CA1189788A - Method of producing a tissue plasminogen activator and composition comprising same - Google Patents

Abstract

ABSTRACT OF THE DISCLOSURE

A method of producing a tissue plasminogen activator is disclosed which comprises adding albumin at a stage of purifying or drying the activator. Also disclosed is a composition comprising such activator and albumin in which the activator retains its activity stably and which is effectively useful as a drug.

Description

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 ~ 1 --.

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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 3`~

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 - 5a -.

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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 . .. ~ . ~
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--- ] l 7~

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.

Claims (15)

1. l. A process for producing a stabilized tissue plasminogen activator composition, comprising the steps of isolating a tissue plasminogen activator from a biological material to obtain a crude activator solution, and admixing at least a stabilizing amount of albumin into the solution to obtain a mixture.
2. 2. A process of claim l, further comprising heating said mixture at around 60°C for about 10 hours.
3. 3. A process of claim l, further comprising purifying said mixture to obtain the same in a pure form.
4. 4. A process of claim l, further comprising removing the amount of said albumin in excess of the effective amount for preventing activator activity-reduction.
5. 5. A process of claim 3, wherein the purification is carried out with said crude activator solution before adding said albumin.
6. 6. A process of claim 31 wherein the purification of said mixture is done by dialysis, ultrafiltration or affinity chromatography, or a combination thereof.
7. 7. A process of claim l, further comprising drying said mixture to obtain a powdered product.
8. 8. A process of claim 7, wherein said mixture is freeze-dried or spray-dried.
9. 9. A stabilized tissue plasminogen activator composi-tion comprising, in admixture, an active tissue plasminogen activator and albumin in an amount effective for preventing activity-reduction in said activator.
10. 10. A composition of claim 9, wherein the amount of said albumin is between 0.001 and 10% w/v based on the crude enzyme solution.
11. 11. A composition of claim 9, wherein said activator is of animal origin.
12. 12. A composition of claim 9, wherein said activator is of human origin.
13. 13. A composition of claim 9, wherein said activator is obtained from a tissue culture medium of normal, cancer or tumor cells.
14. 14. A composition of claim 9 in the form of a powder.
15. 15. A composition of claim 9, further comprising a pharmaceutically acceptable excipient.