U.S. patent application number 14/811572 was filed with the patent office on 2015-11-12 for highly-purified soluble thrombomodulin and method for producing same.
This patent application is currently assigned to ASAHI KASEI PHARMA CORPORATION. The applicant listed for this patent is ASAHI KASEI PHARMA CORPORATION. Invention is credited to Hiroki SHIGEMATSU, Yuji UENO.
Application Number | 20150322133 14/811572 |
Document ID | / |
Family ID | 44861609 |
Filed Date | 2015-11-12 |
United States Patent
Application |
20150322133 |
Kind Code |
A1 |
UENO; Yuji ; et al. |
November 12, 2015 |
HIGHLY-PURIFIED SOLUBLE THROMBOMODULIN AND METHOD FOR PRODUCING
SAME
Abstract
Highly-purified soluble thrombomodulin which has a content of
host cell-originated proteins being in a ratio of less than 10 ng
of the proteins per 10,000 U of the soluble thrombomodulin, wherein
the soluble thrombomodulin is produced by a transformant cell
obtained by transfecting a host cell with a DNA containing a
nucleotide sequence encoding the soluble thrombomodulin.
Inventors: |
UENO; Yuji; (Tokyo, JP)
; SHIGEMATSU; Hiroki; (Tokyo, JP) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
ASAHI KASEI PHARMA CORPORATION |
Tokyo |
|
JP |
|
|
Assignee: |
ASAHI KASEI PHARMA
CORPORATION
Tokyo
JP
|
Family ID: |
44861609 |
Appl. No.: |
14/811572 |
Filed: |
July 28, 2015 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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13641047 |
Dec 21, 2012 |
9127089 |
|
|
PCT/JP2011/060348 |
Apr 28, 2011 |
|
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14811572 |
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Current U.S.
Class: |
514/14.7 ;
530/395 |
Current CPC
Class: |
A61P 29/00 20180101;
A61P 1/16 20180101; A61P 9/00 20180101; A61P 43/00 20180101; A61P
3/10 20180101; A61P 9/08 20180101; A61P 15/00 20180101; C07K
14/7455 20130101; A61K 38/00 20130101; A61P 7/02 20180101; A61P
9/10 20180101; A61P 11/00 20180101 |
International
Class: |
C07K 14/745 20060101
C07K014/745 |
Foreign Application Data
Date |
Code |
Application Number |
Apr 30, 2010 |
JP |
2010-105421 |
Claims
1. Highly-purified soluble thrombomodulin which has a content of
host cell-originated proteins being in a ratio of less than 10 ng
of the proteins per 10,000 U of the soluble thrombomodulin, wherein
the soluble thrombomodulin is produced by a transformant cell
obtained by transfecting a host cell with a DNA containing a
nucleotide sequence encoding the soluble thrombomodulin.
2. The highly-purified soluble thrombomodulin according to claim 1,
which is manufactured on an industrial scale.
3. The highly-purified soluble thrombomodulin according to claim 1
or 2, which is used as a material for a medicament.
4. The highly-purified soluble thrombomodulin according to claim 1
or 2, wherein purity of the highly-purified soluble thrombomodulin
is 99% or higher based on the total proteins.
5. The highly-purified soluble thrombomodulin according to claim 1
or 2, wherein the soluble thrombomodulin is produced by serum-free
culture of the transformant cell.
6. The highly-purified soluble thrombomodulin according to claim 1
or 2, wherein the host cell is a Chinese hamster ovary cell.
7. The highly-purified soluble thrombomodulin according to claim 1
or 2, wherein the soluble thrombomodulin has the following
properties (1) to (5): (1) an action of selectively binding to
thrombin, (2) an action of promoting activation of Protein C by
thrombin, (3) an action of extending thrombin clotting time, (4) an
action of suppressing platelet aggregation caused by thrombin, and
(5) anti-inflammatory action.
8. The highly-purified soluble thrombomodulin according to claim 1
or 2, wherein molecular weight of the soluble thrombomodulin is in
the range of 50,000 to 80,000.
9. The highly-purified soluble thrombomodulin according to claim 1
or 2, wherein the highly-purified soluble thrombomodulin is
produced by a method comprising the following steps: (a) the step
of obtaining a transformant cell by transfecting a host cell with a
DNA encoding soluble thrombomodulin; (b) the step of obtaining a
solution containing soluble thrombomodulin by culturing the
transformant cell, and (c) the step of bringing the solution
containing soluble thrombomodulin into contact with nylon and/or
polyethersulfone to obtain highly-purified soluble thrombomodulin
having a content of host cell-originated proteins being in a ratio
of less than 10 ng of the proteins per 10,000 U of soluble
thrombomodulin.
10. The highly-purified soluble thrombomodulin according to claim 1
or 2, wherein the soluble thrombomodulin is a peptide containing:
(i) the amino acid sequence of the positions 367 to 480 in the
amino acid sequence of SEQ ID NO: 9 or 11, and the amino acid
sequence of (ii-1) or (ii-2) mentioned below, and the peptide is
soluble thrombomodulin having the following properties (1) to (5):
(ii-1) the amino acid sequence of the positions 19 to 244 in the
amino acid sequence of SEQ ID NO: 9 or 11, or (ii-2) the amino acid
sequence of (ii-1) mentioned above, further including substitution,
deletion or addition of one or more amino acid residues, (1) an
action of selectively binding to thrombin, (2) an action of
promoting activation of Protein C by thrombin, (3) an action of
extending thrombin clotting time, (4) an action of suppressing
platelet aggregation caused by thrombin, and (5) anti-inflammatory
action.
11. The highly-purified soluble thrombomodulin according to claim 1
or 2, wherein the soluble thrombomodulin is a peptide containing:
(i-1) the amino acid sequence of the positions 19 to 516 in the
amino acid sequence of SEQ ID NO: 9 or 11, or (i-2) the amino acid
sequence of (i-1) mentioned above, further including substitution,
deletion or addition of one or more amino acid residues, and the
peptide is soluble thrombomodulin having the properties (1) to (5)
mentioned below: (1) an action of selectively binding to thrombin,
(2) an action of promoting activation of Protein C by thrombin, (3)
an action of extending thrombin clotting time, (4) an action of
suppressing platelet aggregation caused by thrombin, and (5)
anti-inflammatory action.
12. The highly-purified soluble thrombomodulin according to claim 1
or 2, wherein the DNA containing a nucleotide sequence encoding
soluble thrombomodulin is a DNA encoding the amino acid sequence of
SEQ ID NO: 9 or 11.
13. A pharmaceutical composition comprising the highly-purified
soluble thrombomodulin according to claim 1 or 2 and a
pharmaceutically acceptable carrier.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application is a Divisional of copending application
Ser. No. 13/641,047, filed on Dec. 21, 2012, which was filed as PCT
International Application No. PCT/JP2011/060348 on Apr. 28, 2011,
which claims the benefit under 35 U.S.C. .sctn.119(a) to Patent
Application No. 2010-105421, filed in Japan on Apr. 30, 2010, all
of which are hereby expressly incorporated by reference into the
present application.
TECHNICAL FIELD
[0002] The present invention relates to highly-purified soluble
thrombomodulin and a method for producing the same.
BACKGROUND ART
[0003] Thrombomodulin is known as a substance having an action of
specifically binding to thrombin to inhibit the blood coagulation
activity of thrombin, and at the same time, significantly promote
the ability of thrombin to activate Protein C, and is also known to
have strong blood coagulation-inhibiting action. It is also known
that thrombomodulin extends the thrombin clotting time, and that it
suppresses platelet aggregation by thrombin. Protein C is a vitamin
K-dependent protein that plays an important role in the blood
coagulation fibrinolytic system, and is activated by the action of
thrombin to become activated Protein C. It is known that the
activated Protein C inactivates activated blood coagulation factor
V and activated blood coagulation factor VIII in vivo, and that it
is involved in generation of plasminogen activator having
thrombolytic action (Non-patent document 1). Therefore,
thrombomodulin is considered to be useful as an anticoagulant agent
or a thrombolytic agent that promotes the activation of Protein C
by thrombin, and there have also been reported animal experiments
demonstrating that thrombomodulin is effective for therapeutic and
prophylactic treatments of diseases associated with acceleration of
coagulation (Non-patent document 2).
[0004] Thrombomodulin was first discovered and obtained as a
glycoprotein expressed on vascular endothelial cells of various
animal species including human, and thereafter successfully cloned.
More specifically, a human thrombomodulin precursor gene containing
a signal peptide was cloned from a human lung cDNA library by
genetic engineering techniques, and the entire gene sequence of
thrombomodulin was analyzed, so that the amino acid sequence
consisting of 575 residues containing a signal peptide (usually 18
amino acid residues are exemplified) was elucidated (Patent
document 1). It is known that mature thrombomodulin, from which the
signal peptide has been cleaved, is constituted by 5 regions,
namely, an N-terminal region (amino acids 1 to 226, these positions
are indicated on the assumption that the signal peptide consists of
18 amino acid residues, and the same shall apply to the other
regions), a region having six EGF-like structures (amino acids 227
to 462), an O-linked glycosylation region (amino acids 463 to 498),
a transmembrane region (amino acids 499 to 521), and an
intracytoplasmic region (amino acids 522 to 557), from the
N-terminal side of the mature peptide, and that a part having the
same activity as that of the full length thrombomodulin (i.e.,
minimum unit for the activity) mainly consists of the 4th, 5th, and
6th EGF-like structure portions from the N-terminal side among the
six EGF-like structures (Non-patent document 3).
[0005] Unless a surfactant is present, the full length
thrombomodulin is hardly dissolved, and therefore addition of a
surfactant is essential for producing a thrombomodulin preparation.
In contrast, there is also soluble thrombomodulin that can be fully
dissolved even in the absence of a surfactant. The soluble
thrombomodulin may be prepared so as not to contain at least a part
of the transmembrane region or the entire transmembrane region. For
example, it has been confirmed that a soluble thrombomodulin
consisting of only 3 regions of the N-terminal region, the region
having six EGF-like structures, and the O-linked glycosylation
region (i.e., soluble thrombomodulin having an amino acid sequence
comprising amino acids at the positions 19 to 516 in SEQ ID NO: 1),
can be obtained by applying recombination techniques, and that such
recombinant soluble thrombomodulin has the same activity as that of
the natural thrombomodulin (Patent document 1). In addition, there
are also some other reports regarding soluble thrombomodulin
(Patent documents 2 to 9). Further, human urine-derived soluble
thrombomodulin and the like are also exemplified as natural
thrombomodulin (Patent documents 10 and 11).
[0006] As recognized in many cases, as a result of spontaneous
mutations or mutations occurring at the time of obtaining
thrombomodulin, polymorphic mutations have been found even in human
genes, and at present, such thrombomodulin genes that the amino
acid at the position 473 of the human thrombomodulin precursor,
that has the aforementioned amino acid sequence consisting of 575
amino acid residues, is Val or Ala have been identified. This
difference corresponds to the difference of the nucleotide at the
position 1418 to T or C in the nucleotide sequences encoding the
amino acid (Non-patent document 4). However, these two
thrombomodulins are completely identical in terms of their
activities and physical properties. Thus, it can be considered that
they are substantially identical.
[0007] It has been reported that thrombomodulin is effective for a
therapeutic treatment of DIC (Non-patent documents 5 and 6). As for
use of thrombomodulin, in addition to the aforementioned uses,
thrombomodulin is expected to be used in therapeutic and
prophylactic treatments of various diseases such as acute coronary
syndrome (ACS), thrombosis, peripheral vessel obstruction,
obstructive arteriosclerosis, vasculitis, functional disorder
occurring after heart surgery, complication caused by organ
transplantation, angina pectoris, transient ischemic attack,
toxemia of pregnancy, diabetes, liver VOD (liver veno-occlusive
disease, e.g., fulminant hepatitis, veno occlusive disease of liver
occurring after bone marrow transplantation), and deep venous
thrombosis (DVT), and further, adult respiratory distress syndrome
(ARDS).
[0008] As a premise of application of thrombomodulin in
pharmaceutical products, it is needless to explain that the soluble
thrombomodulin is required to be manufactured in a large scale and
at a cost as low as possible. However, there is also pointed out a
possibility that heterogenous proteins originated in the production
process, for example, proteins originated in host cells, bovine
serum proteins originated in medium, and mouse IgG and the like
originated in antibody column serve as immunogens to case problems
concerning safety (Non-patent document 7).
[0009] As methods for producing soluble thrombomodulin in an
industrial scale for application as a pharmaceutical product, there
are known, for example, a method of using affinity column
chromatography in a main purification step to which an antibody
that reacts with thrombomodulin is bound, a method for producing
highly purified soluble thrombomodulin substantially free from
serum-originated substances and antibody-originated substances,
which is characterized in that the soluble thrombomodulin is
obtained as a flow-through fraction in a step of bringing the
soluble thrombomodulin obtained by affinity column chromatography
into contact with a cation exchanger under conditions of a specific
conductivity of 25 to 34 ms/cm and pH 3 to 4 (Patent document 12),
and a method for purifying thrombomodulin, wherein affinity column
chromatography as the main purification step is followed by strong
anion exchange chromatography (Patent document 13).
PRIOR ART REFERENCES
Patent Documents
[0010] Patent document 1: Japanese Patent Unexamined Publication
(Kokai) No. 64-6219 [0011] Patent document 2: Japanese Patent
Unexamined Publication No. 2-255699 [0012] Patent document 3:
Japanese Patent Unexamined Publication No. 3-133380 [0013] Patent
document 4: Japanese Patent Unexamined Publication No. 3-259084
[0014] Patent document 5: Japanese Patent Unexamined Publication
No. 4-210700 [0015] Patent document 6: Japanese Patent Unexamined
Publication No. 5-213998 [0016] Patent document 7: WO92/00325
[0017] Patent document 8: WO92/03149 [0018] Patent document 9:
WO93/15755 [0019] Patent document 10: Japanese Patent Unexamined
Publication No. 3-86900 [0020] Patent document 11: Japanese Patent
Unexamined Publication No. 3-218399 [0021] Patent document 12:
Japanese Patent Unexamined Publication No. 11-341990 [0022] Patent
document 13: WO2008/117735
Non-Patent Documents
[0022] [0023] Non-patent document 1: Koji Suzuki, Igaku no Ayumi
(Progress of Medicine), Vol. 125, 901 (1983) [0024] Non-patent
document 2: K. Gomi et al., Blood, 75, 1396-1399 (1990) [0025]
Non-patent document 3: M. Zushi et al., J. Biol. Chem., 264,
10351-10353 (1989) [0026] Non-patent document 4: D. Z. Wen et al.,
Biochemistry, 26, 4350-4357 (1987) [0027] Non-patent document 5: S.
M. Bates et al., Br. J. Pharmacol., 144, 1017-1028 (2005) [0028]
Non-patent document 6: H. Saito et al., J. Thromb Haemost, 5 (1),
31 (2007) [0029] Non-patent document 7: Akio Hayakawa, Development
and Security of Quality and Safety of Biomedical Products, 273-274
(2007)
SUMMARY OF THE INVENTION
Object to be Achieved by the Invention
[0030] An object of the present invention is to provide
highly-purified soluble thrombomodulin in which a concentration of
proteins originated in host cells is a ratio of less than 10 ng of
the proteins per 10,000 U of soluble thrombomodulin, and a method
for producing the same.
Means for Achieving the Object
[0031] Patent document 13 describes purified soluble
thrombomodulin. In particular, it discloses soluble thrombomodulin
in which concentration of proteins originated in the host
(henceforth also abbreviated as "HCP" in the specification) is
indicated as "N.D." (this indication seems to mean "not detected",
although it is not specifically indicated) in Example 14. Those
skilled in the art who read this description would have normally
considered that highly-purified soluble thrombomodulin containing
reduced contamination of HCP was satisfactorily achieved, and would
not have considered to further reduce HCP in soluble
thrombomodulin, in other words, the artisans would not have been
motivated to further reduce HCP in soluble thrombomodulin.
[0032] However, when the purified soluble thrombomodulin is used as
a pharmaceutical product, the HCP, if contaminated in the product,
might possibly cause unexpected condition such as anaphylactic
shock, and might lead to a lethal risk, which the inventors of the
present invention strongly recognized as a serious problem. From
this reason, even under the circumstance that those skilled in the
art would have normally considered that a contamination of HCP was
sufficiently reduced as Patent document 13 mentioned above
describes "N.D." in Example 14, the inventors of the present
invention conducted measurement of a HCP concentration in the
purified soluble thrombomodulin described in Example 14 of Patent
document 13 mentioned above. As a result, although the
concentration was almost near the detection limit, the contaminated
HCP concentration was found to be a ratio of less than 70 to 80 ng
per 10,000 U of the soluble thrombomodulin (henceforth "U" means a
unit of the action for promoting activation of Protein C
(henceforth also abbreviated as APC activity) as later described in
Reference Example 1, unless otherwise specifically indicated), and
thus the inventors of the present invention first recognized that
improvement of the reduction of HCP might still be possibly
achievable. The inventors of the present invention themselves
consider that the HCP concentration can be accurately measured by
employing an additional step of concentration in the HCP
measurement step, which additional step is not disclosed in Patent
document 13.
[0033] The inventors of the present invention who first discovered
the aforementioned fact found a novel object to obtain purified
thrombomodulin consisting of the soluble thrombomodulin having a
further reduced HCP content to minimize the risk of anaphylactic
shock, with the aim of safer use of soluble thrombomodulin as a
pharmaceutical product.
[0034] The inventors of the present invention studied an
application of an additional column chromatography step for
industrial scale production of highly-purified soluble
thrombomodulin in which contamination of HCP was further reduced.
Specifically, they tried to reduce HCP by combining a plurality of
column chromatography steps with affinity column chromatography
considered to have the highest HCP-removing effect. However, such
additional column chromatography steps not only increased the time
and labor for the production, but also caused a problem of
reduction of the yield of soluble thrombomodulin. Moreover, even if
a plurality of column chromatography steps were combined with
affinity column chromatography, highly-purified soluble
thrombomodulin was not obtained in which the HCP concentration was
further reduced compared with the conventional level. Furthermore,
there also arose a problem that, in the column chromatography, only
a small change of pH, ionic strength or the like resulted in a
change of separation of HCP and soluble thrombomodulin, and thus an
expected result was not successfully reproduced. Accordingly, it
was difficult to obtain highly-purified soluble thrombomodulin.
[0035] Therefore, the inventors of the present invention conducted
various researches to find a method for obtaining highly-purified
soluble thrombomodulin containing further reduced contamination of
HCP by efficiently eliminating HCP at an industrially acceptable
level without reducing the yield of soluble thrombomodulin with a
simpler operation compared with column chromatography. As a result,
they found that the aforementioned object of obtaining
highly-purified soluble thrombomodulin having an HCP concentration
corresponding to a ratio of less than 10 ng of HCP per 10,000 U of
soluble thrombomodulin was successfully achieved by using nylon
and/or polyethersulfone, in particular, by using nylon, and thus
accomplished the present invention.
[0036] The present invention is thus embodied as follows.
[1] Highly-purified soluble thrombomodulin which has a content of
host cell-originated proteins being in a ratio of less than 10 ng
of the proteins per 10,000 U of the soluble thrombomodulin, wherein
the soluble thrombomodulin is produced by a transformant cell
obtained by transfecting a host cell with a DNA containing a
nucleotide sequence encoding the soluble thrombomodulin. [1-2] The
highly-purified soluble thrombomodulin according to [1] mentioned
above, wherein purity of the highly-purified soluble thrombomodulin
is 99% or higher based on the total proteins. [1-3] The
highly-purified soluble thrombomodulin according to [1] or [1-2]
mentioned above, wherein the highly-purified soluble thrombomodulin
is in the form of an aqueous solution. [1-4] The highly-purified
soluble thrombomodulin according to [1-3] mentioned above, wherein
a concentration of soluble thrombomodulin in the aqueous solution
of the highly-purified soluble thrombomodulin is 8 mg/mL or higher.
[2] The highly-purified soluble thrombomodulin according to any one
of [1] to [1-4] mentioned above, wherein the soluble thrombomodulin
is thrombomodulin produced by serum-free culture of the
transformant cell.
[0037] When the referred item numbers are indicated with such a
range as "[1] to [1-4]" mentioned above, and the range includes an
item indicated with a number having a subnumber such as [1-2], it
is meant that the item indicated with the number having a subnumber
such as [1-2] is also cited. The same shall apply to the following
definitions.
[2-2] The highly-purified soluble thrombomodulin according to any
one of [1] to [2] mentioned above, wherein the concentration of
host cell-originated proteins of less than 10 ng per 10,000 U of
soluble thrombomodulin is confirmed by measuring content of the
host cell-originated proteins by a method comprising at least the
following steps: (a) the step of preparing host cell-originated
proteins from culture supernatant obtained by carrying out
serum-free culture of a transformant cell obtained by transfecting
the host cell with a DNA containing a nucleotide sequence encoding
the soluble thrombomodulin, or the host cell, (b) the step of
purifying an anti-host cell-originated protein antibody from
antiserum obtained by sensitizing a rabbit with the host
cell-originated proteins obtained in (a) mentioned above, the step
of constructing a measurement system comprising: (c1) the step of
adsorbing the anti-host cell-originated protein antibody obtained
in (b) mentioned above to a solid phase, (c2-1) the step of
bringing a soluble thrombomodulin-containing test solution
suspected to be contaminated with the host cell-originated proteins
into contact with the solid phase to which the anti-host
cell-originated protein antibody is adsorbed, and the step of
bringing a solution containing the host cell-originated proteins of
a known concentration into contact with the solid phase to which
the anti-host cell-originated protein antibody is adsorbed, (c3)
the step of adding a biotinylated anti-host cell-originated protein
antibody to the solid phase, (c4) the step of adding a solution of
avidinylated peroxidase to the solid phase, (c5) the step of adding
an enzyme substrate solution to allow color development, and (c6)
the step of terminating the color development and measuring
absorbance, (d) the step of measuring concentration of the host
cell-originated proteins in the soluble thrombomodulin-containing
test solution suspected to be contaminated with the host
cell-originated proteins in the aforementioned measurement system,
and determining whether the concentration of the host
cell-originated proteins in the soluble thrombomodulin-containing
test solution is within a range that enables quantification of the
proteins in the aforementioned measurement system, which range is
confirmed beforehand by performing measurement using a solution of
the host cell-originated proteins of a known concentration in the
aforementioned measurement system, (e-1) the step of determining
the concentration of the host cell-originated proteins in the
soluble thrombomodulin-containing test solution suspected to be
contaminated with the host cell-originated proteins as the
concentration of the host cell-originated proteins in the solution,
when the concentration is determined to be within the range that
enables the quantification in (d) mentioned above, (e-2-1) the step
of concentrating or diluting the soluble thrombomodulin-containing
test solution suspected to be contaminated with the host
cell-originated proteins, if desired, to make the concentration of
the host cell-originated proteins to be a measurable concentration
within the range that enables the quantification in the
aforementioned measurement system, when the concentration of the
host cell-originated proteins is determined to be not within the
range that enables the quantification in (d) mentioned above, and
recording the concentration ratio or dilution ratio, (e-2-2) the
step of measuring the host cell-originated protein concentration in
the soluble thrombomodulin-containing test solution concentrated or
diluted in (e-2-1) mentioned above in a measurement system
corresponding to the measurement system represented by the steps of
(c1) to (c6) mentioned above in which (c2-1) is replaced with
(c2-2) mentioned below, and obtaining the host cell-originated
protein concentration with taking the concentration ratio or
dilution ratio into consideration, (c2-2) the step of bringing the
soluble thrombomodulin-containing test solution concentrated or
diluted, if necessary, into contact with the solid phase to which
the anti-host cell-originated protein antibody is adsorbed, and the
step of bringing a solution containing the host cell-originated
proteins of a known concentration into contact with the solid phase
to which the anti-host cell-originated protein antibody is
adsorbed, and (f) the step of calculating ratio of the host
cell-originated protein concentration obtained in (e-1) or (e-2-2)
based on APC activity of thrombomodulin per unit volume of the
soluble thrombomodulin-containing test solution measured
separately. [2-3] The highly-purified soluble thrombomodulin
according to [2-2] mentioned above, wherein the host cell mentioned
in [2-2], (a) mentioned above is a cell of Chinese hamster ovary
cell line DXB11. [3] The highly-purified soluble thrombomodulin
according to any one of [1] to [2-3] mentioned above, wherein the
host cell is a Chinese hamster ovary cell. [4] The highly-purified
soluble thrombomodulin according to any one of [1] to [3] mentioned
above, wherein the soluble thrombomodulin has the following
properties (1) to (5): (1) an action of selectively binding to
thrombin, (2) an action of promoting activation of Protein C by
thrombin, (3) an action of extending thrombin clotting time, (4) an
action of suppressing platelet aggregation caused by thrombin, and
(5) anti-inflammatory action. [4-2] The highly-purified soluble
thrombomodulin according to any one of [1] to [3] mentioned above,
wherein the soluble thrombomodulin has the following properties (1)
to (4): (1) an action of selectively binding to thrombin, (2) an
action of promoting activation of Protein C by thrombin, (3) an
action of extending thrombin clotting time, and (4) an action of
suppressing platelet aggregation caused by thrombin. [5] The
highly-purified soluble thrombomodulin according to any one of [1]
to [4-2] mentioned above, wherein molecular weight of the soluble
thrombomodulin is 50,000 to 80,000. [6] The highly-purified soluble
thrombomodulin according to any one of [1] to [5] mentioned above,
wherein the highly-purified soluble thrombomodulin is produced by a
method comprising the following steps: (a) the step of obtaining a
transformant cell by transfecting a host cell with a DNA encoding a
soluble thrombomodulin; (b) the step of obtaining a solution
containing the soluble thrombomodulin by culturing the transformant
cell, and (c) the step of bringing the solution containing the
soluble thrombomodulin into contact with nylon and/or
polyethersulfone to obtain highly-purified soluble thrombomodulin
having a content of host cell-originated proteins being in a ratio
of less than 10 ng of the proteins per 10,000 U of soluble
thrombomodulin. [7] The highly-purified soluble thrombomodulin
according to any one of [1] to [6] mentioned above, wherein the
soluble thrombomodulin is a peptide containing: (i) the amino acid
sequence of the positions 367 to 480 in the amino acid sequence of
SEQ ID NO: 9 or 11, and the amino acid sequence of (ii-1) or (ii-2)
mentioned below, and the peptide is soluble thrombomodulin having
the following properties (1) to (5): (ii-1) the amino acid sequence
of the positions 19 to 244 in the amino acid sequence of SEQ ID NO:
9 or 11, or (ii-2) the amino acid sequence of (ii-1) mentioned
above, further including substitution, deletion or addition of one
or more amino acid residues, (1) an action of selectively binding
to thrombin, (2) an action of promoting activation of Protein C by
thrombin, (3) an action of extending thrombin clotting time, (4) an
action of suppressing platelet aggregation caused by thrombin, and
(5) anti-inflammatory action. [7-2] The highly-purified soluble
thrombomodulin according to any one of [1] to [6] mentioned above,
wherein the soluble thrombomodulin is a peptide containing: (i) the
amino acid sequence of the positions 367 to 480 in the amino acid
sequence of SEQ ID NO: 9 or 11, and the amino acid sequence of
(ii-1) or (ii-2) mentioned below, and the peptide is soluble
thrombomodulin having the following properties (1) to (4): (ii-1)
the amino acid sequence of the positions 19 to 244 in the amino
acid sequence of SEQ ID NO: 9 or 11, or (ii-2) the amino acid
sequence of (ii-1) mentioned above, further including substitution,
deletion or addition of one or more amino acid residues, (1) an
action of selectively binding to thrombin, (2) an action of
promoting activation of Protein C by thrombin, (3) an action of
extending thrombin clotting time, and (4) an action of suppressing
platelet aggregation caused by thrombin. [7-3] The highly-purified
soluble thrombomodulin according to any one of [1] to [6] mentioned
above, wherein the soluble thrombomodulin is a peptide containing
the amino acid sequence of (i-1) or (i-2) mentioned below, and
containing the amino acid sequence of (ii-1) or (ii-2) mentioned
below, and the peptide is soluble thrombomodulin having the
properties (1) to (5) mentioned below: (i-1) the amino acid
sequence of the positions 367 to 480 in the amino acid sequence of
SEQ ID NO: 9 or 11, or (i-2) the amino acid sequence of (i-1)
mentioned above, further including substitution, deletion or
addition of one or more amino acid residues, (ii-1) the amino acid
sequence of the positions 19 to 244 in the amino acid sequence of
SEQ ID NO: 9 or 11, or (ii-2) the amino acid sequence of (ii-1)
mentioned above, further including substitution, deletion or
addition of one or more amino acid residues, (1) an action of
selectively binding to thrombin, (2) an action of promoting
activation of Protein C by thrombin, (3) an action of extending
thrombin clotting time, (4) an action of suppressing platelet
aggregation caused by thrombin, and (5) anti-inflammatory action.
