U.S. patent application number 16/742144 was filed with the patent office on 2020-05-07 for method for monomerizing matrix metalloproteinase 7 (mmp-7) aggregate.
This patent application is currently assigned to THE CHEMO-SERO-THERAPEUTIC RESEARCH INSTITUTE. The applicant listed for this patent is THE CHEMO-SERO-THERAPEUTIC RESEARCH INSTITUTE. Invention is credited to Masaki HIRASHIMA, Reiko MATSUYAMA, Wataru MORIKAWA, Hiroshi NAKATAKE, Hideki TAKEO.
Application Number | 20200140843 16/742144 |
Document ID | / |
Family ID | 54554080 |
Filed Date | 2020-05-07 |
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United States Patent
Application |
20200140843 |
Kind Code |
A1 |
NAKATAKE; Hiroshi ; et
al. |
May 7, 2020 |
METHOD FOR MONOMERIZING MATRIX METALLOPROTEINASE 7 (MMP-7)
AGGREGATE
Abstract
A method for monomerization of MMP-7 aggregates is provided. A
method for monomerization of MMP-7 aggregates which comprises
treating MMP-7 aggregates with a buffer solution comprising a
monovalent cation chloride (sodium chloride, potassium chloride,
etc.) at a low concentration or with a buffer solution not
comprising a monovalent cation chloride, a process for preparing
MMP-7 which involves said method for monomerization, and a
(pharmaceutical) composition comprising MMP-7 in the aforementioned
buffer solution. In case that a (pharmaceutical) composition
comprising MMP-7 at a low concentration is prepared, the
aforementioned buffer solution comprising sugar alcohols or sugars
is used.
Inventors: |
NAKATAKE; Hiroshi;
(Koshi-shi, JP) ; HIRASHIMA; Masaki; (Koshi-shi,
JP) ; TAKEO; Hideki; (Kikuchi-gun, JP) ;
MATSUYAMA; Reiko; (Kumamoto-shi, JP) ; MORIKAWA;
Wataru; (Kumamoto-shi, JP) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
THE CHEMO-SERO-THERAPEUTIC RESEARCH INSTITUTE |
Kumamoto |
|
JP |
|
|
Assignee: |
THE CHEMO-SERO-THERAPEUTIC RESEARCH
INSTITUTE
Kumamoto
JP
|
Family ID: |
54554080 |
Appl. No.: |
16/742144 |
Filed: |
January 14, 2020 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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15310908 |
Nov 14, 2016 |
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PCT/JP2015/064451 |
May 20, 2015 |
|
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16742144 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C12N 9/6491 20130101;
C12N 9/64 20130101; C12N 15/52 20130101; A61P 25/00 20180101; A61K
38/46 20130101; A61K 47/16 20130101; A61K 47/26 20130101; C12N 9/96
20130101; A61P 19/04 20180101; C12Y 304/24023 20130101; A61P 19/00
20180101; A61K 47/02 20130101; A61K 38/4886 20130101; A61K 47/10
20130101 |
International
Class: |
C12N 9/96 20060101
C12N009/96; A61K 47/02 20060101 A61K047/02; A61K 47/26 20060101
A61K047/26; A61K 38/48 20060101 A61K038/48; C12N 9/64 20060101
C12N009/64; C12N 15/52 20060101 C12N015/52; A61K 47/10 20060101
A61K047/10; A61K 47/16 20060101 A61K047/16; A61K 38/46 20060101
A61K038/46 |
Foreign Application Data
Date |
Code |
Application Number |
May 21, 2014 |
JP |
2014-105452 |
Claims
1-25. (canceled)
26. A (pharmaceutical) composition comprising matrix
metalloproteinase 7 (MMP-7) as an active ingredient in a solution
comprising a monovalent cation compound at 130 mM or less or in a
solution not comprising a monovalent cation compound.
27. The (pharmaceutical) composition comprising MMP-7 according to
claim 26 wherein the composition comprises MMP-7 as an active
ingredient in a solution comprising a monovalent cation compound at
130 mM or less.
28. The (pharmaceutical) composition comprising MMP-7 according to
claim 26 wherein the composition comprises MMP-7 as an active
ingredient in a solution not comprising a monovalent cation
compound.
29. The (pharmaceutical) composition comprising MMP-7 according to
claim 26 wherein the monovalent cation compound is selected from
the group consisting of sodium chloride, potassium chloride, sodium
sulfate, potassium sulfate, sodium carbonate, potassium carbonate,
sodium phosphate and potassium phosphate.
30. The (pharmaceutical) composition comprising MMP-7 according to
claim 26 wherein the monovalent cation compound is from a
monovalent cation chloride.
31. The (pharmaceutical) composition comprising MMP-7 according to
claim 30 wherein the monovalent cation compound is selected from
the group consisting of sodium chloride and potassium chloride.
32. The (pharmaceutical) composition comprising MMP-7 according to
claim 26 wherein the composition further comprises calcium
chloride.
33. The (pharmaceutical) composition comprising MMP-7 according to
claim 32 wherein the calcium chloride is at 30 mM or less.
34. The (pharmaceutical) composition comprising MMP-7 according to
claim 26 wherein the solution is a buffer solution.
35. The (pharmaceutical) composition comprising MMP-7 according to
claim 34 wherein the buffer solution is 5 to 25 mM Tris buffer.
36. The (pharmaceutical) composition comprising MMP-7 according to
claim 26 wherein the MMP-7 is at 20 mg/ml or less.
37. The (pharmaceutical) composition comprising MMP-7 according to
claim 36 wherein the MMP-7 is at 1 .mu.g/ml to 1 mg/ml.
38. The (pharmaceutical) composition comprising MMP-7 according to
claim 35 wherein the solution is 5 to 25 mM Tris buffer (pH 6 to 8)
comprising 30 to 40 mM sodium chloride and 5 to 30 mM calcium
chloride.
39. The (pharmaceutical) composition comprising MMP-7 according to
claim 26 wherein the solution further comprises sugar alcohols
and/or sugars.
40. The (pharmaceutical) composition comprising MMP-7 according to
claim 39 wherein the sugar alcohols and/or sugars are selected from
the group consisting of sucrose, lactose, maltose, xylose,
trehalose, mannitol, sorbitol, xylitol, maltitol, lactitol, and
oligosaccharide alcohols.
41. The (pharmaceutical) composition comprising MMP-7 according to
claim 39 wherein the sugar alcohols and/or sugars are at 2% or
more.
42. The (pharmaceutical) composition comprising MMP-7 according to
claim 41 wherein the sugar alcohols and/or sugars are at 2 to
7%.
43. The (pharmaceutical) composition comprising MMP-7 according to
claim 40 wherein the sugar alcohols and/or sugars are mannitol or
sucrose.
44. The (pharmaceutical) composition comprising MMP-7 according to
claim 43 wherein the mannitol is at 2 to 5% and the sucrose is at 2
to 7%.
45. A solid (pharmaceutical) composition comprising MMP-7 wherein
the composition can be dissolved in a solvent and the composition
upon dissolution is the composition as set forth in claim 26.
46. (canceled)
Description
TECHNICAL FIELD
[0001] The present invention relates to a method for monomerizing
aggregates of matrix metalloproteinase 7 (hereinafter also referred
to as "MMP-7"). Specifically, the present invention relates to a
method for monomerizing MMP-7 aggregates which comprises treating
MMP-7 aggregates with a solution comprising a low concentration of
a monovalent cation compound or with a solution not comprising said
compound, a process for preparing MMP-7 which involves said method
for monomerizing MMP-7 aggregates, and a (pharmaceutical)
composition comprising MMP-7 in the aforementioned solution where
sugar alcohols or sugars are further dissolved.
BACKGROUND ART
[0002] MMP-7 is one of matrix metalloproteinases (hereinafter also
referred to as "MMP") belonging to a zinc metalloproteinase family
which possesses a zinc molecule at the active site (cf. for
instance, Non-patent reference 1). MMP is produced as a precursor,
its signal sequence is processed upon extracellular secretion, and
then a pro-sequence is processed to yield an active form. MMP
extracellularly secreted controls metabolism of extracellular
matrix. On the other hand, it is reported that MMP-7 is mainly
secreted from cancer cells and is involved in invasion and
metastasis (cf. for instance, Non-patent reference 2). MMP-7 lacks
the hinge region and the hemopexin-like domain common in many of
the other MMPs and consists of the minimum molecular unit as
compared to the other MMPs. A substrate of MMP-7 is components
constituting collagen or extracellular matrix (fibronectin,
vitronectin, laminin, aggrecan).
