JPS59110625A - Plasminogen activating factor, manufacture and product for manufacture - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

[Detailed Description of the Invention] [Technical Field] The present invention relates to tissue plasminoken activator and/or
or a precursor thereof, a novel precursor of tissue plasminogen activator, and a novel tissue plasminogen activator affinity reagent, and The present invention relates to a pharmaceutical preparation comprising a plasminogen activator produced by the above method and/or a precursor thereof.

[Background of the invention]

Plasminogen activators are substances that produce plasmin by acting on plasminogen (plasmin precursor). Plasmin then acts on fibrin (or blood clots), causing it to liquefy or dissolve.

Plasmin also causes the lysis of fibrinogen, the precursor of fibrin.

The above effects play an important role in the natural fibrinolytic system. They also use plasminogen activators for therapeutic purposes to manage thrombotic diseases or other conditions in which it is desirable to generate local fibrinolytic or proteolytic activity via the plasminogen activation mechanism. enable it to be used.

Two human plasminogen activators are in widespread use. The first is a bacterial protein, streptokinase, which acts by a non-proteolytic mechanism. The second is
Urokinase, a protease obtained from urine or cultured kidney cells, is known to be present in many other mammalian tissues. These two compounds have the therapeutic disadvantage that their action is not limited to fibrin-ζ cooperating sulplasminogen in blood clots. These act on plasminogen, which is generally present in the circulation, resulting in widespread generation of plasmin leading to extensive dissolution of fibrinogen, the precursor of fibrin. This then causes a bleeding condition as a result of the inability of normal blood clotting. Furthermore, urokinase has the drawback of high isolation and preparation costs and only low yields obtained by currently used methods. Since streptokinase is a foreign protein, it causes an immune response in patients. The antibodies produced neutralize the action of streptokinase on plasminogen,
thus reducing its therapeutic efficacy.

Generally called "tissue plasminogen activator",
The existence of a third plasminogen activator, hereinafter abbreviated as 1'-TPAJ, is known. This enzyme is identical or indistinguishable from an enzyme that is found in many large tissues and is a secreted product unique to large melanoma cells cultured in vitro. Although TPA catalyzes the proteolytic activation of plasminogen in much the same way as urokinase,
It differs from urokinase in a number of important respects. first,
These two enzymes are chemically dissimilar. These have different molecular weights and do not react with each other's antibodies.

The catalytic activity of TPA is enhanced over fibrin, but this is not the case with urokinase. TPA has the important property of binding fibrin, but not urokinase.

These facts are summarized in a recent paper entitled "Purification and characterization of plasminogen activator secreted by cultured large melanoma cells" by Deingemann, Rieken et al. [Journal of Biological Chemistry (J. Bio
Chem, Vol. 256, No. 13, No. 7035-4
041 page].

The article also describes the production of TPA from cultured human melanoma cell lines and normal human tissue.

The purification method consists of sequential chromatography using zinc chelate agarose, concanavalin A agarose and Sephadex G-150 in the presence of detergents.

The propensity of TPA to bind fibrin, its significantly enhanced fibrinolytic action in the presence of fibrin, and its inefficient plasminogen activator action in the absence of fibrin compared to urokinase and streptokinase
TPA as the plasmid of choice for the treatment of human thrombosis/
- It is a gene activator. fibrin and TP
The interaction of A largely localizes plasmin production to the clot site, reducing or avoiding the consequential consequences of plasminogen activation observed when using urokinase or streptogenase. Furthermore, streptokinase is an immunochemically foreign protein to humans, whereas TPA is not.

A recent report entitled "Specific lysis of iliac thrombus by administration of exogenous (tissue type) plasminogen activator" by Weimar et al.
9g, Nov. 7, 1, p. 1018] demonstrates the clinical utility of TPA in the management of human thrombosis.

However, no method for easily producing TPA on an industrial scale has been known so far. The precursor of TPA has not been identified or isolated, nor is it known. In fact, conventional methods for also producing TPA are characterized (according to the teachings of this invention) by no or substantially no product or precursor. Until now, no substance was known that had a plasminogen activation effect more localized than that of TPA itself.

