U.S. patent application number 10/206989 was filed with the patent office on 2003-07-17 for method of separating protective components of bordetella pertussis.
This patent application is currently assigned to Takeda Chemical Industries, Ltd.. Invention is credited to Fujii, Shigeo, Suehara, Akihiro, Yamamoto, Eiji.
Application Number | 20030133941 10/206989 |
Document ID | / |
Family ID | 14030044 |
Filed Date | 2003-07-17 |
United States Patent
Application |
20030133941 |
Kind Code |
A1 |
Suehara, Akihiro ; et
al. |
July 17, 2003 |
Method of separating protective components of bordetella
pertussis
Abstract
To provide a method of efficiently separate protective
components of Bordetella pertussis On the basis of differences in
adsorbability to calcium phosphate gel formed by adding calcium
ions to a Bordetella pertussis culture in the presence of excess
phosphate ions, protective components of Bordetella pertussis are
separated from the Bordetella pertussis culture. Traditionally,
protective components of Bordetella pertussis have been separated
using different purification methods for the respective components.
According to the present invention, the use of the same means of
purification for all subject components makes it possible to purify
each component with high efficiency and high recovery rate, an
aspect very advantageous for industrial production. It is also
possible to efficiently produce an improved purified pertussis
component vaccine comprising an effective combination of pertussis
filamentous hemagglutinin (FHA), pertactin (PRN, 69K-OMP),
pertussis fimbriae (FIM) and pertussis toxin (PT).
Inventors: |
Suehara, Akihiro;
(Yamaguchi, JP) ; Yamamoto, Eiji; (Yamaguchi,
JP) ; Fujii, Shigeo; (Yamaguchi, JP) |
Correspondence
Address: |
FOLEY AND LARDNER
SUITE 500
3000 K STREET NW
WASHINGTON
DC
20007
US
|
Assignee: |
Takeda Chemical Industries,
Ltd.
|
Family ID: |
14030044 |
Appl. No.: |
10/206989 |
Filed: |
July 30, 2002 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10206989 |
Jul 30, 2002 |
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09349712 |
Jul 8, 1999 |
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09349712 |
Jul 8, 1999 |
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08530373 |
Oct 13, 1995 |
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6051240 |
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08530373 |
Oct 13, 1995 |
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PCT/JP95/00830 |
Apr 26, 1995 |
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Current U.S.
Class: |
424/185.1 ;
424/190.1; 530/350 |
Current CPC
Class: |
A61P 31/04 20180101;
C07K 14/235 20130101; A61K 39/00 20130101 |
Class at
Publication: |
424/185.1 ;
530/350; 424/190.1 |
International
Class: |
A61K 039/02; C07K
014/235 |
Foreign Application Data
Date |
Code |
Application Number |
Apr 28, 1994 |
JP |
091565-1994 |
Claims
1. A method of separating at least one member of the group
consisting of pertussis filamentous hemagglutinin (FHA), pertactin
(PRN, 69K-OMP), pertussis fimbriae (FIM), and pertussis toxin (PT)
by bringing a Bordetella pertussis culture into contact with
calcium phosphate gel which is formed by adding calcium ions to the
culture in the presence of phosphate ions.
2. A method of separating at least one member of the group
consisting of pertussis filamentous hemagglutinin (FHA), pertactin
(PRN, 69K-OMP), pertussis fimbriae (FIM) and pertussis toxin (PT)
by separating a Bordetella pertussis culture into cells and culture
liquid, and carrying out at least one of processes (A), (B), (C)
and (D): (A) a process in which the separated cells are eluted with
a salt solution, and pertussis filamentous hemagglutinin (FHA) is
separated by bringing the eluted solution into contact with calcium
phosphate gel of claim 1, (B) a process in which the cell residue
resulting from the elution treatment of the above process (A) is
heated in the presence of a salt solution and brought into contact
with calcium phosphate gel, and pertactin (PRN, 69K-OMP) is
separated by bringing the eluted solution into contact with calcium
phosphate gel of claim 1, (C) a process in which the cell residue
resulting from the elution treatment of the above process (A) is
heated in the presence of a salt solution, the supernatant is
brought into contact with calcium phosphate gel and eluted with a
salt solution, and pertussis fimbriae (FIM) is separated by
bringing the eluted solution into contact with calcium phosphate
gel of claim 1, (D) a process in which the culture or the separated
culture liquid is brought into contact with calcium phosphate gel
of claim 1, and pertussis toxin (PT) is separated from the
supernatant.
3. The separation method of claim 2, wherein the supernatant is
brought into contact with calcium phosphate gel and eluted with a
salt solution to separate pertussis filamentous hemagglutinin (FHA)
in process (A).
4. The separation method of claim 2, wherein the supernatant after
being brought into contact with calcium phosphate gel is brought
into contact with ion exchange gel to separate pertactin (PRN,
69K-OMP) in process (B).
5. The separation method of claim 2, wherein the supernatant is
brought into contact with calcium phosphate gel and removed, and
the resulting residue is eluted with a salt solution to separate
pertussis fimbriae (FIM) in process (C).
6. The separation method of claim 2, wherein the supernatant is
brought into contact with ion exchange gel to separate pertussis
toxin (PT) in process (D).
7. The separation method of claim 2, wherein the salt solution used
in processes (A) and (C) is a buffer containing an alkali metal
salt.
8. The separation method of claim 7, wherein the salt solution is a
buffer containing 0.01-1.0 M sodium chloride.
9. The separation method of claim 1 or 2, wherein the calcium
phosphate gel is formed by adding calcium ions to the culture or
the supernatant of pH 7-9 in the presence of phosphate ions.
10. The separation method of claim 9, wherein the equivalent ratio
of phosphate ions and calcium ions is 1.25-30 equivalents of
phosphate ions per equivalent of calcium ions.
11. The separation method of claim 9, wherein the calcium phosphate
gel is formed by adding calcium acetate, as a calcium ion source,
at 0.1-2 w/v % in the presence of a 0.05-0.1 M phosphate
buffer.
12. The separation method of claim 1 or 2, wherein at least one
member of the group consisting of pertussis toxin (PT), pertussis
filamentous hemagglutinin (FHA), pertactin (PRN, 69K-OMP) and
pertussis fimbriae (FIM) is separated, after which endotoxin is
removed by adsorption to aluminum hydroxide gel in the presence of
ammonium sulfate.
13. The separation method of claim 1 or 2, wherein at least one
member of the group consisting of pertussis toxin (PT), pertussis
filamentous hemagglutinin (FHA), pertactin (PRN, 69K-OMP) and
pertussis fimbriae (FIM) is separated, after which endotoxin is
removed by zonal centrifugation.
14. A pertussis vaccine wherein the components PT:FHA:FIM are
admixed in a ratio of 4-6:8-10:1.
15. A pertussis vaccine wherein the components PT:FHA:PRN:FIM are
admixed in a ratio of 2-6:4-10:1-2:1.
Description
TECHNICAL FIELD
[0001] The present invention relates to a method of separating
protective components of Bordetella pertussis. The pertussis
component vaccine can be produced by suitably mixing the protective
components separated by the method of the present invention.
BACKGROUND ART
[0002] Vaccines are widely used to prevent communicable diseases.