[0003] MMP-7 is presumed to be involved in spontaneous remission of
nucleus pulposus existing in the (spinal) epidural space viewing
that a substrate of MMP-7 is aggrecan which is a main component of
the cartilage tissue and that macrophages from specimen of
intervertebral disk displacement surgery express MMP-7 (cf. for
instance, Non-patent reference 3). Thereafter, Haro et al.
administered MMP-7 to the intervertebral disk of hernial dog and
observed the decrease in a volume of nucleus pulposus within the
intervertebral disk to thereby show the possibility of MMP-7 as a
medicament for treating intervertebral disk displacement (cf. for
instance, Non-patent reference 4). Development of MMP-7 as a
medicine is desired. However, MMP-7 occurs in the living body only
in a trace amount and thus it is extremely difficult to isolate and
purify MMP-7 from the living body. Besides, when components from
the living body are used, there is a concern in view of safety such
as potential viral infection. Although MMP-7 can be obtained from
cancer cells, it is not preferable to use cancer cells as a source
for production (cf. for instance, Non-patent reference 5).
[0004] For dissolving such problems, an attempt has been made to
obtain MMP-7 by a recombinant DNA technology. There are the report
by Barnett et al. that MMP-7 is expressed in CHO cells (cf. for
instance, Non-patent reference 6), the report that, by using a
nucleic acid fragment generated by linking a nucleotide sequence of
a signal sequence of alkaline phosphatase to a gene sequence of
pro-matrix metalloproteinase 7 (hereinafter also referred to as
"proMMP-7") optimized for codon usage of E. coli, soluble proMMP-7
was expressed at 34.degree. C. and insoluble proMMP-7 was expressed
at 42.degree. C. (cf. for instance, Patent reference 1) and the
report that, by using a nucleic acid fragment generated by linking
a modified signal peptide to a gene fragment of proMMP-7, proMMP-7
is expressed as an inclusion body in a large amount (cf. for
instance, Patent reference 2).
[0005] For conversion of proMMP-7 to active MMP-7, it is reported
that proMMP-7 is heated at 37.degree. C. in the presence of mM
(4-aminophenyl)mercuric acetate (APMA) or 0.2 .mu.M trypsin or a
solution containing proMMP-7 is heated at 53.degree. C. (cf. for
instance, Non-patent reference 7). They revealed that, after a low
concentration (less than 1 mg/ml) of activated MMP-7 (also known as
Matrilysin) was stored at -20.degree. C. for 6 months and at room
temperature for 28 days, there was no change in its activity and
behavior on electrophoresis. Although there is no definite
description about the change, they appear to suggest that no
decomposition of Matrilysin was observed from the results of
electrophoresis. In addition to these, there are various reports on
purification of proMMP-7 and MMP-7 such as Kihira, and Oneda et al.
(cf. for instance, Patent reference 1, Non-patent reference 8) and
thus a method for the purification of MMP-7 has been established to
some extent at an experimental level. In general, a method for the
purification of MMP-7 at an experimental level is scaled up for
large-scale production. However, a method for the manufacture of
MMP-7 in a large amount has never been established. There is little
report on the problems in the manufacture of MMP-7 in a large
amount and solution therefor.
[0006] For a composition comprising MMP-7, that which comprises
tris(hydroxymethyl)aminomethane hydrochloride (Tris hydrochloride),
calcium chloride and sodium chloride is reported (cf. for instance,
Patent references 3 to 5, Non-patent references 7, 9). It is known
that metalloproteinase such as MMP-7, in a state of a solution
composition, is stabilized in the coexistence of calcium chloride
and sodium chloride (cf. for instance, Patent reference 6). In
particular, a medicine is normally required to have an osmotic
pressure of around that of body fluid in view of safety. Sodium
chloride is commonly used as an osmotic pressure regulator of a
liquid composition. As a practical matter, most of the compositions
disclosed in these documents comprise a monovalent cation compound
such as sodium chloride at the concentration isotonic to or more
than that of body fluid. Besides, the compositions disclosed in
these documents do not comprise sugar alcohols or sugars.
PATENT REFERENCES
[0007] Patent reference 1: JP patent 2938352 [0008] Patent
reference 2: WO 2010/047347A1 [0009] Patent reference 3: JP
2000-344672 [0010] Patent reference 4: JP 2000-226329 [0011] Patent
reference 5: JP 2002-173424 [0012] Patent reference 6: JP
2005-6509
NON-PATENT REFERENCES
[0012] [0013] Non-patent reference 1: Soler et al., Biochem Biophys
Res Commun, 1994, vol. 201, p. 917-923 [0014] Non-patent reference
2: Ii et al., Exp Biol Med (Maywood), 2006, vol. 231, p. 20-27
[0015] Non-patent reference 3: Haro et al., J Spinal Disord, 1999,
vol. 13, p. 245-249 [0016] Non-patent reference 4: Haro et al., J
Orthop Res, 2005, vol. 23, p. 412-419 [0017] Non-patent reference
5: Miyazaki et al., Cancer Research, 1990, vol. 50, p. 7758-7764
[0018] Non-patent reference 6: Barnett et al., Protein Exp Purif,
1994, vol. 5, p. 27-36 [0019] Non-patent reference 7: Crabbe et
al., Biochemistry, 1992, vol. 31, p. 8500-8507 [0020] Non-patent
reference 8: Oneda et al., J Biochem, 1999, vol. 126, p. 905-911
[0021] Non-patent reference 9: Browner et al., Biochemistry, 1995,
vol. 34, p. 6602-6610
DISCLOSURE OF THE INVENTION
Technical Problem to be Solved by the Invention
[0022] In the course of developing a medicine comprising MMP-7, the
present inventors have found MMP-7 forms aggregates under the
circumstances where a monovalent cation compound such as sodium
chloride is at 150 mM or more which is isotonic to that of body
fluid, like in case of the conventional medicinal products,
especially a liquid composition of metalloproteinase as described
above and that MMP-7 is adsorbed to gel in an apparatus normally
used for the manufacture of proteins or their preparations or to a
container such as a vial normally used for storage of preparations.
Thus, the problem was to provide a method for monomerizing MMP-7
aggregates where adsorption of MMP-7 to a manufacturing apparatus
is suppressed at the time when MMP-7 is manufactured, a process for
preparing MMP-7 which involves said method for monomerizing MMP-7
aggregates, and a (pharmaceutical) composition comprising MMP-7
where aggregate formation and adsorption of MMP-7 is
suppressed.
Means for Solving the Problems
[0023] The present inventors have earnestly investigated in order
to solve the above problems and as a result have found the
following (1) to (4) to complete the present invention.
(1) MMP-7 aggregates are dissociated to form monomers by the
treatment with a solution such as a Tris buffer (pH 6 to 8)
comprising a low concentration of a monovalent cation chloride
(sodium chloride and potassium chloride). Also, MMP-7 aggregates
likewise form monomers when they are treated with the solution not
comprising a monovalent cation chloride. (2) By incorporating a
method for monomerizing MMP-7 aggregates with the treatment as
mentioned above (hereinafter also simply referred to as "a method
for monomerization") into a manufacturing process of MMP-7,
efficiency in producing MMP-7 can be increased. In particular, by
incorporating the method for monomerization into a process of
treatment with ultrafiltration membrane immediately after
conversion of proMMP-7 into MMP-7 through self-activation, more
significant effects can be obtained. (3) MMP-7 monomers maintain a
high enzymatic activity. (4) By adding sugars or sugar alcohols
such as mannitol and sucrose to the Tris buffer as mentioned above,
not only formation of MMP-7 aggregates is suppressed but also
adsorption of MMP-7 to gel and the wall of a vial is suppressed.
Namely, by preparing a composition comprising a monovalent cation
chloride at a concentration of around 150 mM which is isotonic to
that of body fluid or less when made to an aqueous solution and
sugar alcohols or sugars, the resulting solution becomes a
pharmaceutical preparation where formation of MMP-7 aggregates and
adsorption of MMP-7 are suppressed.
[0024] Accordingly, the present invention encompasses a method for
monomerizing MMP-7 aggregates (method for monomerization), a
process for preparing MMP-7 which involves said method for
monomerization and a (pharmaceutical) composition comprising MMP-7
prepared by using the aforementioned buffer and relates to the
followings:
[1] A method for monomerization of matrix metalloproteinase 7
(MMP-7) aggregates which comprises treating MMP-7 aggregates with a
solution comprising a monovalent cation compound at 130 mM or less
or with a solution not comprising a monovalent cation compound. [2]
The method for monomerization according to [1] wherein the MMP-7
aggregates are treated with a solution comprising a monovalent
cation compound at 130 mM or less. [3] The method for
monomerization according to [1] wherein the MMP-7 aggregates are
treated with a solution not comprising a monovalent cation
compound. [4] The method for monomerization according to [1] or [2]
wherein the monovalent cation compound is at 100 mM or less. [5]
The method for monomerization according to [1], [2] or [4] wherein
the monovalent cation compound is at 80 mM or less. [6] The method
for monomerization according to [1], [2], [4] or [5] wherein the
monovalent cation compound is at 40 mM or less. [7] The method for
monomerization according to [1], [2], [4], [5] or [6] wherein the
monovalent cation compound is selected from the group consisting of
sodium chloride, potassium chloride, sodium sulfate, potassium
sulfate, sodium carbonate, potassium carbonate, sodium phosphate
and potassium phosphate. [8] The method for monomerization
according to [1], [2], [4], [5] or [6] wherein the monovalent
cation compound is from a monovalent cation chloride. [9] The
method for monomerization according to [8] wherein the monovalent
cation compound is selected from the group consisting of sodium
chloride and potassium chloride. [10] The method for monomerization
according to any one of [1] to [9] wherein the solution further
comprises calcium chloride. [11] The method for monomerization
according to [10] wherein the calcium chloride is at 30 mM or less.