[Summary of the invention]

An object of this invention is to provide a new and relatively simple method for producing TPA.

Another object of this invention is a new substance in itself, TP
TPA precursors with superior therapeutic properties than A itself (
The purpose of the present invention is to provide a method as described above suitable for producing a pro-TPA (hereinafter abbreviated as pro-TPA).

Another object of the present invention is to provide a method as described above which can separate TPA and proTPA from urokinase and which can be adapted to separate and collect urokinase if desired.

Another object of the present invention is to provide a novel formulation consisting of pro-TPA.

Yet another object of this invention is to provide the above method and TPA
and/or to provide novel affinity reagents suitable for selective adsorption of POTPA.

In relation to the above facts, according to one feature of the invention:
This invention provides a method for dissolving dissolved TPA and/or pro-TPA.
and contaminants in the aqueous medium containing EryLhrina latissima.
and other Erythrina plant species, consisting of immobilized KuniLz-type inhibitors that have properties as trypsin, plasmin and TPA inhibitors, but have no effect on urokinase. intimate contact with the reagent, thereby selectively adsorbing TPA and/or proTPA from solution, and contacting the affinity reagent with adsorbed TPA and/or proTPA with the aqueous medium and non-adsorbable contaminants. Released from T
Desorb the PA and/or proT'PA from the affinity reagent, and remove the desorbed TPA and/or proT''PA
TPA and/or ProT consisting of Kokichi who collects
A method for purifying PA is provided.

The seeds of Erythrina latissima (a broad-leaved plant of the genus Erythrina) and other plant species of the genus Erythrina are known to contain two proteinase inhibitors [François et al.
Schubert et al.
Hem.), Vol. 362, pp. 531-538]. These are described as DE-1 and 0E-3 in the above literature. DE-1 inhibits bovine chymotrypsin but not bovine trypsin, whereas DE-3 inhibits trypsin but not chymotrypsin. One of them,
In other words, DE-3 acts as a knit-type phagocytosis inhibitor.
It also has properties as a trypsin, plasmin and TPA inhibitor, but has no effect on urokinase. As a result of screening several enzyme inhibitors, DE-3 was selected as the only substance with the ability to discriminate between TPA and urokinase. If this inhibitor is immobilized, for example by a method known per se, it can be selectively extracted by adsorption of TPA and a hitherto unknown precursor of TPA, namely pro-TPA, leaving the contaminant unadsorbed in the aqueous solution. , was found to produce affinity reagents. DE-
Since 3 is a plasmin inhibitor, it prevents the conversion of pro-TPA to TPA by the action of plasmin, which is normally present in aqueous liquids suitable as TPA and/or pro-TPA sources. Therefore, this immobilized inhibitor exhibits excellent properties when used as an adsorbent for proTPA.

Urokinase is not adsorbed if present in aqueous solution. Therefore, T P A and/or pro T
This affinity reagent can also be used in the method of the invention if the PA is contaminated with urokinase, which is removed along with non-adsorbable contaminants. This means that the above method can be used to recover TPA from a number of sources, such as various tissues in which T'PA and/or pro-T'PA and urokinase are mixed.

However, particularly preferred aqueous liquids for use in the extraction of TPA and/or proTPA are those obtained from tissue cultures of human black lung cells that contain TPA and/or proTPA as cell secretions. , preferably a serum-free harvest fluid.

According to a preferred embodiment, TPA and/or
The aqueous medium containing TPA is combined with a bed of affinity reagents.
), the layer is then washed to desorb the TPA and/or proTPA, and the desorbed 'rPA and/or proTPA is washed out of the layer. The layers may be in the form of columns, for example.

The desorption is preferably carried out using an aqueous solution of potassium thiocyanate, preferably about 1 to 3 molar, such as 1.4 to 2 molar, more preferably 1.6 molar, with the TPA at a stable pH, preferably g# acid. A buffered, approximately neutral saline solution having a concentration of 0.3 to 1 molar, preferably about 0.4 molar, as NaC5, preferably containing a suitable stabilizing amount of a suitable active surfactant, e.g. 0.1% Triton X-10
It is preferably carried out in saline water containing 0 or Tween.

