Note: Descriptions are shown in the official language in which they were submitted.
"` ~2~ L)3
PROCESS FOR THE PREPARATION OF
A PLASMINOGEN ACTIVATOR
FIELD OF THE INVENTION
;This invention relates to a process for the preparation
of a plasminogen activator in a high yield by ~he use of
normal diploid cells derived from human bodies.
BACKGROUND OF THE INVENTION
Urokinase separated and purified from urine or
cultured kidney cells and streptokinase recovered from
streptococci are nowadays practically use~ as Elasminogen
activators. Specifically, these plasminogen activators
are used as thrombolytic agents.
However, it is known that these plasminogen
activators are often administered to patients in large
amounts in order to obtain necessary therapeutic effects
overcoming their poorness in affinity with fibrin and thus
cause side effects such as internal hemorrhage.
Specifically, plasmin which is produced in circulating
~lood by these plasminogen activators tends to lose activity
immediately upon combination with plasmin inhibitors in
the blood. Accordingly, in order to exhibit the neces.sary
therapeutic effects, these plasminogen activators must be
administered in large amounts to produce Flasmin, viz,, in
an amount exceeding that of the plasmin inhibitors in the
1206903
blood. However, the production of a large amount of
plasmin will decompose fibrinogen, resulting in hemorrhage.
Accordingly, if a plasminogen activator which has a hlgh
affinity with fibrin and is capable of producing plasmin
on fibrin can be obtained, it becomes possible to deco~pose
fibrin with a small amount of the plasminogen activator
without being subjected to the influence of plasmin inhibitors
in circulating blood, and also to reduce the effects ~f
decomposing fibrinogen. Therefore, it has been desired to
provide a thrombolytic agent having a high affinity for
f~brin which shows a high thrombolytic activity ~sing a
small amount thereof, and which has less side effects.
One of the inventors, with others, has already
founa a novel plasminogen activator having properties
listed below in a culture liquid of normal diploid cells
or human bodies and endeavored to put it in practical use
(European Patent Application (OPI) No. 0100982).
a) molecular weight: 63,000 + 10,000
b) isoelectric point: 7.0 to 8.5
c) affinity to fibrin: present
d) affinity to Concanavalin A: present
e) optimum pH: 7.0 to 9.S
f) no reactivity with anti-urokinase specific antibody.
However, because of low productivity, this
plasminogen activator has been difficult to provide in
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large amounts on an ~ndustrial scale.
SUL~MAP~Y OF THE INVENTION
As a result of concentrated-studies to find an
efficient process for the preparation of the plasminogen
activator, if has now been found that the presence of a
large amount of peptone derived by the enzymatic decompo-
sition of animal meats in a solution which produces the
plasminogen activator upon contact with normal diploid
cells (i.e., cells having a normal diploid chromosome)
1~ derived from human bodies causes a drastic increase of
the production of the activator. The present invention
has been achieved on the basis of this discovery.
This invention is therefore a process for the
preparation of a plasminogen activator which comprises
bringing normal diploid cells derived from human bodies
capable of producing a plasminogen activator into contact
with a solution containing an enzymatically-decomposed
animal meat peptone.
BXIEF DESCRIPTIO.~ OF THE DRAWINGS
Fig. 1 is a graph showing the optimum pH of a
plasminogen activator obtained by the inventlon; and
Fig~ 2 is a graph showing the results of the
measurement of the yield of the plasminogen activator in
the culture liquid in Example 2.
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DET~ILED DESC~IPTION OF TH~; INVE~ITION
The present invention is achieved by the use of
normal diploid cells derived from human bodies capable of
producing the plasminogen activator. As such normal diploid
cells, there can be used, for example, cells derived from
human kidney, intestines, lung, heart, ureter, skin, foreskin,
tongue, thyroid gland, placenta and womb and cells derived
from the whole embryo, more preferably cells derived from
human lung or foreskin. The cells derived from human
bodies described above herein include cells derived from
fetus and neonate.
rlhese cells can be proliferated in accordance
with a normal method for the culture of animal cells, for
example, as described in P.K. ~ruse and M.K. Patterson,
Tissue Culture Methods and Applications, pp. 220-223,
Academic Press, ~ew York,San Francisco, 1973, and thereafter
they can be brought into contact with a solution containing
a carbon source, a nitrogen source, and, if necessary,
inorganic salts and/or other additives, to produce the
plasminogen activator. ~s additives which are allowed to
coexist in the solution, there can be used amino acids,
vitamins, peptides, saccharides, and organic acids.