[12] The method for monomerization according to any one of [1] to
[11] wherein the solution is a buffer solution. [13] The method for
monomerization according to [12] wherein the buffer solution is 5
to 25 mM Tris buffer. [14] The method for monomerization according
to any one of [1] to [13] wherein the MMP-7 is at 20 mg/ml or less.
[15] The method for monomerization according to [13] or [14]
wherein the solution is 5 to 25 mM Tris buffer (pH 6 to 8)
comprising 30 to 40 mM sodium chloride and 5 to 30 mM calcium
chloride. [16] The method for monomerization according to any one
of [1] to [15] wherein the solution further comprises sugar
alcohols and/or sugars. [17] The method for monomerization
according to [16] wherein the sugar alcohols and/or sugars are
selected from the group consisting of sucrose, lactose, maltose,
xylose, trehalose, mannitol, sorbitol, xylitol, maltitol, lactitol,
and oligosaccharide alcohols. [18] The method for monomerization
according to [16] or [17] wherein the sugar alcohols and/or sugars
are at 2% or more. [19] The method for monomerization according to
[18] wherein the sugar alcohols and/or sugars are at 2 to 7% [20]
The method for monomerization according to any one of [17] to [19]
wherein the sugar alcohols and/or sugars are mannitol or sucrose.
[21] The method for monomerization according to [20] wherein the
mannitol is at 2 to 5% and the sucrose is at 2 to 7%. [22] A
process for preparation of MMP-7 which comprises a step consisting
of the method for monomerization as set forth in any one of [1] to
[21]. [23] The process for preparation according to [22] wherein
the step is carried out after a step of treatment using a solution
comprising a monovalent cation compound at 130 mM or more. [24] The
process for preparation according to [22] or [23] wherein the
process comprises the following steps (1) to (5): (1) a step of
disrupting cells producing proMMP-7 inclusion body; (2) a step of
dissolution/refolding treatment of proMMP-7 inclusion body; (3) a
step of purification of proMMP-7; (4) a step of self-activation of
proMMP-7 into MMP-7; and (5) a step consisting of the method for
monomerization as set forth in any one of [1] to [21]. [25] The
process for preparation according to [24] wherein the step (5) is a
step of concentration using ultrafiltration membrane. [26] A
(pharmaceutical) composition comprising matrix metalloproteinase 7
(MMP-7) as an active ingredient in a solution comprising a
monovalent cation compound at 130 mM or less or in a solution not
comprising a monovalent cation compound. [27] The (pharmaceutical)
composition comprising MMP-7 according to [26] wherein the
composition comprises MMP-7 as an active ingredient in a solution
comprising a monovalent cation compound at 130 mM or less. [28] The
(pharmaceutical) composition comprising MMP-7 according to [26]
wherein the composition comprises MMP-7 as an active ingredient in
a solution not comprising a monovalent cation compound. [29] The
(pharmaceutical) composition comprising MMP-7 according to [26] or
[27] wherein the monovalent cation compound is selected from the
group consisting of sodium chloride, potassium chloride, sodium
sulfate, potassium sulfate, sodium carbonate, potassium carbonate,
sodium phosphate and potassium phosphate. [30] The (pharmaceutical)
composition comprising MMP-7 according to [26] or [27] wherein the
monovalent cation compound is from a monovalent cation chloride.
[31] The (pharmaceutical) composition comprising MMP-7 according to
[30] wherein the monovalent cation compound is selected from the
group consisting of sodium chloride and potassium chloride. [32]
The (pharmaceutical) composition comprising MMP-7 according to any
one of [26] to [31] wherein the composition further comprises
calcium chloride. [33] The (pharmaceutical) composition comprising
MMP-7 according to [32] wherein the calcium chloride is at 30 mM or
less. [34] The (pharmaceutical) composition comprising MMP-7
according to any one of [26] to [33] wherein the solution is a
buffer solution. [35] The (pharmaceutical) composition comprising
MMP-7 according to [34] wherein the buffer solution is 5 to 25 mM
Tris buffer. [36] The (pharmaceutical) composition comprising MMP-7
according to any one of [26] to [35] wherein the MMP-7 is at 20
mg/ml or less. [37] The (pharmaceutical) composition comprising
MMP-7 according to [36] wherein the MMP-7 is at 1 .mu.g/ml to 1
mg/ml. [38] The (pharmaceutical) composition comprising MMP-7
according to [35], [36] or [37] wherein the solution is 5 to 25 mM
Tris buffer (pH 6 to 8) comprising 30 to 40 mM sodium chloride and
5 to 30 mM calcium chloride. [39] The (pharmaceutical) composition
comprising MMP-7 according to any one of [26] to [38] wherein the
solution further comprises sugar alcohols and/or sugars. [40] The
(pharmaceutical) composition comprising MMP-7 according to [39]
wherein the sugar alcohols and/or sugars are selected from the
group consisting of sucrose, lactose, maltose, xylose, trehalose,
mannitol, sorbitol, xylitol, maltitol, lactitol, and
oligosaccharide alcohols. [41] The (pharmaceutical) composition
comprising MMP-7 according to [39] or [40] wherein the sugar
alcohols and/or sugars are at 2% or more. [42] The (pharmaceutical)
composition comprising MMP-7 according to [41] wherein the sugar
alcohols and/or sugars are at 2 to 7%. [43] The (pharmaceutical)
composition comprising MMP-7 according to any one of [40] to [42]
wherein the sugar alcohols and/or sugars are mannitol or sucrose.
[44] The (pharmaceutical) composition comprising MMP-7 according to
[43] wherein the mannitol is at 2 to 5% and the sucrose is at 2 to
7%. [45] A solid (pharmaceutical) composition comprising MMP-7
wherein the composition can be dissolved in a solvent and the
composition upon dissolution is the composition as set forth in any
one of [26] to [44]. [46] A medicament for treating intervertebral
disk displacement which comprises the (pharmaceutical) composition
comprising MMP-7 as set forth in any one of [26] to [45].
Effects of the Invention
[0025] In accordance with the present invention, a method for
monomerization of MMP-7 aggregates with ease is provided. Thus, by
incorporating the method into a manufacturing process of MMP-7,
productivity and recovery efficiency of MMP-7 can be increased.
[0026] Besides, MMP-7 as monomerized by the method has a higher
specific activity of an enzyme as compared to MMP-7 aggregates and
thus can be a suitable material for providing a MMP-7 preparation
of high quality.
[0027] Furthermore, as an embodiment of the present invention, by
storing MMP-7 in a buffer comprising sodium chloride at a low
concentration and sugar alcohols or sugars, MMP-7 not only
maintains its monomeric form but also a high enzymatic activity,
and also adsorption of MMP-7 to a storage container can be
suppressed. Therefore, the buffer used in a method for
monomerization of the present invention is useful as a preservative
solution of a MMP-7 preparation. The MMP-7 composition of the
present invention, which comprises sodium chloride at a low
concentration when made to an aqueous solution and sugar alcohols
or sugars, is useful as a liquid composition for medicinal use and
a composition for preparing the same where the formation of MMP-7
aggregates is suppressed and adsorption of MMP-7 to a container is
suppressed.
BRIEF DESCRIPTION OF DRAWINGS
[0028] FIG. 1 shows the suppressive effects of sodium chloride
(NaCl) and potassium chloride (KCl) to MMP-7 aggregate formation
based on the analysis of molecular weight of MMP-7 measured by
dynamic light scattering.
[0029] FIG. 2 shows the effects of calcium chloride (CaCl.sub.2) on
suppression to MMP-7 aggregate formation based on the analysis of
molecular weight of MMP-7 measured by dynamic light scattering.
[0030] FIG. 3 shows the effects of a concentration of MMP-7 on
MMP-7 aggregate formation based on the analysis of molecular weight
of MMP-7 measured by size exclusion chromatography.
[0031] FIG. 4 shows the effects of sodium chloride (NaCl) on the
enzymatic activity of MMP-7 when MMP-7 at 0.1 mg/ml, 2 mg/ml or 20
mg/ml is diluted with a Tris buffer containing 150 mM NaCl or 40 mM
NaCl.
[0032] FIG. 5 shows the suppressive effects of mannitol to
adsorption of MMP-7 to gel. A: 5 mM Tris buffer (pH 7)/10 mM
CaCl.sub.2/40 mM NaCl; B: 5 mM Tris buffer (pH 7)/10 mM
CaCl.sub.2/40 mM NaCl/3.5% mannitol
[0033] FIG. 6 shows the suppressive effects of mannitol and sucrose
to adsorption of MMP-7 to the wall of a vial.
[0034] FIG. 7 shows the suppressive effects of mannitol and sucrose
at each concentration to adsorption of MMP-7 to the wall of a
vial.