Pertussis, a communicable respiratory disease caused by infection
with Bordetella pertussis, is likely to severely affect patients,
especially infants, due to apneic cough with occasional spasm. To
cope with this disease, it has been common practice to use whole
cultured cells of Bordetella pertussis after inactivation
(inactivated vaccine). However, localized reactions at the site of
vaccination and side reactions, such as fever, have been reported,
creating a social urge to solve this problem. To solve this
problem, there have been a large number of attempts of using
protective components separated from Bordetella pertussis as
vaccine. For example, acellular pertussis vaccine (ACP vaccine),
prepared by extracting protective proteins, such as pertussis toxin
(PT), pertussis filamentous hemagglutinin (FHA), pertactin (PRN,
69K-OMP) and pertussis fimbriae (FIM), from Bordetella pertussis
cells, and removing endotoxin (ET), is being into practical
application, but is not fully satisfactory, due to the drawbacks
described below.
[0003] Pertussis toxin (PT), pertussis filamentous hemagglutinin
(FHA), pertactin (PRN, 69K-OMP) and pertussis fimbriae (FIM), all
protective components of Bordetella pertussis already in practical
application with validated efficacy, are separated by respective
methods.
[0004] Pertussis toxin (PT) can be separated by affinity
chromatography using human haptoglobin as a ligand [Biochimica et
Biophysica Acta, Vol. 580, p. 175 (1979)]. However, human
haptoglobin can be contaminated with hepatitis virus, because it is
collected from human blood; the same applies when animal sera are
used. Another available method is affinity chromatography using
denatured ceruloplasmin as a ligand (Japanese Patent Unexamined
Publication No. 62135/1987). Although this method is free of the
problem of viral contamination, some problems arise, including
vaccine contamination with ceruloplasmin and the high toxicity and
potential body retention of sodium thiocyanate and other eluents
having protein-denaturing effect.
[0005] As for pertussis filamentous hemagglutinin (FHA), a
purification method using hydroxyapatite gel is available
[Infection and Immunity, Vol. 41, p. 313 (1983) and EP-A-231083,
EP-A-427462, EP-A-462534; Japanese Patent Unexamined Publication
Nos. 234031/1987, 169893/1992, 368337/1993). However, it takes long
time for column operation, and is uneconomic due to the high cost
of hydroxyapatite.
[0006] As for pertactin (PRN, 69K-OMP), affinity chromatography
using a mouse serum as a ligand is available [Infection and
Immunity, Vol. 56, p. 3189 (1988)], but has the same drawbacks as
above.
[0007] As for pertussis fimbriae (FIM), Bordetella pertussis cell
extract is purified by salting-out with ammonium sulfate and
magnesium chloride [Infection and Immunity, Vol. 48, p. 442
(1985)], but this method is poor in vaccine production efficiency
due to low yield.
[0008] There is a method of preparing Gram-negative bacterial
vaccine by adosorbing with the aluminum hydroxide gel (WO
93/10216). This method needs the large amount of the aluminum
hydroxide gel, which adsorbs both the protective components and the
endotoxin originated in Gram-negative bacteria. The vaccine
obtained by the method of WO93/10216 has a danger of side effects,
such as fever and endotoxin-shock, by the endotoxin released into
body because of the diluted vaccines.
[0009] As for the pertussis vaccine production included as the
components mixture without separating each protective component
originated in Bordetella pertussis, a method of using calcium
phosphate gel is available (EP-A-291968, Japanese Patent Unexamined
Publication No. 52726/1989). However, this method formed the
calcium phosphate in the presence of a 1M sodium chloride does not
absorb the protective components.
[0010] As stated above, totally different purification methods must
be used to separate the respective protective components of
Bordetella pertussis. This approach is unsuitable to large-scale
vaccine production due to painstaking operation, and difficult to
apply practically. Moreover, the customary methods of separating
protective components disclosed in prior art have some problems
that materials or reagents have pathogenicity or toxity.
DISCLOSURE OF INVENTION
[0011] Against the background described above, the present
inventors investigated methods of efficiently separating protective
components of Bordetella pertussis, and found that protective
components of Bordetella pertussis can be efficiently separated
from Bordetella pertussis culture on the basis of differences in
adsorbability to calcium phosphate gel formed by adding calcium
ions to the Bordetella pertussis culture in the presence of excess
phosphate ions. The inventors made further investigation based on
this finding, and the efficient and safty method of separating the
protective components combined with the calcium phosphate gel
treatment and elution by salt and heating was developed the present
invention. Accordingly, the present invention relates to:
[0012] (1) A method of separating at least one member of the group
consisting of pertussis filamentous hemagglutinin (FHA), pertactin
(PRN, 69K-OMP), pertussis fimbriae (FIM), and pertussis toxin (PT)
by bringing a Bordetella pertussis culture into contact with
calcium phosphate gel which is formed by adding calcium ions to the
culture in the presence of phosphate ions.
[0013] (2) A method of separating at least one member of the group
consisting of pertussis filamentous hemagglutinin (FHA), pertactin
(PRN, 69K-OMP), pertussis fimbriae (FIM) and pertussis toxin (PT)
by separating a Bordetella pertussis culture into cells and culture
liquid, and carrying out at least one of processes (A), (B), (C)
and (D):
[0014] (A) a process in which the separated cells are eluted with a
salt solution, and pertussis filamentous hemagglutinin (FHA) is
separated by bringing the eluted solution into contact with calcium
phosphate gel of the above item (1),
[0015] (B) a process in which the cell residue resulting from the
elution treatment of the above process (A) is heated in the
presence of a salt solution and brought into contact with calcium
phosphate gel, and pertactin (PRN, 69K-OMP) is separated by
bringing the eluted solution into contact with calcium phosphate
gel of the above item (1),
[0016] (C) a process in which the cell residue resulting from the
elution treatment of the above process (A) is heated in the
presence of a salt solution, the supernatant is brought into
contact with calcium phosphate gel and eluted with a salt solution,
and pertussis fimbriae (FIM) is separated by bringing the eluted
solution into contact with calcium phosphate gel of the above item
(1),
[0017] (D) a process in which the culture or the separated culture
liquid is brought into contact with calcium phosphate gel of the
above item (1), and pertussis toxin (PT) is separated from the
supernatant.
[0018] (3) The separation method of the above item (2), wherein the
supernatant is brought into contact with calcium phosphate gel and
eluted with a salt solution to separate pertussis filamentous
hemagglutinin (FHA) in process (A).
[0019] (4) The separation method of the above item (2), wherein the
supernatant after being brought into contact with calcium phosphate
gel is brought into contact with ion exchange gel to separate
pertactin (PRN, 69K-OMP) in process (B).
[0020] (5) The separation method of the above item (2), wherein the
supernatant is brought into contact with calcium phosphate gel and
removed, and the resulting residue is eluted with a salt solution
to separate pertussis fimbriae (FIM) in process (C).
[0021] (6) The separation method of the above item (2), wherein the
supernatant is brought into contact with ion exchange gel to
separate pertussis toxin (PT) in process (D).
[0022] (7) The separation method of the above item (2), wherein the
salt solution used in processes (A) and (C) is a buffer containing
an alkali metal salt.
[0023] (8) The separation method of the above item (7), wherein the
salt solution is a buffer containing 0.01-1.0 M sodium
chloride.
[0024] (9) The separation method of the above item (1) or (2),
wherein the calcium phosphate gel is formed by adding calcium ions
to the culture or the supernatant of pH 7-9 in the presence of
phosphate ions.
[0025] (10) The separation method of the above item (9), wherein
the equivalent ratio of phosphate ions and calcium ions is 1.25-30
equivalents of phosphate ions per equivalent of calcium ions.
[0026] (11) The separation method of the above item (9), wherein
the calcium phosphate gel is formed by adding calcium acetate, as a
calcium ion source, at 0.1-2 w/v % in the presence of a 0.05-0.1 M
phosphate buffer.
