supernatant was adjusted to 8.0
with 1 M NaOH. The supernatant was then filtered on Advantec GF75
0.7 .mu.m GF075090+round filter MN GF5 0.4 .mu.m 001201 art no.
415009 and kept at 4.degree. C. until further use (in Example
2).
Example 2
First anion-exchange chromatography using Source.TM. 30Q
[0250] The conductivity of the filtered supernatant from Example 1
was adjusted to below 2 mS/cm by use of a freshly made buffer A
(20% (v/v) propylene glycol, 20 mM Tris, 1 mM PMSF, pH 8.0). A
Source.TM. 30Q matrix was equilibrated with 5 column volumes (cv)
of buffer A, and a load corresponding to the amount of polypeptide
produced by 1.3 g yeast cells was applied to the column. The column
was washed with 7 cv of buffer A and then with 5 cv of 10% of the
elution buffer (20% (v/v) propylene glycol, 20 mM Tris, 0.2 M NaCl,
1 mM PMSF, pH 8.0). Gradient elution was then performed, going from
10% elution buffer to 100% elution buffer over 25 cv followed by 5
cv of 100% elution buffer at a flow of 12 cv/hour. Fractions were
collected from at about 30% elution buffer to at about 70% elution
buffer. Analysis of factor XIII polypeptide-containing fractions
was performed by HPLC (vide infra) using DEAE-NPR (TosoHaas, cat.
no. 13075, 4.6.times.35 mm) and by SDS-PAGE on a NUpage 4-12%
Bis/Tris Gel (Invitrogen) with MOPS running buffer under reductive
conditions. Fractions containing factor XIII polypeptide were kept
at approx. +4.degree. C. in a refrigerator, or frozen at
-80.degree. C., until further use (in Example 3).
Example 3
Hydrophobic Interaction Chromatography Using Source.TM. 15Phe
[0251] One volume of a buffer containing 400 mM
K.sub.2HPO.sub.4+400 mM KH.sub.2PO.sub.4 was added to the combined
fractions containing factor XIII polypeptide from Example 2, and
the pH was adjusted to 7.5 with NaOH. A Source.TM. 15Phe matrix was
equilibrated with 4 cv of buffer B (100 mM K.sub.2HPO.sub.4, 100 mM
KH.sub.2PO.sub.4, pH 7.5), and a load corresponding to
approximately 2 mg/ml was applied to the column. The column was
then washed with 4 cv of buffer B and then subjected to gradient
elution going from buffer B to 100% elution buffer (5 mM
K.sub.2HPO.sub.4, 5 mM KH.sub.2PO.sub.4, pH 7.5) over 20 cv
followed by 2 cv of 100% elution buffer at a flow of 8 cv/h.
Fractions were collected after elution of approximately 4 cv and
until 18 cv. Analysis of factor XIII polypeptide-containing
fractions was performed by HPLC (vide infra) using DEAE-NPR
(TosoHaas, cat. no. 13075, 4.6.times.35 mm) and by SDS-PAGE on a
NUpage 4-12% Bis/Tris Gel (Invitrogen) with MOPS running buffer
under reductive conditions. A 1/4 volume of propylene glycol was
immediately added to the pool of factor XIII polypeptide-containing
fractions to a final concentration of 20% (v/v) propylene glycol,
and the resulting pool was then kept at approx. +4.degree. C. in a
refrigerator, or frozen, until further use (in Example 5).
Example 4
Hydrophobic Interaction Chromatography Using Phenyl Sepharose.TM.
High Performance High Substitution
[0252] One volume of a buffer containing 400 mM
K.sub.2HPO.sub.4+400 mM KH.sub.2PO.sub.4 was added to the combined
fractions containing factor XIII polypeptide from Example 2, and
the pH was adjusted to 7.5 with NaOH. A Phenyl Sepharose.TM. High
Performance High Substitution matrix was equilibrated with 4 cv of
buffer B (100 mM K.sub.2HPO.sub.4, 100 mM KH.sub.2PO.sub.4, pH
7.5), and a load corresponding to approximately 2 mg/ml was applied
to the column. The column was then washed with 4 cv of buffer B and
then subjected to gradient elution going from buffer B to 100%
elution buffer (5 mM K.sub.2HPO.sub.4, 5 mM KH.sub.2PO.sub.4, pH
7.5) over 20 cv followed by 2 cv of 100% elution buffer at a flow
of 8 cv/h. Fractions were collected after elution of approximately
10 cv and until 20 cv. Analysis of factor XIII
polypeptide-containing fractions was performed by HPLC (vide infra)
using DEAE-NPR (TosoHaas, cat. no. 13075, 4.6.times.35 mm) and by
SDS-PAGE on a NUpage 4-12% Bis/Tris Gel (Invitrogen) with MOPS
running buffer under reductive conditions. A 1/4 volume of
propylene glycol was immediately added to the pool of factor XIII
polypeptide-containing fractions to a final concentration of 20%
(v/v) propylene glycol, and the resulting pool was then kept at
approx. +4.degree. C., or frozen, until used in a procedure as
described in Example 5.