[0035] FIG. 8 shows the effects of mannitol and sucrose at each
concentration on the enzymatic activity of MMP-7.
[0036] FIG. 9 shows the effects of mannitol on suppression to MMP-7
aggregate formation.
BEST MODE FOR CARRYING OUT THE INVENTION
[0037] The present invention provides a method for monomerization
of MMP-7 aggregates, a process for preparing MMP-7 which involves
said method for monomerization and a (pharmaceutical) composition
comprising MMP-7. The present invention is characterized by that
MMP-7 aggregates are treated with a solution comprising a low
concentration of a monovalent cation compound and further
comprising sugar alcohols or sugars (hereinafter also referred to
as "a solution for monomerization"), that MMP-7 is stored in said
solution and that an MMP-7 composition comprises a low
concentration of a monovalent cation compound and sugar alcohols or
sugars when made to an aqueous solution.
[0038] Detection of MMP-7 aggregates is carried out by measuring a
molecular weight of MMP-7 by dynamic light scattering, size
exclusion chromatography, ultra-centrifugation and the like and
comparing the measured weight with the molecular weight of
monomeric MMP-7 (about 19 kDa) calculated from the amino acid
sequence. For the present invention, MMP-7 was determined to be
monomeric in case that the molecular weight of MMP-7 in a sample is
between a range of from 19 kDa to 38 kDa whereas MMP-7 was
determined to form aggregates in case that it is 38 kDa or more.
The above criteria of determination are made by taking into
consideration trueness of measurement by dynamic light scattering
such that MMP-7 evidently formed aggregates when the molecular
weight is more than that of a dimer whereas MMP-7 is monomeric when
it is less than that of a dimer.
[0039] Monomerization of MMP-7 is carried out by treating MMP-7
aggregates with a solution for monomerization. As used herein,
"treating MMP-7 aggregates with a solution for monomerization"
means that MMP-7 aggregates are exposed to a solution for
monomerization and includes dissolution in said solution and
includes that, when MMP-7 aggregates are dissolved in a solution
containing a monovalent cation compound at a high concentration of
130 mM or more, exchange of buffer is conducted with
ultrafiltration membrane or dialysis membrane using a solution for
monomerization. For a buffer to keep the pH of the solution for
monomerization constant, it is possible to use Tris buffer,
phosphate buffer, glycine buffer and carbonic acid buffer, among
which a suitable buffer may be selected depending on the pH at the
monomerization treatment. A concentration of a buffer may be in a
range of 5 to 25 mM, preferably 5 to 20 mM. The pH may be in a
range of 5 to 9, preferably 6 to 8. More preferably, a Tris buffer
is selected that exerts an effect on improvement of stability of
metalloproteinase with the Tris buffer having a concentration of 5
to 10 mM and pH of 6 to 8 (cf. JP 2011-521906).
[0040] As used herein, "a monovalent cation compound" means a
compound consisting of a monovalent cation and a counterpart anion,
including sodium chloride, potassium chloride, sodium sulfate,
potassium sulfate, sodium carbonate, potassium carbonate, sodium
phosphate, potassium phosphate and the like. Also, as used herein,
"a monovalent cation chloride" means a compound consisting of a
monovalent cation and a chloride ion. For a monovalent cation
compound as used herein, any monovalent cation compound may be used
but preferably a monovalent cation chloride is used. Specifically,
sodium chloride and potassium chloride, preferably sodium chloride
is used.
[0041] For dissociating MMP-7 aggregates into monomeric MMP-7, a
low concentration of a monovalent cation compound is used. MMP-7
dissociated into monomers is maintained as monomers in a solution
for monomerization comprising a low concentration of a monovalent
cation compound. The same effect can be obtained by using a
solution for monomerization or water not comprising a monovalent
cation compound. Therefore, the present invention encompasses a
method for monomerization of MMP-7 aggregates using a solution for
monomerization, which solution includes liquid such as water in
accordance with the present invention, not comprising a monovalent
cation compound. Hereinafter, a solution comprising a low
concentration of a monovalent cation compound and the solution not
comprising a monovalent cation compound are also collectively
referred to as merely "a solution for monomerization".
[0042] By adding a polyvalent cation compound to a solution for
monomerization, a range of a concentration of a monovalent cation
compound to be treated can be broadened. A polyvalent cation
compound is preferably a divalent cation compound, more preferably
a divalent calcium ion compound, and most preferably calcium
chloride. Like a monovalent cation compound, "a polyvalent cation
compound" means a compound consisting of a polyvalent cation and a
counterpart anion.
[0043] Specifically, in case that water is used as a solution for
monomerization, a monovalent cation compound at 40 mM or less is
used and, by adding calcium chloride to a solution for
monomerization, treatment with a solution for monomerization
comprising a monovalent cation compound at more than 40 mM becomes
possible. More specifically, a solution for monomerization
comprising a monovalent cation compound at 80 mM or less when it
comprises 5 mM to 10 mM calcium chloride, the solution comprising a
monovalent cation compound at 100 mM or less when it comprises 10
mM to 30 mM calcium chloride, and the solution comprising a
monovalent cation compound at 130 mM or less when it comprises 30
mM calcium chloride, can be used. This result demonstrates that, by
the addition of calcium chloride, the effect of dissociation of
MMP-7 aggregates into monomers and the suppressive effect to MMP-7
aggregate formation were enhanced. Besides, it can be seen from the
results of Example 2 (FIG. 2) that this effect is proportional to a
concentration of calcium chloride. Namely, by using a higher
concentration of calcium chloride, higher effects of monomerization
of MMP-7 and maintenance of MMP-7 monomers can be expected. From
the above, a concentration of a monovalent cation compound is 130
mM or less, preferably 100 mM or less, more preferably 80 mM or
less, and most preferably 40 mM or less, in case of an aqueous
solution. Although a solution for monomerization may not comprise a
monovalent cation compound, the solution comprising a monovalent
cation compound is preferable in view of quality of a
pharmaceutical composition.
[0044] In accordance with the present invention, a solution for
monomerization consisting of 5 to 25 mM Tris buffer (pH 6 to 8)
comprising 30 to 40 mM sodium chloride and 5 to 30 mM calcium
chloride, or the solution for monomerization without sodium
chloride, may preferably be used.
[0045] A method for monomerization of MMP-7 aggregates of the
present invention can be incorporated into a manufacturing process
of MMP-7 so as to efficiently produce MMP-7 monomers of high
quality. A general process for producing MMP-7 by a recombinant DNA
technology is carried out by the steps of culture of cells, release
of inclusion bodies, dissolution and refolding of inclusion bodies,
purification of proMMP-7, conversion of proMMP-7 into MMP-7 by
self-activation, purification and concentration of MMP-7.
[0046] More specifically, a method for monomerization of MMP-7
aggregates of the present invention is used in a manufacturing
process of MMP-7 as described below. Firstly, E. coli producing
proMMP-7 is prepared. In general, it can be prepared by introducing
proMMP-7 gene inserted in an expression vector into E. coli by the
ordinary procedure. However, since proMMP-7 has a potent toxicity
against E. coil, productivity could be decreased drastically. In
order to solve this problem, addition of a signal peptide at the
N-terminus of proMMP-7 is devised. However, in case of proMMP-7,
phenomena are observed that an expression level of proMMP-7 is not
increased merely by adding a signal peptide and a portion of
proMMP-7 expressed is decomposed. Such phenomena can be an obstacle
to establishment of a process for efficient production of MMP-7.
Therefore, as a starting material for production of MMP-7, it is
preferable to use E. coli producing proMMP-7 which has an increased
expression level of proMMP-7 and increased suppression of proMMP-7
decomposition by proteases. Such E. coli producing proMMP-7 can be
prepared by the method described in WO2010/047347 (more
specifically, cf. Preparation therein).
[0047] The thus obtained E. coli producing proMMP-7, after
purification by repeating cloning, is preserved as a working cell
bank and used as a seed for a large-scale culture for production of
MMP-7 preparation. A working cell bank may be preserved under
conditions normally used for preservation of recombinant E. coli,
e.g. in a solution containing 7 to 10% dimethyl sulfoxide or 10 to
50% glycerol in a freezer at -80.degree. to -20.degree. C. or in
liquid nitrogen or lyophilized in an ample to be preserved in a
refrigerator at 2 to 10.degree. C.
[0048] Cultivation of E. coli producing proMMP-7 on a production
scale is carried out in two stages, i.e. preculture on a small
scale and main culture on a large scale. For preculture, LB medium
commonly used for recombinant E. coli, optionally supplemented with
antibiotics such as ampicillin for retention of plasmid, may be
used for propagation of E. coli producing proMMP-7. However, for
main culture, a culture medium where substance causative of side
effects is removed as much as possible is preferably used. Such
culture medium includes, for instance, a glucose medium containing
various trace metals such as magnesium, calcium, copper and sodium,
LB medium, M9 medium and the like. Culture conditions may be those
suited for propagation of E. coli, for instance, culture conditions
of pH (pH 6 to 8), temperature (30.degree. to 45.degree. C.) and
time (4 to 16 hours). These culture conditions may suitably be
adjusted depending on a culture scale and treatment for induction
of expression. An expression inducer is used for efficient
expression of proMMP-7 and includes
isopropyl-.beta.-thiogalactopyranoside (IPTG), lactose and the
like.