[8] The highly-purified soluble thrombomodulin according to any one
of [1] to [6] mentioned above, wherein the soluble thrombomodulin
is a peptide containing: (i-1) the amino acid sequence of the
positions 19 to 516 in the amino acid sequence of SEQ ID NO: 9 or
11, or (i-2) the amino acid sequence of (i-1) mentioned above,
further including substitution, deletion or addition of one or more
amino acid residues, and the peptide is soluble thrombomodulin
having the properties (1) to (5) mentioned below: (1) an action of
selectively binding to thrombin, (2) an action of promoting
activation of Protein C by thrombin, (3) an action of extending
thrombin clotting time, (4) an action of suppressing platelet
aggregation caused by thrombin, and (5) anti-inflammatory action.
[9] The highly-purified soluble thrombomodulin according to any one
of [1] to [6] mentioned above, wherein the DNA containing a
nucleotide sequence encoding soluble thrombomodulin is a DNA
encoding the amino acid sequence of SEQ ID NO: 9 or 11. [10] A
pharmaceutical composition containing the highly-purified soluble
thrombomodulin according to any one of [1] to [9] mentioned above
and a pharmaceutically acceptable carrier. [11] A method for
preparing highly-purified soluble thrombomodulin having a content
of host cell-originated proteins being in a ratio of less than 10
ng of the proteins per 10,000 U of soluble thrombomodulin, which
comprises the step of bringing a solution containing soluble
thrombomodulin produced by a transformant cell obtained by
transfecting a host cell with a DNA containing a nucleotide
sequence encoding soluble thrombomodulin into contact with nylon
and/or polyethersulfone. [12] The preparation method according to
[11] mentioned above, wherein the soluble thrombomodulin is
prepared by serum-free culture of the transformant cell. [13] The
method for preparing highly-purified soluble thrombomodulin
according to [11] or [12] mentioned above, wherein the soluble
thrombomodulin has the following properties (1) to (5); (1) an
action of selectively binding to thrombin, (2) an action of
promoting activation of Protein C by thrombin, (3) an action of
extending thrombin clotting time, (4) an action of suppressing
platelet aggregation caused by thrombin, and (5) anti-inflammatory
action. [14] The preparation method according to any one of [11] to
[13] mentioned above, wherein the host cell is a Chinese hamster
ovary cell. [15] The preparation method according to any one of
[11] to [14] mentioned above, wherein molecular weight of the
soluble thrombomodulin is 50,000 to 80,000. [16] The preparation
method according to any one of [11] to [15] mentioned above,
wherein the soluble thrombomodulin is a peptide containing: (i) the
amino acid sequence of the positions 367 to 480 in the amino acid
sequence of SEQ ID NO: 9 or 11, and the amino acid sequence of
(ii-1) or (ii-2) mentioned below, and the peptide is soluble
thrombomodulin having the following properties (1) to (5): (ii-1)
the amino acid sequence of the positions 19 to 244 in the amino
acid sequence of SEQ ID NO: 9 or 11, or (ii-2) the amino acid
sequence of (ii-1) mentioned above, further including substitution,
deletion or addition of one or more amino acid residues, (1) an
action of selectively binding to thrombin, (2) an action of
promoting activation of Protein C by thrombin, (3) an action of
extending thrombin clotting time, (4) an action of suppressing
platelet aggregation caused by thrombin, and (5) anti-inflammatory
action. [17] The preparation method according to any one of [11] to
[15] mentioned above, wherein the soluble thrombomodulin is a
peptide containing: (i-1) the amino acid sequence of the positions
19 to 516 in the amino acid sequence of SEQ ID NO: 9 or 11, or
(i-2) the amino acid sequence of (i-1) mentioned above, further
including substitution, deletion or addition of one or more amino
acid residues, and the peptide is soluble thrombomodulin having the
properties (1) to (5) mentioned below: (1) an action of selectively
binding to thrombin, (2) an action of promoting activation of
Protein C by thrombin, (3) an action of extending thrombin clotting
time, (4) an action of suppressing platelet aggregation caused by
thrombin, and (5) anti-inflammatory action. [18] The preparation
method according to any one of [11] to [15] mentioned above,
wherein the DNA containing a nucleotide sequence encoding soluble
thrombomodulin is a DNA encoding the amino acid sequence of SEQ ID
NO: 9 or 11. [19] The preparation method according to any one of
[11] to [18] mentioned above, wherein the nylon and/or
polyethersulfone is in the form of a filtration membrane. [20] The
preparation method according to [19] mentioned above, wherein the
filtration membrane has a membrane area of 0.01 to 0.5 m.sup.2 for
1 mg of the host cell-originated proteins. [21] The preparation
method according to any one of [11] to [20] mentioned above,
wherein the nylon and/or polyethersulfone is nylon. [22] The
preparation method according to any one of [11] to [20] mentioned
above, which has the characteristics described in any one of [1-2]
to [1-4], [2-2], [2-3], [7-2] and [7-3]. [23] The preparation
method according to any one of [11] to [22] mentioned above, which
is a method for preparing highly-purified soluble thrombomodulin
having a content of host cell-originated proteins being in a ratio
of less than 10 ng of the proteins per 10,000 U of soluble
thrombomodulin, comprising: (a) the step of obtaining a
transformant cell by transfecting a host cell with a DNA encoding
the amino acid sequence of SEQ ID NO: 9 or 11, (b) the step of
obtaining a solution containing soluble thrombomodulin by culturing
the transformant cell, (c) the step of purifying the solution
containing soluble thrombomodulin to obtain a thrombomodulin purity
of 99% or higher based on the total proteins, and (d) the step of
bringing the solution containing soluble thrombomodulin into
contact with nylon to isolate highly-purified soluble
thrombomodulin having a content of host cell-originated proteins
being in a ratio of less than 10 ng of the proteins per 10,000 U of
soluble thrombomodulin, and wherein the host cell is a Chinese
hamster ovary cell. [24] Highly-purified soluble thrombomodulin
producible by the method according to [23] mentioned above. [25] A
method for removing host cell-originated proteins in soluble
thrombomodulin, which comprises the step of bringing a solution
containing soluble thrombomodulin produced by a transformant cell
obtained by transfecting a host cell with a DNA containing a
nucleotide sequence encoding soluble thrombomodulin into contact
with nylon and/or polyethersulfone. [26] A method for removing host
cell-originated proteins in soluble thrombomodulin, which
comprises: (a) the step of obtaining a transformant cell by
transfecting a host cell with a DNA encoding the amino acid
sequence of SEQ ID NO: 9 or 11, (b) the step of obtaining a
solution containing soluble thrombomodulin by culturing the
transformant cell, (c) the step of purifying the solution
containing soluble thrombomodulin to a thrombomodulin purity of 99%
or higher based on the total proteins, and (d) the step of bringing
the solution containing soluble thrombomodulin into contact with
nylon, and wherein the host cell is a Chinese hamster ovary cell.
[27] The method for removing host cell-originated proteins in
soluble thrombomodulin according to [25] mentioned above, which has
the characteristics mentioned in any one of [1] to [9] mentioned
above.
Effect of the Invention
[0038] By using the preparation method of the present invention,
highly-purified soluble thrombomodulin of reduced contamination of
host cell-originated proteins having a content of host
cell-originated proteins being in a ratio of less than 10 ng of the
proteins per 10,000 U of soluble thrombomodulin can be obtained.
The risk of anaphylactic shock at the time of using soluble
thrombomodulin as a pharmaceutical product can be thereby further
reduced.
MODES FOR CARRYING OUT THE INVENTION
[0039] Hereafter, several preferred embodiments of the present
invention (preferred modes for carrying out the invention,
henceforth also referred to as "embodiments" in this specification)
will be specifically explained. However, the scope of the present
invention is not limited to the specific embodiments explained
below.
[0040] The highly-purified soluble thrombomodulin of this
embodiment can be used as a material for a medicament. The
highly-purified soluble thrombomodulin of this embodiment can also
be combined with another pharmaceutically acceptable carrier and
used as a pharmaceutical product.
[0041] Further, the highly-purified soluble thrombomodulin of this
embodiment can be used as a highly-purified soluble
thrombomodulin-containing pharmaceutical composition not
substantially containing any substances other than the soluble
thrombomodulin. The highly-purified soluble thrombomodulin of this
embodiment can also be combined with another pharmaceutically
acceptable carrier and used as a pharmaceutical composition.
[0042] Hereafter, the term of highly-purified soluble
thrombomodulin may include highly-purified soluble thrombomodulin
as a material for a medicament. The term of highly-purified soluble
thrombomodulin may also include a highly-purified soluble
thrombomodulin-containing pharmaceutical composition not
substantially containing any substances other than the soluble
thrombomodulin.
[0043] The thrombomodulin of this embodiment preferably is known to
have an action of (1) selectively binding to thrombin (2) to
promote activation of Protein C by thrombin. In addition, it is
preferred that the thrombomodulin is confirmed to generally have
(3) an action of extending thrombin clotting time, (4) an action of
suppressing platelet aggregation caused by thrombin, and/or (5)
anti-inflammatory action. Such actions possessed by thrombomodulin
may be referred to as thrombomodulin activities.
[0044] As the thrombomodulin activities, thrombomodulin preferably
has the actions of (1) and (2) mentioned above, and more preferably
has the actions of (1) to (4) mentioned above. As the
thrombomodulin activities, thrombomodulin more preferably has all
of the actions of (1) to (5) mentioned above.
[0045] The action of thrombomodulin to bind with thrombin can be
confirmed by the test methods described in various known
publications such as Thrombosis and Haemostasis, 70(3):418-422
(1993). As for the action of promoting activation of Protein C by
thrombin, degree of the activity of promoting the activation of
Protein C by thrombin or presence or absence of the action can be
easily confirmed by the test methods clearly described in various
known publications including, for example, Japanese Patent
Unexamined Publication No. 64-6219. Further, the action of
extending thrombin clotting time, and/or the action of suppressing
platelet aggregation caused by thrombin can be similarly and easily
confirmed. Furthermore, the anti-inflammatory action can also be
confirmed by the test methods described in various known
publications including, for example, Blood, 112:3361-3670 (2008)
and The Journal of Clinical Investigation, 115, 5:1267-1274
(2005).
[0046] An example of the soluble thrombomodulin includes a soluble
thrombomodulin that is soluble in water in the absence of a
surfactant. For example, the solubility of soluble thrombomodulin
is preferably 1 mg/ml or higher, or 10 mg/ml or higher in water,
for example, in distilled water used for injection (in general,
around a neutral range in the absence of a surfactant such as
Triton X-100 or Polidocanol). The solubility is more preferably 15
mg/ml or higher, or 17 mg/ml or higher; still more preferably 20
mg/ml or higher, 25 mg/ml or higher, or 30 mg/ml or higher; and
mosty preferably 60 mg/ml or higher. In some cases, the solubility
may be 80 mg/ml or higher, or 100 mg/ml or higher. For determining
whether or not soluble thrombomodulin can be dissolved, the
solution can be observed with the naked eye, for example, directly
below white light source, at a position of brightness of
approximately 1,000 lux after the soluble thrombomodulin is
dissolved, and it can be understood that transparency of the
resulting solution and no contamination of apparently observable
insoluble substances may be clear criteria of dissolution. In
addition, it is also possible to filtrate the solution to confirm
the presence or absence of a residue.
[0047] The molecular weight of the soluble thrombomodulin is not
limited so far that it has the thrombomodulin activities and is
soluble as described above. The molecular weight is preferably
100,000 or smaller, more preferably 90,000 or smaller, still more
preferably 80,000 or smaller, most preferably 70,000 or smaller,
and the molecular weight is preferably 50,000 or larger, most
preferably 60,000 or larger. The molecular weight of the soluble
thrombomodulin can be easily measured by ordinary methods for
measuring molecular weight of protein. Measurement by mass
spectrometry is preferred, and MALDI-TOF-MS method is more
preferred.
[0048] For obtaining soluble thrombomodulin having a molecular
weight within a desired range, a soluble thrombomodulin, which is
obtained by culturing a transformant cell prepared by transfecting
a host cell with a DNA encoding soluble thrombomodulin using a
vector, can be subjected to fractionation using column
chromatography or the like as described later.
[0049] As the soluble thrombomodulin, those of the human-type
thrombomodulin are preferred which include the amino acid sequence
of the positions 19 to 132 in SEQ ID NO: 1 together with the amino
acid sequence of the positions 19 to 244 in SEQ ID NO: 9 or 11 or
said amino acid sequence further including substitution, deletion
or addition of one or more amino acid residues. The amino acid
sequence of the positions 19 to 132 in SEQ ID NO: 1 participates in
the action of promoting activation of Protein C by thrombin among
the thrombomodulin activities. The amino acid sequence of the
positions 19 to 244 in SEQ ID NO: 9 or 11 participates in the
anti-inflammatory action among the thrombomodulin activities. So
far that the soluble thrombomodulin has the thrombomodulin
activities as the whole soluble thrombomodulin, the amino acid
sequence of the positions 19 to 132 in SEQ ID NO: 1 may be
naturally or artificially mutated, namely, the amino acid sequence
of the positions 19 to 132 in SEQ ID NO: 1 may include
substitution, deletion or addition of one or more amino acid
residues. Acceptable degree of the mutation is not particularly
limited so far that the soluble thrombomodulin has the
thrombomodulin activities, for example, including homology of 50%
or higher based on an amino acid sequence, preferably homology of
70% or higher, more preferably homology of 80% or higher, further
preferably homology of 90% or higher, further more preferably
homology of 95% or higher, and most preferably homology of 98% or
higher. Such mutated amino acid sequence including substitution,
deletion or addition of one or more amino acid residues is referred
to as homologous mutation sequence. As described later, these
mutated amino acid sequences can be easily produced by using
ordinary gene manipulation techniques. The soluble thrombomodulin
is not particularly limited so far that it has the aforementioned
sequence and the action of selectively binding to thrombin to
promote activation of Protein C by thrombin at least as the whole
soluble thrombomodulin. The soluble thrombomodulin preferably also
has the anti-inflammatory action.
[0050] In the amino acid sequence of SEQ ID NO: 3, Val as the amino
acid at the position 125 in the amino acid sequence of SEQ ID NO: 1
is replaced by Ala. It is also preferred that the thrombomodulin of
this embodiment contains the amino acid sequence of the positions
19 to 132 in SEQ ID NO: 3.
[0051] As described above, the soluble thrombomodulin is not
particularly limited, so far that it has at least the sequence of
the positions 19 to 132 in SEQ ID NO: 1 or 3, or a homologous
mutation sequence thereof, and the amino acid sequence of the
positions 19 to 244 in SEQ ID NO: 9 or 11, or a homologous mutation
sequence thereof, and has at least the action of selectively
binding to thrombin to promote activation of Protein C by thrombin
as the whole soluble thrombomodulin. Preferred examples include a
peptide comprising the sequence of the positions 19 to 132 or the
positions 17 to 132 in SEQ ID NO: 1 or 3, or a homologous mutation
sequence thereof, and the amino acid sequence of the positions 19
to 244 in SEQ ID NO: 9 or 11 or a homologous mutation sequence
thereof, and having the thrombomodulin activities at least as the
whole soluble thrombomodulin, and a peptide comprising the sequence
of the positions 19 to 132 in SEQ ID NO: 1 or 3 and the amino acid
sequence of the positions 19 to 244 in SEQ ID NO: 9 or 11 or a
homologous mutation sequence thereof is more preferred. There is
also another embodiment in which a peptide comprising the sequence
of the positions 19 to 132 or the positions 17 to 132 in SEQ ID NO:
1 or 3 or a homologous mutation sequence thereof, and the amino
acid sequence of the positions 19 to 244 in SEQ ID NO: 9 or 11 or a
homologous mutation sequence, and having the thrombomodulin
activities at least as the whole soluble thrombomodulin, which is
sometimes more preferred.
[0052] It is preferred that the soluble thrombomodulin also has the
anti-inflammatory action as the whole soluble thrombomodulin.
[0053] As another embodiment, the thrombomodulin preferably
contains the amino acid sequence of the positions 19 to 480 in SEQ
ID NO: 5, and such thrombomodulin is not particularly limited so
far that it contains the amino acid sequence of the positions 19 to
480 in SEQ ID NO: 5. The amino acid sequence of the positions 19 to
480 in SEQ ID NO: 5 may be a homologous mutation sequence so far
that it has the thrombomodulin activities.
[0054] The amino acid sequence of SEQ ID NO: 7 corresponds to the
amino acid sequence of SEQ ID NO: 5 in which Val as the amino acid
at the position 473 is replaced with Ala. The soluble
thrombomodulin of this embodiment also preferably contains the
amino acid sequence of the positions 19 to 480 in SEQ ID NO: 7.
[0055] As described above, the soluble thrombomodulin is not
particularly limited so far that it has at least the amino acid
sequence of the positions 19 to 480 in SEQ ID NO: 5 or 7, or a
homologous mutation sequence thereof, and contains a peptide
sequence having at least the thrombomodulin activities. Preferred
examples include a peptide comprising the sequence of the positions
19 to 480 or the positions 17 to 480 in SEQ ID NO: 5 or 7, or a
homologous mutation sequence thereof, and having at least the
thrombomodulin activities, and a peptide comprising the sequence of
the positions 19 to 480 in SEQ ID NO: 5 or 7 is more preferred.
There is also another embodiment in which a peptide comprising a
homologous mutation sequence of the sequence of the positions 19 to
480 or the positions 17 to 480 in SEQ ID NO: 5 or 7 and having at
least the thrombomodulin activity, which is sometime mores
preferred.
[0056] It is preferred that the soluble thrombomodulin also has the
anti-inflammatory action.
[0057] As another particularly preferred embodiment, the soluble
thrombomodulin preferably contains the amino acid sequence of the
positions 19 to 515 in SEQ ID NO: 9, and such soluble
thrombomodulin is not particularly limited so far that it contains
the amino acid sequence of the positions 19 to 515 in SEQ ID NO: 9.
The amino acid sequence of the positions 19 to 515 o in SEQ ID NO:
9 may be a homologous mutation sequence thereof so far that it has
the thrombomodulin activities.
[0058] The amino acid sequence of SEQ ID NO: 11 corresponds to the
amino acid sequence of SEQ ID NO: 9 in which Val as the amino acid
at the position 473 is replaced with Ala. The soluble
thrombomodulin of this embodiment also preferably contains the
amino acid sequence of the positions 19 to 515 in SEQ ID NO:
11.
[0059] As described above, the soluble thrombomodulin is not
particularly limited so far that it contains a peptide having at
least the amino acid sequence of the positions 19 to 515 in SEQ ID
NO: 9 or 11, or a homologous mutation sequence thereof, and having
at least the thrombomodulin activities. More preferred examples
include a peptide comprising the amino acid sequence of the
positions 19 to 516, 19 to 515, 19 to 514, 17 to 516, 17 to 515, or
17 to 514 in SEQ ID NO: 9 or 11, or a peptide comprising a
homologous mutation sequence thereof and having at least the
thrombomodulin activities, and a peptide comprising the amino acid
sequence of the positions 19 to 516, 19 to 515, 19 to 514, 17 to
516, 17 to 515, or 17 to 514 in SEQ ID NO: 9 is particularly
preferred. In addition, a mixture thereof is also a preferred
example. There is another preferred embodiment in which a peptide
comprising the amino acid sequence of the positions 19 to 516, 19
to 515, 19 to 514, 17 to 516, 17 to 515, or 17 to 514 in SEQ ID NO:
11, which is particularly preferred. A mixture thereof is also a
preferred example. Further, a peptide comprising a homologous
mutation sequence thereof and having at least the thrombomodulin
activities is also another preferred example.
[0060] It is preferred that the soluble thrombomodulin also has the
anti-inflammatory action.
[0061] A peptide having a homologous mutation sequence is as
described above. Such a peptide having a homologous mutation
sequence also includes a peptide that may include substitution,
deletion, or addition of one or more, namely, one or multiple, more
preferably several (for example, 1 to 20, preferably 1 to 10, more
preferably 1 to 5, most preferably 1 to 3) amino acid residues in
the amino acid sequence of the target peptide. Acceptable degree of
mutation is not particularly limited so far that the peptide has
the thrombomodulin activities. Examples include, for example,
homology of 50% or higher, preferably homology of 70% or higher,
more preferably homology of 80% or higher, further preferably
homology of 90% or higher, further more preferably homology of 95%
or higher, and most preferably homology of 98% or higher based on
an amino acid sequence.
[0062] As the soluble thrombomodulin, preferred examples further
include a peptide comprising the sequence of SEQ ID NO: 14 (462
amino acid residues), a peptide comprising the sequence of SEQ ID
NO: 8 (272 amino acid residues), and a peptide comprising the
sequence of SEQ ID NO: 6 (236 amino acid residues) described in
Japanese Patent Unexamined Publication No. 64-6219.
[0063] The soluble thrombomodulin is not particularly limited so
far that it is a peptide having at least the sequence of the
positions 19 to 132 in SEQ ID NO: 1 or 3, or a homologous mutation
sequence thereof, and the amino acid sequence of the positions 19
to 244 in SEQ ID NO: 9 or 11, or a homologous mutation sequence
thereof, and has the thrombomodulin activities at least as the
whole soluble thrombomodulin. A peptide comprising at least the
amino acid sequence of the positions 19 to 480 in SEQ ID NO: 5 or 7
is preferred, and a peptide comprising at least the amino acid
sequence of the positions 19 to 515 in SEQ ID NO: 9 or 11 is more
preferred. More preferred examples of the peptide comprising at
least the amino acid sequence of the positions 19 to 515 in SEQ ID
NO: 9 or 11 include a peptide comprising the amino acid sequence of
the positions 19 to 516, 19 to 515, 19 to 514, 17 to 516, 17 to
515, or 17 to 514 in SEQ ID NO: 9 or 11. More preferred examples
also include a mixture of peptides comprising the amino acid
sequence of the positions 19 to 516, 19 to 515, 19 to 514, 17 to
516, 17 to 515, or 17 to 514 for each of SEQ ID NOS: 9 and 11.
[0064] In the case of the aforementioned mixture, the mixing ratio
of a peptide that starts from the position 17 and a peptide that
starts from the position 19 for each of SEQ ID NOS: 9 and 11 is,
for example, 30:70 to 50:50, preferably 35:65 to 45:55.
[0065] Further, the mixing ratio of a peptide that terminates at
the position 514, a peptide that terminates at the position 515,
and a peptide that terminates at the position 516 for each of SEQ
ID NOS: 9 and 11 is, for example, 0:0:100 to 0:90:10, or 0:70:30 to
10:90:0, or 10:0:90 to 20:10:70, if desired.
[0066] The mixing ratio of the peptides can be determined by an
ordinary method.
[0067] The sequence of the positions 19 to 132 in SEQ ID NO: 1
corresponds to the sequence of the positions 367 to 480 in SEQ ID
NO: 9, and the sequence of the positions 19 to 480 in SEQ ID NO: 5
corresponds to the sequence of the positions 19 to 480 in SEQ ID
NO: 9.
[0068] Further, the sequence of the positions 19 to 132 in SEQ ID
NO: 3 corresponds to the sequence of the positions 367 to 480 in
SEQ ID NO: 11, and the sequence of the positions 19 to 480 in SEQ
ID NO: 7 corresponds to the sequence of the positions 19 to 480 in
SEQ ID NO: 11.
[0069] Furthermore, all the sequences of the positions 1 to 18 in
SEQ ID NOS: 1, 3, 5, 7, 9 and 11 are identical sequences.
[0070] These soluble thrombomodulin can be obtained, for example,
from a transformant cell prepared by transfecting a host cell with
a DNA encoding any of those peptides (specifically, a nucleotide
sequence having the nucleotide sequence of the positions 1 to 732
in SEQ ID NO: 10 and the nucleotide sequence of the positions 55 to
396 in SEQ ID NO: 2, a nucleotide sequence having the nucleotide
sequence of the positions 1 to 732 in SEQ ID NO: 10 and the
nucleotide sequence of the positions 55 to 396 in SEQ ID NO: 4, a
nucleotide sequence of SEQ ID NO: 6, 8, 10 or 12) using a vector,
as described later.
