Note: Descriptions are shown in the official language in which they were submitted.
BACKGROUND OF THE INVENTION
Field of the Invention
This invention relates to a method of producing
a tissue plasminogen activator (hereinafter referred
to simply as "TPA") and to a composition comprising
TPA. More particularly, the invention relates to a
method of producing TPA conveniently by which TPA is
stabilized by addition of albumin and also to ~ com-
position in which TPA maintains its activity stably.
even when preserved over an extended period or ~ime.
Description of th2 Prior Art
It is well known that urokinase, which can be
extracted and purified from human urine or a tissue
culture medium of kidney cells, is useful as a plasmi-
nogen activator. However, urokinase has the dxawback
that despite its capability of dissolving thrombus,
fibrinogenl ~2- plasmin and plasminogen lower urokinase
activity in blood, thereby inducing bleeding or tachyphy-
laxis when administered into the blood.
In recent years, it has been found tha~ TPA
which is free of the drawback of urokinase as a
plasminogen activator, exists in the internal organs,
vessel walls, body fluid of human beings ox animals,
tissue culture media of these cells or cancer cells, or
culture media o microorganisms having TPA-producing
efficacy by genetic engineering. There is thereforc
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a continuing need for the development of TPA as a drug.
TPA is different from urokinase in its immunolo~
gical activity and affinity for fibrin. TPA does not
react with an antibody specific for urokinase but combines
firmly with fibrin with the advantage that it develops
strong activity in blood as an activator in the presence
of fibrin. Accordingly, TPA not promoting the side-effects
which urokinase does is greatly expected to be a substitute
for urokinase, which exhibits suitable thrombus-dissolving
activity even in smaller amounts.
TPA has heretofore been produced by isolation
fxom internal organs, vessel walls, body fluid of
human beings or animals, ti sue culture media of these
cells or cancer cells, or culture media of micro-
organisms haviny TPA-producing efficacy by genetic
engineering. Known techniques for the isola~ion and
purification of TPA include salting-out, ion-exchange,
chromatography, gel filtration, hydrophobic chromato-
graphy, affinity chromatography and the like. Among
these techniques, affinity chromatography is ~he most
excellent in which use is made 9 as the ligand, of fibrin
[Biochimica Biophysica Acta, 621, 241 (1980)], arginine
[Thrombos Haemostas, 42, 414 (1973)~ and lysine [Arch.
Biochem. and Biophys., 189, 185 (1978)].
However, TPA is prone to decrease in its activity
as it is purified. Highly purified TPA can only exert
extremely reduced activity. Research has been conducted
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to find stabilizers which might prevent this reductiGn in
the activity of TPA observed during purification. Some
articles report that arginine [J.B.C. 254, 1998 (1979)],
gelatin [Biochemistry, 8, 79 (1969)] and fibrin IThrombos
Haemostas 45, 43 (1981)] are effective stabilizers for
protein.
Axginine and gelatin are still unsatisfactory in
their efficacy, whereas fibrin exhibits an excellent
stabilizing effect but is not pharmaceutically accepta-
ble. Therefore, these stabilizers are far from practi-
cally useful.
Under these circumstances, the present inventors
have made many studies of stabilizing TPA. Though
unstable in higher purity, TPA shows a substantial level
of s~ability in a crude enzyme solution of low purity.
In the studies leading to the present invention, it has
been discov~red that serum albumin is effective to
stabilize TPA in a TPA-containing crude enzyme solution
separated from a perfused ~wine ear. The stabilizing
effect of albumin on TPA has been almost equal to that
of fibrin and substantially high by over 100 times those
of gelatin and protamine sulfate which are known as
excellent stabilizers for urokinase.
SUMMARY OF TH~ INVENTION
Accordinglyp it is an object of the present
invention is to provide a method of producing TPA by
which albumin is added at an arbitrary stage of purify-
ing or drying TPA and which overcomes the difficulties
of the prior art techniques.
Another object of the invention is to provide a
composition comprising TPA and albumin in which the
aetivity of TPA is stably retained.
Thus the present invention provides in a broad
embodiment a proeess for producing a stabilized tissue
plasminogen activator composition, comprising the steps
of isolating a tissue plasminogen activator from a bio-
logical material to obtain a erude aetivator solution,
and admixing at least a stabilizing amount of albumin
into the solution to obtain a mixture.