[0049] For recovery of MMP-7 from E. coli producing proMMP-7
cultured and propagated in a large amount after main culture, the
procedures described below are carried out. Firstly, E. coli
producing proMMP-7 is cultured and the propagated cells are
disrupted by a suitable measure so as to release inclusion bodies
consisting of proMMP-7 out of the cells. For disruption of cells,
lysis with chemical substance (e.g. EDTA as a chelating agent),
surfactant (e.g. Triton X100) and enzyme (e.g. lysozyme) or
physical treatment with French press, sonication and the like may
be used. By combining several of these procedures, cells can be
disrupted more efficiently. A solution obtained after disruption of
cells including inclusion bodies is subject to fractionation with
ultrafiltration membrane or centrifuge for repetition of
concentration and washing to remove most of cell components. For
washing, a buffer commonly used such as Tris buffer, phosphate
buffer, glycine buffer, carbonic acid buffer may be used. For a
pore size of ultrafiltration membrane and the conditions of
centrifuge, there are many reports which may be referred to. In
case of treatment in a large amount, inclusion bodies are
preferably recovered by fractionation with ultrafiltration
membrane.
[0050] The recovered inclusion bodies are temporarily dissolved in
a solution containing a reducing agent and a denaturing agent. For
such reducing agent, cysteine, glutathione, dithiothreitol,
2-mercaptoethanol and the like may be used. Several of these may be
used in combination. A concentration of a reducing agent may depend
on an amount of inclusion bodies to be dissolved and may be in a
range of 10 to 200 mM. For a denaturing agent, urea, guanidine
hydrochloride and the like may be used. Urea and guanidine
hydrochloride may be used at a concentration of 4 to 8M and 2 to
6M, respectively. For a buffer, one that is used for recovery of
inclusion bodies such as, for instance, phosphate buffer and Tris
buffer (pH 7.0 to 9) may be used. Temperature while dissolution is
not particularly limited provided that it is 40.degree. C. or
lower. Dissolution time may be set seeing the conditions of
dissolution of inclusion bodies. Normally, the solution is stirred
for 30 minutes to 1 hour.
[0051] Next, refolding of proMMP-7, i.e. construction of normal
steric structure, is performed by adding a refolding buffer
containing a surfactant and metal ions to the solution of inclusion
bodies. Brij 35 as surfactant and zinc acetate or cobalt chloride
as metal ions are used at a concentration of 0.5 to 2% and 0.05 mM
to 0.2 mM, respectively. A kind and a concentration of a buffer
used for refolding may be the same as those used when inclusion
bodies are dissolved. Refolding treatment is carried out by having
the solution left to stand for a day or more.
[0052] For purification of proMMF-7 from the refolding solution,
purification procedures commonly used in protein chemistry such as
centrifuge, salting-out, ultrafiltration, isoelectric
precipitation, electrophoresis, ion exchange chromatography, gel
filtration chromatography, affinity chromatography, hydrophobic
chromatography, hydroxyapatite chromatography and the like may be
used in combination. proMMP-7 in the present invention can be
purified by the steps consisting of ion exchange chromatography,
hydrophobic chromatography and treatment with ultrafiltration
membrane. Both chromatographies may be done in an ordinary manner.
An amount of the obtained proteins and polypeptides may be measured
with a reagent for measuring protein such as BCA Protein Assay
Reagent Kit (Pierce Biotechnology, Inc) and Protein Assay Kit
(BIO-RAD, Inc).
[0053] Next, conversion of proMMP-7 into MMP-7 is carried out. A
method for conversion includes heating a solution containing
proMMP-7 at 37.degree. C. in the presence of 1 mM
(4-aminophenyl)mercuric acetate (APMA) or 0.2 .mu.M trypsin or
heating a solution containing proMMP-7 at 53.degree. C. (Crabbe et
al., Biochemistry, 1992, vol. 31, 8500-8507), any of which method
may be used. At this time, 30 to 200 mM sodium chloride may
optionally be added (Crabbe et al., Biochemistry, 1992, vol. 31,
8500-8507, WO 2010/047347 A1). Heating time is in a range of 1 to 5
hours and is suitably adjusted depending on a concentration and an
amount to be treated of a reagent and proMMP-7. For trypsin, one
treated with N-tosyl-L-phenylalanine chloromethyl ketone (TPCK) may
be used.
[0054] For measurement of an enzymatic activity of MMP-7, cleavage
of a fluorescent substrate
(Dnp-Pro-Leu-Gly-Leu-Trp-Ala-D-Arg-NH.sub.2; SEQ ID NO: 7) by MMP-7
may be measured with a fluorescence measurement apparatus (Crabbe
et al., Biochemistry, 1992, vol. 31, 8500-8507). Practically, Kit
for measuring MMP-7 activity (ANASPEC) based on the above principle
is commercially available. Thus, the activity may be measured using
this kit in accordance with protocol attached thereto. For
isolation and purification of MMP-7 converted from proMMP-7, the
protein purification procedures as described above may be used.
[0055] MMP-7 as converted forms aggregates in a solution containing
a high concentration of a salt. This more likely occurs in case
that a concentration of MMP-7 in a solution is 1 mg/ml or more. The
presence of MMP-7 aggregates leads to the decrease in productivity
and quality when MMP-7 is manufactured and is made to MMP-7
preparation. As described above, MMP-7 aggregates are monomerized
and MMP-7 monomers are maintained in a solution for monomerization
containing a monovalent cation compound at 130 mM or less in the
presence of calcium chloride. However, in the solution containing a
monovalent cation compound at a concentration of more than 130 mM,
it is suggested that the possibility to form MMP-7 aggregates is
high. Therefore, a method for monomerization of the present
invention is incorporated after treatment with a monovalent cation
compound at 130 mM or more. More specifically, it is incorporated
immediately after conversion of proMMP-7 into MMP-7 by
self-activation.
[0056] The treatment by a method for monomerization of the present
invention is preferably done to MMP-7 at 20 mg/ml or less. When
MMP-7 at 20 mg/ml or more is treated, however, as described above,
the same suppressive effect to MMP-7 aggregate formation can be
expected by adjusting a concentration of calcium chloride.
[0057] A solution containing the thus prepared MMP-7 monomers,
after the steps of purification and concentration of MMP-7 by
ultrafiltration membrane as necessary, is used as a starting
material for producing MMP-7 preparation. In the steps as well, a
solution for monomerization of the present invention may be used.
Once MMP-7 is monomerized, MMP-7 monomers are maintained as far as
they are present in a solution for monomerization and the decrease
of the enzymatic activity is not observed for a long period of
time. Thus, a solution for monomerization of the present invention
may be used for preservation of MMP-7 before production of MMP-7
preparation and for the manufacture of a (pharmaceutical)
composition comprising MMP-7 as an active ingredient to thereby
allow for maintenance of MMP-7 of high quality.
[0058] In a method for monomerization of MMP-7 aggregates and a
process for preparing MMP-7 which involves said method for
monomerization of the present invention, sugar alcohols and sugars
exert the suppressive effect to adsorption of MMP-7 to gel widely
used in the manufacture of MMP-7 and the suppressive effect to
adsorption of MMP-7 to the wall of a vial used for the production
of MMP-7 preparation. Such sugar alcohols and sugars include
sucrose, lactose, maltose, xylose, trehalose, mannitol, sorbitol,
xylitol, maltitol, lactitol, oligosaccharide alcohols, and the
like, preferably mannitol and sucrose, and most preferably
mannitol.
[0059] In case that a low concentration (e.g. 1 .mu.g/ml to 1
mg/ml) of MMP-7 is made to MMP-7 preparation, the loss of MMP-7 due
to its adsorption to the wall of a vial is envisaged and thus it is
particularly effective that a solution for monomerization contains
sugar alcohols and sugars as described above. In case of MMP-7,
sugar alcohols or sugars are added at 2% or more where the
suppressive effect to adsorption to the wall of a vial is exerted
or at 2 to 7% where an osmotic pressure in the body can be
adjusted. In accordance with the present invention, a solution for
monomerization is used which contains 2 to 5% mannitol or 2 to 7%
sucrose where the activity of MMP-7 can be maintained. More
preferably, a solution for monomerization is used which consists of
5 to 25 mM Tris buffer (pH 6 to 8) containing 2 to 5% mannitol, 30
to 40 mM sodium chloride and 5 to 30 mM calcium chloride.
