AU620927B2 - A preparation of plasminogen activator expressed in prokaryotes - Google Patents

Description

43385/ 41, 4 OPI DATE 18/014/90 APPLN. I D*' PC.T NUIMBEPR PrT/FPRq/n-ji-Q INTERNATIONALE ZUSAMMENARBEIT AUF DEM GEBIET DES PATENTWESENS (PCT) (51) Internationale Patentklassirikation 5: (11) Internationale Veriiffen!" ingsnummer: WO 90/03388 C07K 3/08, 3/20, C12N 15/58 Al (43) lnternatio "ns 4-1 nAr90 4,) A61K 37/54 ef,(5 (21) Internationales Aktenzeichen: PCT/EP89/01 139 (74) Anwae: WEICKMAN9,H. 6thlstrage 22, D- 8000 Mflnchen 80 (DE).

(22) Internationales Anmeldedatum: 28. September 1989 (28.09.89) (81) Bestimmungsstaaten: AU, DK, JP, US.

Priorititsdaten: P'13 32 898.4 28. September 1988 (28.09.88) DE Veroffentiicht Mit internationalem Recherchenbericht.

(71) Anmelder (ftir alle Bestimmungsstaaten ausser US): BOEH- RINGER MANNHEIM GMBH [DE/DE]; Standhofer Strage 112-132, D-6800 Mannheim (DE).

(72) Erfinder ;und Erfinder/Anmelder (nurftir US) RUDOLPH, Rainer [DE/ DE]; F~rbergasse 19, D-8120 Weilheim OPITZ, Ulrich [DE/DE]; Anton -Roth-StraRe 2, D-8 170 Bad T6lz KOHNERT, Ulrich [DE/DE]; Heubachweg 6, D- 8121 Habach FISCHER, Stephan [DE/DE]; Jakobifeldweg 11, D-8121 Polling (DE).

(54) Title: A PREPARATION OF PLASMINOGEN ACTIVATOR EXPRESSED IN PROKARYOTE-S (54) Bezeichnung: PRXPAP.AT VON IN PROKARYONTEN EXPRIMIERTEM PLASMINOGENAKTIVATOR (57) Abstract A preparation of recombinant p-t-PA expressed in prokaryonts has an active p-t-PA content of over 90 and a specific activity of more than 0.4 x 106 IU/mg. To produce such a preparation of recombinant p-t-PA expressed in prokaryonts by the opening of the expressing prokaryont cells, by solubilisation in denaturing and reducing conditions and by reactivation in oxidising conditions, concentration is performed in the reactivation unit followed by cleaning by means of affinity chromatography. A p-t-PA preparation of the invention may be used for dissolving blood clots.

(57) Zusammenfassung Emn Prilparat von rekombinantem, in Prokaryonten exprimiertemn p-t-PA weist einen Gehalt an aktivem p-t-PA von mehr als 90 und eine spezifische Aktivittit von mehr als 0,4 x 106 IU/mg auf. Zur Herstellung eines solchen PrAparats von rekombinantem, in Prokaryonten exprimiertemn p-t-PA durch AufschlugI der exprimierenden Prokaryontenzellen, Solubilisierung unter denaturierenden und reduzierenden Bedingungen und Reaktivierung unter oxidierenden Bedingungen konzentriert man im Reaktivierungsansatz und f~hrt dann eine Reinigung mittels Affinitiitschromatographie durch. Emn erfindungsgem1fks p-t-PA- Prllparat kann zur Aufl6sung von Blutgerinnseln verwendet werden.

A

1.

1- 2 Description The invention concerns a preparation of recombinant plasminogen activator (p-t-PA) expressed in prokaryotes which has a particularly high content of active p-t-PA and a high biological activity, a process for the production of the preparation, as well as its application.