Desorption is caused by chaotropic substances other than potassium thiocyanate (
It can also be carried out using a desorbing agent. Desorption can also be carried out at a low pH, for example between 2 and 3.5, preferably between 2.8 and 3. Other suitable desorption conditions include high salt concentration and/or
or the presence of arginine and/or the presence of benzamidine. Since such desorption conditions are well known to those skilled in the art, detailed explanation will be omitted.

In this invention, -f P that does not contain proT P A
If you wish to collect A (2), whether or not it was produced by the above method, for example, by treating pro-TPA with plasmin or an enzyme that has a similar effect on pro-TPA in an aqueous solution. , Pro TPA Easy TPA
can be enzymatically converted to

Such unidentified enzymes are present, for example, in fibrin, so that the conversion of pro-TPA to TPA occurs even in the presence of fibrin. Kallikrein also has this effect, albeit to a lesser extent. .

On the other hand, and according to a further important feature of the invention, it has been found that pro-TPA can be used directly, for example for therapeutic purposes, and that pro-TPA has properties that are considered even more favorable than TPA itself. In the case of TPA,
There is still a small possibility of activating plasminogen in the blood from the fibrin site where a localized effect is desired.

However, the action of pro-TPA is completely selective: it first binds itself to fibrin and only then converts to TPA at the fibrin site. This T P ,A then releases plasmin at the correct fibrin site, which acts to lyse the clot. This is Pro TP
This means that the effect of A is more completely confined to the desired site of action than 1'PA itself.

The difference between PROP A and TPA is that the former is a single-chain compound that has no or no measurable plasminogen viscosity-increasing effect, whereas
TPA (molecular weight 72,000 daltons) is a double-chained compound and has a significant plasmid/-gen activation effect. ProrPA has the same molecular weight as TPA.

The present invention also provides for the production of pro-TPA from an aqueous medium containing pro-TPA and plasmin and/or plasminogen and plasminogen activator.
The present invention provides a method for recovering pro-TPA comprising inhibiting plasmin in an aqueous medium and separating pro-TPA from the aqueous medium while plasmin is inhibited. Here, when TPA is present, it acts as a plasminogen activator that causes the production of plasmin, but then plasmin is inhibited.

ProTPA can be adsorbed from the aqueous medium by an affinity reagent consisting of an immobilized inhibitor of plasmin, a substance with affinity for proTPA, and then desorbed and recovered in pure form. desirable. The proTPA thus recovered can be disinfected and stabilized with physiologically acceptable stabilizers as an aqueous solution for intravenous injection.

Particularly suitable for recovering pro-TPA is one on which the above-mentioned DE-3 inhibitor is immobilized. The reason for this is that in this invention, DE-
3 happens to be an inhibitor of plasmin, which is commonly present in the medium in which TPA and pro-TPA are to be recovered, and has the property of converting pro-TPA to TPA, and this inhibitor is converted into pro-TPA. When used for the isolation of PA, at the same time it serves to preserve pro-TPA in its pro-TPA form, which is an extremely preferred form therapeutically as described above. Other known TPA isolation methods do not provide such results.

According to a further feature of the invention, in addition to being able to separately harvest rPA and/or proTPA from an aqueous medium containing both plasminogen activators (in themselves or as precursors), urokinase can also be harvested. can do. This makes such an aqueous medium, Erythrina
An affinity reagent consisting of an immobilized Kunit type inhibitor that is present in the seeds of Erythrina latissima and other Erythrina species and has properties as trypsin, plasmin, and TPAil harmful substances, but has no effect on urokinase. contact, thereby exposing the TPA and/or
Selectively adsorb TPA from a solution, and use an affinity reagent that has adsorbed TPA and/or Pro1-PA,
This is accomplished by releasing the urokinase from contact with an aqueous medium that is depleted of TP'A and/or proTPA but still contains urokinase and recovering the urokinase from the aqueous medium that is depleted of TPA and/or proTPA. Ru. A suitable aqueous medium for this method is a medium containing an aqueous extract of random sentinel cell cells or secretions of such cells, since such a medium contains both positive and negative This is because it contains a minogen activator. Again, the adsorbed TPA and/or pro-T
PA can be desorbed and recovered, if desired, by adding pro-TPA to T P before or after adsorption to selective affinity reagents.
It can be converted to A.