[0071] It is sufficient that these peptides have any of the
aforementioned amino acid sequences. A sugar chain may be or may
not be added, and the peptides are not particularly limited in this
respect. In addition, in gene manipulation, type of such a sugar
chain, position to which a sugar chain is added, and degree of
addition may vary depending on the type of the host cell used, and
they are not particularly limited. The binding position of such a
sugar chain and the type thereof are described in Japanese Patent
Unexamined Publication No. 11-341990, and in the case of the
thrombomodulin of this embodiment, similar sugar chains may be
added to similar positions. Two types of N-linked sugar chains,
those of fucosyl biantennary type and fucosyl triantennary type,
may bind to the soluble thrombomodulin of this embodiment, and
ratio thereof is, for example, 100:0 to 60:40, preferably 95:5 to
60:40, more preferably 90:10 to 70:30. The ratio of these sugar
chains can be measured on a two-dimensional sugar chain map
described in Biochemical Experimental Methods, Vol. 23, Methods of
Researches on Glycoprotein Sugar Chains, Japan Scientific Societies
Press (1990), and the like. Furthermore, when a sugar composition
of the soluble thrombomodulin of this embodiment is examined,
neutral saccharides, aminosaccharides, and sialic acid is detected,
of which content may be, each independently for example, 1 to 30%,
preferably 2 to 20%, more preferably 5 to 10% in terms of weight
ratio based on a protein content. The sugar contents can be
measured by the methods described in Lecture of New Biochemical
Experiments, Vol. 3, Sugar I, Glycoprotein (Book 1), Tokyo Kagaku
Dojin (1990) (neutral saccharides: phenol-sulfuric acid method,
aminosaccharides: Elson-Morgan method, sialic acid: periodic
acid-resorcinol method).
[0072] As a signal sequence that can be used for expression where
the soluble thrombomodulin is obtained by gene manipulation, a
nucleotide sequence encoding the amino acid sequence of the
positions 1 to 18 in SEQ ID NO: 9, and a nucleotide sequence
encoded by a nucleotide sequence encoding the amino acid sequence
of the positions 1 to 16 in SEQ ID NO: 9 can be used, and other
known signal sequences such as the signal sequence of human tissue
plasminogen activator can also be used (International Publication
WO88/9811).
[0073] When a DNA sequence encoding soluble thrombomodulin is
introduced into a host cell, there is preferably used a method of
incorporating the DNA sequence encoding soluble thrombomodulin into
a vector, more preferably an expression vector that can be
expressed in animal cells, and then introducing the vector into the
host cell. The term "expression vector" means a DNA molecule
constituted by a promoter sequence, a sequence for adding a
ribosome binding site to mRNA, a DNA sequence encoding a protein to
be expressed, a splicing signal, a terminator sequence for
transcription termination, a replication origin sequence, and the
like. Examples of a preferred animal cell expression vector include
pSV2-X reported by R. C. Mulligan et al. (Proc. Natl. Acad. Sci.
U.S.A. 78, 2072 (1981)) and pBP69T (69-6) reported by P. M. Howley
et al. (Methods in Emzymology, 101, 387, Academic Press (1983)).
Further, there is also another preferred embodiment in which DNA is
introduced into an expression vector expressible in a
microorganism.
[0074] Examples of host cell that can be used in production of such
peptides as mentioned above include animal cells.
[0075] Examples of the animal cells include Chinese hamster ovary
(CHO) cells, COS-1 cells, COS-7 cells, VERO (ATCC CCL-81) cells,
BHK cells, canine kidney-derived MDCK cells, hamster AV-12-664
cells, NS0 cells, and the like. In addition, examples of host cell
derived from human include HeLa cells, WI38 cells, human 293 cells,
and PER.C6 cells. Of these cells, CHO cells are very common and
preferred, and among the CHO cells, dihydrofolate reductase
(DHFR)-deficient CHO cells are more preferred.
[0076] In a gene manipulation process or a peptide production
process, microorganisms such as Escherichia coli are also often
used. A host-vector system suitable for each process is preferably
used, and an appropriate vector system can also be selected for the
aforementioned host cells. A thrombomodulin gene used in a genetic
recombination technique has been cloned. Examples of production of
thrombomodulin by such a gene recombination technique have been
disclosed, and further, methods for purifying thrombomodulin to
obtain a purified product thereof are also known (Japanese Patent
Unexamined Publication Nos. 64-6219, 2-255699, 5-213998, 5-310787,
7-155176; and J. Biol. Chem., 264:10351-10353 (1989)). Therefore,
the soluble thrombomodulin used for this embodiment can be produced
by using the methods described in the aforementioned reports, or by
similar methods. For example, Japanese Patent Unexamined
Publication No. 64-6219 discloses the Escherichia coli K-12 strain
DH5 (ATCC Accession No. 67283) containing a plasmid pSV2TMJ2 that
contains a DNA encoding the full-length thrombomodulin. This strain
re-deposited at the former National Institute of Bioscience and
Human-Technology (currently Independent Administrative Institution,
National Institute of Advanced Industrial Science and Technology,
International Patent Organism Depositary) (Escherichia coli
DH5/pSV2TMJ2) (FERM BP-5570) can also be used. The thrombomodulin
of this embodiment can be prepared by a known gene manipulation
technique using a DNA encoding the full-length thrombomodulin as a
starting material.
[0077] The soluble thrombomodulin may be prepared by a
conventionally known method or a similar method. For example, the
aforementioned method of Yamamoto et al. (Japanese Patent
Unexamined Publication No. 64-6219) or the method described in
Japanese Patent Unexamined Publication No. 5-213998 can be referred
to. Specifically, for example, a DNA encoding the amino acid
sequence of SEQ ID NO: 9 is prepared from a human-derived soluble
thrombomodulin gene by a gene manipulation technique, and may be
further modified as required. For such modification, in order to
obtain a DNA encoding the amino acid sequence of SEQ ID NO: 11
(which specifically consists of the nucleotide sequence of SEQ ID
NO: 12), codons encoding the amino acid at the position 473 in the
amino acid sequence of SEQ ID NO: 9 (in particular, the nucleotide
at the position 1418) are mutated by site-directed mutagenesis
according to the method described in Methods in Enzymology, 100:
468 (1983), Academic Press. For example, by using a synthetic DNA
for mutation having the nucleotide sequence of SEQ ID NO: 13, the
nucleotide T at the position 1418 in SEQ ID NO: 10 may be converted
to the nucleotide C to obtain a mutated DNA.
[0078] The DNA prepared as described above is incorporated into,
for example, Chinese hamster ovary (CHO) cells to obtain
transformant cells. Such cells are subjected to appropriate
selection, and soluble thrombomodulin purified by a known method
can be produced from a culture solution obtained by culturing a
selected cell. As described above, the DNA (SEQ ID NO: 10) encoding
the amino acid sequence of SEQ ID NO: 9 is preferably transfected
into the aforementioned host cell.
[0079] For the culture of the aforementioned transformant cell, a
medium used for ordinary cell culture may be used, and it is
preferable to culture the transformant cell in various kinds of
media in advance to choose an optimal medium. For example, a known
medium such as MEM medium, DMEM medium, and 199 medium may be used
as a base medium, and a further improved medium or a medium added
with supplements for various media may be used. Examples of the
culture method include serum culture, in which culture is performed
in a medium containing blood serum, and serum-free culture, in
which culture is performed in a medium not containing blood serum.
Although the culture method is not particularly limited, the
serum-free culture is preferred.
[0080] When serum is added to a medium in the case of the serum
culture, bovine serum is preferred. Examples of bovine serum
include fetal bovine serum, neonate bovine serum, calf bovine
serum, adult bovine serum, and the like, and any of these examples
may be used so far that the serum is suitable for the cell culture.
As the serum-free medium used in the serum-free culture,
commercially available media can be used. Serum-free media suitable
for various cells are marketed, and for example, for the CHO cell,
CD-CHO, CHO-S-SFMII and CHO-III-PFM are sold by Invitrogen, and IS
CHO, IS CHO-CD medium, and the like are sold by Irvine Scientific.
These media may be used without any treatment, or they may be
improved or added with supplements and used. Examples of the
serum-free medium further include the DMEM medium containing 5 mg/L
each of insulin, transferrin, and selenious acid. As described
above, the medium is not particularly limited so far that the
medium can be used to produce the thrombomodulin of this
embodiment. The culture method is not particularly limited, and any
of batch culture, repetitive batch culture, fed-batch culture,
perfusion culture, and the like may be used.
[0081] When the soluble thrombomodulin is prepared by the
aforementioned cell culture method, diversity may be observed in
the N-terminus amino acid due to posttranslational modification of
the protein. For example, the amino acid of the position 17, 18, 19
or 22 in SEQ ID NO: 9 may serve as the N-terminus amino acid.
Further, for example, the N-terminus amino acid may be modified so
that the glutamic acid at the position 22 is changed to
pyroglutamic acid. It is preferred that the amino acid of the
position 17 or 19 serves as the N-terminus amino acid, and it is
more preferred that the amino acid of the position 19 serves as the
N-terminus amino acid. Further, there is also another embodiment in
which the amino acid of the position 17 serves as the N-terminus
amino acid, which is a preferred embodiment. As for the
modification, diversity and the like mentioned above, similar
examples can be mentioned for the sequence of SEQ ID NO: 11.
[0082] Further, when the soluble thrombomodulin is prepared by
using a DNA having the nucleotide sequence of SEQ ID NO: 10,
diversity of the C-terminus amino acid may be observed, and a
peptide shorter by one amino acid residue may be produced.
Specifically, the C-terminus amino acid may be modified so that the
amino acid of the position 515 serves as the C-terminus amino acid,
and further the position 515 is amidated. Further, a peptide
shorter by two amino acid residues may be produced. Specifically,
the amino acid of the position 514 may serve as the C-terminus
amino acid. Therefore, any of peptides having significant diversity
of the N-terminus amino acid and C-terminus amino acid, or a
mixture of them may be produced. It is preferred that the amino
acid of the position 515 or the amino acid of the position 516
serves as the C-terminus amino acid, and it is more preferred that
the amino acid of the position 516 serves as the C-terminus amino
acid. Further, there is also another embodiment in which the amino
acid of the position 514 serves as the C-terminus amino acid, which
is a preferred embodiment. Concerning the modification, diversity
and the like described above, the same shall apply to a DNA having
the nucleotide sequence of SEQ ID NO: 12.
[0083] The thrombomodulin obtained by the method described above
may be a mixture of peptides having diversity in the N-terminus and
C-terminus amino acids. Specific examples include a mixture of
peptides having the sequences of the positions 19 to 516, positions
19 to 515, positions 19 to 514, positions 17 to 516, positions 17
to 515, and positions 17 to 514 in SEQ ID NO: 9.
[0084] Highly-purified soluble thrombomodulin in which
contamination of HCP is reduced is provided by the present
invention.
[0085] Examples of the highly-purified soluble thrombomodulin of
this embodiment include highly-purified soluble thrombomodulin that
does not substantially contain any protein other than soluble
thrombomodulin. Specifically, an example includes a soluble
thrombomodulin not substantially containing HCP, for example.
Preferably, an example includes a soluble thrombomodulin not
substantially containing HCP, mouse IgG, and bovine serum
proteins.
[0086] The highly-purified soluble thrombomodulin of this
embodiment contains no proteins originated from human.
[0087] The content of HCP is not particularly limited so far that
the soluble thrombomodulin is in a state that it does not
substantially contain HCP. The content is preferably a ratio of HCP
being less than 10 ng, more preferably less than 8 ng, still more
preferably less than 7 ng, further more preferably less than 6 ng,
most preferably less than 5 ng per 10,000 U of soluble
thrombomodulin. The highly-purified soluble thrombomodulin of this
embodiment is produced in a transformant cell obtained by
transfecting a host cell with a DNA containing a nucleotide
sequence encoding soluble thrombomodulin, and it is considered that
even the product is purified as highly as possible, there still
actually is a possibility of contamination of HCP in a trace amount
and the like. A content of HCP as low as possible is preferred. An
example of minimum content of HCP includes, for example, a ratio of
0.001 ng of HCP per 10,000 U of soluble thrombomodulin.
[0088] The content of mouse IgG is not particularly limited so far
that the soluble thrombomodulin is in a state that it does not
substantially contain mouse IgG. A ratio of less than 10 ng of
mouse IgG is preferred, a ratio of less than 2 ng is more
preferred, and the ratio of less than 0.6 ng is still more
preferred per 10,000 U of soluble thrombomodulin.
[0089] The content of bovine serum proteins is not particularly
limited so far that the soluble thrombomodulin is in a state that
it does not substantially contain bovine serum proteins. A ratio of
less than 10 ng of bovine serum proteins is preferred, a ratio of
less than 2 ng is more preferred, and a ratio of less than 0.6 ng
is still more preferred per 10,000 U of soluble thrombomodulin.
Concentrations of these proteins are preferably measured by ELISA,
and the measurement can be performed by referring to Biochemical
Experimental Methods, Vol. 11, Enzyme Immunoassay, Tokyo Kagaku
Dojin (1992), and the like.
[0090] As for thrombomodulin purity of soluble thrombomodulin based
on the total proteins in the highly-purified soluble thrombomodulin
of this embodiment, the purity is preferably 99% or higher, more
preferably 99.5% or higher, still more preferably 99.7% or higher,
most preferably 99.9% or higher according to HPLC method. A type of
the chromatography used in the HPLC method is not limited so far
that purity of the soluble thrombomodulin can be measured. Examples
include gel filtration liquid chromatography, ion exchange liquid
chromatography, reverse phase liquid chromatography, and the like,
and gel filtration liquid chromatography is preferred. When gel
filtration liquid chromatography is used, the column to be used may
be chosen depending on the molecular weight of the soluble
thrombomodulin. An example includes, for example, a method of
development by using a phosphate buffer of pH 7.3 using TOSOH
TSKgel G3000SWXL (TOSOH, Japan). The test may be performed
according to the description in Japanese Pharmacopoeia, Liquid
Chromatography <2.01>.
[0091] In the highly-purified soluble thrombomodulin of this
embodiment, DNA components originated from the host is preferably
at a ratio of less than 2 ng, more preferably less than 0.2 ng,
still more preferably less than 0.02 ng per 10,000 U of soluble
thrombomodulin. Amount of DNAs can be easily measured by using
Threshold System (Molecular Devices, U.S.A.).
[0092] A form of the highly-purified soluble thrombomodulin of this
embodiment is not particularly limited so far that the content of
HCP is within a ratio of less than 10 ng per 10,000 U of soluble
thrombomodulin, and it can exist in the form of a solution or
powder. The product preferably exists in the form of a solution.
There is also another embodiment in which the product exists in the
form of powder, which is a preferred embodiment. As for a
concentration in the form of a solution, an upper limit is
preferably 100 mg/mL or lower, more preferably 60 mg/mL or lower,
still more preferably 30 mg/mL or lower, most preferably 15 mg/mL
or lower, and lower limit is preferably 2 mg/mL or higher, more
preferably 4 mg/mL or higher, still more preferably 6 mg/mL or
higher, further more preferably 8 mg/mL or higher, most preferably
10 mg/mL or higher. Further, when the product exists in the form of
a powder, a preferred example includes the form of a lyophilized
powder. Such lyophilized product can be obtained by referring to
the method described in WO03/061687.
[0093] The highly-purified soluble thrombomodulin of this
embodiment can be obtained so as not to substantially contain
endotoxins. The endotoxin content may preferably be less than 1
endotoxin unit (EU), more preferably less than 0.2 EU, still more
preferably less than 0.04 EU, per 10,000 U of soluble
thrombomodulin. Amount of endotoxins can be measured in accordance
with the descriptions in Japanese Pharmacopoeia, General Test
Procedures, Endotoxin Test Method <4.01>. Further, the
highly-purified soluble thrombomodulin of this embodiment can be
obtained in a state that it does not contain any substances harmful
to living bodies such as TFA and almost in a sterile state, and
accordingly, can be used as a material for pharmaceutical
products.
[0094] The highly-purified soluble thrombomodulin of this
embodiment in which contamination of HCP is reduced can be obtained
by bringing a solution containing soluble thrombomodulin into
contact with nylon and/or polyethersulfone. It is preferable to use
nylon. There is also another embodiment in which it is preferable
to use polyethersulfone.
[0095] Examples of nylon with which a solution containing the
soluble thrombomodulin of this embodiment is brought into contact
include, for example, polyamides containing an aliphatic structure
such as Nylon 6, Nylon 66, Nylon 46, and Nylon MXD 6. The type of
nylon is not limited so far that the nylon can adsorb HCP. Nylon 6
is preferred. Nylon is available as, for example, Minisart NY sold
by Sartorius. The form of nylon is not particularly limited so far
that the nylon is in such a form that a solution can be contacted,
such as those of membrane, nonwoven fabric, and beads. The nylon is
preferably molded in the form of membrane and used as a filtration
membrane. In this embodiment, a pore diameter of the filtration
membrane is not limited so far that the diameter allows HCP to pass
through the membrane, for example, 0.01 to 10 .mu.m, preferably 0.1
to 1 .mu.m, more preferably 0.01 to 0.06 .mu.m. The volume of the
solution containing the soluble thrombomodulin to be contacted with
nylon can be easily determined by bringing a part of the solution
into contact with a small amount of nylon beforehand to evaluate
the HCP-removing ability thereof.
[0096] The highly-purified soluble thrombomodulin of this
embodiment can be prepared with confirming that the HCP content in
the solution obtained by bringing a soluble
thrombomodulin-containing solution containing HCP into contact with
nylon is in a ratio of less than 10 ng of HCP per 10,000 U of
soluble thrombomodulin, and when the HCP content becomes 10 ng or
higher per 10,000 U of soluble thrombomodulin, collection of
highly-purified soluble thrombomodulin can be terminated, or after
the used nylon is changed to fresh nylon, the collection of
highly-purified soluble thrombomodulin may be restarted. As for
examples of the relation between the amount of HCP and the area of
nylon, examples where a nylon is in the form of filtration membrane
for example include an upper limit of the area of the membrane
being 50 m.sup.2 or smaller, preferably 5 m.sup.2 or smaller, more
preferably 0.5 m.sup.2 or smaller, still more preferably 0.1
m.sup.2 or smaller, and preferably a lower limit being 0.01 m.sup.2
or larger, more preferably 0.02 m.sup.2 or larger, still more
preferably 0.03 m.sup.2 or larger, per 1 mg of HCP. It is important
to determine the membrane area depending on the amount of HCP
desired to be reduced.
[0097] The polyethersulfone with which a solution containing the
soluble thrombomodulin of this embodiment is brought into contact
is available as, for example, Minisart High-Flow sold by Sartorius.
The form of polyethersulfone is not particularly limited so far
that it is in such a form that a solution can be contacted, such as
those of membrane, nonwoven fabric, and beads. Polyethersulfone is
preferably molded in the form of membrane and used as a filtration
membrane. In this embodiment, a pore diameter of the filtration
membrane is not limited so far that the diameter allows HCP to pass
through the membrane, for example, 0.01 to 10 .mu.m, preferably 0.1
to 1 .mu.m, more preferably 0.01 to 0.06 .mu.m. The volume of the
solution containing the soluble thrombomodulin to be contacted with
polyethersulfone can be easily determined by bringing a part of the
solution into contact with a small amount of polyethersulfone
beforehand to evaluate the HCP-removing ability thereof.
[0098] The highly-purified soluble thrombomodulin of this
embodiment can be collected with confirming that the HCP content in
the solution obtained by bringing a soluble
thrombomodulin-containing solution containing HCP into contact with
polyethersulfone is in a ratio of less than 10 ng per 10,000 U of
soluble thrombomodulin, and when the HCP content becomes 10 ng or
higher per 10,000 U of soluble thrombomodulin, collection of
highly-purified soluble thrombomodulin can be terminated, or after
the used polyethersulfone is changed to fresh polyethersulfone, the
collection of highly-purified soluble thrombomodulin may be
restarted. As for examples of the relation between the amount of
HCP and the area of polyethersulfone, examples where a
polyethersulfone is in the form of filtration membrane for example
include an upper limit of the area of the membrane being 50 m.sup.2
or smaller, preferably 5 m.sup.2 or smaller, more preferably 0.5
m.sup.2 or smaller, still more preferably 0.1 m.sup.2 or smaller,
and preferably a lower limit being 0.01 m.sup.2 or larger, more
preferably 0.02 m.sup.2 or larger, still more preferably 0.03
m.sup.2 or larger, per 1 mg of HCP. It is important to determine
the membrane area depending on the amount of HCP desired to be
reduced.
[0099] The production process of the highly-purified soluble
thrombomodulin of this embodiment in which contamination of HCP is
reduced is not particularly limited, so far that the process
comprises the step of bringing a solution containing soluble
thrombomodulin into contact with nylon and/or polyethersulfone so
that the HCP content can be in a ratio of less than 10 ng per
10,000 U of soluble thrombomodulin. An example includes the
following production process:
A production process comprising the steps of (a) to (g), and the
step of bringing a solution containing soluble thrombomodulin into
contact with nylon and/or polyethersulfone: (a) the step of
culturing a transformant cell and collecting culture medium
(production solution), (b) the step of filtering the production
solution to obtain a filtered production solution, (c) the step of
applying the filtered production solution to anion exchange column
chromatography to obtain a roughly purified solution, (d) the step
of applying the roughly purified solution to affinity column
chromatography using a column carrying anti-thrombomodulin
monoclonal antibody to obtain a purified solution 1, (e) the step
of applying the purified solution 1 to cation exchange column
chromatography to obtain a purified solution 2, (f) the step of
applying the concentrated purified solution 2 to gel filtration
column chromatography, and concentrating the eluate to obtain a
purified solution 3, and (g) the step of filtering the purified
solution 3 with a virus-removing membrane and a sterile filtration
membrane.
[0100] In the aforementioned production process, the step of
bringing a solution containing soluble thrombomodulin into contact
with nylon and/or polyethersulfone may be included in any one of or
two or more of the steps (b) to (g). It is preferred that the
process includes any one of or two or more of the steps (d) to (g).
In order to efficiently remove HCP, it is extremely preferable to
perform the step of bringing the solution into contact with nylon
and/or polyethersulfone after the final step of the production
process, i.e., the step (g).
[0101] Further, in order to completely obviate contamination of
bovine serum proteins, it is more preferred that the culture of the
transformant cell in the step (a) is performed as serum-free
culture.
[0102] Examples of the production process of the highly-purified
soluble thrombomodulin of this embodiment in which contamination of
HCP is reduced also include the following production process:
A production process comprising the steps of (a) to (g), and the
step of bringing a solution containing soluble thrombomodulin into
contact with nylon and/or polyethersulfone: (a) the step of
culturing a transformant cell and collecting culture medium
(production solution), (b) the step of filtering the production
solution to obtain a filtered production solution, (c) the step of
applying the filtered production solution to anion exchange column
chromatography to obtain a roughly purified solution 1, (d) the
step of applying the roughly purified solution to hydrophobic
column chromatography to obtain a roughly purified solution 2, (e)
the step of applying the roughly purified solution 2 to affinity
column chromatography using a column carrying anti-thrombomodulin
monoclonal antibody to obtain a purified solution 1, (f) the step
of applying the concentrated purified solution 1 to gel filtration
column chromatography to obtain a purified solution 2, and (g) the
step of filtering the purified solution 2 with a sterile filtration
membrane.
[0103] In the aforementioned production process, the step of
bringing a solution containing soluble thrombomodulin into contact
with nylon and/or polyethersulfone may be included in any one of or
two or more of the steps (b) to (g). It is preferable that said
step is included in any one of or two or more of the steps (e) to
(g). In order to efficiently remove HCP, it may be preferable to
perform the step of bringing the solution into contact with nylon
and/or polyethersulfone after the final step of the production
process, i.e., the step (g).
[0104] Further, in order to completely obviate contamination of
bovine serum proteins, it is more preferred that the culture of the
transformant cell in the step (a) is performed as serum-free
culture.
[0105] The highly-purified soluble thrombomodulin of this
embodiment can be prepared with confirming that the HCP content in
the solution obtained by bringing a soluble
thrombomodulin-containing solution containing HCP into contact with
nylon and/or polyethersulfone is in a ratio of less than 10 ng per
10,000 U of soluble thrombomodulin, and when the HCP content
becomes 10 ng or larger per 10,000 U of soluble thrombomodulin, the
preparation of highly-purified soluble thrombomodulin may be
terminated, or after the used nylon and/or polyethersulfone is
changed to fresh nylon and/or polyethersulfone, the preparation of
highly-purified soluble thrombomodulin may be restarted. As
described above, the production process of the highly-purified
soluble thrombomodulin of this embodiment in which contamination of
HCP is reduced is not particularly limited so far that the process
comprises the step of bringing a solution containing soluble
thrombomodulin into contact with nylon and/or polyethersulfone, and
HCP content becomes in a ratio of less than 10 ng per 10,000 U of
soluble thrombomodulin. More specifically, a purification step may
be performed after the step of bringing the solution into contact
with nylon and/or polyethersulfone, and as a result, it is
sufficient that highly-purified soluble thrombomodulin having an
HCP content being in a ratio of less than 10 ng of HCP per 10,000 U
of soluble thrombomodulin can be obtained.
[0106] Examples of the purification step that may be performed
after the step of bringing the solution into contact with nylon
and/or polyethersulfone include steps of performing column
chromatography such as anion exchange column chromatography,
affinity column chromatography, cation exchange column
chromatography, gel filtration column chromatography, and
hydrophobic column chromatography, membrane filtration such as
membrane concentration, virus removing, and sterile filtration, or
a combination of two or more of these treatments. A step of
performing cation exchange column chromatography, gel filtration
column chromatography, membrane concentration, virus removing,
sterile filtration, or a combination of two or more of these
treatments is preferred. It may be preferable to perform cation
exchange column chromatography, gel filtration column
chromatography, membrane concentration, virus removing, and sterile
filtration after the step of bringing the solution into contact
with nylon and/or polyethersulfone. As a more preferred example,
the purification step comprising cation exchange column
chromatography, gel filtration column chromatography, membrane
concentration, virus removing, and sterile filtration may be
performed by performing cation exchange column chromatography,
membrane concentration, gel filtration column chromatography,
membrane concentration, virus removing, and sterile filtration in
this order, after the step of bringing the solution into contact
with nylon and/or polyethersulfone. Examples of the material used
for the cation exchange column chromatography include SP Sepharose
Fast Flow, DEAE Sepharose Fast Flow, Capto S, Capto DEAE (GE
Healthcare), S HyperCel (Pall), and TOYOPEARL GigaCap S-650
(TOSOH), and SP Sepharose Fast Flow is preferred. Examples of the
concentration membrane include MICROZA UF (Asahi Kasei Chemicals),
Kvick Flow 10KD (GE Healthcare), and Pellicon 2 (Millipore), and
MICROZA UF is preferred. Examples of the material used for the gel
filtration column chromatography include Sephacryl S-300 HR,
Superose 12 pg (GE Healthcare), and TOYOPEARL HW (TOSOH), and
Sephacryl S-300 HR is preferred. Examples of the virus-removing
membrane include PLANOVA 15N (Asahi Kasei Medical), Biresolve NFP
(Millipore), and Ultipor VF (Pall), and PLANOVA 15N is preferred.
Examples of the sterile filtration membrane include Millipak,
Millidisk (Millipore), Supor EVA (Pall), and Sartopore 2 (Sartorius
Stedim), and Millipak is preferred.