[0027] (12) The separation method of the above item (1) or (2),
wherein at least one member of the group consisting of pertussis
toxin (PT), pertussis filamentous hemagglutinin (FHA), pertactin
(PRN, 69K-OMP) and pertussis fimbriae (FIM) is separated, after
which endotoxin is removed by adsorption to aluminum hydroxide gel
in the presence of ammonium sulfate.
[0028] (13) The separation method of the above item (1) or (2),
wherein at least one member of the group consisting of pertussis
toxin (PT), pertussis filamentous hemagglutinin (FHA), pertactin
(PRN, 69K-OMP) and pertussis fimbriae (FIM) is separated, after
which endotoxin is removed by zonal centrifugation.
[0029] (14) A pertussis vaccine wherein the components PT:FHA:FIM
are admixed in a ratio of 4-6:8-10:1.
[0030] (15) A pertussis vaccine wherein the components
PT:FHA:PRN:FIM are admixed in a ratio of 2-6:4-10:1-2:1.
BEST MODE FOR CARRYING OUT THE INVENTION
[0031] The Bordetella pertussis strain used for the present
invention is not subject to limitation, as long as it is capable of
producing one or more than one member of the group consisting of
pertussis filamentous hemagglutinin (FHA), pertactin (PRN,
69K-OMP), pertussis fimbriae (FIM) and pertussis toxin (PT), all
protective components of Bordetella pertussis. Useful strains
include known strains, such as Bordetella pertussis Tohama phase I
strain (Infection and Immunity, Vol. 6, p. 89, (1972)] (maintained
at the National Institute of Health, Ministry of Social Welfare,
Tokyo, Japan (NIHJ 1052), deposited under accession number IFO
14073 at the Institute for Fermentation, Osaka since Aug. 13,
1980), Bordetella pertussis Yamaguchi phase I strain, Bordetella
pertussis phase I strain 18-323 and Bordetella pertussis phase I
strain 165, with preference given to Bordetella pertussis Tohama
phase I strain (IFO 14073) from the viewpoint of productivity.
Bordetella pertussis can be cultured by known methods. Useful media
include known basal media, such as Cohen-Wheeler medium,
Stainer-Scholte medium and other liquid media, with preference
given to Stainer-Scholte medium. The solution containing protective
components and endotoxin (ET) may be a culture obtained by
stationary culture or tank culture. In the present invention, the
culture means cultured cells or culture liquid resulting from
incubating said Bordetella pertussis. And the present invention,
the supernatant means the culture liquid or the supernatant
resulting from heating the cells in the presence of a salt solution
or eluting with a salt solution from the calcium phosphate gel
adsorbed the protective components as described below. The cells
include the culture cells and the cell residue. In the present
invention, the method used to separate a Bordetella pertussis
culture into cells and culture supernatant may be a known method,
such as centrifugation or filtration.
[0032] The calcium phosphate gel used for the present invention is
not a ready-made gel, but preferably calcium phosphate gel formed
in a culture or a supernatant to be treated by adding calcium ions
to them in the presence of excess phosphate ions (may referred to
as the in-side gel forming method). Although prepared calcium
phosphate gel (e.g., commercially available hydroxyapatite gel). In
comparison with the former method by using the ready-made
hydroxyapatite gel mentioned above (may referred to as the out-side
gel forming method), the present method by using calcium phosphate
gel is higher in both adsorption efficiency for pertussis
filamentous hemagglutinin (FHA) and pertussis fimbriae (FIM) and
recovery efficiency of them, as shown hereafter. Moreover, the
calcium phosphate gel used in the present invention is better in
operational efficiency because of the absence of gel pretreatment
and regeneration process, and more advantageous in cost.
Furthermore, each of protective components of Bordetella pertussis
can be selectively absorbed to the calcium phosphate gel by
properly selecting the ratio of phosphate ions to calcium ions. If
the the culture or the supernatant to be treated with calcium
phosphate gel, does not contain a sufficient amount of phosphate
ions, a phosphate buffer of appropriate concentration is added to
provide phosphate ions before addition of calcium ions. For
example, by adding 1 M phosphate buffer, the final phosphate ion
concentration is adjusted to 0.02-0.2 M, preferably 0.05-0.1 M.
[0033] The calcium ion source added is exemplified by soluble
calcium salts, such as calcium acetate, calcium chloride and
calcium nitrate, with preference given to calcium ions derived from
calcium acetate. Concerning the ratio of phosphate ions and calcium
ions, it is preferable that phosphate ions be in excess, in
comparison with calcium ions. The ratio can be properly selected in
each case of the protective components of Bordetella pertussis, as
mentioned hereafter.
[0034] In process (A) above, pertussis filamentous hemagglutinin
(FHA) is separated as follows: After the culture liquid, i.e. the
culture supernatant, is removed from a Bordetella pertussis culture
by a known method, such as centrifugation or filtration, a
one-tenth to one-twentieth volume (relative to the amount of
culture broth) (corresponding to a final cell concentration of
50-100 billion cells/ml) of a salt solution is added to the cells
to elute the hemagglutinin. In this case, the salt solution used is
preferably a buffer supplemented with an alkali metal salt or an
alkaline earth metal salt, specifically a 0.04-0.08 M phosphate
buffer supplemented with a 0.25-1.0 M alkali metal salt or alkaline
earth metal salt, with greater preference given to a 0.05 M
phosphate buffer supplemented with a 0.5-1.0 M alkali metal salt.
The alkali metal salt or alkaline earth metal salt added to the
buffer is exemplified by sodium chloride, potassium chloride and
magnesium chloride. For example, it is preferable to elute the
hemagglutinin by adding a one-tenth to one-twentieth volume
(relative to the amount of culture broth) of a 0.04-0.08 M
phosphate buffer (pH 7-9) supplemented with 0.5-1.0 M sodium
chloride, more preferably a 0.05 M phosphate buffer (pH 8)
supplemented with 1 M sodium chloride, to the cells collected,
followed by gentle stirring at 4.degree. C. to room temperature,
preferably 8-15.degree. C., for 1-60 minutes, preferably 1-30
minutes, and standing for 1-2 days. The solution containing the
eluted pertussis filamentous hemagglutinin (FHA) is then subjected
to a known method, such as centrifugation or filtration, to recover
the supernatant (the cell residue obtained at the same time by this
treatment is used to isolate pertactin (PRN, 69K-OMP) and pertussis
fimbriae (FIM)). The thus-obtained supernatant is then brought into
contact with calcium phosphate gel.
[0035] Concerning the ratio of phosphate ions and calcium ions, it
is preferable that phosphate ions be in excess, in comparison with
calcium ions. For example, the equivalent ratio of these ions is
preferably 1.25-30 equivalents, more preferably 1.5-7.5 equivalents
of phosphate ions per equivalent of calcium ions. This quantitative
ratio can be expressed in molar ratio as 0.8-20 M of phosphate ions
to 1 M of calcium ions, more preferably 1-5 M of phosphate ions to
1 M of calcium ions. For example, to a solution (pH 7-9) containing
phosphate ions at a concentration within the above-described
concentration range (0.02-0.2 M, preferably 0.05-0.1 M), a calcium
salt is added to a final concentration of 4-70 mM, preferably 8-50
mM (e.g., calcium acetate added to a final concentration of 0.1-0.8
w/v %, preferably 0.2-0.6 w/v %), followed by gentle reaction at
4.degree. C. to room temperature, preferably 8-15.degree. C., for 1
to 4 hours, preferably 1 to 2 hours, to form calcium phosphate
gel.