[0060] MMP-7 dissolved in said solution for monomerization can be
used as it is as a (pharmaceutical) composition comprising MMP-7 of
the present invention. A (pharmaceutical) composition comprising
MMP-7 preferably comprises 2% or more sugar alcohols and/or sugars
and a monovalent cation compound (sodium chloride or calcium
chloride) at 30 to 40 mM in view of suppression to MMP-7 aggregate
formation, maintenance of the activity of MMP-7, suppression to
adsorption of MMP-7 to a container, and ensuring quality as an
aqueous pharmaceutical composition. More specifically, in case that
a concentration of MMP-7 is 20 mg/ml or less, a (pharmaceutical)
composition comprising MMP-7 of the present invention preferably
comprises 2 to 5% mannitol or 2 to 7% sucrose, 30 to 40 mM sodium
chloride and 5 to 30 mM calcium chloride. A (pharmaceutical)
composition comprising MMP-7 of the present invention can be stored
in liquid, lyophilized or frozen form. In doing so, in order to
maintain safety and isotonicity as a medicine, a (pharmaceutical)
composition comprising MMP-7 is further added with a compound that
is admitted for administration to humans and other animals such as
a stabilizing agent, isotonizing agent and a preservative. A
(pharmaceutical) composition comprising MMP-7 of the present
invention may encompass a solid composition which can be dissolved
in a solvent and the composition upon dissolution is the liquid
(pharmaceutical) composition comprising MMP-7 of the present
invention as described above. A solid composition is obtained by
removing a solvent from a liquid (pharmaceutical) composition
comprising MMP-7 of the present invention by lyophilization. A
solvent is one defined as a solvent in Dictionary of Pharmaceutical
Excipients and includes water, ethanol and the like
[0061] The thus obtained (pharmaceutical) composition comprising
MMP-7 of the present invention has a specific enzymatic activity of
MMP-7 with suppression of MMP-7 aggregate formation and suppression
of adsorption and can be used as a medicament for treating or
diagnosing intervertebral disk displacement.
[0062] The present invention is further explained in more detail by
means of the following Examples but is not construed to be limited
thereto.
PREPARATION EXAMPLE
[0063] (1) Construction of proMMP-7 Expression Vector (pETMMP7)
with APSP
[0064] Using primers P1 (SEQ ID NO: 1) and P2 (SEQ ID NO: 2),
proMMP-7 gene in kidney cDNA library (HumanMTC Panel I, Catalog #:
K1420-1 BD) was amplified by PCR. The amplified DNAs were inserted
into a cloning vector (pCRII-TOPO, Invitrogen) and the nucleotide
sequences of the obtained DNAs were determined. The determination
of the nucleotide sequences was carried out with a DNA sequencer.
Homology search between the determined nucleotide sequences and the
nucleotide sequence of proMMP-7 registered in database (Accession
Numbers: NM002423) was carried out to give a plasmid (pCRproMMP-7)
where proMMP-7 gene was inserted.
[0065] Next, using pCRproMMP-7 as a template and primer P3 (SEQ ID
NO: 3), which consists of a restriction enzyme NdeI recognition
sequence, a nucleotide sequence coding for PhoA-alkaline
phosphatase signal peptide (APSP) sequence and the N-terminal
sequence of proMMP-7, and primer P4 (SEQ ID NO: 4), which consists
of a restriction enzyme BamHI recognition sequence and the
C-terminal sequence of proMMP-7, PCR was performed. The DNAs
amplified in the same manner as above were inserted into a cloning
vector and the nucleotide sequences of the obtained DNAs were
determined. After confirming that no change in the nucleotide
sequence occurred, the obtained plasmid was cleaved with
restriction enzymes NdeI and BamHI and the obtained fragment was
inserted into an expression vector pET22b (Merck, product cord;
69744-3) previously cleaved with the same restriction enzymes to
give a plasmid (pETMMP7) where proMMP-7 gene was inserted.
(2) Construction of Expression Vector pETMMP7 (L13P-A21E) with
Modified APSP and Expression
[0066] Using GeneTailor Site-Directed Mutagenesis System
(Invitrogen) in accordance with protocol attached thereto, mutation
was introduced into a signal peptidase recognition sequence of APSE
sequence
(Met-Lys-Gln-Ser-Thr-Ile-Ala-Leu-Ala-Leu-Leu-Pro-Leu-Leu-Phe-Thr-Pro-Val--
Thr-Lys-Ala; SEQ ID NO: 8) in pETMMP7 obtained in (1) above
[leucine (Leu) at position 13 of the amino acid sequence of APSE
was replaced with proline (Pro) and alanine (Ala) at position 21
was replaced with glutamic acid (Glu)]. For modification of APSE,
the sequences of M2 (SEQ ID NO: 5) as a 5' primer and P6 (SEQ ID
NO: 6) as a 3' primer were used. E. coli was transformed with the
obtained pETMMP7 (L13P-A21E) with modified APSP to give a
recombinant E. coli (MMP7L13P-A21E) expressing proMMP-7.
[0067] Induction of expression was carried out with Overnight
Express Autoinduction System 1 (Merck; product cord 71300-3) in
accordance with protocol attached thereto. In brief, each colony
was suspended in 50 mL LB medium containing 50 .mu.g/mL Ampicillin
(Wako Pure Chemical Industries, Ltd.) in 125 mL Erlenmeyer flask,
the reagents of Kit were added and the flask was incubated at
37.degree. C. for 16 hours. OD 600 nm of the cell suspension was
measured and the cells corresponding to OD 600 nm=20, 1 mL were
collected in precipitates by centrifuge. The precipitates were
disrupted with 200 .mu.L. BugBuster and centrifuged to give
precipitates. The precipitates were solubilized with Sample Buffer
for SDS-polyacrylamide gel electrophoresis (SDS-PAGE), subjected to
15% acrylamide gel SDS-PAGE and CBB staining was done. As a result,
the increase in an expression level of proMMP-7 and the enhancement
of the suppressive effects to decomposition were confirmed.
[0068] In this Preparation Example, the primers with the following
sequences were used.
P1: ccataggtcc aagaacaatt gtctctg (SEQ ID NO: 1) P2: caatccaatg
aatgaatgaa tggatg (SEQ ID NO: 2) P3: catatgaaac aaagcactat
tgcactggca ctcttaccgt tactgtttac ccctgtgacc aaggccctgc cgctgcctca g
(SEQ ID NO: 3) P4: ggatccctat ttctttcttg aattac (SEQ ID NO: 4) M2:
ctgtttaccc ctgtgaccaa ggaactgccg ctgcc (SEQ ID NO: 5) P6:
cttggtcaca ggggtaaaca gtggcggtaa gag (SEQ ID NO: 6)
Example 1
[0069] Suppressive effect to MMP-7 aggregate formation by
monovalent cation chloride
(1) Manufacture of MMP-7
[0070] E. coli (MMP7L13P-A21E) expressing proMMP-7 obtained by the
procedures described in Preparation Example was cultured and
propagated as a seed in a glucose medium and induction of proMMP-7
expression was performed with
isopropyl-.beta.-thiogalactopyranoside (IPTG). The cells were
recovered from the culture solution and disrupted with French
press. The solution obtained after disruption of cells was
centrifuged and inclusion bodies were recovered in precipitates.
Next, the inclusion bodies were dissolved in 6M guanidine
hydrochloride containing 0.1 M Tris-HCl (pH 7.5) and 0.1 M
dithiothreitol and refolded with 50 mM HEPES buffer (pH 7.5)
containing 0.1 mM zinc acetate, 10 mM calcium chloride, 0.2 M
sodium chloride and 1.0% Brij 35. Thereafter, proMMP-7 was purified
by ion exchange chromatography and hydrophobic chromatography in an
ordinary manner. The obtained proMMP-7 was heated at 47.degree. to
48.degree. C. for self-activation to give MMP-7. The obtained MMP-7
was subject to repetition of dilution and concentration with
ultrafiltration membrane using 5 mM Tris buffer (pH 7) containing
40 mM NaCl, 10 mM CaCl.sub.2 and 3.5% mannitol and stored at
-80.degree. C.
(2) Measurement of MMP-7 Aggregates
[0071] The solution containing a high concentration of MMP-7
obtained in (1) above was diluted with an aqueous solution
containing 5 mM calcium chloride (CaCl.sub.2) in which sodium
chloride (NaCl) or potassium chloride (K01) was dissolved to
prepare Sample 1 and Sample 2.
Sample 1: 1 mg/ml MMP-7/5 mM CaCl.sub.2/each concentration of NaCl
(10 to 160 mM) Sample 2: 1 mg/ml MMP-7/5 mM CaCl.sub.2/each
concentration of KCl (10 to 160 mM)
[0072] Using each 100 .mu.l of the above samples, the effect of
sodium chloride and potassium chloride on the formation of MMP-7
aggregates was investigated by dynamic light scattering (device;
Wyatt Technology DynaPro (Protein Solutions) Titan, cell; Wyatt
Technology 12 uL Cell 8.5 mm Centre Height, temperature; 20.degree.