In another embodiment the present invention pro-
vides a stabilized tissue plasminogen a~tivator eompo-
sition comprising, in admixture, an aetive tissue
plasminogen aetivator and albumin in an amount effeetive
for pxeventing activity~reduction in said aetivator.
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DETAILED DESCRIPTION OF THE PREFERRED EMBODIM:E:NTS
Albumin useful as a stabilizer for the practice '~
of the present invention may be of any origin, among
which albumin derived from human serum or placental
origin is preferably utilized.
The method of the invention can be suitably
applied to any processes for isolating and purifying TPA
from crude enzyme solutions which are prepared fxom
various starting materials. Such isolation and purifi-
cation processes include dialysis and ultrafiltrationwhich may be used singly or in combination, coupled with
affinity chromatography which is particularly preerable.
Albumin may be added directly to the crude enzyme solu-
tions or at any one sel~cted stage of the production of
TPA.
In the case where TPA originates from tissue
culture media of normal and cancer cells and organ extracts
~uch as those obtained from swine, these media and extracts
should be heated at 60C for abou~ 10 hours for removal
Zo of viruses. However, the heating treatment causes TPA
to be deactivated. It is therefore pre~erable to add
albumin prior to the heat treatment with the result that
albumin hardly affects the activity of TPA.
The amount of albumin to be added at the puri~
fication stage varies depending ~n the type of treatment
and is generally in the range of 0.001 to 10~ /v) based
on the crude enzyme solution.
TPA thus isolated and purifi~d is freeze-dried
or spray-dried to obtain a powdery product. This drying
treatment alsotends to deactivate TPA and hence needs
the addition of alb~min. In such instance, albumin is
added while taking account of the content in the final
product and is generally in the range of 0.003 to 5%
(w/v). If albumin which has been added at the purifica-
tion stage is present in excess in the final product,it is removed by any conventional technique to a suita-
ble extent of concentration and then dried to obtain a
powdery product.
When preserved over a long period of time, TPA
suffers a decrease in activity. However, the addition
of albumin prevents this activity reduction. The amount
of albumin to be added is in the range of 0.003 to 1%
(w/v). Albumin may be added at a step
of purifying, drying or powdering T~A, or freshly
to the final TPA product.
The composition according to the invention which
comprises TPA produced above and albumin is no~ only
much more stable than those compositions containing
other types of stabilizers, but also requires albumin
to be added in very small amounts. The composition of
the invention is thus excellent as a drug.
The above disclosure generally describes the
present invention. A more complete understanding ran
be obtained by reference to the ~ollowing specific
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examples wh:Lch are provided herein for purposes ofillustration only and are not intended to be limiting.
Example 1
Stability Test of TPA
(1) Stablllty in Solutions
To aliquots of a physiological saline aqueous
solution of TPA (60 U/mQ) were added the stabilizers
indicated in Table 1. The mixtures were allowed to
stand at room temperature for 1 or 2 days and provided
for measurement of the residual activities of TPA.
Titration was effected by the following method
both in this example and in the ensuing examples.
An agar-added fibrin plate was made using, as
a starting material, 75% clottable fibrinogen (made by
Miles Inc.: plasminogen content, about 140 casein units/g
of coagulated protein). The fibrinolytic activity of
TPA was determined in terms of its response-in comparison
to that of urokinase. A tissue activator solution was
diluted with a 0.067 M tris-hydrochloric acid buffer
solution (pI-I 8.0) containing 1% gelatin, 0.1 M sodium
chloride and 0.1% sodium azide and applied as a drop of
5 ~Q to the plate. The activity of TPA was culculated
from the dilution factor required to obtain the same
lysis zone as in the case with 5 ~Q of a urokinase
standard solution of 1.5 I~/mQ in the same buffer as
used for TPA.
The results are shown in Table 1.
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Table 1
. _
Resldual
Stabilizer E'inal Conc.(~) After After .
1 day 2 day
_ . _
Human serum 0.001 33 0
albumin 0.003 100 100
O . 01 100 100
O .1 100 100
_
Fibrin 0.001 75 33
0.003 100 100
O . 01 100 100
O . 1 100 100
. _ .