[0036] Although pertussis filamentous hemagglutinin (FHA) is
adsorbed to the calcium acetate gel added, provided that the amount
of calcium phosphate gel added to a final concentration exceeding
0.8 w/v %, it is preferable to add the calcium acetate gel in an
amount such that the final concentration falls within the above
concentration range, for selectively adsorbing pertussis
filamentous hemagglutinin (FHA) only. After completion of the
reaction, the supernatant is removed by a known method, such as
centrifugation or filtration; the resulting gel precipitate is
collected. To this precipitate, a one-tenth to one-twentieth volume
(relative to the amount of culture broth) of a salt solution is
added, to elute the pertussis filamentous hemagglutinin (FHA). In
this case, the salt solution used may be the same salt solution as
used to elute pertussis filamentous hemagglutinin (FHA) from the
above-described cells. It is preferable to add a one-tenth to
one-twentieth volume of 0.05-0.1 M phosphate buffer (pH 7-9)
supplemented with 1-2 M sodium chloride, more preferably 0.1 M
phosphate buffer (pH 8) supplemented with 1-1.5 M sodium chloride,
to the above-described gel precipitate, followed by gentle stirring
at 4.degree. C. to room temperature for 1 to 2 hours, to elute the
hemagglutinin. After completion of the stirring, the precipitate is
removed by a known method, such as centrifugation or filtration, to
recover pertussis filamentous hemagglutinin (FHA) in the
supernatant. The supernatant, if necessary, can be concentrated and
desalinized, by ammonium sulfate salting-out or using an
ultrafiltration membrane. By subjecting the supernatant obtained by
the above-described treatment to the aluminum hydroxide gel
treatment or zonal centrifugation described below, pertussis
filamentous hemagglutinin (FHA) having endotoxin selectively
removed can be separated with substantially no loss.
[0037] In process (B) or (C) above, pertactin (PRN, 69K-OMP) and
pertussis fimbriae (FIM) are separated as follows: The cell residue
resulting from elution of the solution containing pertussis
filamentous hemagglutinin (FHA) is heated in the presence of a
one-tenth to one-twentieth volume (relative to the amount of
culture broth) (corresponding to a final cell concentration of
500-100 billion cells/ml) of a salt solution to extract the
pertactin (PRN, 69K-OMP) and pertussis fimbriae (FIM). In this
case, the salt solution used may be the same as used in process (A)
above. However, it is preferable to use a one-tenth to
one-twentieth volume (relative to the amount of culture broth) of
0.01-0.05 M phosphate buffer (pH 7-9) supplemented with 0.15-0.25 M
sodium chloride, with greater preference given to 0.01 M phosphate
buffer (pH 7) supplemented with 0.15-0.25 M sodium chloride. It is
preferable that heating be achieved in warm water at 40-80.degree.
C., preferably 50-60.degree. C., for 60 to 120 minutes, preferably
80 to 90 minutes. The heated extracted pertactin (PRN, 69K-OMP) and
pertussis fimbriae (FIM) are recovered in the supernatant by a
known method, such as centrifugation or filtration. The
thus-obtained supernatant is then brought into contact with calcium
phosphate gel. In this case, calcium phosphate gel treatment can be
performed in accordance with process (A) above; however, it is
preferable to perform it within the following concentration range.
For example, to a solution (pH 7-9) containing phosphate ions,
adjusted as necessary to a final phosphate ion concentration of
0.05-0.1 M, preferably 0.1 M, by adding a 1 M phosphate buffer, or
the like, a calcium salt is added to a final concentration of
40-180 mM, preferably 55-150 mM (e.g., calcium acetate added to a
final concentration of 1-2 w/v %, preferably 1.3-1.7 w/v %),
followed by gentle reaction at 4.degree. C. to room temperature,
preferably 8-15.degree. C., for 1 to 4 hours, preferably 1 to 2
hours, to form calcium phosphate gel. After completion of the
reaction, the resulting precipitate and supernatant are separated
from each other by a known separation method, such as filtration or
centrifugation, to recover pertactin (PRN, 69K-OMP) in the
supernatant and pertussis fimbriae (FIM) in the gel residue, with
substantially no loss.
[0038] The crudely purified pertactin (PRN, 69K-OMP) obtained by
the above-described treatment can be further purified by a known
method, preferably by ion exchange gel treatment; it is preferable
that the crudely purified pertactin (PRN, 69K-OMP) be previously
concentrated and desalinized by ammonium sulfate salting-out or
using an ultrafiltration membrane. In the present invention, useful
ion exchange gels include anion exchange gel and cation exchange
gel, with preference given to cation exchange gel. Contact with ion
exchange gel may be achieved by the column chromatography method or
the batch method. By this treatment, impurities, i.e., substances
other than pertactin (PRN, 69K-OMP) in the crudely purified
pertactin (PRN, 69K-OMP), are adsorbed; the effluent is collected
to yield a solution containing pertactin (PRN, 69K-OMP). In the
column chromatography method, the column is packed with ion
exchange gel, through which the starting material, i.e., crudely
purified pertactin (PRN, 69K-OMP), is passed at a flow rate of
100-500 ml/cm.sup.2/hr. In the batch method, crudely purified
pertactin (PRN, 69K-OMP) is placed in a container, to which ion
exchange gel is added directly, followed by stirring for about 30
minutes to 3 hours, preferably about 1 hour, to adsorb impurities,
i.e., substances other than pertactin (PRN, 69K-OMP). Such impurity
adsorption is achieved using a buffer of a pH value of 5.0-8.0 and
an electroconductivity of 100-300 umho (0.1-0.3 mS), e.g., a
0.01-0.02 M phosphate buffer (pH 5.5-6.0). By subjecting the
supernatant obtained by the above-described treatment to the
aluminum hydroxide gel treatment or zonal centrifugation treatment
described below, pertactin (PRN, 69K-OMP) having endotoxin removed
can be separated with substantially no loss.
[0039] To the gel residue containing crude pertussis fimbriae (FIM)
obtained by the above-described treatment, a one-tenth to
one-twentieth volume (relative to the amount of culture broth) of a
salt solution is added, to elute the pertussis fimbriae (FIM). In
this case as well, the salt solution may be the same as used in
process (A) above. For example, it is preferable to add a one-tenth
to one-twentieth volume (relative to the amount of culture broth)
of a 0.05-0.1 M phosphate buffer (pH 7-9) supplemented with 1-2 M
sodium chloride, preferably a 0.1 M phosphate buffer (pH 8)
supplemented with 1-1.5 M sodium chloride, followed by gentle
stirring at 4.degree. C. to room temperature for 1 to 2 hours, to
elute the pertussis fimbriae (FIM). After completion of the
stirring, the precipitate is removed by a known method, such as
centrifugation or filtration, to recover pertussis fimbriae (FIM)
in the supernatant. By subjecting the supernatant obtained by the
above-described treatment to the above-described aluminum hydroxide
gel treatment or zonal centrifugation treatment, pertussis fimbriae
(FIM) having endotoxin selectively removed can be separated with
substantially no loss.
[0040] In process (D) above, pertussis toxin (PT) is separated as
follows: Although a Bordetella pertussis culture can be used
without separation into cultured cells and culture supernatant in
this process, it is preferable in respect of efficiency to recover
the supernatant from the Bordetella pertussis culture by a known
method, such as centrifugation or filtration, concentrate the
supernatant about 10-20 fold using an ultrafiltration membrane, or
the like, and collect the supernatant by centrifugation or another
method before this process. This supernatant is then brought into
contact with calcium phosphate gel. In this case, calcium phosphate
gel treatment can be carried out in the same manner as in process
(A) above, but it is preferable to carried out this treatment
within the following concentration range. For example, to a
solution (pH 7-9) containing phosphate ions, adjusted as necessary
to a final phosphate ion concentration of 0.05-0.1 M, preferably
0.1 M, by adding a 1 M phosphate buffer, or the like, a calcium
salt is added to a final concentration of 40-180 mM, preferably
55-150 mM (e.g., calcium acetate added to a final concentration of
1-2 w/v %, preferably 1.3-1.7 w/v %), followed by gentle reaction
at 4.degree. C. to room temperature, preferably 8-15.degree. C.,
for 1 to 4 hours, preferably 1 to 2 hours, to form calcium
phosphate gel. After completion of the reaction, the resulting
precipitate and supernatant are separated from each other by a
known method, such as centrifugation or filtration, to recover
pertussis toxin (PT) in the supernatant with substantially no loss.