[0107] As described above, the highly-purified soluble
thrombomodulin of this embodiment obtained by bringing a solution
containing soluble thrombomodulin into contact with nylon and/or
polyethersulfone contains HCP being in a ratio of less than 10 ng
of HCP per 10,000 U of soluble thrombomodulin.
[0108] One U of the soluble thrombomodulin of this embodiment is
defined as an amount that can generate 0.1 .mu.mol of
p-nitroaniline per 1 minute in the APC assay using activation of
Protein C as an index, and can be measured by the method comprising
the following steps according to the method described in
Biologicals, 30, 69-76 (2002):
(a) the step of adding human thrombin to a test solution containing
soluble thrombomodulin, and warming the mixture, (b) the step of
adding human Protein C, and warming the mixture, (c) the step of
adding heparin-antithrombin III, and warming the mixture, (d) the
step of adding a synthetic substrate S-2366
(pyroGlu-Pro-Arg-pNA.HCl), and warming the mixture, (e) the step of
adding acetic acid to terminate the substrate cleaving reaction,
(f) the step of measuring absorbance at 405 nm, and (g) the step of
determining activity of the soluble thrombomodulin-containing test
solution in accordance with the following equation:
Activity
(U/mL)=[(A.sub.sample.times.A.sub.blank).times.V.sub.1]/(M.time-
s.T.times.k.times.V.sub.2).times.Dilution time of sample [Equation
1]
A.sub.sample: Absorbance of sample solution A.sub.blank: Absorbance
of blank (water) M: Molar absorption coefficient of p-nitroaniline:
9.6.times.10.sup.-3 [1/.mu.M] V.sub.1: Volume at the time of
spectrometry (L) V.sub.2: Volume of sample solution (mL) T:
Substrate cleaving reaction time (minute) k: Molar number of
p-nitroaniline released by activated Protein C generated by 1 U of
thrombomodulin: 0.1 (.mu.mol/minute/U)
[0109] The highly-purified soluble thrombomodulin of this
embodiment has the activity of, for example, 3,000 U, preferably
4,000 to 9,000 U, more preferably 5,000 to 8,000 U, still more
preferably 6,000 to 7,000 U, per 1 mg of the protein. Concentration
of the protein can be measured in accordance with a known method
for measuring protein concentration by using bovine serum albumin
as a standard sample. Examples of the method include, for example,
Lowry method, Bradford method, BCA method, and the like.
[0110] The HCP content of the highly-purified soluble
thrombomodulin of this embodiment is measured by a method
comprising at least the following steps:
(a) the step of preparing host cell-originated proteins from
culture supernatant obtained by carrying out serum-free culture of
a transformant cell obtained by transfecting a host cell with a DNA
containing a nucleotide sequence encoding soluble thrombomodulin,
or the host cell, (b) the step of purifying an anti-host
cell-originated protein antibody from antiserum obtained by
sensitizing a rabbit with the host cell-originated proteins
obtained in (a) mentioned above, the step of constructing a
measurement system comprising: (c1) the step of adsorbing the
anti-host cell-originated protein antibody obtained in (b)
mentioned above to a solid phase, (c2-1) the step of bringing a
soluble thrombomodulin-containing test solution suspected to be
contaminated with the host cell-originated proteins into contact
with the solid phase to which the anti-host cell-originated protein
antibody is adsorbed, (c3) the step of adding a biotinylated
anti-host cell-originated protein antibody to the solid phase, (c4)
the step of adding a solution of avidinylated peroxidase to the
solid phase, (c5) the step of adding an enzyme substrate solution
to allow color development, and (c6) the step of terminating the
color development and measuring absorbance, (d) the step of
measuring concentration of the host cell-originated proteins in the
soluble thrombomodulin-containing test solution suspected to be
contaminated with the host cell-originated proteins in the
aforementioned measurement system, and determining whether the
concentration of the host cell-originated proteins in the soluble
thrombomodulin-containing test solution is within a range that
enables quantification of the proteins in the aforementioned
measurement system, which range is confirmed beforehand by
performing measurement using a solution of the host cell-originated
proteins of a known concentration in the aforementioned measurement
system, (e-1) the step of determining the concentration of the host
cell-originated proteins in the soluble thrombomodulin-containing
test solution suspected to be contaminated with the host
cell-originated proteins as the concentration of the host
cell-originated proteins in the solution, when the concentration is
determined to be within the range that enables the quantification
in (d) mentioned above, (e-2-1) the step of concentrating or
diluting the soluble thrombomodulin-containing test solution
suspected to be contaminated with the host cell-originated
proteins, if desired, to make the concentration of the host
cell-originated proteins to be a measurable concentration within
the range that enables the quantification in the aforementioned
measurement system, when the concentration of the host
cell-originated proteins is determined to be not within the range
that enables the quantification in (d) mentioned above, and
recording the concentration ratio or dilution ratio, (e-2-2) the
step of measuring the host cell-originated protein concentration in
the soluble thrombomodulin-containing test solution concentrated or
diluted in (e-2-1) mentioned above in a measurement system
corresponding to the measurement system represented by the steps of
(c1) to (c6) mentioned above in which (c2-1) is replaced with
(c2-2) mentioned below, and obtaining the host cell-originated
protein concentration with taking the concentration ratio or
dilution ratio into consideration, (c2-2) the step of bringing the
soluble thrombomodulin-containing test solution concentrated or
diluted, if necessary, into contact with the solid phase to which
the anti-host cell-originated protein antibody is adsorbed, and the
step of bringing a solution containing the host cell-originated
proteins of a known concentration into contact with the solid phase
to which the anti-host cell-originated protein antibody is
adsorbed, and (f) the step of calculating ratio of the host
cell-originated protein concentration obtained in (e-1) or (e-2-2)
based on APC activity of thrombomodulin per unit volume of the
soluble thrombomodulin-containing test solution measured
separately.
[0111] In this specification, HCP means proteins originated from
the host cells used for preparing gene recombinant cells that
produce soluble thrombomodulin, and does not mean to include
soluble thrombomodulin. HCP can be prepared from culture
supernatant obtained by culturing host cells of the same type as
those of the cells used for the transfection with a DNA containing
the nucleotide sequence encoding thrombomodulin. In the case of CHO
cell, for example, the term of host cell of the same type means a
concept encompassing cells of strains classified into CHO cells,
such as those of the cell lines CHO-K1 (ATCC No. CCL-61), CHO-S
(Invitrogen, U.S.A., Catalog No. 11619-012), CHO-DXB11, and
CHO-DG44 (Invitrogen, U.S.A., Catalog No. 12610-010), and the
preparation may be performed by using any of cell lines classified
into CHO cells. As for the CHO cell, it is preferable to use cells
of the cell line DXB11 or CHO-K1, more preferably cells of the cell
line DXB11, as the host cell of the same type. There is also
another embodiment in which it is preferable to use cells of the
cell line CHO-K1.
[0112] HCP means proteins originated from the host cells used for
preparing the gene recombinant cells that produce soluble
thrombomodulin, and is defined to be measurable by the method
including at least the aforementioned steps (a) to (f). Examples of
constituents of HCP include, as shown in Test Example 6, histone
H2B (Biochimie, 61 (1), 61-69 (1979)).
[0113] Further, when preparation is carried out from the culture
supernatant obtained by culturing the transformant cell obtained by
transfecting a host cell of the same type with a DNA containing a
nucleotide sequence encoding thrombomodulin, the culture
supernatant can be applied to an antibody column using an antibody
that specifically binds to thrombomodulin as the ligand, and a
non-adsorbed fraction can be collected. After it is confirmed that
the APC activity of thrombomodulin is not detected in this
non-adsorbed fraction, the fraction can be used as HCP. HCP is
preferably concentrated by using an ultrafiltration membrane, as
required. In addition, in order to avoid contamination of other
proteins, the medium used for culturing the host cell or the
transformant cell is preferably a serum-free medium, and it is more
preferred that the serum-free medium is a protein-free medium. For
the purification of the anti-HCP antibody from an anti-HCP
antiserum obtained by sensitizing a rabbit with HCP, column
chromatography can be used, and for example, a combination of
ammonium sulfate salting-out and column chromatography can be used.
For the column chromatography for the purification of the anti-HCP
antibody, it is preferable to use a Protein A column. There is also
another embodiment in which, when a transformant cell obtained by
transfecting a host cell with a DNA containing a nucleotide
sequence encoding thrombomodulin is used for the preparation of
HCP, it is preferred that a thrombomodulin column is used for
purification of the anti-HCP antibody after the purification with a
Protein A column, and the non-adsorbed fraction is collected and
used as the anti-HCP antibody.
[0114] When an HCP concentration measurement system is constructed,
it is necessary to clarify the quantifiable range thereof, and the
quantifiable range is not limited so far that an HCP content of
less than 10 ng of HCP per 10,000 U of thrombomodulin can be
measured. It is more preferable that a lower concentration can be
measured. The quantifiable range is defined to be, for example, 100
ng/mL or higher, preferably 50 ng/mL or higher, more preferably 25
ng/mL or higher, and for example, 500 ng/mL or lower.
[0115] When the test solution is concentrated, it can be
concentrated by a usual protein concentration method, and the
method is not particularly limited. However, it is preferably
concentrated with an ultrafiltration membrane. Further, there is
also another embodiment in which it is preferable to concentrate
the test solution by lyophilizing the solution, and then dissolving
the product with a small volume of water or buffer. Components
other than HCP are also concentrated by the concentration and may
affect the HCP measurement system. Accordingly, it is necessary to
concentrate the test solution in such a degree that the HCP
measurement system is not affected. For example, upper limit of the
concentration of the soluble thrombomodulin-containing test
solution not affecting the HCP measurement system is, for example,
5 mg/mL.
[0116] The HCP content per 10,000 U of thrombomodulin is calculated
in accordance with the following equation.
a/b.times.10,000
a: HCP content per 1 mL of sample (ng/mL) b: APC activity of
thrombomodulin per 1 mL of sample (U/mL)
[0117] The highly-purified soluble thrombomodulin of this
embodiment promotes the activation of Protein C by thrombin to
provide generation of a large amount of active Protein C that has
an anti-blood coagulation action and a thrombolysis action.
Therefore, the highly-purified soluble thrombomodulin of this
embodiment greatly contributes to anti-blood coagulation and
thrombolysis in a living body. The highly-purified soluble
thrombomodulin of this embodiment has an anti-blood coagulation
action, a platelet aggregation inhibition action, and a
thrombolysis action. Accordingly, the product can be used for a
pharmaceutical composition for controlling blood coagulation, or
controlling platelet aggregation. Specifically, it can be used for
prophylactic and therapeutic treatments of diseases including, for
example, myocardial infarction, thrombosis, embolism, peripheral
vessel obstruction, obstructive arteriosclerosis, disseminated
intravascular coagulation (DIC), angina pectoris, transient
cerebral ischemic attack, toxemia of pregnancy, and the like.
[0118] When the pharmaceutical composition of this embodiment is
prepared, the highly-purified soluble thrombomodulin of this
embodiment and a pharmaceutically acceptable carrier can be mixed.
More specifically, a pharmaceutical composition suitable for
effective administration to patients can be prepared by mixing the
highly-purified soluble thrombomodulin of this embodiment in an
amount effective for a prophylactic or therapeutic treatment of any
of the diseases mentioned above with an appropriate amount of
carrier. As the pharmaceutical composition of this embodiment, a
lyophilized preparation can be preferably prepared. Further, the
pharmaceutical composition of this embodiment is preferably used as
a preparation for intravascular injection. The composition can also
be preferably prepared as a preparation for intravenous infusion. A
lyophilized preparation can be prepared by referring to the method
described in WO03/061687.
[0119] When the composition is used as an injection, the
aforementioned carrier is preferably a carrier that can be
administered as a pharmaceutical, and can be dissolved in
physiological saline or glucose injection. Examples of the carrier
include one or more selected from the group consisting of sucrose,
purified gelatin, albumin, mannitol, glucose, and sodium chloride.
For example, a pH adjustor consisting of any of various mineral
salts, and the like are also preferably added. In such a case, the
whole pharmaceutical composition as a combination with the
highly-purified soluble thrombomodulin of this embodiment is
soluble, and can be finely lyophilized, and thus such a composition
is preferred. Further, in this embodiment, it is also preferred
that the aforementioned carrier is glycerol. The aforementioned
carrier is preferably added at the time of preparing the
composition. The carriert may also be added when the composition is
dissolved before use.
[0120] A dose of the highly-purified soluble thrombomodulin of this
embodiment for one time of administration to an adult may change
depending on age, sex, body weight, symptoms, and the like. The
doses may generally be about 0.1 to 200 mg, and may be
administered, for example, once or several times, as required, per
day by intravascular injection, preferably intravenous drip
infusion. The pharmaceutical composition of this embodiment may
also be administered so that a dose can be 0.1 to 200 mg of the
soluble thrombomodulin as the active ingredient, for example, once
or several times, as required, per day by intravascular injection,
preferably intravenous drip infusion.
EXAMPLES
[0121] The present invention will be more specifically explained
with reference to the following examples. However, the scope of the
present invention is not limited at all by these.
Reference Example 1
Method for Measuring APC Activity of Thrombomodulin
[0122] According to the description of Biologicals, 30, 69-76
(2002), the APC activity of thrombomodulin is measured on the basis
of activation of Protein C as an index.
[0123] A 20 mM calcium chloride solution (75 .mu.L) is added with
25 .mu.L of a sample solution diluted with a Tris-imidazole buffer
containing 0.05% polysorbate 20, the mixture is cooled on ice, and
then added with 25 .mu.L of a 40 U/mL solution of human thrombin
(Sigma, U.S.A.), and the mixture is stirred and warmed at
37.degree. C. Ten minutes after the addition of the human thrombin
solution, the mixture was added with 25 .mu.L of a 12 U/mL solution
of human Protein C (Enzyme Research, U.S.A.), and the mixture was
stirred and warmed at 37.degree. C. Ten minutes after the addition
of the human Protein C solution, the mixture was added with 100
.mu.L of a heparin-antithrombin III solution, and the mixture was
stirred and warmed at 37.degree. C. Ten minutes after the addition
of the heparin-antithrombin III solution, the mixture was added
with 250 .mu.L of a synthetic substrate S-2366 (ChromoGenics,
Sweden) solution warmed at 37.degree. C. beforehand, and the
mixture is stirred and warmed at 37.degree. C. Ten minutes after
the addition of the substrate solution, the mixture was added with
1.5 mL of 50% acetic acid, the mixture is stirred, and absorbance
of the mixture is measured at 405 nm by using water as a blank.
[0124] The APC activity of thrombomodulin is calculated in
accordance with the following equation. One U of thrombomodulin is
defined as an amount that can generate 0.1 .mu.mol of
p-nitroaniline per 1 minute.
Activity
(U/mL)=[(A.sub.sample-A.sub.bank).times.V.sub.1]/(M.times.T.tim-
es.k.times.V.sub.2).times.Dilution time of sample [Equation 2]
A.sub.sample: Absorbance of sample solution A.sub.blank: Absorbance
of blank (water) M: Molar absorption coefficient of p-nitroaniline:
9.6.times.10.sup.-3 [1/.mu.M] V.sub.1: Volume at the time of
spectrometry: 2.0.times.10.sup.-3 (L) V.sub.2: Volume of sample
solution: 0.025 (mL) T: Substrate cleaving reaction time: 10
(minute) k: Molar number of p-nitroaniline released by activated
Protein C generated with 1 U of thrombomodulin: 0.1
(.mu.mol/minute/U)
[0125] The reagents are as follows.
<Tris-Imidazole Buffer>
[0126] Solution B (100 mL) is added with Solution A, and the
mixture is adjusted to pH 8.4, and diluted 10 times with water.
Solution A: 2-Amino-2-hydroxymethyl-1,3-propanediol (3.03 g) and
imidazole (1.70 g) are dissolved in 1 M hydrochloric acid (50 mL),
the solution was added with water to a volume of 100 mL, and sodium
chloride (11.7 g) is added to the solution and dissolved. Solution
B: 2-Amino-2-hydroxymethyl-1,3-propanediol (4.04 g), imidazole
(2.27 g), and sodium chloride (1.95 g) are dissolved in water to
obtain a volume of 100 mL, and sodium chloride (11.7 g) is added to
the solution and dissolved.
<20 mM Calcium Chloride Solution>
[0127] A 60 mM calcium chloride solution (1 mL) is added with a
Tris-imidazole buffer (2 mL).
<Heparin-Antithrombin III Solution>
[0128] An antithrombin III solution (2 U/mL, 7.5 .mu.L, Mitsubishi
Pharma, Japan), a Tris-imidazole buffer (42.5 .mu.L), and a 30 U/mL
heparin solution (50 .mu.L, Mochida Pharmaceutical, Japan) are
mixed by shaking. This solution is prepared before use, and cooled
on ice until just before use.
Reference Example 2
Method for Measuring HCP Concentration
[0129] Serum-free culture of gene recombinant CHO cells introduced
with the thrombomodulin gene is performed. The culture supernatant
is applied on an anti-thrombomodulin antibody column to obtain a
non-adsorbed fraction. According to the description of Reference
Example 1, the APC activity of thrombomodulin in this non-adsorbed
fraction is measured to confirm that the activity is not detected,
then the fraction is concentrated with an ultrafiltration membrane,
and the concentrate is used as HCP. Anti-HCP antiserum obtained by
sensitizing a rabbit with HCP as an antigen is purified by ammonium
sulfate salting-out and with a Protein A column, and then applied
on an affinity column using thrombomodulin as the ligand to obtain
a non-adsorbed fraction. As described above, a rabbit anti-HCP
antibody that does not recognize thrombomodulin is obtained.
[0130] A sample solution is obtained by dilution with PBS
containing 0.05% polysorbate 80 so that expected HCP concentration
becomes 0 to 500 ng/mL. When the HCP concentration of a sample
solution is low, the solution is concentrated to an appropriate
concentration by using an ultrafiltration membrane or the like.
Separately, HCP is added with PBS containing 0.05% polysorbate 80
to prepare eight kinds of solutions containing 500, 400, 300, 200,
100, 50, 25, and 0 ng of HCP in 1 mL as standard solutions.
[0131] A 25 .mu.g/mL rabbit anti-HCP antibody solution diluted with
a sodium carbonate buffer is added to a 96-well polystyrene plate
in a volume of 100 .mu.L per well, and the plate is left standing
at 25.degree. C. for about 2 hours. Then, each well is washed 5
times with 250 .mu.L of PBS containing 0.05% polysorbate 80, PBS
containing 1% gelatin (200 .mu.L) is added to each well, and the
plate is left standing at 25.degree. C. for about 1 hour. Each well
is washed 5 times with 250 .mu.L of PBS containing 0.05%
polysorbate 80, then a sample solution and the standard solutions
(100 .mu.L) are added to the wells, and the plate is left standing
at 25.degree. C. for about 16 hours. Then, each well is washed 5
times with 250 .mu.L of PBS containing 0.05% polysorbate 80, then a
biotinylated rabbit anti-HCP antibody solution (100 .mu.L) is added
to each well, and the plate is left standing at 25.degree. C. for
about 2 hours. Each well is washed 5 times with 250 .mu.L of PBS
containing 0.05% polysorbate 80, then an avidin-peroxidase solution
(100 .mu.L) is added to each well, and the plate is left standing
at 25.degree. C. for about 2 hours. Each well is washed 5 times
with 250 .mu.L of PBS containing 0.05% polysorbate 80, then an
enzyme substrate solution (100 .mu.L) is added to each well, and
the plate is left standing at room temperature in a dark place.
When a color is appropriately developed, 50 .mu.L of 25% sulfuric
acid is added to each well to terminate the reaction, and
absorbance of the mixture is measured at 492 nm with an
absorptiometer for 96-well plates (Tecan Japan, Japan). By using a
calibration curve prepared with the standard solutions, HCP content
in the sample (1 mL) is calculated. The measurement limit of this
measurement method is, for example, 25 ng/mL. In accordance with
the following equation, HCP content per 10,000 U of thrombomodulin
is calculated, as required.
HCP content per 10,000 U of thrombomodulin (ng/10,000
U)=a/b.times.10,000
a: HCP content per 1 mL of sample (ng/mL) b: APC activity of
thrombomodulin per 1 mL of sample (U/mL)
[0132] The reagents are as follows.
<Sodium Carbonate Buffer>
[0133] Anhydrous sodium carbonate (0.16 g), and sodium
hydrogencarbonate (0.29 g) are added to water and dissolved to
obtain a volume of 100 mL.
<Avidin-Peroxidase Solution>
[0134] A stock solution of horseradish peroxidase bound with avidin
D (Vector Laboratories, U.S.A.) is diluted about 30,000 times with
PBS containing 0.05% polysorbate 80.
<Enzyme Substrate Solution>
[0135] Ortho-phenylenediamine dihydrochloride (10 mg) is added to
20 mL of a citrate/phosphate buffer (citric acid monohydrate (2.56
g) and disodium hydrogenphosphate dodecahydrate (9.12 g) are
dissolved in water to obtain a volume of 500 mL) and dissolved, and
aqueous hydrogen peroxide (10 .mu.L) is added immediately before
use.
Reference Example 3
Method for Measuring Mouse IgG Concentration
[0136] Anti-mouse IgG antiserum obtained by sensitizing a rabbit
with mouse IgG as an antigen is purified by ammonium sulfate
salting-out and with a Protein A column to obtain a rabbit
anti-mouse IgG antibody.
[0137] A sample solution is obtained by dilution with PBS
containing 0.05% polysorbate 80 so that expected mouse IgG
concentration becomes 0 to 25 ng/mL. When the IgG concentration of
a sample solution is low, the solution is concentrated to an
appropriate concentration by using an ultrafiltration membrane or
the like. Separately, mouse IgG is added with PBS containing 0.05%
polysorbate 80 to prepare eight kinds of solutions containing 25,
20, 15, 10, 5, 2.5, 1.25, 0.63, and 0 ng of IgG in 1 mL as standard
solutions.
[0138] A 1.5 .mu.g/mL rabbit anti-mouse IgG antibody solution
diluted with a sodium carbonate buffer is added to a 96-well
polystyrene plate in a volume of 100 .mu.L per well, and the plate
is left standing at 25.degree. C. for about 2 hours. Then, each
well is washed 5 times with 250 .mu.L of PBS containing 0.05%
polysorbate 80, PBS containing 1% gelatin (200 .mu.L) is added to
each well, and the plate is left standing at 25.degree. C. for
about 1 hour. Each well is washed 5 times with 250 .mu.L of PBS
containing 0.05% polysorbate 80, then a sample solution and the
standard solutions (100 .mu.L) are added to the wells, and the
plate is left standing at 25.degree. C. for about 16 hours. Then,
each well is washed 5 times with 250 .mu.L of PBS containing 0.05%
polysorbate 80, and then a biotinylated rabbit anti-mouse IgG
antibody solution (100 .mu.L) is added to each well, and the plate
is left standing at 25.degree. C. for about 2 hours. Each well is
washed 5 times with 250 .mu.L of PBS containing 0.05% polysorbate
80, then an avidin-peroxidase solution (100 .mu.L) is added to each
well, and the plate is left standing at 25.degree. C. for about 2
hours. Each well is washed 5 times with 250 .mu.L of PBS containing
0.05% polysorbate 80, then an enzyme substrate solution (100 .mu.L)
is added to each well, and the plate is left standing at room
temperature in a dark place. When a color is appropriately
developed, 50 .mu.L of 25% sulfuric acid is added to each well to
terminate the reaction, and absorbance of the mixture is measured
at 492 nm with an absorptiometer for 96-well plates (Tecan Japan,
Japan). By using a calibration curve prepared with the standard
solutions, mouse IgG content in the sample (1 mL) is calculated.
The measurement limit of this measurement method is, for example,
0.63 ng/mL. In accordance with the following equation, mouse IgG
content per 10,000 U of thrombomodulin is calculated, as
required.
Mouse IgG content per 10,000 U of thrombomodulin (ng/10,000
U)=a/b.times.10,000
a: Mouse IgG content per 1 mL of sample (ng/mL) b: APC activity of
thrombomodulin per 1 mL of sample (U/mL)
[0139] The reagents are as follows.
<Sodium Carbonate Buffer>
[0140] Anhydrous sodium carbonate (0.16 g), and sodium
hydrogencarbonate (0.29 g) are added to water and dissolved to
obtain a volume of 100 mL.
<Avidin-Peroxidase Solution>
[0141] A stock solution of horseradish peroxidase bound with avidin
D (Vector Laboratories, U.S.A.) is diluted about 30,000 times with
PBS containing 0.05% polysorbate 80.
<Enzyme Substrate Solution>
[0142] Ortho-phenylenediamine dihydrochloride (10 mg) is added to
20 mL of a citrate/phosphate buffer (citric acid monohydrate (2.56
g) and disodium hydrogenphosphate dodecahydrate (9.12 g) are
dissolved in water to obtain a volume of 500 mL) and dissolved, and
aqueous hydrogen peroxide (10 .mu.L) is added immediately before
use.
Reference Example 4
Method for Measuring Bovine Serum Protein Concentration
[0143] Anti-bovine serum protein antiserum obtained by sensitizing
a rabbit with bovine serum as an antigen is purified by ammonium
sulfate salting-out and with a Protein A column to obtain a rabbit
anti-bovine serum protein antibody.
[0144] A sample solution is obtained by dilution with PBS
containing 0.05% polysorbate 80 so that expected bovine serum
protein concentration becomes 0 to 25 ng/mL. When the bovine serum
protein concentration of a sample solution is low, the solution is
concentrated to an appropriate concentration by using an
ultrafiltration membrane or the like. Separately, bovine serum is
added with PBS containing 0.05% polysorbate 80 to prepare eight
kinds of solutions containing 25, 20, 15, 10, 5, 2.5, 1.25, and 0
ng of bovine serum proteins in 1 mL as standard solutions.
[0145] A 10 .mu.g/mL rabbit anti-bovine serum protein antibody
solution diluted with a sodium carbonate buffer is added to a
96-well polystyrene plate in a volume of 100 .mu.L per well, and
the plate is left standing at 25.degree. C. for about 2 hours.