Examples of such additives include the natural amino acids,
p-aminobenzoic acid, D-biotin, calciferol, calcium D-
panthotenate, cholesterol, choline chloride, folic acid,
lZ069(~3
i-inositol, menadione, nicotinamide, nicotinic acid,
pyridoxal, pyridoxine, riboflavin, thiamine, DL-~-tocopherol,
~een 30 (trademark of ~ao Atlas for polyoxyethylene
monooleate), vitamin A, adenine, ATP, ~lP, deoxyribose,
6 ribose, glutathione, guanine, thymine, hypoxanthine, uracil,
xanthine, hydrolyzate of lactalbumine, polypeptone, hydrolyzate
of casein, glucose, maltose, fructose, mannitol, aextran,
fumaric acid, malic acid, oxalacetic acid, citric acid,
succinic acid, pyruvic acid, NaCl, KCl, l~gC12, I~gSO4,
2 4' 2 P4/ ~H2PO4, CuSO4, Fe(NO3)3, FeSO4 ~InCl
(~IH4)2~1OO4, and ZnSO4. The addition of the enzymatically-
decomposed animal nleat peptone in accordance with the
present invention can drastically improve the yield of the
plasminogen activator.
Mass culture on an industrial scale according to
the present invention can be achieved by the use of roller
bottle culture process, a multi-layer plate culture process,
hollow fiber culture process, plastic bag culture process,
and microcarrier culture process, as described in R.T. Acton
and J.D. Iynn, Cell Culture and Its Application, pp. 191-216,
Academic Press, `.~ew York, 1977. For mass culture on a
greater scale, a microcarrier culture process is desirable.
As the peptone used in the present invention
there may be employed so-called proteose peptone, protease
peptone and meat peptone, which are typical bacterial culture
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medium. The process for the preparation of the peptone
is ~ell known in the art and can be performed in accord-
ance with the method described in Study of Bacterial
Culture ~edium, Vol. 2, by Toshikazu Sakazaki, published
by Uaya Bookshop Co., Ltd., 1967. Examples of animal
meats that can be used include internal organs of cattle,
pig, chicken, sheep and whale, among which beef is most
often used. Examples of the decomposition enz~me are
trypsin, papain, pepsin and pancreatin. These animal
meats are minced, mixed with water, and then adjusted
with sodium carbonate or concentrated hydrochloric acid
to the p~; value suitable for the enzymatic decomposition
Tne pEi in the enzymatic decomposition varies depending
upon tne kind of enzyme used, and it is generally 7 to 9
for trypsin, 5 to 7 papain, 2 to 4 for pepsin and 6 to
for pancreatin. Then an enzyme is added to the mixture.
The mixture is then subjected to an enzymatic decomposition
at a temperature of 20 to 40C for 1 to 20 days, usually
at a temperature of 37C for 2 to 3 days. After being
digested, the mixture is heated to a temperature of 100C
or more to inactivate the decomposition enzyme and
coagulate the undigested protein. 'l'he undigested protein
thus coagulated is removed by filtration, and the filtrate
is concentrated, dried, and pulverized. '~he concentration,
drying and pulveriæation can be achieved by boiling and
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pulverizing or by concentrating at a low te~perature in
a vacuurn device and pulverizing. ~xamples ~f commercially
available peptone are Proteose Peptone, Proteose Peptone
No. 2, Proteose Pep~one No. 3, and Thiopeptone from Difco,
Proteose Peptone and Peptone PL 46 from Oxoid, Thiotone
from BBL and Proteose Peptone from ~aigo ~iyo Kagaku Co.,
Lta.
The added concentration of the peptone varies
depending upon the type and concentration of cells employed,
1~ and aMinO acids, vitamins, peptides, saccharides and
organic acids which are allowed to coexist t?herewith, but
is preferably fxom ~.1 to 4% twt/V) and more~ preferably
0.1 to 2% ~wt/v).
As described above, t~ere can be used as suitable
peptone various peptones which are different in preparation
method. These peptones can be used singly o~ in combination.
These peptones must be sterilized before being
added to the solution. The sterilization can be achieved
by directly sterilizing the peptone pow~er with ethylene
oxide or y-ray, by sterilizing a solution of the peptone
powder in an autoclave, or by passing a solution of the
peptone powdex through a steriliza~ion filter.
The sterilization method is not specifically limited but
is preferably achieved by heating at a tempe~ature of
120C in an autoclave for 10 to 6~ minutes.