Urokinase can be recovered from the aqueous medium by any suitable method, such as by adsorption from the aqueous medium using an affinity reagent that has affinity for urokinase.

Such affinity reagents include immobilized inhibitors that have no selectivity for T'PA over urokinase. Furthermore, as an affinity reagent, it is also possible to use an antibody to urokinase, preferably immobilized by a method known per se.

Further, according to the present invention, Kunitz-type inhibitors are present in the seeds of Erythrina latissima and other plant species of the genus Erythrima and have properties as trypsin, plasmin, and TPA inhibitors, but have no effect on urokinase. Novel affinity reagents are provided, comprising an inhibitor immobilized by binding onto a solid support, such as by covalent bonding to the carrier. The inhibitor may, for example, be coupled to bromcyan-activated agarose by methods well known to those skilled in the art. However, other solid supports can also be used, and other coupling reagents can also be used depending on the chemical nature of the surface to which the inhibitor is to be attached. Examples include: (a) Carbodiimide coupling of a free amino group to a free carboxy group.

(b) Cultaraldehyde coupling to agarose in which the NH2 groups have been previously converted to aminoalkyl type.

(C) generates an aldehyde functionality, which in turn pH 4
Periodate activation of agarose, which reacts with an inhibitory amine to form a Schiff base, which is reduced by sodium borohydride or sodium cyanoborohydride.

(d) A method of converting acarose into hydrazide succinyl derivative 4, and bonding a carboxyl ligand to this with E I) C, or bonding an amine with cyanation.

(e) Converting cellulose fibers or beads into hydrazide derivatives, using the method of Parikh et al.
34, pp. 77-102].

(f) A method in which polyacrylamide beads are coupled directly by the gel taraldehyde method or by diazotization of a P-aminobenzamide ethyl derivative or a hydrazide derivative.

(g) Methods for coupling proteins, such as albumin or trypsin or gelatin, for example in the form of hydrazides.

Finally, glass peas (or other solid materials) which may be coated with proteins such as gelatin, have been described in South African patent application no.゜8
6. A method of crosslinking and coupling with an inhibitor by a method similar to the coupling method described in British Patent Application No. 821907 (l and US Patent Application No. 392111).

Since the above-mentioned methods are known to those skilled in the art and are described in the above-mentioned documents, a detailed description will be omitted.

Included within the scope of this invention is a process for making an affinity reagent comprising covalently bonding a Kunitz-type inhibitor as described above onto a solid support, eg, by any of the methods described above.

The affinity reagent according to the invention can be used for the production of TPA and/or proTPA and for the production of formulations consisting of TPA and/or proTPA, eg by the methods described above. However, affinity reagents also have use as diagnostic or analytical reagents.

The scope of this invention further includes, in addition to such pro-TPA, purified mixtures of TPA and pro-TPA, in particular, in which the ratio of pro-TPA to TPA is greater than 1:2, and even greater than 1:1. Contains a good mixture.

To obtain even higher ratios, e.g. 9:1 or higher, cultures of pro-TPA producing cells, e.g. melanoma cells, in a suitable medium may be cultured with an enzyme lacking the enzyme that catalyzes the conversion of pro-TPA to TPA. By culturing in the presence or in a medium containing the enzyme, for example containing serum, and containing an inhibitor suitable for inhibiting the enzyme (specifically plasmin) that catalyzes the conversion of pro-TPA to TPA. , it is proposed to produce pro-TPA. Suitable inhibitors include trypsin inhibitor from soybean or l-antiplasmin. Pro1'PA is recovered from the medium in the absence of the enzyme or in the presence of one or more inhibitors.