Then, each well is washed 5 times with 250 .mu.L of PBS containing
0.05% polysorbate 80, PBS containing 1% gelatin (200 .mu.L) is
added to each well, and the plate is left standing at 25.degree. C.
for about 1 hour. Each well is washed 5 times with 250 .mu.L of PBS
containing 0.05% polysorbate 80, then a sample solution and the
standard solutions (100 .mu.L) are added to the wells, and the
plate is left standing at 25.degree. C. for about 16 hours. Then,
each well is washed 5 times with 250 .mu.L of PBS containing 0.05%
polysorbate 80, then a biotinylated rabbit anti-bovine serum
protein antibody solution (100 .mu.L) is added to each well, and
the plate is left standing at 25.degree. C. for about 2 hours. Each
well is washed 5 times with 250 .mu.L of PBS containing 0.05%
polysorbate 80, then an avidin-peroxidase solution (100 .mu.L) is
added to each well, and the plate is left standing at 25.degree. C.
for about 2 hours. Each well is washed 5 times with 250 .mu.L of
PBS containing 0.05% polysorbate 80, then an enzyme substrate
solution (100 .mu.L) is added to each well, and the plate is left
standing at room temperature in a dark place. When a color is
appropriately developed, 50 .mu.L of 25% sulfuric acid is added to
each well to terminate the reaction, and absorbance of the mixture
is measured at 492 nm with an absorptiometer for 96-well plates
(Tecan Japan, Japan). By using a calibration curve prepared with
the standard solutions, bovine serum protein content in the sample
(1 mL) is calculated. The measurement limit of this measurement
method is, for example, 1.25 ng/mL. In accordance with the
following equation, bovine serum protein content per 10,000 U of
thrombomodulin is calculated, as required.
Bovine serum protein content per 10,000 U of thrombomodulin
(ng/10,000 U)=a/b.times.10,000
a: Bovine serum protein content per 1 mL of sample (ng/mL) b: APC
activity of thrombomodulin per 1 mL of sample (U/mL)
[0146] The reagents are as follows.
<Sodium Carbonate Buffer>
[0147] Anhydrous sodium carbonate (0.16 g), and sodium
hydrogencarbonate (0.29 g) are added to water and dissolved to
obtain a volume of 100 mL.
<Avidin-Peroxidase Solution>
[0148] A stock solution of horseradish peroxidase bound with avidin
D (Vector Laboratories, U.S.A.) is diluted about 30,000 times with
PBS containing 0.05% polysorbate 80.
<Enzyme Substrate Solution>
[0149] Ortho-phenylenediamine dihydrochloride (10 mg) is added to
20 mL of a citrate/phosphate buffer (citric acid monohydrate (2.56
g) and disodium hydrogenphosphate dodecahydrate (9.12 g) are
dissolved in water to obtain a volume of 500 mL) and dissolved, and
aqueous hydrogen peroxide (10 .mu.L) is added immediately before
use.
Comparative Example 1
Preparation of Soluble Thrombomodulin 1
[0150] A gene recombinant CHO cell into which a DNA encoding the
amino acid sequence of SEQ ID NO: 9 was introduced was prepared by
a genetic manipulation technique according to Japanese Patent
Unexamined Publication No. 11-341990, Example 1, then inoculated
into the DMEM medium (Invitrogen, U.S.A.) containing 150 mg/L of
L-proline (Ajinomoto, Japan), 60 mg/L of kanamycin sulfate (Meiji
Seika, Japan), 1 mg/L of tylosin tartrate (Mercian, Japan), and 10%
bovine serum (HyClone, U.S.A.), and cultured at 37.degree. C. in a
CO.sub.2 incubator. The cells obtained by centrifuging the culture
medium were suspended in the DMEM medium (Invitrogen, U.S.A.)
containing 150 mg/L of L-proline (Ajinomoto, Japan), 60 mg/L of
kanamycin sulfate (Meiji Seika, Japan), 1 mg/L of tylosin tartrate
(Mercian, Japan), 10% dimethyl sulfoxide (Merck, Germany), and 10%
bovine serum (HyClone, U.S.A.), and the medium was dispensed into
vials (4.times.10.sup.7 cells/vial), and cryopreserved in liquid
nitrogen.
[0151] Cell culture was performed by using, as the base medium, the
medium described in Japanese Patent Unexamined Publication No.
11-341990, Ingredient Table 2, provided that NaHCO.sub.3
concentration was changed to 5,700 mg/L, and NaCl concentration was
changed to 2,410 mg/L. Growth medium was obtained by adding 60 mg/L
of kanamycin sulfate (Invitrogen, U.S.A.), 1 mg/L of tylosin
tartrate (Sigma-Aldrich, U.S.A.), and 8% bovine serum (HyClone,
U.S.A.) to the base medium, and used. Further, production medium
was the same as the growth medium, provided that the serum
concentration was changed to 3%.
[0152] The cells of one vial were thawed, inoculated into 100 mL of
the growth medium, and cultured with stirring at 37.degree. C. for
5 days by using a spinner flask in a CO.sub.2 incubator. When the
living cell density became 7.0.times.10.sup.5 cells/mL or more, the
entire volume of the culture medium was transferred to 0.9 L of the
growth medium, and the cells were cultured with stirring at
37.degree. C. for 5 days by using a spinner flask in a CO.sub.2
incubator. When the living cell density became 7.0.times.10.sup.5
cells/mL or more, the entire volume of the culture medium was
transferred to 9 L of the growth medium, and the cells were
cultured with stirring at 37.degree. C., pH 7.2 and 50% of
dissolved oxygen for 5 days by using a culture tank. When the
living cell density became 7.0.times.10.sup.5 cells/mL or more, the
entire volume of the culture medium was transferred to 120 L of the
growth medium, and the cells were cultured with stirring at
37.degree. C., pH 7.2 and 50% of dissolved oxygen for 7 days by
using a perfusion culture tank. When the living cell density became
7.0.times.10.sup.5 cells/mL or more, perfusion culture was started,
in which the production medium was continuously added, and the
culture supernatant was continuously collected. The culture
conditions consisted of 37.degree. C., pH 7.2, dissolved oxygen:
50%, medium exchange: 130 to 200 L/day, and surface pressurization:
0 to 0.2 MPa. After the living cell density reached
7.5.times.10.sup.6 cells/mL, the culture was further continued for
36 days, and the culture supernatant was collected as a production
solution. The collected production solution was clarified by using
filtration filters, SUPRAdisc II (Pall, U.S.A.) and Supor EBV
(Pall, U.S.A.), and stored at 2 to 10.degree. C. as a filtered
production solution.
[0153] About 700 L of the filtered production solution was applied
to a Q-Sepharose Fast Flow (GE Healthcare, U.S.A.) column
(diameter: 63 cm, height: 25 cm) equilibrated with a 20 mM
Tris-hydrochloric acid buffer (pH 7.7) containing 150 mM sodium
chloride. Then, the column was washed with 6 column volumes (CV) of
a 20 mM acetate buffer (pH 5.5) containing 180 mM sodium chloride,
and further washed with a 20 mM Tris-hydrochloric acid buffer (pH
7.7) containing 180 mM sodium chloride until absorbance at 280 nm
returned to the baseline. Elution was started with a 20 mM
Tris-hydrochloric acid buffer (pH 7.7) containing 300 mM sodium
chloride, and 0.5 column volume of the eluate from the start of the
peak of absorbance at 280 nm was obtained as a roughly purified
solution. The same operation was repeated 6 times to obtain 6 lots
of the roughly purified solution. The operation was performed at a
temperature of 2 to 10.degree. C., and a chromatography flow rate
of 109 L/hour.
[0154] An anti-thrombomodulin monoclonal antibody was prepared by
using human lung-originated thrombomodulin as the antigen according
to Japanese Patent Unexamined Publication No. 11-341990, Example
10, contacted and reacted with CNBr-activated Sepharose 4 Fast Flow
(GE Healthcare, U.S.A.) to couple the anti-thrombomodulin
monoclonal antibody and thereby prepare anti-thrombomodulin
monoclonal antibody-bound Sepharose 4 Fast Flow, which was filled
in a column to obtain a monoclonal antibody column. About 40 L of
the roughly purified solution was applied to the monoclonal
antibody column (diameter: 44 cm, height: 13 cm) equilibrated with
a 20 mM phosphate buffer (pH 7.3) containing 0.3 M sodium chloride.
6 CV of a 20 mM phosphate buffer (pH 7.3) containing 1.0 M sodium
chloride was poured into the column, 3 CV of 0.1 M acetate buffer
(pH 5.0) was further poured to wash the column, and elution was
started with a 0.1 M glycine-hydrochloric acid buffer (pH 3.0)
containing 0.3 M sodium chloride. The eluate corresponding to the
start to the end of the peak of absorbance at 280 nm was obtained,
and added with 1/10 volume of a 0.5 M phosphate buffer (pH 7.3) to
obtain a purified solution 1. The same operation was repeated 6
times to obtain 6 lots of the purified solution 1. The operation
was performed at a temperature of 2 to 10.degree. C., and a
chromatography flow rate of 46 L/hour.
[0155] About 170 L of the purified solution 1 of the 6 lots was
adjusted to pH 3.5 with a 1.0 M glycine-hydrochloric acid buffer
(pH 2.0), and applied to an SP-Sepharose FF (GE Healthcare
Bioscience, U.S.A.) column (diameter: 45 cm, height: 10 cm)
equilibrated with a 0.1 M glycine-hydrochloric acid buffer (pH 3.5)
containing 0.3 M NaCl. Washing was started with a 0.1 M
glycine-hydrochloric acid buffer (pH 3.5) containing 0.3 M NaCl,
and a flow-through fraction corresponding to the start to the end
of the peak of absorbance at 280 nm was obtained, and immediately
neutralized to pH 7 with a 0.5 M phosphate buffer (pH 7.3) to
obtain a purified solution 2. The operation was performed at a
temperature of 2 to 10.degree. C., and a chromatography flow rate
of 160 L/hour.
[0156] About 200 L of the purified solution 2 was concentrated to
about 10 L by using an ultrafiltration membrane, Microza UF Module
SIP-2013 (Asahi Kasei Chemicals, Japan), and then applied to a
Sephacryl S-300 HR (GE Healthcare Bioscience, U.S.A.) column
(diameter: 63 cm, height: 94 cm) equilibrated with a 20 mM
phosphate buffer (pH 7.3) containing 50 mM sodium chloride. An
elution peak with the maximum absorbance at 280 nm was separated,
and concentrated to about 12 L by using an ultrafiltration
membrane, Microza UF Module SIP-1013 (Asahi Kasei Chemicals,
Japan), to obtain a purified solution 3. The operation was
performed at a temperature of 2 to 10.degree. C., and a
chromatography flow rate of 6.2 L/hour.
[0157] The purified solution 3 was passed through a virus-removing
membrane, PLANOVA 15N (membrane area: 1 m.sup.2, Asahi Kasei
Medical, Japan), equilibrated with a 20 mM phosphate buffer (pH
7.3) containing 50 mM sodium chloride at room temperature and a
pressure lower than 0.1 MPa, and then further passed through a
0.22-.mu.m PVDF filtration membrane (Millipore, U.S.A.), and the
entire volume of the solution was collected. The result was used as
a purified product of soluble thrombomodulin.
[0158] By performing the same operation, 3 lots (A1, A2, A3) of the
purified product were obtained.
[0159] The APC activities of thrombomodulin of A1, A2, and A3 were
69000 U/mL, 68000 U/mL, and 72000 U/mL, respectively.
[0160] Soluble thrombomodulin concentrations in the solutions of
A1, A2, and A3 were 10.5 mg/mL, 10.2 mg/mL, and 10.3 mg/mL,
respectively.
Comparative Example 2
Preparation of Soluble Thrombomodulin 2
[0161] The medium described in Japanese Patent Unexamined
Publication No. 11-341990, Ingredient Table 2 was used as the base
medium. Growth medium was obtained by adding 60 mg/L of kanamycin
sulfate (Invitrogen, U.S.A.), 1 mg/L of tylosin tartrate
(Sigma-Aldrich, U.S.A.), and 8% bovine serum (HyClone, U.S.A.) to
the base medium, and used. Further, production medium was the same
as the growth medium, provided that the serum concentration was
changed to 4%.
[0162] The cells of one vial prepared in Comparative Example 1 were
thawed, inoculated into 100 mL of the growth medium, and cultured
with stirring at 37.degree. C. for 3 days by using a spinner flask
in a CO.sub.2 incubator. When the living cell density became
5.0.times.10.sup.5 cells/mL or more, the entire volume of the
culture medium was transferred to 400 mL of the growth medium, and
the cells were cultured with stirring at 37.degree. C. for 3 days
by using a spinner flask in a CO.sub.2 incubator. When the living
cell density became 5.0.times.10.sup.5 cells/mL or more, the entire
volume of the culture medium was transferred to 2 L of the growth
medium, and the cells were cultured with stirring at 37.degree. C.
for 3 days by using a spherical bottle in a CO.sub.2 incubator.
When the living cell density became 5.0.times.10.sup.5 cells/mL or
more, the entire volume of the culture medium was transferred to
7.5 L of the growth medium, and the cells were cultured with
stirring at 37.degree. C. for 4 days by using a spherical bottle in
a CO.sub.2 incubator. When the living cell density became
5.0.times.10.sup.5 cells/mL or more, perfusion culture was started,
in which the production medium was continuously added, and the
culture supernatant was continuously collected. The culture
conditions consisted of 37.degree. C., pH 7.2, dissolved oxygen:
50%, medium exchange: 10 L/day, and surface pressurization: 0 to
0.2 MPa. After the living cell density reached 7.5.times.10.sup.6
cells/mL, the culture was further continued for 40 days, and the
culture supernatant was collected as a production solution.
[0163] The collected production solution was clarified by using
filtration filters having pore diameters of 0.7 .mu.m and 0.22
.mu.m (Pall, U.S.A.), and stored at 2 to 10.degree. C. as a
filtered production solution.
[0164] About 400 L of the filtered production solution was applied
to a Q-Sepharose Fast Flow (GE Healthcare, U.S.A.) column
(diameter: 44 cm, height: 26 cm) equilibrated with a 20 mM
Tris-hydrochloric acid buffer (pH 7.4) containing 150 mM sodium
chloride. Then, the column was washed with 6 CV of a 20 mM acetate
buffer (pH 5.5) containing 180 mM sodium chloride, and further
washed with a 20 mM Tris-hydrochloric acid buffer (pH 7.4)
containing 180 mM sodium chloride until absorbance at 280 nm
returned to the baseline. Elution was started with a 20 mM
Tris-hydrochloric acid buffer (pH 7.4) containing 300 mM sodium
chloride, and 0.5 column volume of the eluate from the start of the
peak of absorbance at 280 nm was obtained as a roughly purified
solution. The operation was performed at a temperature of 2 to
10.degree. C., and a chromatography flow rate of 45 L/hour.
[0165] About 20 L of the roughly purified solution was applied to a
monoclonal antibody column (diameter: 44 cm, height: 12 cm)
equilibrated with a 20 mM phosphate buffer (pH 7.3) containing 0.3
M sodium chloride. 6 CV of a 20 mM phosphate buffer (pH 7.3)
containing 1.0 M sodium chloride was poured into the column, 3 CV
of a 0.1 M acetate buffer (pH 5.0) was further poured to wash the
column, and elution was started with a 0.1 M glycine-hydrochloric
acid buffer (pH 3.0) containing 0.3 M sodium chloride. The eluate
corresponding to the start to the end of the peak of absorbance at
280 nm was obtained, and added with 1/10 volume of a 0.5 M
phosphate buffer (pH 7.3) to obtain a purified solution 1. The
operation was performed at a temperature of 2 to 10.degree. C., and
a chromatography flow rate of 45 L/hour.
[0166] About 12 L of the purified solution 1 was adjusted to pH 3.5
with a 1.0 M glycine-hydrochloric acid buffer (pH 2.0), and applied
to an SP-Sepharose FF (GE Healthcare Bioscience, U.S.A.) column
(diameter: 14 cm, height: 13 cm) equilibrated with a 0.1 M
glycine-hydrochloric acid buffer (pH 3.5) containing 0.3 M NaCl.
Washing was started with a 0.1 M glycine-hydrochloric acid buffer
(pH 3.5) containing 0.3 M NaCl, and a flow-through fraction
corresponding to the start to the end of the peak of absorbance at
280 nm was obtained, and immediately neutralized to pH 7 with a 0.5
M phosphate buffer (pH 7.3) to obtain a purified solution 2. The
operation was performed at a temperature of 2 to 10.degree. C., and
a chromatography flow rate of 15 L/hour.
[0167] About 16 L of the purified solution 2 was concentrated to
about 1.2 L by using an ultrafiltration membrane, Microza UF Module
SIP-1013 (Asahi Kasei Chemicals, Japan), and then applied to a
Sephacryl S-300 HR (GE Healthcare Bioscience, U.S.A.) column
(diameter: 25 cm, height: 85 cm) equilibrated with a 20 mM
phosphate buffer (pH 7.3) containing 50 mM sodium chloride. An
elution peak with the maximum absorbance at 280 nm was separated,
and concentrated to about 0.8 L by using an ultrafiltration
membrane, Microza UF Module SIP-1013 (Asahi Kasei Chemicals,
Japan), to obtain a purified solution 3. The operation was
performed at a temperature of 2 to 10.degree. C., and a
chromatography flow rate of 1 L/hour.
[0168] The purified solution 3 was passed through a virus-removing
membrane, PLANOVA 15N (membrane area: 0.3 m.sup.2, Asahi Kasei
Medical, Japan), equilibrated with a 20 mM phosphate buffer (pH
7.3) containing 50 mM sodium chloride at room temperature and a
pressure lower than 0.1 MPa, and then further passed through a
0.22-.mu.m PVDF filtration membrane (Millipore, U.S.A.), and the
entire volume of the solution was collected. The result was used as
a purified product of soluble thrombomodulin (lot: B1).
[0169] The APC activity of thrombomodulin of B1 was 79000 U/mL.
[0170] Soluble thrombomodulin concentration in the solution of B1
was 12.6 mg/mL.
Comparative Example 3
Preparation of Soluble Thrombomodulin 3
[0171] The DMEM medium (Invitrogen, U.S.A.) was used as the base
medium.
[0172] Growth medium was obtained by adding 150 mg/L of L-proline
(Ajinomoto, Japan), 60 mg/L of kanamycin sulfate (MeijiSeika
Pharma, Japan), 1 mg/L of tylosin tartrate (Mercian, Japan), and
10% bovine serum (HyClone, U.S.A.) to the base medium, and used.
Further, production medium was the same as the growth medium,
provided that the serum concentration was changed to 1 to 3%.
[0173] The cells of one vial prepared in Comparative Example 1 were
thawed, inoculated into 100 mL of the growth medium, and cultured
with stirring at 37.degree. C. for 5 days by using a spinner flask
in a CO.sub.2 incubator. The entire volume of the culture medium
was transferred to 400 mL of the growth medium, and the cells were
cultured with stirring at 37.degree. C. for 5 days by using a
spinner flask in a CO.sub.2 incubator. The entire volume of the
culture medium was transferred to 1.6 L of the growth medium, and
the cells were cultured with stirring at 37.degree. C. for 5 days
by using a spherical bottle with bubbling air and CO.sub.2 into the
medium. The entire volume of the culture medium was transferred to
6 L of the growth medium, and the cells were cultured with stirring
at 37.degree. C. for 5 days by using a spherical bottle with
bubbling air and CO.sub.2 into the medium. The entire volume of the
culture medium was transferred to 56 L of the growth medium, and
the cells were cultured with stirring at 37.degree. C. for 4 days
by using a spherical bottle with bubbling air and CO.sub.2 into the
medium. After the entire medium was exchanged, the cells were
further cultured for 3 days. Further, after the entire medium was
exchanged, and when the living cell density reached
1.0.times.10.sup.6 cells/mL, the medium was changed to the
production medium. The production solution was collected every day
by using a continuous centrifugation machine CC-100 (Alfa Laval,
Sweden), and the fresh medium was supplemented. The production
culture was carried out for 100 days. The collected production
solution was clarified by using filtration filters having pore
diameters of 0.7 .mu.m and 0.22 .mu.m (Pall, U.S.A.), and stored at
2 to 10.degree. C. as a filtered production solution.
[0174] About 2400 L of the filtered production solution was applied
to a Q-Sepharose Fast Flow (GE Healthcare, U.S.A.) column
(diameter: 44 cm, height: 25 cm) equilibrated with a 20 mM
Tris-hydrochloric acid buffer (pH 7.4) containing 150 mM sodium
chloride. Then, the column was washed with 6 CV of a 20 mM acetate
buffer (pH 5.5) containing 180 mM sodium chloride, and further
washed with 2 CV of a 20 mM Tris-hydrochloric acid buffer (pH 7.4)
containing 180 mM sodium chloride. Elution was started with a 20 mM
Tris-hydrochloric acid buffer (pH 7.4) containing 300 mM sodium
chloride, and about 15 L of the eluate from the start of the peak
of absorbance at 280 nm was obtained as a roughly purified
solution. The operation was performed at a temperature of 2 to
10.degree. C., and a chromatography flow rate of 45 L/hour.
[0175] The aforementioned roughly purified solution 1 was applied
to a Butyl-Sepharose FF (GE Healthcare Bioscience, U.S.A.) column
(diameter: 25 cm. height: 10 cm) equilibrated with a 20 mM
phosphate buffer (pH 7.0) containing 0.3 M NaCl. Washing was
started with a 20 mM phosphate buffer (pH 7.0) containing 0.3 M
NaCl, and a flow-through fraction corresponding to the start to the
end of the peak of absorbance at 280 nm was obtained as a roughly
purified solution 2. The operation was performed at a temperature
of 2 to 10.degree. C., and a chromatography flow rate of 13
L/hour.
[0176] About 20 L of the roughly purified solution was applied to a
monoclonal antibody column (diameter: 44 cm, height: 18 cm)
equilibrated with a 20 mM phosphate buffer (pH 7.3) containing 0.3
M sodium chloride. 6 CV of a 20 mM phosphate buffer (pH 7.3)
containing 1.0 M sodium chloride was poured into the column, 3 CV
of a 0.1 M acetate buffer (pH 5.0) was further poured to wash the
column, and elution was started with a 0.1 M glycine-hydrochloric
acid buffer (pH 3.0) containing 0.3 M sodium chloride. The eluate
corresponding to the start to the end of the peak of absorbance at
280 nm was obtained, and added with 1/10 volume of a 1 M
glycine-sodium hydroxide buffer (pH 9.0) and 1/25 volume of a 0.5 M
phosphate buffer (pH 7.3) to obtain a purified solution 1. The
operation was performed at a temperature of 2 to 10.degree. C., and
a chromatography flow rate of 50 L/hour.
[0177] About 15 L of the purified solution 1 was concentrated to
about 1 L by using an ultrafiltration membrane, Microza UF Module
SIP-1013 (Asahi Kasei Chemicals, Japan), and then applied to a
Sephacryl S-300 HR (GE Healthcare Bioscience, U.S.A.) column
(diameter: 25 cm, height: 80 cm) equilibrated with a 20 mM
phosphate buffer (pH 7.3) containing 150 mM sodium chloride. The
operation was performed at a temperature of 2 to 10.degree. C., and
a chromatography flow rate of 1 L/hour. An elution peak with the
maximum absorbance at 280 nm was separated, and passed through a
0.22-.mu.m PVDF filtration membrane (Millipore, U.S.A.) to collect
about 3 L of the eluate. The result was used as a purified product
of soluble thrombomodulin (lot: B2).
[0178] The APC activity of thrombomodulin of B2 was 28000 U/mL.
[0179] Soluble thrombomodulin concentration in the solution of B2
was 3.8 mg/mL.
Comparative Example 4
Preparation of Soluble Thrombomodulin 4
[0180] The cells of one vial cryopreserved in Comparative Example 1
were thawed, inoculated into a serum-free medium IS CHO-CD (Irvine
Scientific, U.S.A.) containing 8 mM L-glutamine (Invitrogen,
U.S.A.), 50 .mu.M hypoxanthine (Invitrogen, U.S.A.), and 8 .mu.M
thymidine (Invitrogen, U.S.A.), and cultured at 37.degree. C. in a
CO.sub.2 incubator. The cells obtained by centrifuging the culture
medium was suspended in the serum-free medium IS CHO-CD (Irvine
Scientific, U.S.A.) containing 8 mM L-glutamine (Invitrogen,
U.S.A.), 50 .mu.M hypoxanthine (Invitrogen, U.S.A.), 8 .mu.M
thymidine (Invitrogen, U.S.A.), and 10% dimethyl sulfoxide
(Sigma-Aldrich, U.S.A.), and then the medium was dispensed into
vials (2.times.10.sup.7 cells/vial), and cryopreserved in liquid
nitrogen.
[0181] Growth medium was prepared by dissolving 20.78 g of IS
CHO-CD-A3 (Irvine Scientific, U.S.A.), 4.06 g of sodium chloride
(Tomita Pharmaceutical, Japan), and 2.20 g of sodium
hydrogencarbonate (Wako Pure Chemical Industries, Japan) in 1 L of
water. Production medium was prepared by dissolving 20.78 g of IS
CHO-CD-A3 (Irvine Scientific, U.S.A.), 2.63 g of sodium chloride
(Tomita Pharmaceutical, Japan), and 4.40 g of sodium
hydrogencarbonate (Wako Pure Chemical Industries, Japan) in 1 L of
water.
[0182] The cells of one vial were thawed, inoculated into 100 mL of
the growth medium, and cultured at 36.degree. C. for 5 days as
stationary culture by using a T-flask in a CO.sub.2 incubator. When
the living cell density became 7.0.times.10.sup.5 cells/mL or more,
40 mL of the culture medium was transferred to 360 mL of the growth
medium, and the cells were cultured with stirring at 36.degree. C.
for 7 days by using a spinner flask in a CO.sub.2 incubator. When
the living cell density became 7.0.times.10.sup.5 cells/mL or more,
80 mL of the culture medium was transferred to 720 mL of the growth
medium, and the cells were cultured with stirring at 36.degree. C.
for 6 days by using a spinner flask in a CO.sub.2 incubator. When
the living cell density became 7.0.times.10.sup.5 cells/mL or more,
the entire volume of the culture medium was transferred to 9.2 L of
the growth medium, and the cells were cultured with stirring at
36.degree. C., pH 7.1 and 50% of dissolved oxygen for 8 days by
using a perfusion culture tank. When the living cell density became
7.0.times.10.sup.5 cells/mL or more, perfusion culture was started,
in which the production medium was continuously added, and the
culture supernatant was continuously collected. The culture
conditions consisted of 36.degree. C., pH 7.1, dissolved oxygen:
50%, medium exchange: 10 L/day, and surface pressurization: 0 to
0.2 MPa. After the living cell density reached 7.5.times.10.sup.6
cells/mL, the culture was further continued for 26 days, and the
culture supernatant was collected as a production solution.
[0183] The collected production solution was clarified by using
filtration filters, SUPRAcap (Pall, U.S.A.) and Supor EBV (Pall,
U.S.A.), and stored at 2 to 10.degree. C. as a filtered production
solution.