C.). A molecular weight of MMP-7 in each sample was measured and
analyzed. MMP-7 with a molecular weight of 38 kDa or less was
determined to be monomeric based on the molecular weight of MMP-7
monomers (about 19 kDa). As a result, a molecular weight of MMP-7
in 10 mM to 80 mM NaCl solution was 20 to 29 kDa to reveal that
MMP-7 was present as a monomer. Likewise, when potassium chloride
was used, the results were obtained that MMP-7 was present as a
monomer in 10 mM to 80 mM KCl solution (FIG. 1). The dotted line in
the figure shows a molecular weight of 38 kDa.
Example 2
Effect of Calcium Chloride on Suppression to MMP-7 Aggregate
Formation
[0073] The MMP-7 solution obtained in Example 1-(1) was diluted
with an aqueous solution in which calcium chloride and sodium
chloride were dissolved to prepare Sample 3.
Sample 3: 1 mg/ml MMP-7/each concentration of CaCl.sub.2 (0 to 30
mM)/each concentration of NaCl (0 to 160 mM)
[0074] The effect of the presence of each concentration of calcium
chloride on suppression to MMP-7 aggregate formation was
investigated as described in Example 1-(2).
[0075] As a result, it was shown that MMP-7 monomer was formed with
40 mM or less NaCl in case of 0 mM CaCl.sub.2, with 80 mM or less
NaCl in case of 5 mM CaCl.sub.2, with 100 mM or less NaCl in case
of 10 mM CaCl.sub.2, with 120 mM or less NaCl in case of 20 mM
CaCl.sub.2, and with 130 mM or less NaCl in case of 30 mM
CaCl.sub.2. Besides, also in case of dilution with water alone, the
formation of MMP-7 monomer was observed. Thus, it was revealed that
MMP-7 aggregates were formed in the presence of 130 mM or more NaCl
but the coexistence of 30 mM or less (up to 30 mM) CaCl.sub.2
suppressed the formation of MMP-7 monomers. Namely, this
concentration of calcium chloride has the effect of maintaining and
stabilizing MMP-7 monomers more effectively (FIG. 2).
Example 3
Effect of Concentration of MMP-7 on MMP-7 Aggregate Formation
[0076] The MMP-7 solution obtained in Example 1-(1) was diluted
with a solution containing 10 mM CaCl.sub.2 and each concentration
of sodium chloride to prepare Sample 4 containing each
concentration of MMP-7.
Sample 4: each concentration of MMP-7 (10, 15, 20 mg/ml)/10 mM
CaCl.sub.2/each concentration of NaCl (50 to 250 mM)
[0077] The effect of a concentration of MMP-7 on the suppressive
effect to MMP-7 aggregate formation was investigated by size
exclusion chromatography (HPLC device; HEWLETT PACKED 1100 series,
carrier; TOYOPARL HW50S, temperature; 25.degree. C., flow rate 0.5
mL/min, wavelength 280 nm). A column size was 5 mm diameter and 150
mm length and equilibration of column was conducted with a solution
containing 5 mM Tis-HCl (pH 7), 10 mM CaCl.sub.2, 3.5% mannitol and
40 to 500 mM NaCl.
[0078] Under these conditions, proteins with a molecular weight 60
kDa to 80 kDa appear in void volume (1.8 minutes after initiation
of chromatography). Thus, a peak in the vicinity of 2 minutes after
initiation of chromatography (or leading peak) was considered to be
MMP-7 aggregates and, using an area of the peak as an index, a
concentration of sodium chloride with which MMP-7 aggregates were
formed were calculated.
[0079] As a result, it was found that, although the suppressive
effect of sodium chloride to MMP-7 aggregate formation got weakened
depending on a concentration of MMP-7, MMP-7 was present as
monomers with 0 to 80 mM NaCl in case of MMP-7 at 20 mg/ml or less
(FIG. 3). From the results of Example 2 (FIG. 2), it is evident
that a concentration of sodium chloride with which MMP-7 monomers
are maintained increases proportionally with the increase of a
concentration of calcium chloride. In the present Example, if 30 mM
CaCl.sub.2 is used, it is assumed that MMP-7 at 20 mg/ml exists as
monomers even in the presence of 100 mM NaCl. Besides, when MMP-7
at more than 20 mg/ml is used, it is assumed that MMP-7 can be
maintained as monomers by increasing a concentration of calcium
chloride.
[0080] The same experiment as above was conducted after Sample 4
was left to stand at 4.degree. C. overnight to give the same
results as in FIG. 3.
Example 4
Effect of MMP-7 Aggregate Formation on Enzymatic Activity
[0081] The MMP-7 solution obtained in Example 1-(1) was diluted
with 5 mM Tris buffer (pH 7) containing 40 mM NaCl and 10 mM
CaCl.sub.2 to prepare Sample 5 containing each concentration of
MMP-7.
Sample 5: each concentration of MMP-7 (0.1, 2, 20 mg/ml)/10 mM
CaCl.sub.2/40 mM NaCl/5 mM Tris buffer (pH7)
[0082] The solutions containing each concentration of MMP-7 were
diluted to 0.1 mg/ml with 50 mM Tris buffer (pH 7) containing 150
mM NaCl and 10 mM CaCl.sub.2 or 10 mM Tris buffer (pH 7) containing
40 mM NaCl and 10 mM CaCl.sub.2 (primary dilution) and further
diluted to 5 ng/ml with 50 mM Tris buffer (pH 7) containing 150 mM
NaCl and 10 mM CaCl.sub.2 (secondary dilution). For the resulting
dilutions, a cleavage activity to a fluorescent substrate
(Dnp-Pro-Leu-Gly-Leu-Trp-Ala-D-Arg-NH.sub.2; SEQ ID NO: 7) was
measured using Kit for measuring MMP-7 activity (ANASPEC) in
accordance with protocol attached thereto.
[0083] As a result, the enzymatic activity was decreased for MMP-7
at 2 mg/ml or more diluted with Tris buffer (pH7) containing 150 mM
NaCl in the primary dilution (FIG. 4). This decrease in the
enzymatic activity is consistent with MMP-7 aggregate formation. On
the other hand, at a concentration of 0.1 mg/ml or less, the
decrease in the enzymatic activity was not observed, suggesting
that aggregate formation by 150 mM NaCl did not occur at this
concentration of MMP-7.
Example 5
Effect of Concentration of Sodium Chloride on Step of Concentration
of MMP-7 by Ultrafiltration Membrane
[0084] The MMP-7 solution obtained in Example 1-(1) was diluted
with 5 mM Tris buffer (pH 7) containing 10 mM CaCl.sub.2 to prepare
Sample 6 containing each concentration of sodium chloride.
Sample 6: 4 mg/ml MMP-7/10 mM CaCl.sub.2/each concentration of NaCl
(40 mM, 80 mM, 200 mM, 500 mM)/5 mM Tris buffer (pH 7)
[0085] Each 4 ml of Sample 6 was concentrated with centrifuge (2500
g) using ultrafiltration membrane (Amicon Ultra-4 10K) and a volume
of filtrate and absorbance of the concentrate at a constant time
interval.
[0086] As a result, MMP-7 could be concentrated in a shorter time
when a lower concentration of sodium chloride was used whereas it
took a longer time for concentration when a higher concentration of
sodium chloride was used (Table 1). In Table 1, the symbol (-)
denotes the termination of concentration.
TABLE-US-00001 TABLE 1 Permeation rate of centrifuge filtrate (%:
filtrate/total amount) Time of centrifuge (min.) 10 15 18 23 33 50
64 40 mM NaCl (%) 87.5 95 -- -- -- -- -- 80 mM NaC1 (%) 75 87.5
87.5 95 -- -- -- 0.2M NaCl (%) 40 55 65 72.5 82.5 92.5 -- 0.5M NaCl
(%) 32.5 45 50 55 65 75 82.5
Example 6
Suppressive Effect of Mannitol on Adsorption of MMP-7
(1) Suppression to Adsorption of MMP-7 to Gel
[0087] The MMP-7 solution obtained in Example 1-(1) was diluted
with 5 mM Tris buffer (pH 7) containing 10 mM CaCl.sub.2 and 40 mM
NaCl to prepare Sample 7.
Sample 7: 5.5 mg/ml MMP-7/10 mM CaCl.sub.2/40 mM NaCl
[0088] Sample 7 (1 ml) was applied to a column (HW40F, 26.times.6
cm, Tosoh Corporation) previously equilibrated with 5 mM Tris
buffer (pH 7) containing 10 mM CaCl.sub.2 and 40 mM NaCl, and after
washing with the Tris buffer, eluted with the Tris buffer
containing 3.5% mannitol (flow rate: 5 ml/min).
[0089] As a result, MMP-7 adsorbed to gel was eluted by mannitol
(FIG. 5). This result suggests that mannitol has a suppressive
effect to adsorption of MMP-7 to gel.
(2) Suppression to Adsorption of MMP-7 to Wall of Container
[0090] The MMP-7 solution obtained in Example 1-(1) was diluted
with 5 mM Tris buffer (pH 7) containing 10 mM CaCl.sub.2, 40 mM
NaCl and mannitol or sucrose to prepare solutions (1 ml) of Samples
8 to 10 in vials. The concentrations were adjusted so that each
Sample has the same osmotic pressure. A control was the MMP-7
solution diluted with the Tris buffer not containing mannitol and
sucrose (Sample 10).