If a mixture of pro-TP A and T i' A is obtained and it is desired to limit its enzymatic activity to that of pro-1' P A, 'l' l) an inhibitor that irreversibly inhibits A. TPA can be removed using A suitable example is the irreversible proteinase inhibitor diisopropylphosphofluoridate. Inhibitors can later be removed by dialysis or gel chromatography. Irreversibly inhibited ``■'' PA may remain in the mixture.

The scope of this invention further includes pharmaceutical compositions consisting of TPA produced or purified by the method of this invention and/or pro-TPA dissolved in a physiologically compatible injectable medium. Particularly preferred compositions are those comprising pro-TPA mixed with TPA, and even more preferred are those containing pro-TPA as the only active ingredient. This composition is particularly for the management of conditions in which the generation of local fibrinolytic or proteolytic activity via thrombosis or other plasminogen activation mechanisms is desirable.

The scope of this invention includes locally generated TPA from pro-TPA.
Included are local fibrinolytic or proteolytic methods by the action of plasmin, which consist in liberating plasmin locally from plasminogen by activation of plasminogen by PA.

[Specific description of the invention]

The invention will now be illustrated by way of examples, ie non-limiting embodiments.

Example 1 Production of affinity reagent according to the invention The 0E-3 inhibitor was produced as follows.

Seeds of Erythrina latissima are collected and processed by the method of Schubert et al. Powder the seeds, defatte, 0.5
Extract with molar sodium chloride solution at 10°C overnight. The extract was centrifuged, and the supernatant was precipitated with ammonium sulfate and Sephadex G50. DEA-Cellulose and DEA
-DE by chromatography on Sepharose-
3. Collect inhibitory substances. The final purified material has an apparent molecular weight of 1 when electrophoresed on a 15% polyacrylamide gel containing 0.1% sodium dodecyl sulfate (SDS).
22o, oo Dalton migrates as a single band.

Purified DE-3 (26wuj) is bound to commercially available bromcyan-activated agarose 5tnl using a conventional method.

(Sepharose 4b is sold in fractions, and the company also distributes instructions on how to use the composition.) The affinity reagents are 0.4 molar sodium chloride, 0.1% Triton
Equilibrate with a phosphate buffered saline solution, pH 7.4, containing -100C (commercial detergents commonly used in the art) and 0.02% sulfur azide as a stabilizer. This affinity reagent is then packed into a 5 me column made from the body of a disposable plastic tube.

Example 2 Production and Purification of TPA and ProTPA A medium containing TPA and proTPA is produced as follows.

Balanis cells RPMI-7272 (obtained from Temper Hospital, Denver, Colorado, Journal of Biological Chemistry, Vol. 256, No. 7035-7041)
Large melanoma cells of a cell line known as P.
10% and antibiotics (henicillin 300μ9/ml
, RPMI-1640 with streptomycin 200μf/ml and tylosin 10μQ/ld)
Grow as an adherent monolayer in tissue tissue. Cells are confused by trypsinization (
Approximately 4 X 10” cells/cm) passage
L. Re-seeding 5 x 105 cells in 100 dishes. Aspirate the medium and incubate the cells in 0.25% trypsin in Tris.
Dulbecco saline (24.8mM Tris HCl 1 pH 7)
,4,0,l rnMN a2HP 04.5 mMK
c4.0.14 MNa (J' containing gold) and incubate at 37°C for 5 minutes. Gently aspirate the separated cells with a pipette to disperse them, and add the suspension to an equal volume of fetal bovine serum-containing medium. In addition, proteases are neutralized. Cells are then washed by centrifugation at 350 g for 5 minutes, resuspended and reseeded into new dishes.

From these stock cultures, cells were incubated at 75CI or 150c.
Grow as an adherent monolayer in tissue culture flasks for 1
Grow until nearly confluent in medium supplemented with 0% tallow serum. Wash the culture once and cover with 20 ml of serum-free medium. After 24 hours, collect the medium and add fresh medium.