The crudely purified pertussis toxin (PT) obtained by the
above-described treatment is further purified by ion exchange gel
treatment; it is preferable that the crudely purified pertussis
toxin (PT) be previously concentrated and desalinized by ammonium
sulfate salting-out or using an ultrafiltration membrane. The ion
exchange gel used here is exemplified by anion exchange gel and
cation exchange gel, with preference given to cation exchange gel.
Contact with ion exchange gel may be achieved by the column
chromatography method or the batch method. By this treatment,
pertussis toxin (PT) in the crudely purified pertussis toxin (PT)
is adsorbed to the gel, followed by washing with an appropriate
buffer to elute and remove impurities, after which pertussis toxin
(PT) is eluted and isolated with a buffer of appropriate pH and
ionic strength. In the column chromatography method, the column is
packed with ion exchange gel, through which the starting material,
i.e., crudely purified pertussis toxin (PT), is passed at a flow
rate of 100-500 ml/cm.sup.2/hr to cause toxin adsorption. In the
batch method, the crudely purified pertussis toxin (PT) is placed
in a container, to which ion exchange gel is added directly,
followed by stirring for about 30 minutes to 3 hours, preferably
about 1 hour, to cause toxin adsorption. Such adsorption of the
crudely purified pertussis toxin (PT) is achieved using a buffer of
a pH level of 5.0-6.0 and an electroconductivity of 100-300 umho
(0.1-0.3 mS), e.g., a 0.01-0.02 M phosphate buffer (pH 5.5-6.0).
Elution from the ion exchange gel to which the pertussis toxin (PT)
has been adsorbed can be achieved using a buffer of a pH level of
7.0-7.5 and an electroconductivity of 1,000-2,000 umho (1-2 mS),
e.g., a 0.1-0.2 M phosphate buffer (pH 7.0-7.5). By subjecting the
eluate obtained by the above-described treatment to the aluminum
hydroxide gel treatment or zonal centrifugation treatment described
below, pertussis toxin (PT) having endotoxin selectively removed
can be separated with substantially no loss.
[0041] In the present invention, the aluminum hydroxide gel
treatment for endotoxin removal is carried out to adsorb only the
endotoxin selectively by bringing the subject into contact with
previously prepared aluminum hydroxide gel in the presence of
ammonium sulfate. But, the aluminum hydroxide gel, whose amount to
be used is less than one-tenth of that used in WO93/10216, hardly
absorbs any amounts of protective components of Bordetella
pertussis. It is normally preferable that this treatment be carried
out after concentration by a known method, such as ammonium sulfate
salting-out or an ultrafiltration membrane method. Aluminum ions
useful for the previously prepared aluminum hydroxide gel include
those of soluble aluminum compounds, such as aluminum sulfate and
aluminum chloride, with preference given to the aluminum ions of
aluminum chloride. It is preferable that aluminum hydroxide gel be
prepared by adding a 2 M sodium hydroxide solution to a 25-190 mM
aluminum salt solution (e.g., 0.9-4.5% aluminum chloride solution)
to a pH level of 7.0-7.5, followed by gentle reaction at 4.degree.
C. to room temperature for 1 to 3 hours, to form the desired
aluminum hydroxide gel. The aluminum hydroxide gel obtained by the
above-described treatment is then treated to recover the resulting
gel precipitate by a known method, such as filtration or
centrifugation, to remove free aluminum ions after completion of
the reaction. The protective component of Bordetella pertussis
concentrated by a known method, such as ammonium sulfate
salting-out, is recovered by centrifugation; the precipitate is
dissolved in a 0.25 M phosphate buffer (pH 7.0-7.5) supplemented
with 0.25 M sodium chloride. To this protective component of
Bordetella pertussis, a saturated ammonium sulfate solution is
added to a final concentration of 2.0-8.0 v/v %, followed by
addition of previously prepared, recovered aluminum hydroxide gel
to a final concentration of 0.1-1.0 mg/ml, preferably 0.2-0.5
mg/ml, and gentle reaction at 4.degree. C. to room temperature for
30 minutes to 1 hour. After completion of the reaction, the
aluminum hydroxide gel is removed by a known method, such as
filtration or centrifugation, to separate the protective component
of Bordetella pertussis having endotoxin removed, with
substantially no loss.
[0042] In the present invention, zonal centrifugation treatment is
carried out to remove endotoxin, and is preferably carried out
after concentration by a known method, such as ammonium sulfate
salting-out. Zonal centrifugation methods include sucrose density
gradient centrifugation, cesium chloride density gradient
centrifugation and potassium tartrate density gradient
centrifugation, with preference given to sucrose density gradient
centrifugation. For example, when sucrose density gradient
centrifugation is carried out on a sucrose density gradient of 0-30
w/v % at an R.sub.max of 60,000 to 122,000 G for about 10 to 24
hours, the protective component of Bordetella pertussis having
endotoxin removed can be separated.
[0043] PT is detoxified by using a conventional detoxification
technique as described in British Journal of Experimental
Pathslogy, vol. 44, p. 177, (1963). FHA, PRN and FIM may be
inactivated, for example, by the method as described in Japanese
Patent Unexamined Publication No. 52726/1989. An improved purified
pertussis component vaccine which is superior to a known pertussis
vaccine can be produced by blending in any desireded ratio of
protective components of Bordetella pertussis obtained by the
method of present invention. Namely, it's not possible to change
the ratio of each component which is stable in whole cell or
co-purified acellular vaccine without obtaining furified component
respectively, while an antigen ratio can be chosen in the method of
present invention which gives the optimal which gives the optimal
response in humans as a pertussis vaccine since each component is
efficiently purified in the present invention. The purified
pertussis component vaccine is desirable to blend the protective
components in as little amount of total protein as possible and in
a way of giving more effective immunogenicity. The purified
pertussis component vaccine of the present invention preferably
includes all of three components, i.e. FHA, FIM and PT, and may
also include other pharmaceutically acceptable components such as
PRN which does not give undesired side effects.
[0044] When blending these components to produce a purified
pertussis component vaccine of the present invention, the ratio of
it may be examplified in Examples metioned hereinafter. The
component vaccine of the present invention has a PT:FHA:FIM ratio
of approximate 4-6:8-10:1, preferably 5-6:8-10:1, and comprise, for
example, 20-30 .mu.g-protein/ml of PT, 40-50 .mu.g-protein/ml of
FHA and 5-10 .mu.g-protein/ml of FIM, preferably 25-30
.mu.g-protein/ml of PT, 40-50 .mu.g-protein/ml of FHA and 5
.mu.g-protein/ml of FIM. The component vaccine mentioned above may
further include 5-10 .mu.g-protein/ml of PRN, and has a
PT:FHA:PRN:PT ratio of 2-6:4-10:1-2:1, preferably 5-6:8-10:2:1.
Namely, it preferably comprise 25-30 .mu.g-protein/ml of PT, 40-50
.mu.g-protein/ml of FHA, 10 .mu.g-protein/ml of PRN and 5
.mu.g-protein/ml of FIM.