The activation of the fibrinolytic system proceeds under physiological conditions in a reaction cascade which is similar to that of blood coagulation. The central reaction of this cascade is the activation of plasminogen to plasmin. Plasmin, in turn, dissolves fibrin, the main component of the protein matrix of clotted blood. Due to its ability to specifically bind to fibrin the tissue-type plasminogen activator t-PA is generally regarded as the physiologically active plasminogen activator (Matsuo et al., Nature 291 (1981), 590). This focus of the enzymatic activity on the fibrin surface appears to make it a suitable agent for the treatment of pathological vascular occlusions in myocardial infarction). The hopes placed in the application of t-PA were confirmed to a large extent in extensive clinical studies (Collen et al., Circulation (1984), 1012; Circulation 73, (1986), 511).

Several methods have therefore already been suggested for the isolation of t-PA in large quantities. Different mammalian cell lines were for example described which could produce the necessary amounts of t-PA for therapeutic applications (Riiken and Collen, J. Biol.

Chem. 256, (1981), 7035; Collen et al., J. Pharm. Exp.

Ther. 231, (1984), 146; Kaufman et al., Mol. Cell Biol.

(1985), 1750). The culture of such cells was however n 3 relatively complicated and expensive. Since the cloning of t-PA cDNA in E. coli (Pennica et al., Nature (1983), 214 -221) a way has been opened for the expression of the enzyme in a prokaryotic system.

Plasminogen activator produced in prokaryotes is unglycosylated in contrast to t-PA from mammalian cells which is glycosylated in 2 to 3 positions. In the following it is therefore referred to as p-t-PA (prokaryotic t-PA) to distinguish it from eukaryotic t- PA. p-t-PA accumulates, however, in bacteria, like many other eukaryotic proteins expressed in bacteria, in an inactive aggregated form, the so-called inclusion bodies (IB's) and the correct folding to form the native active structure does not take place. These protein aggregates are often also contaminated by bacterial proteins. In the case of p-t-PA these are primarily outer membrane proteins and the elongation factor EF-Tu (Brinkmann, 1987, Diplomarbeit, Universitat Munchen). Such impure protein preparations which, in addition, accumulate in the form of insoluble inclusion bodies must, after their expression in the prokaryotic cells, be first solubilized, denatured and then renatured in order to effect the correct folding of the proteins. However, only a relatively small portion of the available material in the inclusion bodies could be obtained in an active form using the processes known up to now, so that only a relatively low specific activity of p-t-PA could be obtained after renaturation. In addition the content of active p-t-PA was limited by the presence of foreign proteins.

It is therefore the object of the present invention to prepare p-t-PA, which has been expressed in prokaryotes, in as high a yield as possible and of the highest purity possible.

4- This object is achieved according to the present invention by a preparation of recombinant p-t-PA expressed in prokaryotes, which has a content of active p-t-PA of more than 90 with respect to total protein and a specific activity of more than 0.4 x 106 IU/mg.

In a preferred embodiment the p-t-PA preparation according to the present invention has a content of active p-t-PA of more than 95 and in particular of more than 98 A further embodiment of the invention is a process for the production of a preparation of recombinant p-t-PA expressed in prokaryotes with a content of active p-t-PA of more than 90 with respect to total protein and a specific activity of at least 0.4 x 106 TU/mg by lysis of the prokaryotic cells which express the p-t-PA, isolation of IB's, solubilization of the IB's under denaturing and reducing conditions, derivatization to mixed disulphides and reactivation under controlled redox conditions, which is characterized in that after reactivation p-t-PA is concentrated in the reactivation mixture and this is followed by a chromatographic purification by affinity chromatography.

By use of the process according to the present invention it was established that, surprisingly, p-t-PA expressed in prokaryotes could be successfully produced in a particularly pure form and free of foreign proteins and with an active p-t-PA content of more than 90 All prokaryotes which contain t-PA plasmids are suitable for the process. Such prokaryotes and plasmids are known to the expert. The plasmids described in EP-A-0 242 835 I i 5 are particularly suitable whereby the plasmid pePA 133 is preferably used.

Cell lysis of the prokaryotes which express p-t-PA is carried out according to conventional methods such as for example by treatment with lysozyme in the presence of Triton-X-100 and NaC1 followed by high pressure dispersion.

The solubilization under denaturing or reducing conditions, as well as the reactivation under oxidizing conditions can be carried out using known methods as described for example in EP-A-0 253 823.