-` 121~69(~3
The production of the plasminogen activator is
normally carried out in 0. 2 ml or more o~ a culture liquid
per 100,000 cells at a temperature of 25 to 40C, and
preferably at 31 to 37C, in the pI; range o~ 6.0 to 8.0,
and prefera~ly 7.0 to 7.a The maintenance of the above
pH value can be achieved by tne use of a buffer system
of CO2/HCO3. ~owever, if the cells produce a large amount
of CO2lor organic acid such as lactic acid which obstructs
the maintenance of the above p~ value, a buffer such as
E'EP~S (N-2-hydroxyethylpiperazine-.~'-2-ethane sulfonic
acid~ may be used. The production normally takes from
4 to 30 days, but may take more than 30 days. Since the
production rate of the activator of the present invention
is graaually decreased in the latter half of the production
stage/ the industrial production process employs the
number of days which, are the most efficient for the total
production. The plasminogen activator is released from
the cells into the c~lture liquid under the above.conditions.
Tne measurement of the yield of the activator was carried
out as follows: .
An ayar-added fibrin plate prepared from a 95%
coagulated fibrinogen (,plasminogen content: about S0 casein
unit/y coagulated protein) was used to conduct a plate
methodusing urokinase as a standard r.laterial. The liquid
of the plasminogen activator was diluted with 0.067 M
. lZC~69(~3
tris-~CQ buffer solution (pl~ 8.Q~ containing 1~ gelatin,
O.1 M sodium chioride and 0.1~ sodium nitride. The
concentration of the liquid of the activator showing
the same dissolution window as 10 IU/ml of urokinase on
the fibrin plate was set at 10 U/ml. When the measurement
was made of the solution containing urokinase, an anti-
urokinase IgG obtained from rabbits was added to the test
solution so that the concentration thereof was 100 ~g/r.ll.
~hen the desired yield or time is reached, the culture
liquid is collected to recover the activator.
The recovery of the Plasminogen activator can be
achieved by any one of or a combination or an adsorption
process, salting-out process, dialysis process, chromato-
graphy process and gel filtration process, which are normally
applied for recovery of protein. Examples of such purifi-
cation processes are fibrin Sepharose*column chromatography
using Sepharose*having fibrin bonded thereto, CM Sepharose*
column chromatography using Sepharose*having carboxymethyl
group bonded thereto, lysine Sepharose*column chromato-
graphy using Sepharo5e*having lysine bonded thereto, ligandexchanginy chromatography using zinc-chelated Sepharose*
lectin column chromatography using Sepharose*having
Concanavalin A bonded thereto, antibody affinity chromato-
graphy using antibody which specifically combines with
the plasminogen activator, and gel filtration using
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~Z069(D3
crosslinked dextran particles.
.~s an example of the purification process, the
tissue culturP liquid is adsorbed into a CM cephalose*
column equilibrated with a 20 mM acetate buffer solution
(pH 4.0~ containing 0.1% Tween*80 and 0.15 M sodium
chloride. After being washed with a 20 mM acetate buffer
solution (pE 4.0) containing 0.1% Tween*80 and 0.15 M
sodium chloride, the column is treated with a 20mM tris-HCQ
buffer solution (pH 8.9~ containing 0.1~ Tween*80 and 1 M
sodium chloride to effect an elution; whereby the solution
of the part having the plasminogen activator activit~ is
collected. The solution thus collected is dialyzed over-
night at 4c a~ainst a 20 mM tris-HCQ ~uffersolution containing
0.1 M potassium rhodanide, 0.1% Tween*80, and 0.05 M
sodium chloride. The solution thus dialyzed is adsorbed
into a lysine Sepharosé*COlUmn equilibrated with the same
buffer solution. After being washed with an equilibrated
buffer solution, the column is treated with a 20 mM tris-
HCQ buffer solution containing 0.05 M sodium chloride,
1 M potassium rhodanide, 0.2 M ~-amino-n-caproic acid and
0.1% Tween*80 to effect an elution. The solution thus
eluted is concentrated through hollow fibers for ultra-
filtration, and then gel-filtrated through a column of
Sephacry~ S-200 to obtain the desired plasminogen activator.