The harvest fluid is centrifuged at 200 rpm for 5 minutes to remove intact cells and cell debris. Add solution to Triton X-100 (0.1%) or Tween 8
0 (0,1%) and acidified with glacial acetic acid (pH 5,
5 to 6.0)t, stored at -20°C.

2 liters of the harvest liquid was adjusted to a NaC, 5 concentration of 0.4M, and
．． E was passed through a 45 μm P membrane. The filtered harvest liquid is passed through a column of affinity reagent at a flow rate of 45 ml V/hr at room temperature. The effluent is collected at 4°C and tested for plasminogen-dependent fibrinolytic activity. TPA or ProTP
A is not accepted.

After passing the entire harvest liquid through the column, the column is
xQ, 4M NaCC and 0.1% Triton X-
Wash with 100% phosphate buffered saline. In this process, TP
No A or proTPA elutes.

Next, the adsorbed protein was mixed with 1.6M potassium thiocyanate, Q
, 4 M NaCg and 0.1% Trito:/X-Z
o.

Elute with containing PBS. The protein concentration is determined by measuring the absorption at 2 g Q nm. We found that when potassium thiocyanate was added to the elution buffer, total plasminogen-dependent fibrinolytic activity eluted as a sharp peak;
This peak corresponds to a small protein peak.

The fractions showing the highest activity are collected to give a 6-8d solution, which is stored at -20'C. This represents 70 to 80% of the activity passed through the column. Fractions showing low activity are collected separately.

The total recovery rate of both activities is usually 90 to 100.
reach %. Potassium thiocyanate eluent was pure TPA
and a molecular weight of 72,000 corresponding to a mixture of pro-TPA.
A single band due to Dalton's protein is shown, of which the latter can be seen to account for 39-50% or more of the total in separate experiments.

The yield increases when the above column is used repeatedly. In this way, the column can not only be reused many times, but actually improves with use.

This is the inhibitor D when TPA is not immobilized.
E-3. j, − Considering that it has the property of cutting,
That's surprising.

Example 3 Pharmaceutical composition containing TPA and/or proTPA.

The combined fraction containing TPA and/or POTPA was combined with 0.01% by volume Tween 80.
．． Dialyze against 30 sodium chloride solution and store at -80°C. Before administration, the concentration, 2TPA plus prober
P A 75 μQ/ml (T P A therapeutic dose is the same as pro T P A) and Na (4' 0.3
Adjust to molar. The solution is sterilized by filtering through a τ 0.22 μm filter membrane.

This solution is infused intravenously at a dose of 3 to 15/24 hours, typically 7.5 to/24 hours.

Ideally, the dosage should be such that the TPA blood concentration during treatment is between 0.6 and 1 International Mo/d, preferably about 0.8 International Mo/ml. It can be seen that at this concentration, the treatment against thrombosis is highly effective and no systemic fibrinolysis occurs.

If pro-TPA is predominant in the formulation, the risk of systemic fibrinolysis is reduced and thrombotic clearance can be hastened by increasing the dose.

Example 4 Mass culture of melanoma cells Rosswell Park Memorial Institute (Rosswell Park Memorial I)
Melanoma cells are inoculated at a concentration of 5 x 105 cells/ml into spinner flasks containing medium 1640/10% tallow serum. Keep the flask at 37°C and stir with a magnetic stirrer at 3 Q rpm.

The medium is aspirated from the flask at a rate of 1% of flask volume/hour and replaced at the same rate with fresh medium equilibrated with air containing 5% CO2. Cells are left in the culture vessel by using an exit glass tube plugged with glass wool. The sucked out medium is filtered using a Millipore filter. The collected liquid is pasteurized.
)do.

Example 5 Melanoma Cell Culture to Increase Pro-TPA Yield Sufficient firefly soybean trypsin inhibitor or alpha-1-antiplasmin is added to the medium to inhibit serum plasmin.
Example 2 is modified.

All other operations were performed as in Example 2. The pro-TPA:TPA ratio in the final product increases to approximately 9:1.