Gelatin 0.1 17 0
0.3 67 42
0.5 100 100
. _
Fibrinogen 0.1 100 100
Pro-tamine sulfate 0.5 75 33
Dextran T-40 1 25 0
Mannitol 3 25 0
.._ ........................ __ . __
Con-trol 16 0
(2) Stability on Freeze-drying
To aliquots of a physiological saline aqueous
solution of TPA (60 U/mQ) were added the stabilizers
indicated in Table 2, and the mixtures were adjusted in
their pH to 7Ø The samples were preliminarily frozen
at a temperature ranging from 40 to -50C for 3 hours
and then subjected to primary drying at a temperature
ran~in~ from -40 to -~30C under a vacuum of 0.4 xlO 3
to 0.6 xlO 3 mmHg for 2 hours and subsequently to
secondary drying at 30C under a vacuum of 0.1 x 10-3 to
0.2:x 10-3 mmHg for 3 hours. The residual activities
of the resulting powder were compared to determine -the
stabilizing effect of each of the test stabilizers.
The results are shown in Table 2.
Table 2
Stabilizer Final Conc. Residual Activity
_ . . . _ .
Human serum albumin 0 001 72
0.5 100
Gelatin 0 1 85
. _ _
Dextran 1 96
Protamin sulfate 0.5 58
Mannitol 3 83
. _ . . _ _ .
Control 71
(3) Stability on Virus-removing Treatment
To aliquots of a physiological saline aqueous
solu-tion of TPA (60 U/m~) were added the stabilizers
indicated in Table 3, and the mixtures were adjusted in
their pH to 7Ø The samples were thermally treated at
60C for 10 hours and measured in terms of their residual
activities for comparatively determining the stabilizing
effect of each of the test stabilizers.
The results are shown in Table 3.
Table 3
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Stabilizer Conc. (4) Residual Acltivity (~)
Human serum albumin O.001 30
0.003 34
0.01 62
O .1 100
0.5 100
_ _ _ . . ~
Gelatin 0 1 28
_ _
Control 23
Example 2
An ear of swine was perfused with Tyrode's solu-
tion and added intermittently with acetylcholine tabout
1 ug) to allow an activator of its vascular wall to be
released. The perfusion liquid was fractionated in a
fraction collector to collect a fraction of high fibrino-
lytic activity. As a result, there was obtained about
500 mQ of a solution per ear of swine.
100 ears of swine (400 g/ear on the average) were
perfused to obtain a solution of about 50 Q (3U/mQ).
The solution was added with 300 g/Q of ammonium sulfate,
adjusted in its pH to 7.0 and allowed to stand over-
night. The resulting precipitate was separated by
filtration using celite and charged into a column
(4 x 25 cm) along with the celite. The column was
washed with a 0.01 M phosphate buffer solution (pH 7.2)
containing 2 M ammonium sulfate and 1 M sodium chloride, b
whereupon an activator was eluted at a concentration
gradient of from the buffer solution just stated through
a 0.01 M phosphate buffer solution ~p~ 7.2) containing
1 M sodium chloride. The resul-ting eluate was found to
ha~e a volume of 2 ~, an activity of 50 U/m~ and a
specific activity of 125 U/A280. The solution was
subjected to adsorption on an octyl sepharose column
(2.5 x 10 cm), after which ethylene glycol was increased
up to about 50% by a concentration gradient technique to
elute an activator. The eluate had a volume of 3 Q an
activity of 25 U/mQ and a specific activity of 900U/A280.
One liter of the thus obtained solution was used
and aclmixed with human serum albumin at a rate of 100
g/mQ (0.01%), followed by adsorption on a fibrin
sepharose column (2 x 20 cm), suffieient washing with a
1 M sodium chloride solution and elution with a 0.005 M
phosphate buffer solution (pH 7.2) eontaining 0.01%
human serum albumin and 0.5 M arginine. The resulting
solution had a volume of 230 mQ and an activity of
110 IU/mQ. The solution was concentrated by means of
Diafilter A-15T ancl subjected to gel filtration on a
Sephadex G-150 column (3 x 100 cm) which had been equil-
ibrated with a 0.01 M phosphate buffer solution (pH 7.0)
eontaining 0.01~ human serum albumin, 105 M sodium
chloride and 0.01 M EDTA, thereby collecting an active
fraction containing the activator. The fraction was
dialyzed against a physiological saline solution. The
resulting solution had a volume of 70 mQ and an activit~
of 300 IU/mQ. The specific activity of the solut,ion
from which albumin added had been removed was found to
be 17,000 U/A230 (yield 42~).