Sample 8: 50 .mu.g/ml MMP-7/10 mM CaCl.sub.2/40 mM NaCl/5 mM Tris
buffer (pH 7)/3.5% mannitol Sample 9: 50 .mu.g/ml MMP-7/10 mM
CaCl.sub.2/40 mM NaCl/5 mM Tris buffer (pH 7)/6.6% sucrose Sample
10: 50 .mu.g/ml MMP-7/10 mM CaCl.sub.2/40 mM NaCl/5 mM Tris buffer
(pH 7)
[0091] After left to stand at room temperature for 3 hours, each
solution in vials was diluted in two steps. For primary dilution, 5
mM Tris buffer (pH 7) containing 10 mM CaCl.sub.2 and 40 mM NaCl
was used. For secondary dilution, 1% Block Ace (Block Ace powder:
DS Pharma Biomedical)/TBS-T (0.05% Tween20/50 mM Tris/150 mM NaCl)
was used. MMP-7 in the solution was quantitated by ELISA. For
ELISA, rabbit anti-MMP-7 antibody obtained by immunizing rabbit
with MMP-7, biotin-labeled rabbit anti-MMP-7 antibody wherein the
rabbit anti-MMP-7 antibody was labeled with biotin labelling
reagent (Biotin (Long Arm) NHS-Water Soluble: Vector Laboratories),
Horseradish Peroxidase (HRP)-labeled Streptavidin (Horseradish
Peroxidase Streptavidin Concentrate: Vector Laboratories) and HRP
substrate solution (Peroxidase Substrate Solution B: KPL). As a
control for ELISA reaction, MMP-7 standard for ELISA with a fixed
concentration of MMP-7 was used. A concentration of MMP-7 was
calculated by measuring absorption.
[0092] As a result, as compared to no addition (Sample 10), a
higher rate of recovery of MMP-7 was shown when mannitol (Sample 8)
or sucrose (Sample 9) was added to confirm the suppressive effect
thereof (FIG. 6).
[0093] Next, an effective concentration of mannitol or sucrose for
suppression to absorption of MMP-7 to the wall of a container and
effect of mannitol or sucrose on the enzymatic activity of MMP-7
was investigated. The MMP-7 solution obtained in Example 1-(1) was
diluted with 5 mM Tris buffer (pH 7) containing 10 mM CaCl.sub.2,
40 mM NaCl and mannitol or sucrose to prepare solutions (1 ml) of
Samples 12 to 16 in vials. A control was the MMP-7 solution diluted
with the Tris buffer not containing mannitol and sucrose (Sample
11).
Sample 11: 50 .mu.g/ml MMP-7/10 mM CaCl.sub.2/40 mM NaCl/5 mM Tris
buffer (pH 7)
[0094] Sample 12: 50 .mu.g/ml MMP-7/10 mM CaCl.sub.2/40 mM NaCl/5
mM Tris buffer (pH7)/1% mannitol
Sample 13: 50 .mu.g/ml MMP-7/10 mM CaCl.sub.2/40 mM NaCl/5 mM Tris
buffer (pH7)/2% mannitol Sample 14: 50 .mu.g/ml MMP-7/10 mM
CaCl.sub.2/40 mM NaCl/5 mM Tris buffer (pH7)/2% sucrose Sample 15:
50 .mu.g/ml MMP-7/10 mM CaCl.sub.2/40 mM NaCl/5 mM Tris buffer
(pH7)/5% mannitol Sample 16: 50 .mu.g/ml MMP-7/10 mM CaCl.sub.2/40
mM NaCl/5 mM Tris buffer (pH7)/7% sucrose
[0095] After left to stand at room temperature for 3 hours, each
solution in vials was diluted in two steps. Each solution in vials
was diluted with primary dilution solution of 50 mM Tris buffer (pH
7) containing 0.01% Briji35, 0.01% BSA, 150 mM NaCl and 10 mM
CaCl.sub.2 to 5 ng/ml and the cleavage activity to a fluorescent
substrate (the enzymatic activity of MMP-7) was measured as in
Example 4. In this experiment, a fluorescent substrate obtained
from PEPTIDE INSTITUTE, INC.
(MOCAc-Pro-Leu-Gly-Leu-A.sub.2pr(Dnp)-Ala-Arg-NH.sub.2;
(7-Methoxycoumarin-4-yl)acetyl-L-prolyl-L-leucylglycyl-L-leucyl-[.sup..be-
ta.-(2,4-dinitrophenyl)-L-2,3-diaminopropionyl]-L-alanyl-L-arginine
amide; SEQ ID NO: 9) was used. For secondary dilution, 1% Block Ace
(Block Ace powder: DS Pharma Biomedical)/TBS-T (0.05% Tween20/50 mM
Tris/150 mM NaCl) was used and a concentration of MMP-7 in the
solution was measured by ELISA as above.
[0096] As a result, as compared to no addition (Sample 11), a
higher rate of recovery of MMP-7 was shown when 2 to 5% mannitol
(Samples 13, 15) or 2 to 7% sucrose (Samples 14, 15) was added to
confirm the suppressive effect thereof to adsorption to the wall of
a container (FIG. 7). No decrease in the enzymatic activity of
MMP-7 was observed after addition of mannitol or sucrose (FIG.
8).
Example 7
Effect of Mannitol on Suppression to MMP-7 Aggregate Formation
[0097] The MMP-7 solution obtained in Example 1-(1) was subject to
buffer exchange with 5 mM Tris buffer (pH 7) containing each
concentration of NaCl, 5 mM CaCl.sub.2 and 3.5% mannitol using spin
filter and then was diluted with the Tris buffer to prepare Samples
17 to 22. A control was the MMP-7 solution treated with the Tris
buffer not containing mannitol (Sample 17).
Sample 17: 10 mM NaCl/1 mg/ml MMP-7/5 mM CaCl.sub.2 Sample 18: 10
mM NaCl/1 mg/ml MMP-7/5 mM CaCl.sub.2/3.5% mannitol Sample 19: 40
mM NaCl/1 mg/ml MMP-7/5 mM CaCl.sub.2/3.5% mannitol Sample 20: 80
mM NaCl/1 mg/ml MMP-7/5 mM CaCl.sub.2/3.5% mannitol Sample 21: 120
mM NaCl/1 mg/ml MMP-7/5 mM CaCl.sub.2/3.5% mannitol Sample 22: 180
mM NaCl/1 mg/ml MMP-7/5 mM CaCl.sub.2/3.5% mannitol
[0098] A molecular weight of MMP-7 in each Sample was measured by
dynamic light scattering as described in Example 1-(2). As a
result, it was shown that MMP-7 formed aggregates with 40 mM or
more sodium chloride (FIG. 9).
INDUSTRIAL APPLICABILITY
[0099] A method for monomerizing MMP-7 aggregates of the present
invention can be used for the manufacture of MMP-7 and the
production of MMP-7 preparation.
Sequence CWU 1
1
9127DNAArtificialPrimer used for the cloning of a pro-matrix
metalloproteinase-7 gene 1ccataggtcc aagaacaatt gtctctg
27226DNAArtificialPrimer used for the cloning of a pro-matrix
metalloproteinase-7 gene 2caatccaatg aatgaatgaa tggatg
26381DNAArtificialPrimer used to obtain a DNA fragment consisting
of an alkaline phosphatase signal peptide and a pro-matrix
metalloproteinase-7 3catatgaaac aaagcactat tgcactggca ctcttaccgt
tactgtttac ccctgtgacc 60aaggccctgc cgctgcctca g
81426DNAArtificialPrimer used to obtain a DNA fragment consisting
of an alkaline phosphatase signal peptide and a pro-matrix
metalloproteinase-7 4ggatccctat ttctttcttg aattac
26535DNAArtificialPrimer used to obtain a DNA fragment consisting
of a modified alkaline phosphatase signal peptide and a pro-matrix
metalloproteinase-7 5ctgtttaccc ctgtgaccaa ggaactgccg ctgcc
35633DNAArtificialPrimer used to obtain a DNA fragment consisting
of a modified alkaline phosphatase signal peptide and a pro-matrix
metalloproteinase-7 6cttggtcaca ggggtaaaca gtggcggtaa gag
3377PRTArtificialA DNP-protein of fluorescent substrate used for
measuring the activity of MMP-7 7Pro Leu Gly Leu Trp Ala Arg1
5821PRTArtificialAmino acid sequence of a signal peptide of
alkaline phosphatase 8Met Lys Gln Ser Thr Ile Ala Leu Ala Leu Leu
Pro Leu Leu Phe Thr1 5 10 15Pro Val Thr Lys Ala 2097PRTArtificialA
MOCAc-protein of fluorescent substrate used for measuring the
activity of
MMP-7misc_feature(5)..(5)Xaa=Nbeta-(2,4-dinitrophenyl)-L-2,3-
diaminopropionyl 9Pro Leu Gly Leu Xaa Ala Arg1 5
* * * * *