The physicochemical properties of the plasminogen
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activator thus obtained are illustrated below:
a) molecular weight: 63,000 + 10,000
The molecular weight measurement was effected by
means of a gel filtration process using Sephadex*G-150
equilibrated with a 0.01 ~l phosphate buffer solution
~pH 7.0) containing 1.5 II sodium chloride, 0.1 M E~TA,
0.1 M arginine and 0.1% Tween*~0. Tl1e measurement of
molecular weight in unreduced state by the S~S (sodi~
dodecysulphate~ electrophoresis process indicated a
molecular weight of about 70,000.
b) isoelectric point: 7.0 to 8.5
. An isoelectric point electrophoresis process using
ampholyte was applied to effect a fractionation at the
isoelectric point, wnereby the isoelectric point was
5 measured.
c) affinity to fibrin
To 950 ~1 of a 0.2~ plasminogen-free fibrinogen
solution in physiological saline was added 20 ~1 of a 500 U/ml
plasminogen activator solution obtained by the present
invention. The admixture was allowed to stand at room
temperature for 1 hour. The resulting fibrin was separated
an collected from the admixture, dehydrated, and washed
with physiological saline. The extraction of the activator
in fibrin with 1 ml of a 2 M ammonium rhodanide solution
showed ~hat about 70% of the activator had been incorporated
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12069(~3
into fibrin. On the other hand, the tissue culture urokinase
was not incorporated into fibrin at all.
d) affinity to Concanavalin A
2 ml of the plasminogen activator (30 U/ml) obtained
by the present invention was dissolved into physiological
saline. The solution thus prepared was adsorbed into
a column ~0.5 x 4 cm) filled with Concanavalin A~epharose*
(manufactured by Pharmacia). The column was washed with
a 1 M sodium chloride, with the result that almost 100%
of tIle plasminogen activator was adsorbed.
e) optimum pH value: 7 to 9.5
50 ~1 of the solution of the plasminogen activator
in physiological saline was mixed with 50 ~1 of a 8 CU/ml
-physiological saline solution of plasminogen containing
10% glycerin and 100 ~1 of various buffer solutions conta~ng a
0.10 M sodium chloride; the buffers being a 0.05 M citrate buffer
solution ~pH 5.0 or 6.0), a phosphate buffer solution (pH 6.0, 7.0 or
8.0) and a glycine-sodium hydroxide buffer solution (pH 8.0,
9.0, ~0.0 or 11.0) ~i.e., seven buffers, each at a different
pH of 5.0, 6.0, 7.0, 8.0, 9.0, 10.0 or 11.0).
The admixtures were preincubated at a temperature of 37C
for 30 minutes. To the admixtures thus pre-incubated were
added 500 ~1 of Boc-Glu-Lys-~ys-.~CA dissolved in a 0.15 M
tris-~iCQ buffer solution (pH 8.0). The admixtures were
fuxther incubated at a temperature of 37C for 15 minutes.
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1 ml of acetic acid was added to the admixtures thus
incubated to stop reaction. ~he resulting aminomethyl-
cumarine was measured by fluorescence-process to determine
the optimum p~ value. Tne results are shown in Fig. 1.
f) reactivity with anti-urokinase specific antibody
~abbits were immunized by tne in~ection of purifi~d
urokinase (specific activity: 150,000 IU/mg protein~ with
Freund's complete ad~uvant every 7 days in 35 days.
Elood was collected from the rabbits and purified to
obtain 50 ~g/ml of a solution of anti-urokinase specific
antiboay. The solution of the anti-urokinase specific
antibody and a 20 U/ml solution of plasminogen activator
obtained by the present invention were mixed with each
other in a mixing ratio of 1:1. The activity of the
admixture was measured according to the above mentioned
method. As a result, no activity drop was recognized.
On the contrary, the urokinase activity of the
mixture of the solution of the anti-urokinase antibody
and a 20 IU/ml urokinase solution as a control was 100
inhibited.
As described above, the plasminogen activator
obtained by the present invention does not react with
anti-urokinase antibody.
The plasminogen activator thus obtained can be
applied not only for medicines as thrombolytic agent but
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also for chemicals for prevention of thrombus adapted
to be bonded to artificial vein, artifioial intestines
or the like and diagno~tic medicine or thrombus.
The process of the present invention is well
suited for ~he stable mass production on the industrial
scale of plasminogen activator which proves effective
with less amount than familiar urokinase or streptokinase
and has higher thrombolytic activity and less side effects
than urokinase or streptokinase.