Example 6 Removal of TPA remaining in a mixture of pro-TPA and TPA TP
Diisopropylphosphofluoridate is added to the solution of A and proTPA to a concentration of 1 Q mM. p
Adjust I-1 to 8. After 4 hours, TPA is permanently inhibited. Residual inhibitors are later removed by dialysis.

Example 7 Conversion of Pro-TPA to TPA An aqueous solution containing 100 μf/me of pro-TPA protein was mixed with an equal volume of phosphate buffered saline containing 5 μQ of plus 1 me and incubated at 20°C for 16 hours. Sodium dotecyl sulfate is added to a final concentration of 0.1% and the proteins are precipitated with 6% trifluoroacetic acid. The precipitate is washed in acetone and redissolved in 0.06 M TolJS hydrochloride (pH 6,8) containing 1% sodium dodecyl sulfate and 10% glycerin.

After this treatment, electrophoresis shows that pro-TPA was completely converted to the active 1''PA form.

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Claims (1)

[Scope of Claims] (1) An aqueous medium containing dissolved tissue plasminogen activator and/or tissue plasminogen activator precursor and contaminants is purified from Erythrina latissima and other Erythrina plant species. It is present in the seeds of and closely associated with an affinity reagent consisting of an immobilized Knicks-type inhibitor that has properties as a trypsin, plasmin, and tissue plasminogen activator inhibitor, but has no effect on urokinase. contacting the tissue plasminogen activator and/or the tissue plasminogen activator precursor to selectively adsorb the tissue plasminogen activator and/or the tissue plasminogen activator precursor from the solution; Desorbing the precursor-adsorbed affinity reagent from contact with the aqueous medium and non-adsorbed contaminants, and desorbing tissue plasminogen activator and/or tissue plasminogen activator precursor from the affinity reagent. and recovering the desorbed tissue plasminogen activator and/or tissue plasminogen activator precursor. Purification method. (2) The tissue plasminogen activator and/or tissue plasminogen activator precursor is contaminated with urokinase, and the urokinase is removed together with other non-adsorbable contaminants. the method of. (3) The method according to claim 1, wherein the aqueous medium is obtained by culturing a melanoma cell culture. (4) The method according to claim 1, wherein the tissue plasminogen activator precursor is treated with plasmin and converted into the tissue plasminogen activator before or after the adsorption step. (5) passing an aqueous medium containing tissue plasminogen activator and/or tissue plasminogen activator precursor through a layer consisting of an affinity reagent to adsorb the factor and/or precursor; The tissue plasminogen activator and/or the tissue plasminogen activator precursor are then desorbed, and the desorbed tissue plasminogen viscosity factor and/or tissue plasminogen activator precursor is removed. 2. A method according to claim 1, wherein the layer is washed out. (6) The method according to claim 1, wherein the desorption is carried out using an aqueous potassium thiocyanate solution. (7) A method according to claim 1 in a pharmaceutical composition for managing a condition in which local fibrinolytic or proteolytic activity is desired to be produced by thrombosis or other plasminogen activation mechanisms. A pharmaceutical composition comprising a tissue plasminogen activator and/or a tissue plasminogen activator precursor purified in an injectable medium. (8) The tissue plasminogen activator precursor present in the aqueous solution is subjected to the action of plasmin or an equivalent enzyme that catalyzes the conversion of the tissue plasminogen activator precursor to tissue plasminogen activator. A method for producing a tissue plasminogen activator, the method comprising: (9) A method according to claim 8, in a pharmaceutical composition for managing a condition in which the production of local fifurinolytic or proteolytic activity is desired due to thrombosis or other plasminogen activation mechanisms. A pharmaceutical composition comprising a tissue plasminogen activator prepared by dissolving in an injectable medium. QO) Adding an aqueous medium containing dissolved tissue plasminogen activator and/or tissue plasminogen activator precursor and urokinase to Erythrina