[0184] About 250 L of the filtered production solution was applied
to a Q-Sepharose Fast Flow (GE Healthcare, U.S.A.) column
(diameter: 25 cm, height: 25 cm) equilibrated with a 20 mM
Tris-hydrochloric acid buffer (pH 7.7) containing 150 mM sodium
chloride. Then, the column was washed with 6 CV of a 20 mM acetate
buffer (pH 5.6) containing 180 mM sodium chloride, and further
washed with 4 CV of a 20 mM Tris-hydrochloric acid buffer (pH 7.7)
containing 180 mM sodium chloride. Elution was started with a 20 mM
Tris-hydrochloric acid buffer (pH 7.7) containing 290 mM sodium
chloride, and 0.5 column volume of the eluate from the start of the
peak of absorbance at 280 nm was obtained as a roughly purified
solution. The operation was performed at a temperature of 2 to
10.degree. C., and a chromatography flow rate of 18 L/hour.
[0185] About 6 L of the roughly purified solution was applied to a
monoclonal antibody column (diameter: 44 cm, height: 8 cm)
equilibrated with a 20 mM phosphate buffer (pH 7.3) containing 0.3
M sodium chloride. 6 CV of a 20 mM phosphate buffer (pH 7.3)
containing 1.0 M sodium chloride was poured into the column, 3 CV
of a 0.1 M acetate buffer (pH 5.0) was further poured to wash the
column, and elution was started with a 0.1 M glycine-hydrochloric
acid buffer (pH 3.0) containing 0.3 M sodium chloride. The eluate
corresponding to the start to the end of the peak of absorbance at
280 nm was obtained, and added with 1/10 volume of a 0.5 M
phosphate buffer (pH 7.3) to obtain a purified solution 1. The
operation was performed at a temperature of 2 to 10.degree. C., and
a chromatography flow rate of 46 L/hour.
[0186] About 14 L of the purified solution 1 was adjusted to pH 3.5
with a 1.0 M glycine-hydrochloric acid buffer (pH 2.0), and applied
to an SP-Sepharose FF (GE Healthcare Bioscience, U.S.A.) column
(diameter: 14 cm, height: 13 cm) equilibrated with a 0.1 M
glycine-hydrochloric acid buffer (pH 3.5) containing 0.3 M NaCl.
Washing was started with a 0.1 M glycine-hydrochloric acid buffer
(pH 3.5) containing 0.3 M NaCl, and a flow-through fraction
corresponding to the start to the end of the peak of absorbance at
280 nm was obtained, and immediately neutralized to pH 7 with a 0.5
M phosphate buffer (pH 7.3) to obtain a purified solution 2. The
operation was performed at a temperature of 2 to 10.degree. C., and
a chromatography flow rate of 15 L/hour.
[0187] About 20 L of the purified solution 2 was concentrated to
about 1 L by using an ultrafiltration membrane, Microza UF Module
SIP-1013 (Asahi Kasei Chemicals, Japan), and then applied to a
Sephacryl S-300 HR (GE Healthcare Bioscience, U.S.A.) column
(diameter: 25 cm, height: 79 cm) equilibrated with a 20 mM
phosphate buffer (pH 7.3) containing 50 mM sodium chloride. An
elution peak with the maximum absorbance at 280 nm was separated,
and concentrated to about 0.7 L by using an ultrafiltration
membrane, Microza UF Module SIP-1013 (Asahi Kasei Chemicals,
Japan), to obtain a purified solution 3. The operation was
performed at a temperature of 2 to 10.degree. C., and a
chromatography flow rate of 1 L/hour.
[0188] The entire volume of the purified solution 3 was passed
through a virus-removing membrane, PLANOVA 15N (membrane area: 0.12
m.sup.2, Asahi Kasei Medical, Japan), equilibrated with a 20 mM
phosphate buffer (pH 7.3) containing 50 mM sodium chloride at room
temperature and a pressure lower than 0.1 MPa, and then further
passed through a 0.22-.mu.m PVDF filtration membrane (Millipore,
U.S.A.), and the entire volume of the solution was collected. The
result was used as a purified product of soluble thrombomodulin
(lot: B3).
Example 1
Preparation of Highly-Purified Soluble Thrombomodulin 1
[0189] Cell culture was performed by using, as the base medium, the
medium described in Japanese Patent Unexamined Publication No.
11-341990, Ingredient Table 2, provided that NaHCO.sub.3
concentration was changed to 5,700 mg/L, and NaCl concentration was
changed to 2,410 mg/L. Growth medium was obtained by adding 60 mg/L
of kanamycin sulfate (Invitrogen, U.S.A.), 1 mg/L of tylosin
tartrate (Sigma-Aldrich, U.S.A.), and 8% bovine serum (HyClone,
U.S.A.) to the base medium, and used. Further, production medium
was the same as the growth medium, provided that the serum
concentration was changed to 3%.
[0190] The cells of one vial prepared in Comparative Example 1 were
thawed, inoculated into 100 mL of the growth medium, and cultured
with stirring at 37.degree. C. for 5 days by using a spinner flask
in a CO.sub.2 incubator. When the living cell density became
7.0.times.10.sup.5 cells/mL or more, the entire volume of the
culture medium was transferred to 0.9 L of the growth medium, and
the cells were cultured with stirring at 37.degree. C. for 5 days
by using a spinner flask in a CO.sub.2 incubator. When the living
cell density became 7.0.times.10.sup.5 cells/mL or more, the entire
volume of the culture medium was transferred to 9 L of the growth
medium, and the cells were cultured with stirring at 37.degree. C.,
pH 7.2 and 50% of dissolved oxygen for 5 days by using a culture
tank. When the living cell density became 7.0.times.10.sup.5
cells/mL or more, the entire volume of the culture medium was
transferred to 120 L of the growth medium, and the cells were
cultured with stirring at 37.degree. C., pH 7.2 and 50% of
dissolved oxygen for 7 days by using a perfusion culture tank. When
the living cell density became 7.0.times.10.sup.5 cells/mL or more,
perfusion culture was started, in which the production medium was
continuously added, and the culture supernatant was continuously
collected. The culture conditions consisted of 37.degree. C., pH
7.2, dissolved oxygen: 50%, medium exchange: 130 to 200 L/day, and
surface pressurization: 0 to 0.2 MPa. After the living cell density
reached 7.5.times.10.sup.6 cells/mL, the culture was further
continued for 40 days, and the culture supernatant was collected as
a production solution. The collected production solution was
clarified by using filtration filters, SUPRAdisc II (Pall, U.S.A.)
and Supor EBV (Pall, U.S.A.), and stored at 2 to 10.degree. C. as a
filtered production solution.
[0191] About 700 L of the filtered production solution was applied
to a Q-Sepharose Fast Flow (GE Healthcare, U.S.A.) column
(diameter: 63 cm, height: 25 cm) equilibrated with a 20 mM
Tris-hydrochloric acid buffer (pH 7.7) containing 150 mM sodium
chloride. Then, the column was washed with 6 CV of a 20 mM acetate
buffer (pH 5.5) containing 180 mM sodium chloride, and further
washed with a 20 mM Tris-hydrochloric acid buffer (pH 7.7)
containing 180 mM sodium chloride until absorbance at 280 nm
returned to the baseline. Elution was started with a 20 mM
Tris-hydrochloric acid buffer (pH 7.7) containing 300 mM sodium
chloride, and 0.5 column volume of the eluate from the start of the
peak of absorbance at 280 nm was obtained as a roughly purified
solution. The same operation was repeated 3 times to obtain 3 lots
of the roughly purified solution. The operation was performed at a
temperature of 2 to 10.degree. C., and a chromatography flow rate
of 109 L/hour.
[0192] About 20 L of the roughly purified solution was applied to a
monoclonal antibody column (diameter: 44 cm, height: 13 cm)
equilibrated with a 20 mM phosphate buffer (pH 7.3) containing 0.3
M sodium chloride. 6 CV of a 20 mM phosphate buffer (pH 7.3)
containing 1.0 M sodium chloride was poured into the column, 3 CV
of a 0.1 M acetate buffer (pH 5.0) was further poured to wash the
column, and elution was started with a 0.1 M glycine-hydrochloric
acid buffer (pH 3.0) containing 0.3 M sodium chloride. The eluate
corresponding to the start to the end of the peak of absorbance at
280 nm was obtained, and added with 1/10 volume of a 0.5 M
phosphate buffer (pH 7.3) to obtain a purified solution 1. The same
operation was repeated 6 times to obtain 6 lots of the purified
solution 1. The operation was performed at a temperature of 2 to
10.degree. C., and a chromatography flow rate of 46 L/hour.
[0193] About 130 L of the purified solution 1 of the 6 lots was
passed through a nylon filtration membrane (pore diameter: 0.4
.mu.m+0.2 .mu.m, membrane area: 1.8 m.sup.2, SARTOLON Maxi Caps
5101307H3, Sartorius, Germany) at a flow rate of 5 L/minute (about
0.07 m.sup.2 of membrane area was used for 1 mg of HCP), adjusted
to pH 3.5 with a 1.0 M glycine-hydrochloric acid buffer (pH 2.0),
and applied to an SP-Sepharose FF (GE Healthcare Bioscience,
U.S.A.) column (diameter: 45 cm, height: 10 cm) equilibrated with a
0.1 M glycine-hydrochloric acid buffer (pH 3.5) containing 0.3 M
NaCl. Washing was started with a 0.1 M glycine-hydrochloric acid
buffer (pH 3.5) containing 0.3 M NaCl, and a flow-through fraction
corresponding to the start to the end of the peak of absorbance at
280 nm was obtained, and immediately neutralized to pH 7 with a 0.5
M phosphate buffer (pH 7.3) to obtain a purified solution 2. The
operation was performed at a temperature of 2 to 10.degree. C., and
a chromatography flow rate of 160 L/hour.
[0194] About 160 L of the purified solution 2 was concentrated to
about 10 L by using an ultrafiltration membrane, Microza UF Module
SIP-2013 (Asahi Kasei Chemicals, Japan), and then applied to a
Sephacryl S-300 HR (GE Healthcare Bioscience, U.S.A.) column
(diameter: 63 cm, height: 94 cm) equilibrated with a 20 mM
phosphate buffer (pH 7.3) containing 50 mM sodium chloride. An
elution peak with the maximum absorbance at 280 nm was separated,
and concentrated to about 6 L by using an ultrafiltration membrane,
Microza UF Module SIP-1013 (Asahi Kasei Chemicals, Japan), to
obtain a purified solution 3. The operation was performed at a
temperature of 2 to 10.degree. C., and a chromatography flow rate
of 6.2 L/hour.
[0195] The purified solution 3 was passed through a virus-removing
membrane, PLANOVA 15N (membrane area: 1 m.sup.2, Asahi Kasei
Medical, Japan), equilibrated with a 20 mM phosphate buffer (pH
7.3) containing 50 mM sodium chloride at room temperature and a
pressure lower than 0.1 MPa, and then further passed through a
0.22-.mu.m PVDF filtration membrane (Millipore, U.S.A.), and the
entire volume of the solution was collected. The result was used as
a highly-purified soluble thrombomodulin purified product (lot:
A4).
[0196] The APC activity of thrombomodulin of A4 was 60000 U/mL.
[0197] Soluble thrombomodulin concentration in the solution of A4
was 9.3 mg/mL.
Example 2
Preparation of Highly-Purified Soluble Thrombomodulin 2
[0198] About 2,000 L of the filtered production solution obtained
in Example 1 was applied to a Q-Sepharose Fast Flow (GE Healthcare,
U.S.A.) column (diameter: 63 cm, height: 25 cm) equilibrated with a
20 mM Tris-hydrochloric acid buffer (pH 7.7) containing 150 mM
sodium chloride. Then, the column was washed with 6 CV of a 20 mM
acetate buffer (pH 5.45) containing 170 mM sodium chloride, and
further washed with 4 CV of a 20 mM Tris-hydrochloric acid buffer
(pH 7.7) containing 170 mM sodium chloride. Elution was started
with a 20 mM Tris-hydrochloric acid buffer (pH 7.7) containing 300
mM sodium chloride, and 0.5 column volume of the eluate from the
start of the peak of absorbance at 280 nm was obtained as a roughly
purified solution. The same operation was repeated twice to obtain
2 lots of the roughly purified solution. The operation was
performed at a temperature of 2 to 10.degree. C., and a
chromatography flow rate of 109 L/hour.
[0199] About 10 L of the roughly purified solution was applied to a
monoclonal antibody column (diameter: 44 cm, height: 13 cm)
equilibrated with a 20 mM phosphate buffer (pH 7.3) containing 0.3
M sodium chloride. 6 CV of a 20 mM phosphate buffer (pH 7.3)
containing 1.0 M sodium chloride was poured into the column, 3 CV
of a 0.1 M acetate buffer (pH 5.0) was further poured to wash the
column, and elution was started with a 0.1 M glycine-hydrochloric
acid buffer (pH 3.0) containing 0.3 M sodium chloride. The eluate
corresponding to the start to the end of the peak of absorbance at
280 nm was obtained, and added with 1/10 volume of a 0.5 M
phosphate buffer (pH 7.3) to obtain a purified solution 1. The same
operation was repeated 8 times to obtain 8 lots of the purified
solution 1. The operation was performed at a temperature of 2 to
10.degree. C., and a chromatography flow rate of 46 L/hour.
[0200] About 180 L of the purified solution 1 for 6 lots was passed
through a nylon filtration membrane (pore diameter: 0.4 .mu.m+0.2
.mu.m, membrane area: 1.8 m.sup.2, SARTOLON Maxi Caps 5101307H3,
Sartorius, Germany) at a flow rate of 5 L/minute (about 0.05
m.sup.2 of membrane area was used for 1 mg of HCP), adjusted to pH
3.5 with a 1.0 M glycine-hydrochloric acid buffer (pH 2.0), and
applied to an SP-Sepharose FF (GE Healthcare Bioscience, U.S.A.)
column (diameter: 45 cm, height: 10 cm) equilibrated with a 0.1 M
glycine-hydrochloric acid buffer (pH 3.5) containing 0.3 M NaCl.
Washing was started with a 0.1 M glycine-hydrochloric acid buffer
(pH 3.5) containing 0.3 M NaCl, and a flow-through fraction
corresponding to the start to the end of the peak of absorbance at
280 nm was obtained, and immediately neutralized to pH 7 with a 0.5
M phosphate buffer (pH 7.3) to obtain a purified solution 2. The
operation was performed at a temperature of 2 to 10.degree. C., and
a chromatography flow rate of 160 L/hour.
[0201] About 220 L of the purified solution 2 was concentrated to
about 5 L by using an ultrafiltration membrane, Microza UF Module
SIP-2013 (Asahi Kasei Chemicals, Japan), and then applied to a
Sephacryl S-300 HR (GE Healthcare Bioscience, U.S.A.) column
(diameter: 63 cm, height: 94 cm) equilibrated with a 20 mM
phosphate buffer (pH 7.3) containing 50 mM sodium chloride. An
elution peak with the maximum absorbance at 280 nm was separated,
and concentrated to about 10 L by using an ultrafiltration
membrane, Microza UF Module SIP-1013 (Asahi Kasei Chemicals,
Japan), to obtain a purified solution 3. The operation was
performed at a temperature of 2 to 10.degree. C., and a
chromatography flow rate of 6.2 L/hour.
[0202] The purified solution 3 was passed through a virus-removing
membrane, PLANOVA 15N (membrane area: 1 m.sup.2, Asahi Kasei
Medical, Japan), equilibrated with a 20 mM phosphate buffer (pH
7.3) containing 50 mM sodium chloride at room temperature and a
pressure lower than 0.1 MPa, and then further passed through a
0.22-.mu.m PVDF filtration membrane (Millipore, U.S.A.), and the
entire volume of the solution was collected. The result was used as
a highly-purified soluble thrombomodulin purified product (lot:
A5).
[0203] The APC activity of thrombomodulin of A5 was 69000 U/mL.
[0204] Soluble thrombomodulin concentration in the solution of A5
was 10.9 mg/mL.
Example 3
Preparation of Highly-Purified Soluble Thrombomodulin 3
[0205] Cell culture was performed by using, as the base medium, the
medium described in Japanese Patent Unexamined Publication No.
11-341990, Ingredient Table 2, provided that NaHCO.sub.3
concentration was changed to 5,700 mg/L, and NaCl concentration was
changed to 2,410 mg/L. Growth medium was obtained by adding 60 mg/L
of kanamycin sulfate (Invitrogen, U.S.A.), 1 mg/L of tylosin
tartrate (Sigma-Aldrich, U.S.A.), and 8% bovine serum (HyClone,
U.S.A.) to the base medium, and used. Further, production medium
was the same as the growth medium, provided that the serum
concentration was changed to 3%.
[0206] The cells of one vial prepared in Comparative Example 1 were
thawed, inoculated into 100 mL of the growth medium, and cultured
with stirring at 37.degree. C. for 5 days by using a spinner flask
in a CO.sub.2 incubator. When the living cell density became
7.0.times.10.sup.5 cells/mL or more, the entire volume of the
culture medium was transferred to 0.9 L of the growth medium, and
the cells were cultured with stirring at 37.degree. C. for 5 days
by using a spinner flask in a CO.sub.2 incubator. When the living
cell density became 7.0.times.10.sup.5 cells/mL or more, the entire
volume of the culture medium was transferred to 9 L of the growth
medium, and the cells were cultured with stirring at 37.degree. C.,
pH 7.2 and 50% of dissolved oxygen for 5 days by using a culture
tank. When the living cell density became 7.0.times.10.sup.5
cells/mL or more, the entire volume of the culture medium was
transferred to 120 L of the growth medium, and the cells were
cultured with stirring at 37.degree. C., pH 7.2 and 50% of
dissolved oxygen for 7 days by using a perfusion culture tank. When
the living cell density became 7.0.times.10.sup.5 cells/mL or more,
perfusion culture was started, in which the production medium was
continuously added, and the culture supernatant was continuously
collected. The culture conditions consisted of 37.degree. C., pH
7.2, dissolved oxygen: 50%, medium exchange: 130 to 200 L/day, and
surface pressurization: 0 to 0.2 MPa. After the living cell density
reached 7.5.times.10.sup.6 cells/mL, the culture was further
continued for 36 days, and the culture supernatant was collected as
a production solution. The collected production solution was
clarified by using filtration filters, SUPRAdisc II (Pall, U.S.A.)
and Supor EBV (Pall, U.S.A.), and stored at 2 to 10.degree. C. as a
filtered production solution.
[0207] About 700 L of the filtered production solution was applied
to a Q-Sepharose Fast Flow (GE Healthcare, U.S.A.) column
(diameter: 63 cm, height: 25 cm) equilibrated with a 20 mM
Tris-hydrochloric acid buffer (pH 7.7) containing 150 mM sodium
chloride. Then, the column was washed with 6 CV of a 20 mM acetate
buffer (pH 5.5) containing 180 mM sodium chloride, and further
washed with a 20 mM Tris-hydrochloric acid buffer (pH 7.7)
containing 180 mM sodium chloride until absorbance at 280 nm
returned to the baseline. Elution was started with a 20 mM
Tris-hydrochloric acid buffer (pH 7.7) containing 300 mM sodium
chloride, and 0.5 column volume of the eluate from the start of the
peak of absorbance at 280 nm was obtained as a roughly purified
solution. The same operation was repeated 6 times to obtain 6 lots
of the roughly purified solution. The operation was performed at a
temperature of 2 to 10.degree. C., and a chromatography flow rate
of 109 L/hour.
[0208] About 20 L of the roughly purified solution was applied to a
monoclonal antibody column (diameter: 44 cm, height: 13 cm)
equilibrated with a 20 mM phosphate buffer (pH 7.3) containing 0.3
M sodium chloride. 6 CV of a 20 mM phosphate buffer (pH 7.3)
containing 1.0 M sodium chloride was poured into the column, 3 CV
of a 0.1 M acetate buffer (pH 5.0) was further poured to wash the
column, and elution was started with a 0.1 M glycine-hydrochloric
acid buffer (pH 3.0) containing 0.3 M sodium chloride. The eluate
corresponding to the start to the end of the peak of absorbance at
280 nm was obtained, and added with 1/10 volume of a 0.5 M
phosphate buffer (pH 7.3) to obtain a purified solution 1. The same
operation was repeated 12 times to obtain 12 lots of the purified
solution 1. The operation was performed at a temperature of 2 to
10.degree. C., and a chromatography flow rate of 46 L/hour.
[0209] About 270 L of the purified solution 1 of the 12 lots was
passed through a nylon filtration membrane (pore diameter: 0.4
.mu.m+0.2 .mu.m, membrane area: 1.8 m.sup.2, SARTOLON Maxi Caps
5101307H3, Sartorius, Germany) at a flow rate of 5 L/minute (about
0.05 m.sup.2 of membrane area was used for 1 mg of HCP), adjusted
to pH 3.5 with a 1.0 M glycine-hydrochloric acid buffer (pH 2.0),
and applied to an SP-Sepharose FF (GE Healthcare Bioscience,
U.S.A.) column (diameter: 45 cm, height: 10 cm) equilibrated with a
0.1 M glycine-hydrochloric acid buffer (pH 3.5) containing 0.3 M
NaCl. Washing was started with a 0.1 M glycine-hydrochloric acid
buffer (pH 3.5) containing 0.3 M NaCl, and a flow-through fraction
corresponding to the start to the end of the peak of absorbance at
280 nm was obtained, and immediately neutralized to pH 7 with a 0.5
M phosphate buffer (pH 7.3) to obtain a purified solution 2. The
operation was performed at a temperature of 2 to 10.degree. C., and
a chromatography flow rate of 160 L/hour.
[0210] About 300 L of the purified solution 2 was concentrated to
about 11 L by using an ultrafiltration membrane, Microza UF Module
SIP-2013 (Asahi Kasei Chemicals, Japan), and then applied to a
Sephacryl S-300 HR (GE Healthcare Bioscience, U.S.A.) column
(diameter: 63 cm, height: 94 cm) equilibrated with a 20 mM
phosphate buffer (pH 7.3) containing 50 mM sodium chloride. An
elution peak with the maximum absorbance at 280 nm was separated,
and concentrated to about 13 L by using an ultrafiltration
membrane, Microza UF Module SIP-1013 (Asahi Kasei Chemicals,
Japan), to obtain a purified solution 3. The operation was
performed at a temperature of 2 to 10.degree. C., and a
chromatography flow rate of 6.2 L/hour.
[0211] The purified solution 3 was passed through a virus-removing
membrane, PLANOVA 15N (membrane area: 1 m.sup.2, Asahi Kasei
Medical, Japan), equilibrated with a 20 mM phosphate buffer (pH
7.3) containing 50 mM sodium chloride at room temperature and a
pressure lower than 0.1 MPa, and then further passed through a
0.22-.mu.m PVDF filtration membrane (Millipore, U.S.A.), and the
entire volume of the solution was collected. The result was used as
a highly-purified soluble thrombomodulin purified product (lot:
A6).
[0212] The APC activity of thrombomodulin of A6 was 81000 U/mL.
[0213] Soluble thrombomodulin concentration in the solution of A6
was 11.9 mg/mL.
Example 4
Preparation of Highly-Purified Soluble Thrombomodulin 4
[0214] Growth medium was prepared by dissolving 20.78 g of IS
CHO-CD-A3 (Irvine Scientific, U.S.A.), 4.06 g of sodium chloride
(Tomita Pharmaceutical, Japan), and 2.20 g of sodium
hydrogencarbonate (Wako Pure Chemical Industries, Japan) in 1 L of
water. Production medium was prepared by dissolving 20.78 g of IS
CHO-CD-A3 (Irvine Scientific, U.S.A.), 2.63 g of sodium chloride
(Tomita Pharmaceutical, Japan), and 4.40 g of sodium
hydrogencarbonate (Wako Pure Chemical Industries, Japan) in 1 L of
water.
[0215] The cells of one vial prepared in Comparative Example 4 were
thawed, inoculated into 100 mL of the growth medium, and cultured
at 36.degree. C. for 5 days as stationary culture by using a
T-flask in a CO.sub.2 incubator. When the living cell density
became 7.0.times.10.sup.5 cells/mL or more, the entire volume of
the culture medium was transferred to 0.9 L of the growth medium,
and the cells were cultured with stirring at 36.degree. C. for 5
days by using a spinner flask in a CO.sub.2 incubator. When the
living cell density became 7.0.times.10.sup.5 cells/mL or more, the
entire volume of the culture medium was transferred to 9 L of the
growth medium, and the cells were cultured with stirring at
36.degree. C., pH 7.1 and 50% of dissolved oxygen for 5 days by
using a culture tank. When the living cell density became
7.0.times.10.sup.5 cells/mL or more, the entire volume of the
culture medium was transferred to 120 L of the growth medium, and
the cells were cultured with stirring at 36.degree. C., pH 7.1 and
50% of dissolved oxygen for 7 days by using a perfusion culture
tank. When the living cell density became 7.0.times.10.sup.5
cells/mL or more, perfusion culture was started, in which the
production medium was continuously added, and the culture
supernatant was continuously collected. The culture conditions
consisted of 36.degree. C., pH 7.1, dissolved oxygen: 50%, medium
exchange: 130 L/day, and surface pressurization: 0 to 0.2 MPa.
After the living cell density reached 7.5.times.10.sup.6 cells/mL,
the culture was further continued for 20 days, and the culture
supernatant was collected as a production solution.
[0216] The collected production solution was clarified by using
filtration filters, SUPRAdisc II (Pall, U.S.A.) and Supor EBV
(Pall, U.S.A.), and stored at 2 to 10.degree. C. as a filtered
production solution.
[0217] About 1,400 L of the filtered production solution was
applied to a Q-Sepharose Fast Flow (GE Healthcare, U.S.A.) column
(diameter: 63 cm, height: 25 cm) equilibrated with a 20 mM
Tris-hydrochloric acid buffer (pH 7.7) containing 150 mM sodium
chloride. Then, the column was washed with 6 CV of a 20 mM acetate
buffer (pH 5.6) containing 180 mM sodium chloride, and further
washed with 4 CV of a 20 mM Tris-hydrochloric acid buffer (pH 7.7)
containing 180 mM sodium chloride. Elution was started with a 20 mM
Tris-hydrochloric acid buffer (pH 7.7) containing 290 mM sodium
chloride, and 0.5 column volume of the eluate from the start of the
peak of absorbance at 280 nm was obtained as a roughly purified
solution. The same operation was performed also for about 900 L of
the filtered production solution, and thus 2 lots of the roughly
purified solution were obtained. The operation was performed at a
temperature of 2 to 10.degree. C., and a chromatography flow rate
of 109 L/hour.