All the conventional denaturing agents or even arginine can be used as the denaturing agent for the solubilization step. The known denaturing agents guanidine hydrochloride,-urea-or its derivatives are preferred. In addition, mixtures of these denaturing agents can be used. Furthermore, denaturing agents which are for example known from EP-A-0 114 506 can be used.

It is preferable to use reducing agents from the thiol-group such as for example reduced glutathione (GSH) or 2-mercapto-ethanol as the reducing agent. These can be used in concentrations from about 50 to 400 mmol/l. Further preferred reducing agents are DTE (dithioerythritol) and DTT (dithiothreitol) for example in a concentration from about 80 to 400 mmol/l. It is expedient to solubilize for a period of from 1 to several hours, preferably for 2 hours. To prevent the oxidation of the reducing agent by atmospheric oxygen it can also be advisable to add EDTA.

m*T y 6 As described in the above mentioned European Application EP-A-0 253 823 the renaturation can be carried out directly or via conversion of the p-t-PA into the mixed disulphide of p-t-PA and GSSG. The reaction conditions necessary for this are also described in detail in the European Application.

According to the present invention, in the process for the production of the p-t-PA preparation, p-t-PA is concentrated in the reactivation mixture after its reactivation and following this foreign proteins are removed by affinity chromatography.

The concentration of p-t-PA in the reactivation mixture can be carried out by known methods; however, for smaller volumes an ammonium sulphate precipitation is preferred, whereas, for larger volumes concentration in a haemodialyzer is'preferred.

In the case of ammonium sulphate precipitation it is expedient to wash the pellet again after centrifugation and after a renewed centrifugation to dissolve it in reoxidation buffer.

The process according to the present invention is preferably carried out in the presence of L-arginine, especially at a concentration of 25 to 1000 mmol/l.

The removal of foreign proteins according to the present invention by affinity chromatography is carried out in a preferred embodiment of the invention by chromatography on an ETI (erythrina-trypsin-inhibitor) adsorber column.

For this purpose, the ETI is fixed on a supporting material (adsorber) such as for example Sepharose.

11 7 Purification on an ETI adsorber column has the advantage that the ETI adsorber material of the column can be loaded directly from the concentrated reoxidation solution even in the presence of arginine concentrations as high as 0.8 .Mol/l arginine. An aggregation of p-t-PA which can occur at low arginine concentrations under mmol/1 is therefore avoided. The purification of the p-t-PA preparation over an ETI adsorber column is particularly preferably carried out in the presence of to 1.0 M arginine. In the course of this the solution containing p-t-PA has a pH of more than particularly preferably of more than 7.

Elution from the ETI column is effected by lowering the pH in the presence as well as absence of arginine under conditions which allow a good solubility of t-PA which has been expressed in prokaryotes. In the preferred elution the pH is in the weakly acid range, particularly preferably in the range from 5.5 to A p-t-PA preparation produced according to the present invention has a specific t-PA activity of >0.4 x 106 IU/mg which can be stimulated more than ten-fold by fibrinogen cleavage products (activity in the presence of fibrinogen peptides/activity in the absence of fibrinogen peptides). The purity of the preparation according to the present invention, which is more than and preferably more than 95% and in particular more than 98 was detected by SDS-PAGE and RP-HPLC (reversed-phase HPLC). The ability of the p-t-PA preparation produced according to the present invention to lyse blood clots was detected in the rabbit model. In addition the rate of clearance of the p-t-PA preparation according to the present invention was measured in the rabbit model. Compared to t-PA expressed in eukaryotes _1 8 the p-t-PA preparation according to the present invention disappears at a much slower rate from the plasma.

A further embodiment of the invention is the use of the p-t-PA preparation according to the present invention in high purity and with a high content of active p-t-PA in an agent to dissolve blood clots in man and animals.

According to the present invention such a preparation can contain further pharmaceutically acceptable additives and adjuvants such as e.g. arginine, urea 100 mmol/l of each) and buffer components.

The invention is elucidated further by the following examples in conjunction with the figures.

Fig. 1 shows a SDS-PAGE before and after purification by affinity chromatography on ETI-Sepharose; Fig. 2 shows a RP-HPLC after purification of p-t-PA from E. coli by affinity chromatography on ETI-Sepharose.