An immobilized Kunitzz-type inhibitor present in the seeds of Erythrina latissima and other Erythrina species and having properties as a trypsin, plasmin and tissue plasminogen activator inhibitor, but with no effect on urokinase. tissue plasminogen activator and/or tissue plasminogen activator precursor from solution, thereby selectively adsorbing tissue plasminogen activator and/or tissue plasminogen activator precursor from solution; or an affinity reagent adsorbed with tissue plasminogen activator precursor with an aqueous medium that has lost tissue plasminogen activator and/or tissue plasminogen activator precursor or still contains urokinase. release from contact with tissue plasminogen activator and/or tissue plasminogen activator.
or recovering urokinase from an aqueous medium depleted of tissue plasminogen activator precursor,
A method for separating urokinase from tissue plasminogen activator and/or tissue plasminogen activator precursor. 11. The method according to claim 10, wherein the adsorbed tissue plasminogen activator and/or tissue plasminogen activator precursor is desorbed and recovered at time 0. (4) A method according to claim 10, characterized in that urokinase is characterized from an aqueous medium using an affinity reagent having an affinity for urokinase. A Kunitz-type inhibitor that has properties as an inhibitor of trypsin, plasmin, and tissue plasminogen activator, or has no effect on urokinase, and is immobilized by being bound to a solid carrier. (14) The affinity reagent according to claim 13, wherein the inhibitory substance is bound to the carrier by a covalent bond. Affinity reagent according to claim 14, which comprises covalently binding a Kunitz-type inhibitor present in the seeds of Erythrina latissima and other Erythrina species to a solid support. , a method for producing an affinity reagent. Alpha power for the production of tissue plasminogen activator and/or tissue plasminogen activator precursor and for the production of preparations consisting of tissue plasminogen activator and/or tissue plasminogen activator precursor. Use of an affinity reagent according to claim 13. Use of the affinity reagent according to claim 13 as a diagnostic and analytical reagent. (19) Tissue plasmid/-ken activator precursor. KEN Tissue Plasmi/-Purified mixture of KEN activator and tissue plasminogen activator precursor. 21. The mixture of claim 20, wherein the ratio of Qυ tissue plasminogen activator precursor to tissue plasminogen activator is greater than 1:2. (4) The mixture of claim 21, wherein the ratio of tissue plasminogen activator precursor to tissue plasminogen activator is 9:1 or more. B. Claim 19 characterized by an irreversibly inhibited tissue plasminogen activator. A medicament comprising a tissue plasminogen activator precursor dissolved in a physiologically compatible injection medium. Composition. □□□ Plasmin consists of locally releasing plasmin from plasminogen through activation of plasminogen by the tissue plasminogen activator produced locally from the tissue plasminogen activator precursor. Local fibrinolysis and proteolysis method by the action of ■ A culture of cells producing tissue plasminogen activator precursor is transformed from tissue plasminogen activator precursor to tissue plasminogen activator. The tissue plasminogen activator precursor is cultured in a suitable medium in the absence of an enzyme that catalyzes the conversion or, if present, in the presence of an inhibitor suitable for inhibiting the enzyme. A method for producing and purifying a tissue plasminogen activator precursor, comprising recovering the tissue plasminogen from a culture medium in the absence of the enzyme or in the presence of one or more of the inhibitors. Melanoma cells producing activator precursors are stimulated with one or more serum enzymes suitable for inhibiting the serum enzyme that catalyzes the conversion of tissue plasminogen activator precursors to tissue plasminogen activator precursors. The method according to claim 26, wherein the method is cultured in a serum-containing medium in the presence of an inhibitor. ■ Containing a tissue plasminogen activator precursor and containing plasmin and/or plasminogen and plasminol. In a method for recovering tissue plasminogen activator precursor from an aqueous medium that also contains gene activator, inhibiting plasmin in the aqueous medium, and while plasmin is inhibited, tissue plasminogen activator A method for recovering tissue plasminogen activator precursor comprising separating the precursor from an aqueous medium. 29. A method according to claim 28, comprising adsorption from an aqueous medium by an affinity reagent consisting of an immobilized plasmin inhibitor with affinity for plasmin, followed by desorption and recovery in pure form. ) sterilizing the obtained tissue plasminogen activator precursor and stabilizing it in the form of an aqueous solution for intravenous injection using a physiologically compatible stabilizer. Method.