[0218] About 13 L of the roughly purified solution was applied to a
monoclonal antibody column (diameter: 44 cm, height: 13 cm)
equilibrated with a 20 mM phosphate buffer (pH 7.3) containing 0.3
M sodium chloride. 6 CV of a 20 mM phosphate buffer (pH 7.3)
containing 1.0 M sodium chloride was poured into the column, 3 CV
of a 0.1 M acetate buffer (pH 5.0) was further poured to wash the
column, and elution was started with a 0.1 M glycine-hydrochloric
acid buffer (pH 3.0) containing 0.3 M sodium chloride. The eluate
corresponding to the start to the end of the peak of absorbance at
280 nm was obtained, and added with 1/10 volume of a 0.5 M
phosphate buffer (pH 7.3) to obtain a purified solution 1. The same
operation was repeated 5 times to obtain 5 lots of the purified
solution 1. The operation was performed at a temperature of 2 to
10.degree. C., and a chromatography flow rate of 46 L/hour.
[0219] About 110 L of the purified solution 1 of the 5 lots was
passed through a nylon filtration membrane (pore diameter: 0.4
.mu.m+0.2 .mu.m, membrane area: 1.8 m.sup.2, SARTOLON Maxi Caps
5101307H3, Sartorius, Germany) at a flow rate of 5 L/minute (about
0.03 m.sup.2 of membrane area was used for 1 mg of HCP), adjusted
to pH 3.5 with a 1.0 M glycine-hydrochloric acid buffer (pH 2.0),
and applied to an SP-Sepharose FF (GE Healthcare Bioscience,
U.S.A.) column (diameter: 45 cm, height: 10 cm) equilibrated with a
0.1 M glycine-hydrochloric acid buffer (pH 3.5) containing 0.3 M
NaCl. Washing was started with a 0.1 M glycine-hydrochloric acid
buffer (pH 3.5) containing 0.3 M NaCl, and a flow-through fraction
corresponding to the start to the end of the peak of absorbance at
280 nm was obtained, and immediately neutralized to pH 7 with a 0.5
M phosphate buffer (pH 7.3) to obtain a purified solution 2. The
operation was performed at a temperature of 2 to 10.degree. C., and
a chromatography flow rate of 160 L/hour.
[0220] About 120 L of the purified solution 2 was concentrated to
about 5 L by using an ultrafiltration membrane, Microza UF Module
SIP-2013 (Asahi Kasei Chemicals, Japan), and then applied to a
Sephacryl S-300 HR (GE Healthcare Bioscience, U.S.A.) column
(diameter: 63 cm, height: 94 cm) equilibrated with a 20 mM
phosphate buffer (pH 7.3) containing 50 mM sodium chloride. An
elution peak with the maximum absorbance at 280 nm was separated,
and concentrated to about 5 L by using an ultrafiltration membrane,
Microza UF Module SIP-1013 (Asahi Kasei Chemicals, Japan), to
obtain a purified solution 3. The operation was performed at a
temperature of 2 to 10.degree. C., and a chromatography flow rate
of 6.2 L/hour.
[0221] The entire volume of the purified solution 3 was passed
through a virus-removing membrane, PLANOVA 15N (membrane area: 1
m.sup.2, Asahi Kasei Medical, Japan), equilibrated with a 20 mM
phosphate buffer (pH 7.3) containing 50 mM sodium chloride at room
temperature and a pressure lower than 0.1 MPa, and then further
passed through a 0.22-.mu.m PVDF filtration membrane (Millipore,
U.S.A.), and the entire volume of the solution was collected. The
result was used as a highly-purified soluble thrombomodulin
purified product (lot: A7).
[0222] The APC activity of thrombomodulin of A7 was 69000 U/mL.
[0223] Soluble thrombomodulin concentration in the solution of A7
was 10.4 mg/mL.
Test Example 1
Evaluation of Removal of HCP Using Various Filtration Membranes
[0224] The purified solution 1 obtained in Comparative Example 1
(HCP concentration: 462 ng/ml) was passed through filtration
membranes of different materials, and HCP concentrations of the
filtrates were compared. Specifically, 5 ml of the purified
solution 1 was passed at a flow rate of 1 ml/minute through each of
filtration membranes made of (1) PVDF (polyvinylidene fluoride)
(Millex GV, Millipore, U.S.A.), (2) CA (cellulose acetate)
(Minisart, Sartorius, Germany), (3) PES (polyethersulfone)
(Minisart High-Flow, Sartorius, Germany), (4) nylon (NALGENE
Syringe Filter, Thermo Fisher Scientific, U.S.A.), and (5) CA+GF
(cellulose acetate+glass fiber) (Minisart Plus, Sartorius), and the
entire volume was collected as a filtrate.
[0225] Protein concentration and HCP concentration of the solution
were measured before and after the filtration. The protein
concentration was obtained on the basis of absorbance at 280 nm,
and the HCP concentration was measured according to the method
described in Reference Example 2. As a result, substantial
difference was not observed between the protein concentrations
measured before and after the filtration for all the filtration
membranes, but high HCP-removing effect was observed for the
filtration membrane made of nylon and the filtration membrane made
of PES, and it was found that they reduced the HCP concentration to
28% and 36%, respectively (Table 1). Further, degree of the
reduction of the HCP concentration significantly differed depending
on the material of the membrane in spite of the same pore diameter.
Accordingly, it was not considered that HCP insolubilized by
aggregation was removed, but it was considered that HCP was removed
by adsorption by the membranes. The diameters of the evaluated
filtration membranes were 25 mm or 26 mm, and the membrane areas
for 1 mg of HCP calculated on the basis of effective membrane areas
of the filtration membranes described in the data sheets of the
manufacturers were 0.17 m.sup.2 or 0.23 m.sup.2.
TABLE-US-00001 TABLE 1 Re- Re- Mem- Effective Membrane covery
covery Pore brane membrane area for of of Filtration diameter
diameter area 1 mg of HCP proteins membrane (.mu.m) (mm) (cm.sup.2)
HCP (m.sup.2) (%) (%) (1) PVDF 0.22 25 3.9 0.17 93 101 (2) CA 0.2
26 5.3 0.23 69 100 (3) PES 0.2 26 5.3 0.23 36 100 (4) Nylon 0.2 25
Unknown Unknown 28 99 (5) CA + 0.2 26 5.3 0.23 44 99 GF
Test Example 2
Comparison of HCP-Removing Abilities of Nylon and PES Filtration
Membranes
[0226] It was found in Test Example 1 that the nylon filtration
membrane and the PES filtration membrane had high HCP-removing
abilities. Accordingly, change of HCP-removing abilities of these
filtration membranes depending on the volume of solution passed
through them was evaluated. Products of two manufacturers were
prepared for each material of the filtration membrane, and the
purified solution 1 of a lot different from the lots used in Test
Example 1 was passed through the membranes (HCP concentration: 303
ng/ml). The filtration membranes used were a PVDF filtration
membrane (pore diameter: 0.22 .mu.m, membrane diameter: 25 mm,
effective membrane area: 3.9 cm.sup.2, Millex GV, Millipore,
U.S.A.) as a control, (1) Acrodisc AP-4436T, Pall, U.S.A., pore
diameter: 0.2 .mu.m, membrane diameter: 25 mm, effective membrane
area: 3.9 cm.sup.2, and (2) Minisart NY25, Sartorius, Germany, pore
diameter: 0.2 .mu.m, membrane diameter 25 mm, effective membrane
area: 4.8 cm.sup.2 as nylon filtration membranes, as well as (3)
Acrodisc PN4612, Pall, U.S.A., pore diameter: 0.2 inn, membrane
diameter 25 mm, effective membrane area: 2.8 cm.sup.2, and (4)
Minisart High-Flow, Sartorius, Germany, pore diameter: 0.2 .mu.m,
membrane diameter 26 mm, effective membrane area: 5.3 cm.sup.2 as
PES filtration membranes. The purified solution 1 was passed
through each filtration membrane in a volume of 100 ml at a flow
rate of 10 ml/minute, and the filtrate was sampled for every 20 ml
of the solution passed through the filtration membrane, of which
HCP concentration was measured according to the method described in
Reference Example 2, and of which protein concentration was
obtained on the basis of absorbance at 280 nm.
[0227] As a result, change of the protein concentration was not
observed for all the samples, but HCP-removing effect was observed
for both the nylon and PES filtration membranes (Table 2). The
nylon filtration membrane gave especially high HCP-removing effect,
and reduced the HCP concentration to 25% at most. It was found that
when volume of the solution passed through the filtration membrane
was smaller, i.e., filtration membrane area for 1 mg of HCP was
larger, higher HCP-removing effect was obtained.
TABLE-US-00002 TABLE 2 Membrane area for 1 mg of Recovery of HCP
(%) Filtration membrane HCP (m.sup.2) 20 ml 40 ml 60 ml 80 ml 100
ml PVDF 0.013~0.064 94 98 109 89 92 Nylon (1) Pall 0.013~0.064 50
62 60 69 75 (2) Sartorius 0.016~0.079 25 34 30 49 47 PES (3) Pall
0.009~0.046 77 84 98 88 107 (4) Sartorius 0.017~0.087 68 66 75 62
63
Test Example 3
Evaluation of HCP-Removing Ability of Nylon Filtration Membrane for
Different Solution Compositions
[0228] Effects of differences in buffer composition and soluble
thrombomodulin concentration on the HCP-removing ability of a nylon
filtration membrane were evaluated. Each of the purified solution
1, the purified solution 2, the purified solution 2 after the
concentration, the purified solution 3, and the purified product
obtained in Comparative Example 4 in a volume of 5 mL was passed
through a nylon filtration membrane having a membrane diameter of
25 mm, an effective membrane area of 4.8 cm.sup.2, and a pore
diameter of 0.2 .mu.m (Minisart NY25, Sartorius, Germany) at a flow
rate of 1 mL/minute, and the entire volume was collected as a
filtrate. The filtration membrane areas for 1 mg of HCP contained
in the samples were as follows: 0.77 m.sup.2 for the purified
solution 1, 1.7 m.sup.2 for the purified solution 2, 0.43 m.sup.2
for the purified solution 2 after the concentration, 0.72 m.sup.2
for the purified solution 3, and 0.81 m.sup.2 for the purified
product. HCP concentration of each obtained filtrate was measured
according to the method described in Reference Example 2, and
protein concentration of the same was obtained on the basis of
absorbance at 280 nm. As a result, the nylon membrane gave high
HCP-removing effect for all the samples, and provided high protein
recovery higher than 90% (Table 3).
TABLE-US-00003 TABLE 3 HCP Protein Before After Re- Before After
Re- filtration filtration covery filtration filtration covery
Sample (ng/mL) (ng/mL) (%) (mg/mL) (mg/mL) (%) Purified 125 65 52
0.47 0.44 94 solution 1 Purified 56 35 63 0.40 0.37 93 solution 2
Purified 222 115 52 8.28 8.28 100 solution 2 after concentration
Purified 134 <25 <19 11.1 11.4 103 solution 3 Purified 119
<25 <21 10.1 10.3 102 product
Test Example 4
Evaluation of Removal of HCP from Purified Products of Soluble
Thrombomodulin Obtained by Various Preparation Methods
[0229] It was examined whether or not HCP was successfully removed
from soluble thrombomodulin products obtained by different methods
by further passing them through a nylon filtration membrane. The
soluble thrombomodulin purified products obtained in Comparative
Examples 1 to 3 (A1, B1 and B2, respectively) in a volume of 5 mL
each were passed through a nylon filtration membrane having a
membrane diameter 25 mm, an effective membrane area: 4.8 cm.sup.2,
and a pore diameter: 0.2 .mu.m (Minisart NY25, Sartorius, Germany)
at a flow rate of 1 mL/minute, and the entire volume of each was
collected as a filtrate. The filtration membrane areas for 1 mg of
HCP contained in the soluble thrombomodulin purified products were
0.34 m.sup.2 for A1, 0.46 m.sup.2 for B1, and 1.7 m.sup.2 for B2.
HCP contents, mouse IgG contents, and bovine serum protein contents
of the solutions were measured before and after the filtration by
the methods described in Reference Examples 2 to 4. The nylon
membrane gave high removing effect for only HCP for all the
purified products (Table 4). On the basis of this result, it was
considered that the nylon filtration membrane did not have an
action of non-specifically adsorbing proteins, but had an action of
specifically adsorbing HCP.
TABLE-US-00004 TABLE 4 Bovine serum HCP content Mouse IgG content
protein content (ng/10,000 U) (ng/10,000 U) (ng/10,000 U) Purified
Before After Before After Before After product filtration
filtration filtration filtration filtration filtration Comparative
40.9 <8.2 <0.18 <0.18 0.67 1.03 Example 1 (A1) Comparative
26.0 <6.7 <0.16 <0.16 0.24 <0.16 Example 2 (B1)
Comparative 20.0 7.0 0.65 0.59 13.5 13.2 Example 3 (B2)
Test Example 5
Comparison of Purities of Thrombomodulin Purified Products Obtained
with or without Use of Nylon Filtration Membrane
[0230] HCP contents, mouse IgG contents, and bovine serum protein
contents of the thrombomodulin purified products obtained by the
industrial level production using no nylon filtration membrane
(Comparative Example 1) and the industrial level production using a
nylon filtration membrane (Examples 1 to 4) were measured by the
methods described in Reference Examples 2 to 4.
[0231] The three lots of Comparative Example 1 (A1, A2 and A3) not
passed through any nylon filtration membrane had high HCP contents
higher than 10 ng/10,000 U, whilst the HCP contents of the four
lots of Examples 1 to 4 (A4, A5, A6 and A7) passed through a nylon
filtration membrane were lower than the quantification limit. Any
significant difference was not observed in contents of mouse IgG
and bovine serum proteins as other impurities in the products
obtained by using or not using the nylon filtration membrane (Table
5). As described above, the nylon filtration membrane gave specific
removing ability for HCP also in industrial level production, and
enabled production of highly-purified soluble thrombomodulin having
an HCP content less than 10 ng/10,000 U of thrombomodulin.
[0232] Thrombomodulin purities based on the total proteins of the
highly-purified soluble thrombomodulin products obtained in
Examples 1 to 4 were measured by gel filtration liquid
chromatography and ion exchange liquid chromatography. The
measurement by gel filtration liquid chromatography was performed
by using TOSOH TSKgel G3000SWXL (TOSOH, Japan), and a 50 mM
phosphate buffer (pH 7.3) containing 0.1 M sodium sulfate under the
conditions of a temperature of 40.degree. C. and a flow rate of 0.9
mL/minute. As a result, purities of the purified products (A4, A5,
A6 and A7) were all higher than 99%. Further, the measurement by
ion exchange liquid chromatography was performed by using TOSOH
DEAE 5PW (TOSOH, Japan) with elution using a linear gradient of
from a 20 mM piperazine-hydrochloric acid buffer (pH 5.6)
containing 50 mM sodium chloride to a 20 mM piperazine-hydrochloric
acid buffer (pH 5.6) containing 350 mM sodium chloride over 30
minutes under the conditions of a temperature of 40.degree. C. and
a flow rate of 0.9 mL/minute. As a result, the purities of the
purified products (A4, A5, A6 and A7) were all higher than 99%.
[0233] Further, when molecular weights of a soluble thrombomodulin
purified product prepared as described in Comparative Example 1, of
which molecular weight had been already confirmed to be 64,000 by
MALDI-TOF-MS, and the highly-purified soluble thrombomodulin
purified products obtained in Examples 1 to 4 were compared by
SDS-PAGE, the bands were detected at the same position. On the
basis of this result, the molecular weight of the highly-purified
soluble thrombomodulin was considered to be 64,000.
[0234] Furthermore, endotoxin contents determined by the gelling
method described in Japanese Pharmacopoeia, General Test
Procedures, Endotoxin Test Method <4.01> were 0.004 to 0.03
EU/10,000 U, and thus were at an extremely low level (Table 6).
TABLE-US-00005 TABLE 5 Application Mouse Bovine serum or non- HCP
IgG protein application content content content Lot of filtration
(ng/10.sup.4 U) (ng/10.sup.4 U) (ng/10.sup.4 U) Comparative A1 Not
used 40.9 <0.18 0.67 Example 1 Comparative A2 Not used 17.2
<0.19 0.25 Example 1 Comparative A3 Not used 22.3 <0.18
<0.23 Example 1 Example 1 A4 Used <8.3 <0.21 1.42 Example
2 A5 Used <7.2 <0.18 0.42 Example 3 A6 Used <6.2 <0.16
0.71 Example 4 A7 Used <7.2 <0.18 Not measured
TABLE-US-00006 TABLE 6 Application or non- Endotoxin content Lot
application of filtration (EU/10.sup.4 U) Example 1 A4 Used 0.0050
Example 2 A5 Used 0.0043 Example 3 A6 Used 0.0105 Example 4 A7 Used
0.0245
Test Example 6
Analysis of HCP Removed with Nylon Filtration Membrane
[0235] Highly-purified soluble thrombomodulin was prepared in the
same manner as that described in Example 3, and the nylon
filtration membrane used for the preparation (pore diameter: 0.4
.mu.m+0.2 .mu.m, membrane area: 1.8 m.sup.2, SARTOLON Maxi Caps
5101307H3, Sartorius, Germany) was taken out from the housing, and
cut into pieces of about 3 g each. Five of the pieces were
sufficiently washed with a 20 mM phosphate buffer (pH 7.3)
containing 50 mM sodium chloride, and then each piece was put into
a test tube containing 40 mL of a 50 mM Tris-hydrochloric acid
buffer (pH 8.0) containing 0.5% CHAPS and 200 mM sodium chloride.
The proteins adsorbed on the membrane were extracted in the buffer
by shaking overnight at room temperature. The entire volume of the
extract was concentrated to 15 .mu.L by using ultrafiltration
membranes, Vivaspin 20 (Sartorius, Germany) and Amicon Ultra-0.5 mL
(Millipore, U.S.A.). 1/5 Volume of this concentrate was subjected
to SDS-PAGE (e-PAGEL5/20, Atto, Japan) and CBB staining (Quick-CBB,
Wako Pure Chemical Industries, Japan). A band detected around a
molecular weight of 10,000 was excised, and the gel portion was
reduced with dithiothreitol, then carbamidomethylated with
iodoacetamide, and subjected to enzymatic digestion with trypsin
overnight. The enzymatic digestion product was subjected to
LC/MS/MS, and Mascot search was performed on the basis of the
obtained mass-spectrum data by using the database of NCBI to
analyze the amino acid sequence of the enzymatic digestion
product.
Measurement Conditions of LC/MS/MS
[0236] LC/MS/MS (measurement apparatus): DiNa-2A Multi-dimensional
Autoinjector System (KYA Technologies, Japan) MS measurement range:
MS1 (m/z 400-1500), MS2 (m/z 50-1500).times.3 (data dependent
scanning mode) Ionization mode: nanoESI.sup.+
Column: PicoFrit Column BataBasic C18 (New Objective, U.S.A.)
Mobile Phase:
[0237] Mobile phase A: 0.1% formic acid/2% acetonitrile Mobile
phase B: 0.1% formic acid/80% acetonitrile Gradient: 0 to 30
minutes: Mobile phase B, 5 to 40% [0238] 30 to 40 minutes: Mobile
phase B, 40 to 100% [0239] 40 to 60 minutes: Mobile phase B, 100%
Flow rate: 300 nL/minute
[0240] The amino acid sequences of the fragments expected from the
mass-spectrum data were as shown in (1) to (7) mentioned below, and
agreed with partial sequences of histone H2B (Biochimie, 61 (1),
61-69 (1979)) shown below. This result revealed that one of
constituents of HCP removed with the nylon filter was histone
H2B.
TABLE-US-00007 (SEQ ID NO: 14) (1) KESYSVYVYK (SEQ ID NO: 15) (2)
VLKQVHPDTGISSK (SEQ ID NO: 16) (3) STITSREIQTAVR (SEQ ID NO: 17)
(4) EIQTAVR (SEQ ID NO: 18) (5) EIQTAVRLLLPGELAK (SEQ ID NO: 19)
(6) LLLPGELAK (SEQ ID NO: 20) (7) LLLPGELAKHAVSEGTK
TABLE-US-00008 TABLE 7 Amino acid sequence of histone H2B (SEQ ID
NO: 21) ##STR00001##
(The boxed sequences are amino acid sequence regions of the amino
acid sequence of histone H2B identical with the amino acid
sequences of (1) to (7) mentioned above, which were deduced from
the mass-spectrum data.)