3,0 This invention will be further illustrated in
the following examples:
E~PLE
500 g of well-minced beef was put into 1,000 ml
of purified water. 12.5 ml of concentrated hydrochloric
acid was added to the admixture. 6 g of,pepsin was then
added to the ad~ixture. The admixture was allowed to
effect digestion at a temperature of 37C for 2 days while
being,shaken occasionally. This digested li~uid was heated
to a temperature of 100C for 5 minutes. The liquid was
filtered, and the filtrate was treated with sodium hydroxide
to obtain pH value around neutrality (i.e., about 7.0).
While maintained at a temperature of 45C, the liquid was
concentrated by means of a rotary evaporator. The liquid
thus concentrated was then dried to obtain ~30 g of a
light-yellowish brown pepsin-decomposed bee peptone.
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~2069C?3
Ne~t, the e~fect of the pepsin-decompo~ed beef
peptone over normal diploid cells derived from human fetus
lung (manufactured by Flow Laboratory~ on the promotion
of the production of the plasminogen activator was studied.
A plastic laboratory dish (diameter: 100 mm) was planted
with the normal diploid cells in a density of 7 x 104
cells/ml. 10 ml of Medium MEM (minimum essential medium:
see Eagle H., Science, 130, 432, C1959)) containing 10%
fetal calf serum was added to the dish as a culture medium.
The dish thus prepared was then treated in air containing
5% carbon ~io~idc at a temperature o~ 37C to effect a
full proliferation. The dish thus treated was washed with
physiological saline. ~0 ml of ~ledium 199 containing 1%
(wt/v) of pepsin-decomposed beef peptone was added to the
dish. The contents of the dish was sampled every 5 days
for the measurement of the activity of the plasminogen
activator.- ~he results are shown in Table 1 along with
that of a control experiment free of the pepsin-decomposed
beef peptone. ThR medium containing the pepsin-decomposed
beef peptone showed higher yield than that free af the
peptone.
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Table
Yield (U~mI)
25 (day)
Medium containing 8 18 32 39 47
1~ (wt/v) peptone
l~edi~un free of 0,3 0,~ 1 1.2 1.5
peptone
EXAMPLE 2
Normal diploid cells derived from human fe,us
lung and human fetus foreskin tmanufactured ~y Flow Laboratory)
were proliferated as in Example 1. To these proliferated
diploidc,ells was added Medium MEM with different concentrations
of Proteose Peptone No. 3, which is a pepsin-decomposed
animal meat peptone commercially available from Difco.
The diploid cells were maintained in air containing 5%
carbon dioxide,at a temperature of 37C for 10 days.
The results of the measurement of the activity of the
plasminogen activator in the culture medium are shown in
Fig. 2. A rapid increase of yield appears in the peptone
content range of from 1 to 4% (wt/v).
E~PLE 3
-
In this exarnple, the effect of different enzymatically-
decomposed animal meat peptones over normal diploid cells
derived from human fetus lung or human fetus foreskin on
the promotion of the production of the plasminogen activator
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was studied.
84 g or pepsin-decomposed whale meat peptone
was prepared from 500 g of whale meat as in E.~ample 1.
110 ~ of pancreatin-decomposed beef peptone was
obtained as follows:
500 g of minced beef was put into 1,000 ml of
purified water. The admixture was treated with sodium
carbonate to obtain a pEI value of about 8. 15 g of
pancreatin was added to the admixture. The admixture was
then allowed to effect digestion at a temperature of 37C
for 2 days. The liquid thus digested was treated as in
Example 1 to obtain the peptone desired.
Various Medium 199 containing 1% by weight of
these peptones or various commercially available enzymatically-
decomposed animal meat peptones were prepared. The abovenormal diploid cells were proliferated as in Example 1.
To the diploid cells thus proliferated were added these
culture mediums respectively. These culture medium were
maintained in air containing 5% carbon dioxide at a
temperature of 37C for 7 days to produce the plasminogen
activator. The results of the measurement of the yield
of the activator are shown in Table 2.
As control, the hydrolyzate of lactalbumin and
polypeptone which had been not made from animal meats were
used. Although these materials proved effective in the
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promotion of the production of the activator, the peptones
made from animal meats proved far more effective.
Tabl e 2
Yield (U/ml)
: Human fetus Human fetus
Additive lung foreskin
Nothing added 0.4 0,3
l~(wt/v) Pepsin-~ecomposed 14 3 12 6
beef peptone -
1%(wt/v) Pepsin-decomposed lO 6 14 2
whale meat peptone
1%(w~/v) pancreat.in-ll 2
decomposed beef peptone 15.6
1%(wt/v) Proteose Peptone 12.4 14.4
No. 2 (Difco) 4.8 9.6
No. 3 ~Difco) 15.6 15.2
1%(w~/v) Proteose Peptone 4 9 4.6
(Daigo Elyo)
1%(wt/v) lactalbumin 2.8 2.4
hydrolyzate (Difco)
1%~wt/v) Polypeptone 4.8 5.4
(Daigo Eiyo)
EXAMPLE 4
In this example, the effect of the pancreatin-
decomposed beef peptone over various cells on the promotion
of the production of the plasminoaen activator is illustrated.