[0045] The above-described effect of the present invention can be
summarized as follows: The method of the present invention is
characterized by the use of the same means of purification for all
subject protective components of Bordetella pertussis. This
obviates the necessity of different painstaking procedures for the
respective components as in prior art methods, thus permitting
component purification with high efficiency and high recovery rate,
an aspect very advantageous for industrial production. In addition,
the endotoxin content, as determined by the Limulus test, is not
more than 1 ng per 100 .mu.g total protein, for all protective
components of Bordetella pertussis obtained by the present
invention, providing very high practical value. It is also possible
to produce an improved purified pertussis component vaccine
comprising an effective combination of pertussis filamentous
hemagglutinin (FHA), pertactin (PRN, 69K-OMP), pertussis fimbriae
(FIM) and pertussis toxin (PT).
EXAMPLES
[0046] The present invention is hereinafter described in more
detail by means of, but is not limited to, the following working
examples and reference examples. In the following description,
pertussis toxin (PT), pertussis filamentous hemagglutinin (FHA),
pertactin (PRN, 69K-OMP), pertussis fimbriae (FIM) and endotoxin
are also referred to as PT, FHA, 69K-OMP, FIM and ET,
respectively.
Example 1
[0047] Bordetella pertussis Tohama phase I strain was cultured to a
final concentration of 2 billion cells/ml by Roux bottle stationary
culture (450 ml, 35.degree. C., 5 days) and tank agitating culture
(40l, 35.degree. C., 2 days) using Stainer-Scholte medium, to yield
a Bordetella pertussis culture.
[0048] The cell culture was concentrated to a one-tenth volume
using an ultrafiltration membrane, after which it was centrifuged
to separate the supernatant and cells. To the supernatant, a 1 M
phosphate buffer (pH 8.0) was added to a final concentration of 0.1
M, followed by addition of an calcium acetate solution to a final
concentration of 1.6 w/v % and stirring at room temperature for 1
hour. This calcium phosphate gel solution was filtered. The
resulting filtrate was concentrated and desalinized to an
electroconductivity of 200 umho using an ultrafiltration membrane,
passed through a sulfopropyl cation exchange chromatography column
(produced by Tosoh Corporation), washed with a 0.01 M phosphate
buffer (pH 6.0), and eluted with a 0.1 M phosphate buffer (pH 7.0),
to yield pertussis toxin (PT). Next, cells were dispersed in a
one-tenth volume (relative to the amount of culture broth) of a
0.05 M phosphate buffer (pH 8.0) supplemented with 1 M sodium
chloride, followed by centrifugation to yield the supernatant and
cells. To the supernatant, a calcium acetate solution was added to
a final concentration of 0.5 w/v %, followed by stirring at room
temperature for 1 hour. This calcium phosphate gel solution was
filtered; the resulting gel layer was collected. The gel layer was
eluted with a 0.1 M phosphate buffer (pH 8.0) supplemented with 1 M
sodium chloride to yield a solution containing pertussis
filamentous hemagglutinin (FHA). Separately, cells were dispersed
in a one-tenth volume (relative to the amount of culture broth) of
a 0.01 M phosphate buffer (pH 7.0) supplemented with 0.15 M sodium
chloride, after which it was heated in 60.degree. C. warm water for
90 minutes, followed by centrifugation to yield the supernatant. To
the supernatant, a 1 M phosphate buffer (pH 8.0) was added to a
final concentration of 0.1 M, after which a calcium acetate
solution was added to a final concentration of 1.6 w/v %, followed
by stirring at room temperature for 1 hour. This calcium phosphate
gel solution was filtered; the filtrate and the gel layer were
collected. The filtrate was concentrated and desalinized to an
electroconductivity of 200 umho using an ultrafiltration membrane
and passed through a sulfopropyl cation exchange chromatography
column (produced by Tosoh Corporation); the effluent was collected
to,yield a solution containing pertactin (PRN, 69K-OMP).
Separately, the gel layer was eluted with a 0.1 M phosphate buffer
(pH 8.0) supplemented with 1 M sodium chloride to yield a solution
containing pertussis fimbriae (FIM).
[0049] Control sample was prepared as follows: Ammonium sulfate was
added at 220 g per liter of culture broth, followed by sufficient
stirring. After being kept standing at 4.degree. C. for about 14
days, the mixture was centrifuged; the supernatant was discarded,
and the precipitate was collected. To the precipitate thus
obtained, a one-tenth volume (relative to the amount of culture
broth) of a 0.05 M phosphate buffer (pH 8.0) supplemented with 1 M
sodium chloride was added, followed by sufficient stirring. After
being kept standing at 4.degree. C. for 4 days, the mixture was
again centrifuged; the supernatant was collected to yield a
solution containing pertussis toxin (PT), pertussis filamentous
hemagglutinin (FHA), pertactin (PRN, 69K-OMP) or pertussis fimbriae
(FIM).
[0050] The pertussis toxin (PT), pertussis filamentous
hemagglutinin (FHA), pertactin (PRN, 69K-OMP) or pertussis fimbriae
(FIM) content in each sample was determined by ELISA, with purified
products of pertussis toxin (PT), pertussis filamentous
hemagglutinin (FHA), pertactin (PRN, 69K-OMP) and pertussis
fimbriae (FIM) as references. Results are expressed in .mu.g
protein/ml unit.
[0051] Protein content determination: Protein precipitated with
heated trichloroacetic acid was quantitated by the Lowry method,
with bovine serum albumin (Fraction V, produced by Wako Pure
Chemical Industries) as a reference. Results are expressed in .mu.g
protein/ml unit.
[0052] The results for Roux bottle culture broth and those for tank
culture broth are shown in Tables 1 and 2, respectively.
1TABLE 1 Purity (%) Active (active Ingredient Total ingredient
Protein Protein protein Content Recovery* Content content/total
Sample (.mu.g protein/ml) (%) (.mu.g protein/ml) protein content)
PT 2656.8 90.0 2662.1 99.8 FHA 9161.7 85.0 9339.1 98.1 FIM 474.7
244.6 495.5 95.8 69K-OMP 3683.8 244.6 3607.2 102.1 *Each figure
represents a percent value relative to the control group.
[0053]
2TABLE 2 Purity (%) Active (active Ingredient Total ingredient
Protein Protein protein Content Recovery* Content content/total
Sample (.mu.g protein/ml) (%) (.mu.g protein/ml) protein content)
PT 3242.2 78.9 3359.8 96.5 FHA 9527.0 98.0 9752.9 97.7 FIM 675.0
1184.0 714.7 95.0 69K-OMP 4333.5 2364.0 4505.1 96.3 *Each figure
represents a percent value relative to the control group.
[0054] It is evident from these figures that each protective
component was efficiently isolated, and that in the case of tank
culture broths, pertactin (PRN, 69K-OMP) and pertussis fimbriae
(FIM), both produced at low productivity in the case of Roux bottle
culture broths, were recovered in large amounts.
Reference Example 1
[0055] To a control solution prepared by the method described in
Example 1, calcium acetate was added to a final concentration of
0.5 w/v %, followed by stirring at room temperature for 1 hour. To
the filtrate obtained by filtering this calcium solution, a half
amount of a saturated ammonium sulfate solution was added; the
mixture was kept standing at 4.degree. C. for 7 days. This ammonium
sulfate salting-out product was centrifuged; the resulting
precipitate was collected and resuspended in a 0.025 M phosphate
buffer (pH 7.0) supplemented with 0.25 M sodium chloride to yield a
starting material. To this starting material, aluminum hydroxide
gel, previously prepared to a final concentration of 0.4 mg/ml, was
added; to the aluminum hydroxide gel recovered by centrifugation,
ammonium sulfate was added to a final concentration of 0, 2, 4 or 8
w/v %, followed by gentle stirring at room temperature for 30
minutes. After completion of the reaction, the aluminum hydroxide
gel was removed by centrifugation to separate the supernatant. Each
supernatant was assayed for hemagglutination activity and endotoxin
content by the methods described below. The results are shown in
Table 3.