Example 5
Second Anion-Exchange Chromatography Using Source.TM. 30Q
(Alternative 1)
[0253] The pH of the pool of factor XIII polypeptide-containing
fractions from Example 3 was adjusted to 7.5, and buffer C (20%
(v/v) propylene glycol, 10 mM glycyl-glycine, pH 7.5) was added to
a conductivity of below 2 mS/cm. A Source.TM. 30Q matrix was
equilibrated with 5 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 8 cv of buffer C, and a buffer containing 67% buffer C
and 33% elution buffer (20% (v/v) propylene glycol, 10 mM
glycyl-glycine, 0.5 M NaCl, pH 7.5) was used to elute the factor
XIII polypeptide in small fractions. The conductivity of the pool
containing the factor XIII polypeptide fractions was adjusted to
about 10 mS/cm with a buffer containing 20% (v/v) propylene glycol,
10 mM glycyl-glycin, 2 M NaCl (pH 7.5).
Example 6
Second Anion-Exchange Chromatography Using Source.TM. 30Q
(Alternative 2)
[0254] The pH of the pool of factor XIII polypeptide-containing
fractions from Example 3 was adjusted to 7.5, and buffer D (1.5%
weight per volume (w/v) sucrose, 3.5% (w/v) mannitol, 10 mM
glycyl-glycine, pH 7.5) was added to a conductivity of below 2
mS/cm. A Source.TM. 30Q matrix was equilibrated with 5 cv of buffer
D, and then a load corresponding to 5 mg/ml gel was applied to the
column. The column was then washed with 8 cv of buffer D, and a
buffer containing 67% buffer D and 33% elution buffer (1.5% (w/v)
sucrose, 3.5% (w/v) mannitol, 0.5 M NaCl, pH 7.5) was used to elute
the factor XIII polypeptide in small fractions. The conductivity of
the pool containing the factor XIII polypeptide fractions was
adjusted to about 10 mS/cm with a buffer containing 1.5% (w/v)
sucrose, 3.5% (w/v) mannitol, 2 M NaCl (pH 7.5).
HPLC Analysis Procedure
[0255] High-Performance Liquid Chromatography (HPLC; referred to in
Examples 2-4, above) was performed using DEAE-NPR column matrix
material from TosoHaas (cat. No. 13075, 4.6.times.35 mm) and
employing buffers as follows:
Buffer I: 20 mM K.sub.2HPO.sub.4, pH 8.0 (adjusted with HCl) Buffer
II: 20 mM K.sub.2HPO.sub.4, 0.4 M KCl, pH 8.0 (adjusted with
HCl).
[0256] Equilibration of the column was carried out using a mixture
of 95% (v/v) Buffer I with 5% (v/v) Buffer II for 2.5 minutes (flow
rate 0.5 ml/min.).
[0257] Elution of the column took place using a gradient going from
95% Buffer I/5% Buffer 11 to 60% Buffer I/40% Buffer II over a
period of 17 minutes (flow rate 0.5 ml/min.).
[0258] Regeneration of the column was performed by washing with
100% Buffer II for 1.4 minutes (flow rate 0.5 ml/min.). Each day
before use of the column it was found advantageous to inject 100
.mu.l 0.2 M NaOH followed by 100 .mu.l 1 M K.sub.2HPO.sub.4, pH
8.0, onto the column before performing a new cycle of
equilibration, elution and regeneration. 1 M citric acid (pH 3.0)
may alternatively be employed.
[0259] The detection wavelength employed was 220 nm.
[0260] Samples of from 2 to 30 .mu.g were loaded onto the column;
by four-fold dilution of samples with water it was possible to load
up to 30 .mu.g of sample irrespective of injection volume up to 100
.mu.l. The use of greater injection volumes and higher sample
loadings was not examined.
[0261] Examples of results from 3 purifications carried out
according to examples 1, 2, 3 and 5
TABLE-US-00001 Fermentation No. H913 H932 H933 Cell mass/gram 1057
1630 1830 Yield (mg) after source 30Q (Ex. 2) 529 699 977 Purity
(HPLC %) after source 30Q (Ex. 2) 79 84 64 Yield (mg) after
source15phe (Ex. 3) 514 314 638 Yield (%) after source15phe (Ex. 3)
97 45 65 Purity (HPLC) after source15phe (Ex. 3) 90 93 82 Yield
(mg) after concentration on Source30Q, 363 270 456 (UV), (Ex. 5)
Yield after concentration on Source30Q (%) 70.6 86 71 (Ex. 5)
Purity (HPLC) after concentration on 90.2 91.5 86 Source30Q (Ex.
5)
* * * * *