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In the same manner, 1 Q of the solution was used
and treated wi-thout use of albumin. The solution had a
volume of 130 mQ, an activity of 80 U/mQ and a specific
activity of 12,000 U/A280 (yield 28%).
Example 3
Five kilograms of a heart of swine was ground in
a meat grinder and added with acetone (-20C) at a rate
of 2 Q/kg, followed by agitation in a belnder and
filtration. This defatting procedure was repeated
several times to obtain 700 g of an acetone-treated
powder. The powder was added with 800 mQ of a 0.3 M
potassium acetate solution (pH 4.2) per 100 g of powder,
followed by agitation in a blender, thereby extracting
a tissue activator at 4C for 3 hours. The resulting
extract was collected by centrifugal separation, and
the residue was re-extracted by addition of a 0.3 M
potassium acetate so]ution (pH 4.2) in an amount of
400 mQ/100 g. The re-extract was admixed with ammonium
sulfate at a rate of 300 g/Q, adjusted in its pH to 7O0
and allowed to stand overnight at 4C. The resulting
precipitate was collected by centrifugal separation,
dissolved in a 0.3 M potassium acetate solution (ph 4.2)
and adjusted in its p~ to 7.0 to obtain a crude enzyme
solution. The solution had a volume of 2 Q and an
activity of 300 U/mQ. The solution was added with
albumin according to the procedure of Example 2, purified
by affinity column chromatography of fibrin sepharose
and dialyzed using a 0.001 N hydrochloric acid solution
containing 0.9% sodium chloride, Thereafter, neutrali-
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7.ation was effected with use of an aqueous sodiumhydroxide sol.ution to obtain a solu-tion having a volume
of 1 Q,an activity of 350 U/mQ and a specific activity
of 8,000 U/~2~0.
Example 4
Blood was collected, using a sodium citrate solu-
tion as an anticoagulant, from healthy volunteers just
compressed vascularly by means of a haenadynamometer.
Immediately after being collected, the blood was centri- ,~
fuged at 3,000 rpm for 10 minutes to give 1 Q of blood
plasma in total. The plasma was added with benzamidine
and EDTA each in an amount of 5 mM, followed by adsorp
tion on a lysine sepharose column (3 x 25 cm) over each
of three divided portions. Each column was sufficiently
washed with a 0.005 M phosphate buffer solution (pH 7.4)
containing 0.6 M sodium chloride and eluted with a
0.005 M phosphate buffer solution (pH 7.4) containing
1.5 M sodium chloride. The resulting eluate was diluted
to a factor of three times with water and admixed with
albumin according to the procedure of Example 2,followed
by purification by affinity column chromatography of
fibrin sepharose and concentration by ultrafiltration.
The resulting solution had a volume of 4 mQ, an activity
of 30 U/mQ and a specific activity of 3,000 U/A280.
Example 5
Cells isolated from human tumor cells were
sufficiently grown in a DME medium to which 100 U/mQ of
penicillin G and 100 Y/mQ of streptomycin were added
and then in a medium admixed with 20% of FCS.
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Thereafter, the culture solution was cultivated in a
FCS-free medium at 37C for 30 hours. At this stage,
the resulting solution was found to have an activity of
about 10 U/mQ. One liter of the culture solution was
separated by centrifuge at 5,000 rpm for 20 minutes.
The resulting supernatant liquid was trea-ted in the same
manner as in Example 2 to obtain a final tissue activa-
tor having a volume of 80 mQ and an activity of 50 U/mQ.
The invention now being fully described, it will
be apparent to one of ordinary skill in the art that
many changes and modifications can be made thereto
without departing from the spirit or scope of the inven-
tion as set forth herein.
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