~2069(~3
Specifically, ~Jarious cells were subjected to
- a full proliferation in a plastic laborato~y dish (dia~.eter:100mm)
according to the method of Example l. The medium of the
cells thus proliferated was replaced by Medium l99 to
which 1% lactalbumin hydrolyzate or 1% pancreatin-
decomposed beef peptone had been added. The medium was
maintained in air containing 5% carbon dioxide at a
temperature of 37C for 7 days. The results of the measure-
ment of the yield of the activator are shown in Table 3.
Table 3
1% pancreatin-
Nothing l~ lactalbumin decomposed
Cells addedhydr~lyzatebeef peptone
Human fetus 1ung 0.4 14.0 25.0
human fetus 0 22.4 18.8
foreskin
lS Eluman fetus 0 O.l 3.0
kidney
Human fetus skin 0 0.2 3.6
Human fetus small
intestines (Flow 0.2 l.6 5.4
Laboratory)
EXAMPLE 5
A 12 liter spinner flask was planted with human
fetus lung cells of a density of lO cells/ml along with
Cytouex I(beads carrier for cell culture; Pharmacia's
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registered trademark) of a concentration of 3 mg/ml.
8 liters of Medium MEM containing 10% fetal calf serum
as a culture me~i~m was added to the flask. The flask
thus prepared was treated in air containing 5% carbon
dioxide at a temperature of 37C while being rotated at
a speed of 30 rpm to effect a suspension culture. The
culture was effected for 6 days so that the cells were
fully proliferated. The beads carrier to which the cells
had ahered was washed with physiological saline and
replaced by 8 liters of Medium 1~9 containing 1% serum~free
Proteose Peptone Wo. 3 (Difco). Another culture was
effected with stirring at a rotation speed of 30 rpm.
The medium liquid containing the plasminogen activator
of the present invention was then recovered at the time ,
of the replacement of the medium every 5 days.
10 liters of the,thus obtained medium liquid containing
the activator at the concentration of 35 U/ml
was absorbed into a CM SePharse*column (1.5~ x 10 cm)
equilibrated with a 20 m~l acetate buffer solution (pH 4.0)
containing 0.1% Tween*80 and 0.15 M sodium chloride.
After being washed with a 20 ~1 acetate buffer solution
(pH 4.0) containing 0.1% Tween*80 and 0.15 M sodium chloride,
the CM Sepharose*_olumn was treated with a 20 mM tris-~CQ
buffer solution (pH 8.9) containing 0.1% Tween*80 and 1 M
sodium chloride to effect an elution,whereby the solution
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of the part having the plasminogen activator activity was
recovered in an amount of 103 ml. The solution thus
recovered was dialyzed overnight at a temperature of 4C
against 5 liters of a 20 mM tris-HCQ buffer solution
containing 0.1 M potassium rhodanide, 0.1~ Tween*80, and
0.05 ~1 sodium chloride. The solution thus dialyzed was
adsorbed into a lysine Sepharose*column (2.6~ x 12 cm)
equilibrated with the same buffer solution. After being
washed with an equilibrated buffer solution, the lysine
Sepharose*column was treated with a 20 mM tris-HC~ buffer
solution containing 0.05 M sodium chloride, 1 M potassium
` rhodanide, 0.2 M ~-amino-n-caproic acid and 0.1% Tween*80
to effect an elution. 130 ml of the liguid thus eluted
was concentrated to 12 ml through hollow fibers for ultra-
filtration. The liquid thus concentrated was gel-filtered
through a column (2.6~x 94 cm ) of Sephacryl*S-200 to recover
45 ml of the solution of the part having the plasminogen
activator activity of the pr~sent invention. The plasminogen
activator solution thus obtained had a concentration of
4, sao U/ml and showed a specific activity of 38,000 U/mg
protein.
~Jhile the invention has been described in detail
and with reference to specific embodiments thereof, it will
be apparent to one skilled in the art that various changes
and modifications can be made therein without departing
from the spirit and scope thereof.
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