Sequence CWU 1
1
211132PRThuman 1Met Leu Gly Val Leu Val Leu Gly Ala Leu Ala Leu Ala
Gly Leu Gly 1 5 10 15 Phe Pro Asp Pro Cys Phe Arg Ala Asn Cys Glu
Tyr Gln Cys Gln Pro 20 25 30 Leu Asn Gln Thr Ser Tyr Leu Cys Val
Cys Ala Glu Gly Phe Ala Pro 35 40 45 Ile Pro His Glu Pro His Arg
Cys Gln Met Phe Cys Asn Gln Thr Ala 50 55 60 Cys Pro Ala Asp Cys
Asp Pro Asn Thr Gln Ala Ser Cys Glu Cys Pro 65 70 75 80 Glu Gly Tyr
Ile Leu Asp Asp Gly Phe Ile Cys Thr Asp Ile Asp Glu 85 90 95 Cys
Glu Asn Gly Gly Phe Cys Ser Gly Val Cys His Asn Leu Pro Gly 100 105
110 Thr Phe Glu Cys Ile Cys Gly Pro Asp Ser Ala Leu Val Arg His Ile
115 120 125 Gly Thr Asp Cys 130 2396DNAhuman 2atgcttgggg tcctggtcct
tggcgcgctg gccctggccg gcctggggtt ccccgacccg 60tgcttcagag ccaactgcga
gtaccagtgc cagcccctga accaaactag ctacctctgc 120gtctgcgccg
agggcttcgc gcccattccc cacgagccgc acaggtgcca gatgttttgc
180aaccagactg cctgtccagc cgactgcgac cccaacaccc aggctagctg
tgagtgccct 240gaaggctaca tcctggacga cggtttcatc tgcacggaca
tcgacgagtg cgaaaacggc 300ggcttctgct ccggggtgtg ccacaacctc
cccggtacct tcgagtgcat ctgcgggccc 360gactcggccc ttgtccgcca
cattggcacc gactgt 3963132PRThuman 3Met Leu Gly Val Leu Val Leu Gly
Ala Leu Ala Leu Ala Gly Leu Gly 1 5 10 15 Phe Pro Asp Pro Cys Phe
Arg Ala Asn Cys Glu Tyr Gln Cys Gln Pro 20 25 30 Leu Asn Gln Thr
Ser Tyr Leu Cys Val Cys Ala Glu Gly Phe Ala Pro 35 40 45 Ile Pro
His Glu Pro His Arg Cys Gln Met Phe Cys Asn Gln Thr Ala 50 55 60
Cys Pro Ala Asp Cys Asp Pro Asn Thr Gln Ala Ser Cys Glu Cys Pro 65
70 75 80 Glu Gly Tyr Ile Leu Asp Asp Gly Phe Ile Cys Thr Asp Ile
Asp Glu 85 90 95 Cys Glu Asn Gly Gly Phe Cys Ser Gly Val Cys His
Asn Leu Pro Gly 100 105 110 Thr Phe Glu Cys Ile Cys Gly Pro Asp Ser
Ala Leu Ala Arg His Ile 115 120 125 Gly Thr Asp Cys 130
4396DNAhuman 4atgcttgggg tcctggtcct tggcgcgctg gccctggccg
gcctggggtt ccccgacccg 60tgcttcagag ccaactgcga gtaccagtgc cagcccctga
accaaactag ctacctctgc 120gtctgcgccg agggcttcgc gcccattccc
cacgagccgc acaggtgcca gatgttttgc 180aaccagactg cctgtccagc
cgactgcgac cccaacaccc aggctagctg tgagtgccct 240gaaggctaca
tcctggacga cggtttcatc tgcacggaca tcgacgagtg cgaaaacggc
300ggcttctgct ccggggtgtg ccacaacctc cccggtacct tcgagtgcat
ctgcgggccc 360gactcggccc ttgcccgcca cattggcacc gactgt
3965480PRThuman 5Met Leu Gly Val Leu Val Leu Gly Ala Leu Ala Leu
Ala Gly Leu Gly 1 5 10 15 Phe Pro Ala Pro Ala Glu Pro Gln Pro Gly
Gly Ser Gln Cys Val Glu 20 25 30 His Asp Cys Phe Ala Leu Tyr Pro
Gly Pro Ala Thr Phe Leu Asn Ala 35 40 45 Ser Gln Ile Cys Asp Gly
Leu Arg Gly His Leu Met Thr Val Arg Ser 50 55 60 Ser Val Ala Ala
Asp Val Ile Ser Leu Leu Leu Asn Gly Asp Gly Gly 65 70 75 80 Val Gly
Arg Arg Arg Leu Trp Ile Gly Leu Gln Leu Pro Pro Gly Cys 85 90 95
Gly Asp Pro Lys Arg Leu Gly Pro Leu Arg Gly Phe Gln Trp Val Thr 100
105 110 Gly Asp Asn Asn Thr Ser Tyr Ser Arg Trp Ala Arg Leu Asp Leu
Asn 115 120 125 Gly Ala Pro Leu Cys Gly Pro Leu Cys Val Ala Val Ser
Ala Ala Glu 130 135 140 Ala Thr Val Pro Ser Glu Pro Ile Trp Glu Glu
Gln Gln Cys Glu Val 145 150 155 160 Lys Ala Asp Gly Phe Leu Cys Glu
Phe His Phe Pro Ala Thr Cys Arg 165 170 175 Pro Leu Ala Val Glu Pro
Gly Ala Ala Ala Ala Ala Val Ser Ile Thr 180 185 190 Tyr Gly Thr Pro
Phe Ala Ala Arg Gly Ala Asp Phe Gln Ala Leu Pro 195 200 205 Val Gly
Ser Ser Ala Ala Val Ala Pro Leu Gly Leu Gln Leu Met Cys 210 215 220
Thr Ala Pro Pro Gly Ala Val Gln Gly His Trp Ala Arg Glu Ala Pro 225
230 235 240 Gly Ala Trp Asp Cys Ser Val Glu Asn Gly Gly Cys Glu His
Ala Cys 245 250 255 Asn Ala Ile Pro Gly Ala Pro Arg Cys Gln Cys Pro
Ala Gly Ala Ala 260 265 270 Leu Gln Ala Asp Gly Arg Ser Cys Thr Ala
Ser Ala Thr Gln Ser Cys 275 280 285 Asn Asp Leu Cys Glu His Phe Cys
Val Pro Asn Pro Asp Gln Pro Gly 290 295 300 Ser Tyr Ser Cys Met Cys
Glu Thr Gly Tyr Arg Leu Ala Ala Asp Gln 305 310 315 320 His Arg Cys
Glu Asp Val Asp Asp Cys Ile Leu Glu Pro Ser Pro Cys 325 330 335 Pro
Gln Arg Cys Val Asn Thr Gln Gly Gly Phe Glu Cys His Cys Tyr 340 345
350 Pro Asn Tyr Asp Leu Val Asp Gly Glu Cys Val Glu Pro Val Asp Pro
355 360 365 Cys Phe Arg Ala Asn Cys Glu Tyr Gln Cys Gln Pro Leu Asn
Gln Thr 370 375 380 Ser Tyr Leu Cys Val Cys Ala Glu Gly Phe Ala Pro
Ile Pro His Glu 385 390 395 400 Pro His Arg Cys Gln Met Phe Cys Asn
Gln Thr Ala Cys Pro Ala Asp 405 410 415 Cys Asp Pro Asn Thr Gln Ala
Ser Cys Glu Cys Pro Glu Gly Tyr Ile 420 425 430 Leu Asp Asp Gly Phe
Ile Cys Thr Asp Ile Asp Glu Cys Glu Asn Gly 435 440 445 Gly Phe Cys
Ser Gly Val Cys His Asn Leu Pro Gly Thr Phe Glu Cys 450 455 460 Ile
Cys Gly Pro Asp Ser Ala Leu Val Arg His Ile Gly Thr Asp Cys 465 470
475 480 61440DNAhuman 6atgcttgggg tcctggtcct tggcgcgctg gccctggccg
gcctggggtt ccccgcaccc 60gcagagccgc agccgggtgg cagccagtgc gtcgagcacg
actgcttcgc gctctacccg 120ggccccgcga ccttcctcaa tgccagtcag
atctgcgacg gactgcgggg ccacctaatg 180acagtgcgct cctcggtggc
tgccgatgtc atttccttgc tactgaacgg cgacggcggc 240gttggccgcc
ggcgcctctg gatcggcctg cagctgccac ccggctgcgg cgaccccaag
300cgcctcgggc ccctgcgcgg cttccagtgg gttacgggag acaacaacac
cagctatagc 360aggtgggcac ggctcgacct caatggggct cccctctgcg
gcccgttgtg cgtcgctgtc 420tccgctgctg aggccactgt gcccagcgag
ccgatctggg aggagcagca gtgcgaagtg 480aaggccgatg gcttcctctg
cgagttccac ttcccagcca cctgcaggcc actggctgtg 540gagcccggcg
ccgcggctgc cgccgtctcg atcacctacg gcaccccgtt cgcggcccgc
600ggagcggact tccaggcgct gccggtgggc agctccgccg cggtggctcc
cctcggctta 660cagctaatgt gcaccgcgcc gcccggagcg gtccaggggc
actgggccag ggaggcgccg 720ggcgcttggg actgcagcgt ggagaacggc
ggctgcgagc acgcgtgcaa tgcgatccct 780ggggctcccc gctgccagtg
cccagccggc gccgccctgc aggcagacgg gcgctcctgc 840accgcatccg
cgacgcagtc ctgcaacgac ctctgcgagc acttctgcgt tcccaacccc
900gaccagccgg gctcctactc gtgcatgtgc gagaccggct accggctggc
ggccgaccaa 960caccggtgcg aggacgtgga tgactgcata ctggagccca
gtccgtgtcc gcagcgctgt 1020gtcaacacac agggtggctt cgagtgccac
tgctacccta actacgacct ggtggacggc 1080gagtgtgtgg agcccgtgga
cccgtgcttc agagccaact gcgagtacca gtgccagccc 1140ctgaaccaaa
ctagctacct ctgcgtctgc gccgagggct tcgcgcccat tccccacgag
1200ccgcacaggt gccagatgtt ttgcaaccag actgcctgtc cagccgactg
cgaccccaac 1260acccaggcta gctgtgagtg ccctgaaggc tacatcctgg
acgacggttt catctgcacg 1320gacatcgacg agtgcgaaaa cggcggcttc
tgctccgggg tgtgccacaa cctccccggt 1380accttcgagt gcatctgcgg
gcccgactcg gcccttgtcc gccacattgg caccgactgt 14407480PRThuman 7Met
Leu Gly Val Leu Val Leu Gly Ala Leu Ala Leu Ala Gly Leu Gly 1 5 10
15 Phe Pro Ala Pro Ala Glu Pro Gln Pro Gly Gly Ser Gln Cys Val Glu
20 25 30 His Asp Cys Phe Ala Leu Tyr Pro Gly Pro Ala Thr Phe Leu
Asn Ala 35 40 45 Ser Gln Ile Cys Asp Gly Leu Arg Gly His Leu Met
Thr Val Arg Ser 50 55 60 Ser Val Ala Ala Asp Val Ile Ser Leu Leu
Leu Asn Gly Asp Gly Gly 65 70 75 80 Val Gly Arg Arg Arg Leu Trp Ile
Gly Leu Gln Leu Pro Pro Gly Cys 85 90 95 Gly Asp Pro Lys Arg Leu
Gly Pro Leu Arg Gly Phe Gln Trp Val Thr 100 105 110 Gly Asp Asn Asn
Thr Ser Tyr Ser Arg Trp Ala Arg Leu Asp Leu Asn 115 120 125 Gly Ala
Pro Leu Cys Gly Pro Leu Cys Val Ala Val Ser Ala Ala Glu 130 135 140
Ala Thr Val Pro Ser Glu Pro Ile Trp Glu Glu Gln Gln Cys Glu Val 145
150 155 160 Lys Ala Asp Gly Phe Leu Cys Glu Phe His Phe Pro Ala Thr
Cys Arg 165 170 175 Pro Leu Ala Val Glu Pro Gly Ala Ala Ala Ala Ala
Val Ser Ile Thr 180 185 190 Tyr Gly Thr Pro Phe Ala Ala Arg Gly Ala
Asp Phe Gln Ala Leu Pro 195 200 205 Val Gly Ser Ser Ala Ala Val Ala
Pro Leu Gly Leu Gln Leu Met Cys 210 215 220 Thr Ala Pro Pro Gly Ala
Val Gln Gly His Trp Ala Arg Glu Ala Pro 225 230 235 240 Gly Ala Trp
Asp Cys Ser Val Glu Asn Gly Gly Cys Glu His Ala Cys 245 250 255 Asn
Ala Ile Pro Gly Ala Pro Arg Cys Gln Cys Pro Ala Gly Ala Ala 260 265
270 Leu Gln Ala Asp Gly Arg Ser Cys Thr Ala Ser Ala Thr Gln Ser Cys
275 280 285 Asn Asp Leu Cys Glu His Phe Cys Val Pro Asn Pro Asp Gln
Pro Gly 290 295 300 Ser Tyr Ser Cys Met Cys Glu Thr Gly Tyr Arg Leu
Ala Ala Asp Gln 305 310 315 320 His Arg Cys Glu Asp Val Asp Asp Cys
Ile Leu Glu Pro Ser Pro Cys 325 330 335 Pro Gln Arg Cys Val Asn Thr
Gln Gly Gly Phe Glu Cys His Cys Tyr 340 345 350 Pro Asn Tyr Asp Leu
Val Asp Gly Glu Cys Val Glu Pro Val Asp Pro 355 360 365 Cys Phe Arg
Ala Asn Cys Glu Tyr Gln Cys Gln Pro Leu Asn Gln Thr 370 375 380 Ser
Tyr Leu Cys Val Cys Ala Glu Gly Phe Ala Pro Ile Pro His Glu 385 390
395 400 Pro His Arg Cys Gln Met Phe Cys Asn Gln Thr Ala Cys Pro Ala
Asp 405 410 415 Cys Asp Pro Asn Thr Gln Ala Ser Cys Glu Cys Pro Glu
Gly Tyr Ile 420 425 430 Leu Asp Asp Gly Phe Ile Cys Thr Asp Ile Asp
Glu Cys Glu Asn Gly 435 440 445 Gly Phe Cys Ser Gly Val Cys His Asn
Leu Pro Gly Thr Phe Glu Cys 450 455 460 Ile Cys Gly Pro Asp Ser Ala
Leu Ala Arg His Ile Gly Thr Asp Cys 465 470 475 480 81440DNAhuman
8atgcttgggg tcctggtcct tggcgcgctg gccctggccg gcctggggtt ccccgcaccc
60gcagagccgc agccgggtgg cagccagtgc gtcgagcacg actgcttcgc gctctacccg
120ggccccgcga ccttcctcaa tgccagtcag atctgcgacg gactgcgggg
ccacctaatg 180acagtgcgct cctcggtggc tgccgatgtc atttccttgc
tactgaacgg cgacggcggc 240gttggccgcc ggcgcctctg gatcggcctg
cagctgccac ccggctgcgg cgaccccaag 300cgcctcgggc ccctgcgcgg
cttccagtgg gttacgggag acaacaacac cagctatagc 360aggtgggcac
ggctcgacct caatggggct cccctctgcg gcccgttgtg cgtcgctgtc
420tccgctgctg aggccactgt gcccagcgag ccgatctggg aggagcagca
gtgcgaagtg 480aaggccgatg gcttcctctg cgagttccac ttcccagcca
cctgcaggcc actggctgtg 540gagcccggcg ccgcggctgc cgccgtctcg
atcacctacg gcaccccgtt cgcggcccgc 600ggagcggact tccaggcgct
gccggtgggc agctccgccg cggtggctcc cctcggctta 660cagctaatgt
gcaccgcgcc gcccggagcg gtccaggggc actgggccag ggaggcgccg
720ggcgcttggg actgcagcgt ggagaacggc ggctgcgagc acgcgtgcaa
tgcgatccct 780ggggctcccc gctgccagtg cccagccggc gccgccctgc
aggcagacgg gcgctcctgc 840accgcatccg cgacgcagtc ctgcaacgac
ctctgcgagc acttctgcgt tcccaacccc 900gaccagccgg gctcctactc
gtgcatgtgc gagaccggct accggctggc ggccgaccaa 960caccggtgcg
aggacgtgga tgactgcata ctggagccca gtccgtgtcc gcagcgctgt
1020gtcaacacac agggtggctt cgagtgccac tgctacccta actacgacct
ggtggacggc 1080gagtgtgtgg agcccgtgga cccgtgcttc agagccaact
gcgagtacca gtgccagccc 1140ctgaaccaaa ctagctacct ctgcgtctgc
gccgagggct tcgcgcccat tccccacgag 1200ccgcacaggt gccagatgtt
ttgcaaccag actgcctgtc cagccgactg cgaccccaac 1260acccaggcta
gctgtgagtg ccctgaaggc tacatcctgg acgacggttt catctgcacg
1320gacatcgacg agtgcgaaaa cggcggcttc tgctccgggg tgtgccacaa
cctccccggt 1380accttcgagt gcatctgcgg gcccgactcg gcccttgccc
gccacattgg caccgactgt 14409516PRThuman 9Met Leu Gly Val Leu Val Leu
Gly Ala Leu Ala Leu Ala Gly Leu Gly 1 5 10 15 Phe Pro Ala Pro Ala
Glu Pro Gln Pro Gly Gly Ser Gln Cys Val Glu 20 25 30 His Asp Cys
Phe Ala Leu Tyr Pro Gly Pro Ala Thr Phe Leu Asn Ala 35 40 45 Ser
Gln Ile Cys Asp Gly Leu Arg Gly His Leu Met Thr Val Arg Ser 50 55
60 Ser Val Ala Ala Asp Val Ile Ser Leu Leu Leu Asn Gly Asp Gly Gly
65 70 75 80 Val Gly Arg Arg Arg Leu Trp Ile Gly Leu Gln Leu Pro Pro
Gly Cys 85 90 95 Gly Asp Pro Lys Arg Leu Gly Pro Leu Arg Gly Phe
Gln Trp Val Thr 100 105 110 Gly Asp Asn Asn Thr Ser Tyr Ser Arg Trp
Ala Arg Leu Asp Leu Asn 115 120 125 Gly Ala Pro Leu Cys Gly Pro Leu
Cys Val Ala Val Ser Ala Ala Glu 130 135 140 Ala Thr Val Pro Ser Glu
Pro Ile Trp Glu Glu Gln Gln Cys Glu Val 145 150 155 160 Lys Ala Asp
Gly Phe Leu Cys Glu Phe His Phe Pro Ala Thr Cys Arg 165 170 175 Pro
Leu Ala Val Glu Pro Gly Ala Ala Ala Ala Ala Val Ser Ile Thr 180 185
190 Tyr Gly Thr Pro Phe Ala Ala Arg Gly Ala Asp Phe Gln Ala Leu Pro
195 200 205 Val Gly Ser Ser Ala Ala Val Ala Pro Leu Gly Leu Gln Leu
Met Cys 210 215 220 Thr Ala Pro Pro Gly Ala Val Gln Gly His Trp Ala
Arg Glu Ala Pro 225 230 235 240 Gly Ala Trp Asp Cys Ser Val Glu Asn
Gly Gly Cys Glu His Ala Cys 245 250 255 Asn Ala Ile Pro Gly Ala Pro
Arg Cys Gln Cys Pro Ala Gly Ala Ala 260 265 270 Leu Gln Ala Asp Gly
Arg Ser Cys Thr Ala Ser Ala Thr Gln Ser Cys 275 280 285 Asn Asp Leu
Cys Glu His Phe Cys Val Pro Asn Pro Asp Gln Pro Gly 290 295 300 Ser
Tyr Ser Cys Met Cys Glu Thr Gly Tyr Arg Leu Ala Ala Asp Gln 305 310
315 320 His Arg Cys Glu Asp Val Asp Asp Cys Ile Leu Glu Pro Ser Pro
Cys 325 330 335 Pro Gln Arg Cys Val Asn Thr Gln Gly Gly Phe Glu Cys
His Cys Tyr 340 345 350 Pro Asn Tyr Asp Leu Val Asp Gly Glu Cys Val
Glu Pro Val Asp Pro 355 360 365 Cys Phe Arg Ala Asn Cys Glu Tyr Gln
Cys Gln Pro Leu Asn Gln Thr 370 375 380 Ser Tyr Leu Cys Val Cys Ala
Glu Gly Phe Ala Pro Ile Pro His Glu 385 390 395 400 Pro His Arg Cys
Gln Met Phe Cys Asn Gln Thr Ala Cys Pro Ala Asp 405 410 415 Cys Asp
Pro Asn Thr Gln Ala Ser Cys Glu Cys Pro Glu Gly Tyr Ile 420 425 430
Leu Asp Asp Gly Phe Ile Cys Thr Asp Ile Asp Glu Cys Glu Asn Gly 435
440 445 Gly Phe Cys Ser Gly Val Cys His Asn Leu Pro Gly Thr Phe Glu
Cys 450 455 460 Ile Cys Gly Pro Asp Ser Ala Leu Val Arg His Ile Gly
Thr Asp Cys 465 470 475 480 Asp Ser Gly Lys Val Asp Gly Gly Asp Ser
Gly Ser Gly Glu Pro Pro 485
490 495 Pro Ser Pro Thr Pro Gly Ser Thr Leu Thr Pro Pro Ala Val Gly
Leu 500 505 510 Val His Ser Gly 515 101548DNAhuman 10atgcttgggg
tcctggtcct tggcgcgctg gccctggccg gcctggggtt ccccgcaccc 60gcagagccgc
agccgggtgg cagccagtgc gtcgagcacg actgcttcgc gctctacccg
120ggccccgcga ccttcctcaa tgccagtcag atctgcgacg gactgcgggg
ccacctaatg 180acagtgcgct cctcggtggc tgccgatgtc atttccttgc
tactgaacgg cgacggcggc 240gttggccgcc ggcgcctctg gatcggcctg
cagctgccac ccggctgcgg cgaccccaag 300cgcctcgggc ccctgcgcgg
cttccagtgg gttacgggag acaacaacac cagctatagc 360aggtgggcac
ggctcgacct caatggggct cccctctgcg gcccgttgtg cgtcgctgtc
420tccgctgctg aggccactgt gcccagcgag ccgatctggg aggagcagca
gtgcgaagtg 480aaggccgatg gcttcctctg cgagttccac ttcccagcca
cctgcaggcc actggctgtg 540gagcccggcg ccgcggctgc cgccgtctcg
atcacctacg gcaccccgtt cgcggcccgc 600ggagcggact tccaggcgct
gccggtgggc agctccgccg cggtggctcc cctcggctta 660cagctaatgt
gcaccgcgcc gcccggagcg gtccaggggc actgggccag ggaggcgccg
720ggcgcttggg actgcagcgt ggagaacggc ggctgcgagc acgcgtgcaa
tgcgatccct 780ggggctcccc gctgccagtg cccagccggc gccgccctgc
aggcagacgg gcgctcctgc 840accgcatccg cgacgcagtc ctgcaacgac
ctctgcgagc acttctgcgt tcccaacccc 900gaccagccgg gctcctactc
gtgcatgtgc gagaccggct accggctggc ggccgaccaa 960caccggtgcg
aggacgtgga tgactgcata ctggagccca gtccgtgtcc gcagcgctgt
1020gtcaacacac agggtggctt cgagtgccac tgctacccta actacgacct
ggtggacggc 1080gagtgtgtgg agcccgtgga cccgtgcttc agagccaact
gcgagtacca gtgccagccc 1140ctgaaccaaa ctagctacct ctgcgtctgc
gccgagggct tcgcgcccat tccccacgag 1200ccgcacaggt gccagatgtt
ttgcaaccag actgcctgtc cagccgactg cgaccccaac 1260acccaggcta
gctgtgagtg ccctgaaggc tacatcctgg acgacggttt catctgcacg
1320gacatcgacg agtgcgaaaa cggcggcttc tgctccgggg tgtgccacaa
cctccccggt 1380accttcgagt gcatctgcgg gcccgactcg gcccttgtcc
gccacattgg caccgactgt 1440gactccggca aggtggacgg tggcgacagc
ggctctggcg agcccccgcc cagcccgacg 1500cccggctcca ccttgactcc
tccggccgtg gggctcgtgc attcgggc 154811516PRThuman 11Met Leu Gly Val
Leu Val Leu Gly Ala Leu Ala Leu Ala Gly Leu Gly 1 5 10 15 Phe Pro
Ala Pro Ala Glu Pro Gln Pro Gly Gly Ser Gln Cys Val Glu 20 25 30
His Asp Cys Phe Ala Leu Tyr Pro Gly Pro Ala Thr Phe Leu Asn Ala 35
40 45 Ser Gln Ile Cys Asp Gly Leu Arg Gly His Leu Met Thr Val Arg
Ser 50 55 60 Ser Val Ala Ala Asp Val Ile Ser Leu Leu Leu Asn Gly
Asp Gly Gly 65 70 75 80 Val Gly Arg Arg Arg Leu Trp Ile Gly Leu Gln
Leu Pro Pro Gly Cys 85 90 95 Gly Asp Pro Lys Arg Leu Gly Pro Leu
Arg Gly Phe Gln Trp Val Thr 100 105 110 Gly Asp Asn Asn Thr Ser Tyr
Ser Arg Trp Ala Arg Leu Asp Leu Asn 115 120 125 Gly Ala Pro Leu Cys
Gly Pro Leu Cys Val Ala Val Ser Ala Ala Glu 130 135 140 Ala Thr Val
Pro Ser Glu Pro Ile Trp Glu Glu Gln Gln Cys Glu Val 145 150 155 160
Lys Ala Asp Gly Phe Leu Cys Glu Phe His Phe Pro Ala Thr Cys Arg 165
170 175 Pro Leu Ala Val Glu Pro Gly Ala Ala Ala Ala Ala Val Ser Ile
Thr 180 185 190 Tyr Gly Thr Pro Phe Ala Ala Arg Gly Ala Asp Phe Gln
Ala Leu Pro 195 200 205 Val Gly Ser Ser Ala Ala Val Ala Pro Leu Gly
Leu Gln Leu Met Cys 210 215 220 Thr Ala Pro Pro Gly Ala Val Gln Gly
His Trp Ala Arg Glu Ala Pro 225 230 235 240 Gly Ala Trp Asp Cys Ser
Val Glu Asn Gly Gly Cys Glu His Ala Cys 245 250 255 Asn Ala Ile Pro
Gly Ala Pro Arg Cys Gln Cys Pro Ala Gly Ala Ala 260 265 270 Leu Gln
Ala Asp Gly Arg Ser Cys Thr Ala Ser Ala Thr Gln Ser Cys 275 280 285
Asn Asp Leu Cys Glu His Phe Cys Val Pro Asn Pro Asp Gln Pro Gly 290
295 300 Ser Tyr Ser Cys Met Cys Glu Thr Gly Tyr Arg Leu Ala Ala Asp
Gln 305 310 315 320 His Arg Cys Glu Asp Val Asp Asp Cys Ile Leu Glu
Pro Ser Pro Cys 325 330 335 Pro Gln Arg Cys Val Asn Thr Gln Gly Gly
Phe Glu Cys His Cys Tyr 340 345 350 Pro Asn Tyr Asp Leu Val Asp Gly
Glu Cys Val Glu Pro Val Asp Pro 355 360 365 Cys Phe Arg Ala Asn Cys
Glu Tyr Gln Cys Gln Pro Leu Asn Gln Thr 370 375 380 Ser Tyr Leu Cys
Val Cys Ala Glu Gly Phe Ala Pro Ile Pro His Glu 385 390 395 400 Pro
His Arg Cys Gln Met Phe Cys Asn Gln Thr Ala Cys Pro Ala Asp 405 410
415 Cys Asp Pro Asn Thr Gln Ala Ser Cys Glu Cys Pro Glu Gly Tyr Ile
420 425 430 Leu Asp Asp Gly Phe Ile Cys Thr Asp Ile Asp Glu Cys Glu
Asn Gly 435 440 445 Gly Phe Cys Ser Gly Val Cys His Asn Leu Pro Gly
Thr Phe Glu Cys 450 455 460 Ile Cys Gly Pro Asp Ser Ala Leu Ala Arg
His Ile Gly Thr Asp Cys 465 470 475 480 Asp Ser Gly Lys Val Asp Gly
Gly Asp Ser Gly Ser Gly Glu Pro Pro 485 490 495 Pro Ser Pro Thr Pro
Gly Ser Thr Leu Thr Pro Pro Ala Val Gly Leu 500 505 510 Val His Ser
Gly 515 121548DNAhuman 12atgcttgggg tcctggtcct tggcgcgctg
gccctggccg gcctggggtt ccccgcaccc 60gcagagccgc agccgggtgg cagccagtgc
gtcgagcacg actgcttcgc gctctacccg 120ggccccgcga ccttcctcaa
tgccagtcag atctgcgacg gactgcgggg ccacctaatg 180acagtgcgct
cctcggtggc tgccgatgtc atttccttgc tactgaacgg cgacggcggc
240gttggccgcc ggcgcctctg gatcggcctg cagctgccac ccggctgcgg
cgaccccaag 300cgcctcgggc ccctgcgcgg cttccagtgg gttacgggag
acaacaacac cagctatagc 360aggtgggcac ggctcgacct caatggggct
cccctctgcg gcccgttgtg cgtcgctgtc 420tccgctgctg aggccactgt
gcccagcgag ccgatctggg aggagcagca gtgcgaagtg 480aaggccgatg
gcttcctctg cgagttccac ttcccagcca cctgcaggcc actggctgtg
540gagcccggcg ccgcggctgc cgccgtctcg atcacctacg gcaccccgtt
cgcggcccgc 600ggagcggact tccaggcgct gccggtgggc agctccgccg
cggtggctcc cctcggctta 660cagctaatgt gcaccgcgcc gcccggagcg
gtccaggggc actgggccag ggaggcgccg 720ggcgcttggg actgcagcgt
ggagaacggc ggctgcgagc acgcgtgcaa tgcgatccct 780ggggctcccc
gctgccagtg cccagccggc gccgccctgc aggcagacgg gcgctcctgc
840accgcatccg cgacgcagtc ctgcaacgac ctctgcgagc acttctgcgt
tcccaacccc 900gaccagccgg gctcctactc gtgcatgtgc gagaccggct
accggctggc ggccgaccaa 960caccggtgcg aggacgtgga tgactgcata
ctggagccca gtccgtgtcc gcagcgctgt 1020gtcaacacac agggtggctt
cgagtgccac tgctacccta actacgacct ggtggacggc 1080gagtgtgtgg
agcccgtgga cccgtgcttc agagccaact gcgagtacca gtgccagccc
1140ctgaaccaaa ctagctacct ctgcgtctgc gccgagggct tcgcgcccat
tccccacgag 1200ccgcacaggt gccagatgtt ttgcaaccag actgcctgtc
cagccgactg cgaccccaac 1260acccaggcta gctgtgagtg ccctgaaggc
tacatcctgg acgacggttt catctgcacg 1320gacatcgacg agtgcgaaaa
cggcggcttc tgctccgggg tgtgccacaa cctccccggt 1380accttcgagt
gcatctgcgg gcccgactcg gcccttgccc gccacattgg caccgactgt
1440gactccggca aggtggacgg tggcgacagc ggctctggcg agcccccgcc
cagcccgacg 1500cccggctcca ccttgactcc tccggccgtg gggctcgtgc attcgggc
15481321DNAArtificial SequenceDescription of Artificial Sequence
Synthetic DNA 13aatgtggcgg gcaagggccg a 211410PRThuman 14Lys Glu
Ser Tyr Ser Val Tyr Val Tyr Lys 1 5 10 1514PRThuman 15Val Leu Lys
Gln Val His Pro Asp Thr Gly Ile Ser Ser Lys 1 5 10 1613PRThuman
16Ser Thr Ile Thr Ser Arg Glu Ile Gln Thr Ala Val Arg 1 5 10
177PRThuman 17Glu Ile Gln Thr Ala Val Arg 1 5 1816PRThuman 18Glu
Ile Gln Thr Ala Val Arg Leu Leu Leu Pro Gly Glu Leu Ala Lys 1 5 10
15 199PRThuman 19Leu Leu Leu Pro Gly Glu Leu Ala Lys 1 5
2017PRThuman 20Leu Leu Leu Pro Gly Glu Leu Ala Lys His Ala Val Ser
Glu Gly Thr 1 5 10 15 Lys 21125PRThuman 21Pro Glu Pro Ala Lys Ser
Ala Pro Ala Pro Lys Lys Gly Ser Lys Lys 1 5 10 15 Ala Val Thr Lys
Ala Gln Lys Lys Asp Gly Lys Lys Arg Lys Arg Ser 20 25 30 Arg Lys
Glu Ser Tyr Ser Val Tyr Val Tyr Lys Val Leu Lys Gln Val 35 40 45
His Pro Asp Thr Gly Ile Ser Ser Lys Ala Met Gly Ile Met Asn Ser 50
55 60 Phe Val Asn Asp Ile Phe Glx Arg Ile Ala Gly Glu Ala Ser Arg
Leu 65 70 75 80 Ala His Tyr Asn Lys Arg Ser Thr Ile Thr Ser Arg Glu
Ile Gln Thr 85 90 95 Ala Val Arg Leu Leu Leu Pro Gly Glu Leu Ala
Lys His Ala Val Ser 100 105 110 Glu Gly Thr Lys Ala Val Thr Lys Tyr
Thr Ser Ser Lys 115 120 125
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