[0056] Determination of hemagglutination activity: After the sample
was serially diluted 2 folds with a 0.01 M phosphate buffered
saline, 0.6 v/v % chick immobilized red blood cells were added to
cause hemagglutination. The maximum dilution rate of each sample
showing hemagglutination was taken as the hemagglutinin titer HA.
Determination of endotoxin (ET) content: Using Escherichia coli
(Difico 055-B5) as a reference strain, ET content was determined by
the Limulus test (Wako Pure Chemical kit). Results are expressed in
ng/ml unit. It is evident from Table 3 that endotoxin can be
selectively removed, without active ingredient loss, by treating
the sample with previously prepared aluminum hydroxide gel in the
presence of ammonium sulfate.
3TABLE 3 Amount of Ammonium Sulfate Endotoxin HA Recovery Added
Content HA Value Rate* (w/v %) (ng/ml) (HAU/ml) (%) 0 15.8 16000
50.0 2 11.1 32000 100.0 4 <9.0 32000 100.0 8 <9.0 24000 75.0
*Each figure for endotoxin removal rate or HA recovery rate is a
percent value relative to the pretreatment value.
Example 2
[0057] To each of pertussis toxin (PT), pertussis filamentous
hemagglutinin (FHA), pertactin (PRN, 69K-OMP) and pertussis
fimbriae (FIM) as obtained in Example 1, a half amount of a
saturated ammonium sulfate solution was added, followed by
sufficient stirring. After being kept standing at 4.degree. C. for
1 week, the mixture was again centrifuged; the resulting
precipitate was collected.
[0058] This precipitate was dissolved in a 0.025 M phosphate buffer
(pH 7.0) supplemented with 0.25 M sodium chloride to yield a
solution of pertussis toxin (PT), pertussis filamentous
hemagglutinin (FHA), pertactin (PRN, 69K-OMP) or pertussis fimbriae
(FIM). To each solution, a saturated ammonium sulfate solution was
added to a final concentration of 4.0 v/v %. To this mixture,
previously prepared, recovered aluminum hydroxide gel was added to
a final concentration of 0.4 mg/ml, followed by gentle stirring for
30 minutes at room temperature. After completion of the reaction,
the aluminum hydroxide gel was removed by centrifugation to yield
pertussis toxin (PT), pertussis filamentous hemagglutinin (FHA),
pertactin (PRN, 69K-OMP) and pertussis fimbriae (FIM).
[0059] Pertussis toxin (PT) content, pertussis filamentous
hemagglutinin (FHA) content, pertactin (PRN, 69K-OMP) content and
pertussis fimbriae (FIM) content were determined in the same manner
as in Example 1; and endotoxin content, in the same manner as in
Reference Example 1. The results are shown in Table 4.
4 TABLE 4 Endotoxin Content Active Ingredient (ng/100 .mu.g Protein
Content Recovery Sample protein) (.mu.g protein/ml) Rate* (%) PT
0.01 2999.0 82.5 FHA 0.11 9060.2 95.1 FIM 0.54 478.6 70.9 69K-OMP
0.08 3505.8 80.9 *Each figure for recovery rate represents a
percent ratio relative to the pretreatment value.
[0060] It is evident from this table that endotoxin was selectively
removed, with substantially no loss of any protective component,
the endotoxin content per 100 .mu.g protein/ml being not more than
1 ng/ml for all components.
Example 3
[0061] To each of pertussis toxin (PT), pertussis filamentous
hemagglutinin (FHA), pertactin (PRN, 69K-OMP) and pertussis
fimbriae (FIM) as obtained in Example 1, a half amount of a
saturated ammonium sulfate solution was added, followed by
sufficient stirring. After being kept standing at 4.degree. C. for
1 week, the mixture was again centrifuged; the resulting
precipitate was collected. This precipitate was dissolved in a 0.05
M phosphate buffer (pH 8.0) supplemented with 1 M sodium chloride,
after which it was dialyzed by the tube method using a 0.05 M
phosphate buffer (pH 8.0) supplemented with 1 M sodium chloride as
the external fluid, to yield a solution of pertussis toxin (PT),
pertussis filamentous hemagglutinin (FHA), pertactin (PRN, 69K-OMP)
or pertussis fimbriae (FIM). The concentrate dialyzate was
subjected to sucrose gradient density centrifugation on a sucrose
density gradient of 1-30 w/w % and at an R.sub.max of 64,900 G for
about 18 hours. After completion of the centrifugation, 34 w/w %
sucrose was fed into the rotor at a low rate of rotation to collect
fractions.
[0062] Pertussis toxin (PT) content, pertussis filamentous
hemagglutinin (FHA) content, pertactin (PRN, 69K-OMP) content and
pertussis fimbriae (FIM) content were determined in the same manner
as in Example 1; and endotoxin content, in the same manner as in
Reference Example 1. The results are shown in Table 5.
5 TABLE 5 Endotoxin Content Active Ingredient (ng/100 .mu.g Protein
Content Recovery Sample protein) (.mu.g protein/ml) Rate* (%) PT
0.04 231.2 82.5 FHA 0.01 849.4 79.3 FIM 0.20 49.1 80.3 69K-OMP 0.03
319.8 92.0 *Each figure for recovery rate represents a percent
ratio relative to the pretreatment value.
[0063] It is evident from this table that endotoxin was selectively
removed, with substantially no loss of any protective component,
the endotoxin content per 100 .mu.g protein/ml being not more than
1 ng/ml for all components.
Example 4
[0064] To the PT as obtained in Example 3, with addition of amino
acid such as Lysine, was added formalin to a final concentration of
0.4 v/v %, and after through mixing, was allowed to stand in an
incubator at 39.degree. C. for 21-35 days.
[0065] To each of FHA, 69K-OMP and FIM as obtained in Example 3,
was added formaline to a final concentration of 0.4 v/v %, and
after through mixing, was allowed to stand in an incubator at
39.degree. C. for 7 days.
[0066] Each of these components as treated above was dialyzed
against 4 mM phosphate buffer (pH 7.0) supplemented with 0.15M
sodium chloride to yield detoxificated PT, inactivated FHA,
inactivated 69K-OMP and inactivated FIM.
[0067] These detoxificated or inactivated components were blended
in several ratios shown in Table 6 and 7, and followed by addition
of aluminum chloride to a final concentration of 0.2 mg/ml to give
a vaccine respectively.
[0068] The results for the experiments of mouse intracerebral
potency with these blended vaccines, are shown in Table 6 and 7.
The experiments were performed according to the method of Japanese
Minimum Requirements for Biological Products (Association of
Biologicals Manufactures of Japan).
6 TABLE 6 Mouse intracerebral Protein content of respective potency
protective components 50% effective (.mu.g protein/ml) dose PT FHA
FIM 69K-OMP IU/ml (.mu.g protein) 10 40 0 0 10.9 1.06 20 30 0 0
13.4 0.89 20 40 0 0 11.6 1.18 20 50 0 0 13.6 1.18 20 80 0 0 12.6
1.81 30 40 0 0 19.3 0.85 40 40 0 0 18.7 1.04
[0069]
7 TABLE 7 Mouse intracerebral Protein content of respective potency
protective components 50% effective (.mu.g protein/ml) dose PT FHA
FIM 69K-OMP IU/ml (.mu.g protein) 25 25 0 0 19.5 1.18 25 25 5 0
26.0 0.98 25 25 0 10 24.1 1.18 25 25 5 10 19.3 1.60 25 50 0 0 24.1
1.47 25 50 5 0 22.2 1.70 25 50 0 10 23.7 1.68 25 50 5 10 24.8
1.71
[0070] It is evident from these figures that both inactivated
69K-OMP and inactivated FIM had small effects on the mouse
intracerebral potency, and no significant difference were observed
among the blended vaccines which contain more than 25 .mu.g
protein/ml of detoxificated PT.
Example 5
[0071] The experiment of mouse aerozol infection protecting potency
were performed with the blended vaccines as obtained in Example 4.
Each vaccine diluted to one-third was subcutaneously administered
to 4 week-old mouse respectively with 0.2 ml of each diluted one.
Four weeks later after the administration, each mouse was subjected
to airway infection with 18-323 phase I strain of Bordetella
pertussis by using the aerozol chamber, and 10 days later after the
infection, the abdomen of each mouse was opened and the trachea and
lung were picked out from each infected mice.
[0072] The specinen of each homoginized tissue applied to
Bordet-Gengou agar. The agar was cultured at 35.degree. C. for 5
days and the colonies of Bordetella pertussis were counted.
[0073] Based on the colony counts of the non-administered mice, the
protective dose was calculated.
[0074] The 75% protective dose was calculated in the case of
trachea, and the 50% protective dose was calculated in the case of
lung. Results were expressed in .mu.g protein.
[0075] And growth inhibitory rate was calculated in the high dose
administered group. Equation of the growth inhibitory rate was as
follows. 1 Growth inhibitory rate ( % ) = ( 1 - Colony counts of
High - dose administered mice Colony counts of non - administered
mice ) .times. 100
[0076] Results are shown in Table 8.
8 TABLE 8 Trachea Lungs 75% 50% Protein content of respective
protective Protective Growth Protective Growth components (.mu.g
protein/ml) dose inhibitory dose inhibitory PT FHA FIM 69K-OMP
(.mu.g protein) rate (%) (.mu.g protein) rate (%) 40 40 0 0 3.75
97.5 1.00 89.9 20 80 0 0 4.76 87.7 1.10 92.6 25 50 0 0 4.14 89.5
0.96 89.7 25 50 5 0 1.07 100 0.49 100 25 50 0 10 1.74 100 0.49 100
25 50 5 10 0.52 100 0.26 100
[0077] It is evident from this table that both inactivated 69K-OMP
and inactivated FIM ahad small effects on the mouse intracerebral
potency, but they showed protective effect on the aerozol infection
potency.
Test Example 1
[0078] Differences of adsorption performance between the calcium
phosphate gel (In-side Gel) forming and the hydroxyapatite gel
(Out-side Gel) on FHA and FIM.
[0079] Roux bottle stationary culture prepared by method described
in Example 1. The cell culture was concentrated to a one-tenth
volume using an ultrafiltration membrane, after which it was
centrifuged to separate the supernatant (Sample a) and cells. The
cells were dispersed in a one-tenth volume of 0.05 M phosphate
buffer (pH 8.0) supplemented with 1 M sodium chloride and stirred
well. It was kept standing at 4.degree. C. for 4 days, followed by
centrifugation to yield the eluted supernatant (Sample b) included
PT, FHA, 69K-OMP and FIM.
[0080] The culture supernatant (Sample a) and eluted supernatant
(Sample b) described above were treated as following 1) or 2).
[0081] 1) Calcium Phosphate Gel (In-side Gel) Forming Treatment
[0082] To the samples, a 1 M phosphate buffer (pH 8) was added,
followed by addition of a calcium acetate solution to a final
concentration of 0.5w/v %, 1.0 w/v % or 2.0 w/v % and gently
stirred at room temperature for 1 hour, followed by centrifugation
at 1000 rpm for 10 minutes to supernatant and gel residue. The
eluted solution was obtained by eluting the gel residue with a 0.1
M phosphate buffer (pH 8.0) supplemented with a 1M sodium
chloride.
[0083] 2) Hydroxyapatite Gel (Out-side Gel) Treatment
[0084] Hydroxyapatite gel (produced by BDH Chemicals Ltd) was
equilibrated with a 0.01M phosphate buffer (pH 8.0). The gel was
added to 20w/v %, 10 w/v % or 50w/v % to the sample volume and
gently stirred at room temperature for 1 hour, followed by
centrifugation at 1000 rpm for 10 minutes to seperate supernatant
from gel residue. The eluted solution was obtained by eluting the
gel residue with a 0.1 M phosphate buffer (pH 8.0) supplemented
with a 1M sodium chloride.
[0085] The content of FHA or FIM cotent in each sample was
determined by ELISA, with FHA or FIM as the house references. The
assay results are expressed in .mu.g protein/ml unit. Protein
content; Protein precipitated with heated trichloroacetic acid was
quantitated by the Lowry method, with bovine serum albumin
(Fraction V, produced by Wako Pure Chemical Industries) as a
refference. Results are expressed in .mu.g protein/ml unit.
[0086] Adsorption rate and recovery rate to the gel on FHA and FIM
were calculated by following equations respectively. 2 Absorption
rate ( % ) = ( 1 - Supernatant of post - gel treatment Pre - gel
treatment Sample ) .times. 100 Recovery rate ( % ) = Eluted
solution of post - gel treatment Pre - gel treatment Sample .times.
100
[0087] Results are shown in Table 9 and Table 10
9TABLE 9 a) Culture supernatant FHA FIN Absorp- Absorp- tion
Recovery tion Recovery rate (%) rate (%) rate (%) rate (%)
Concentration 0.5 89.7 69.6 0.0 0.0 of calcium 1.0 90.4 63.0 95.0
77.9 acetate added 2.0 89.5 44.4 94.3 98.4 (w/v %) Concentration
2.0 25.4 27.5 0.0 0.0 of 10.0 49.5 26.4 7.9 4.1 hydroxyapatite 50.0
90.8 48.9 22.1 5.3 added (w/v %)
[0088]
10TABLE 10 b) The eluted solution from the cell with 0.05 M
phosphate buffer (pH 8.0) supplemented with 1M-NaCl FHA FIN Absorp-
Absorp- tion Recovery tion Recovery rate (%) rate (%) rate (%) rate
(%) Concentration 0.5 96.3 87.2 18.7 12.5 of calcium 1.0 98.7 61.0
99.9 74.3 acetate added 2.0 98.7 55.1 99.8 94.3 (w/v %)
Concentration 2.0 10.8 1.0 4.6 1.1 of 10.0 4.7 3.8 9.7 3.9
hydroxyapatite 50.0 15.6 16.7 13.5 8.2 added (w/v %)
[0089] The calcium phosphate gel (In-side gel) strongly adsorbs
both FHA and FIM, but the hydroxyapatite gel has small adsorption
effect on the FIM. Also the Hydroxyapatite gel compared with the
calcium phosphate gel, has less adsorption effect on the FHA and
depend on the volume added.
Industrial Applicability
[0090] The method of the present invention is characterized by the
use of the same means of purification for all subject protective
components of Bordetella pertussis. Each component can therefore be
purified with high efficiency and high recovery rate, an aspect
very advantageous for industrial production. It is also possible to
efficiently produce an improved purified pertussis component
vaccine comprising an effective combination of pertussis
filamentous hemagglutinin (FHA), pertactin (PRN, 69K-OMP),
pertussis fimbriae (FIM) and pertussis toxin (PT).
* * * * *