Fig. 3 shows the binding in vitro of p-t-PA from E.

coli to fibrin and Fig. 4 shows the time-course of p-t-PA activity during and after infusion in rabbits.

i A 9- Example 1 Preparation of active p-t-PA from E. coli Cell lysis and preparation of the inclusion bodies (IB's) 1.6 kg cell wet-weight coli, DSM 3689, transformed with the plasmid pePA 133 described in EP-A-0242 835) was suspended in 10 1 0.1 mol/l Tris-HCl, 20 mmol/l EDTA, pH 6.5, 4°C. 2.5 g lysozyme was added to this and incubated for 30 minutes at 4*C; afterwards complete cell lysis was carried out by high pressure dispersion.

1 0.1 mol/l Tris-HCl, 20 mmol/l EDTA, 6 Triton-X-100 and 1.5 mol/l NaCl, pH 6.5 was mixed with the lysate solution and incubated for a further 30 minutes at 4°C.

Following this the insoluble components (IB's) were separated by centrifugation.

The pellet was suspended in 10 1 0.1 mol/l Tris-HCl, mmol/1 EDTA, pH 6.5, incubated for 30 minutes at 4°C and the IB-preparation was isolated by subsequent centrifugation.

Solubilization of the IB's 100 g IB's (wet weight) was suspended in 450 ml S0.1 mol/1 Tris-HC1, 6 mol/l guanidinium HC1, 0.2 mol/1 DTE, 1 mmol/l EDTA, pH 8.6 and stirred for 2.5 hours at 1 10 After adjustment of the pH to pH 3 with HC1 (25 the solution was dialyzed against 10 mmol/1 HC1 (3 x 50 1, 24 hours, 4"C).

Derivatization Solid guanidinium HC1 was added in such a quantity that after final dilution of the above dialysate with mmol/l HC1 the guanidinium HC1 concentration was 6 mol/l.

The preparation was preincubated for 1.5 hours at afterwards the GSSG concentration was adjusted to 0.1 mol/l and the Tris-HCl concentration to 0.05 mol/l and the pH was titrated with 5 mol/l NaOH to pH 9.3. The preparation was stirred for 3.5 hours at After adjustment of the pH to pH 3 with HC1 (25 the solution was dialyzed against 10 mmol/l HC1 (3 x 100 1, 48 hours, After the dialysis the preparation was centrifuged and the clear supernatant was processed further.

Renaturation A 10 1 reaction vessel was filled with 0.1 mol/l Tris- HC1, 0.8 mol/1 L-arginine, 2 mmol/l GSH, 1 mmol/l EDTA, pH 8.5. The renaturation was carried out at 20°C by a three-fold addition of 100 ml derivative (mixed disulphide, see above) at 24 hour intervals.

After the renaturation a preparation was obtained with a specific activity of 10000 to 20000 IU/mg (test 0 k 11 according to J.H. Verheihen et al (Thromb. Haemostas.

48, 266, 1982), modified according to H. Lill Ges.

Inn. Med. 42, 478, 1987)).

Concentration If required the renaturation preparation can be concentrated by precipitation with ammonium sulphate or by ultra-filtration.

In the ammonium sulphate precipitation the pH of the renaturation solution was adjusted to pH 7.5. The precipitation was carried out by adding solid (NH 4 2

SO

4 to a final concentration of 60% at 4°C. After stirring for one hour at 0°C the suspension was centrifuged and the pellet was redissolved in 1/10 to 1/100 of the original volume with 0.8 mol/l L-arginine, 2 mmol/l GSH, 1 mmol/l EDTA, pH E x a mp 1 e 2 Purification of p-t-PA from E. coli by affinity chromatography using erythrina trypsin inhibitor (ETI).

Preparation of the affinity adsorber ETI was coupled to BrCN-activated Sepharose from Pharmacia according to the manufacturer's instructions.

-1-1~1 i r; 12 Affinity chromatography without previous concentration of the renaturation preparation An ETI-Sepharose column (V 5 ml) was equilibrated with 0.1 mol/l Tris-HCl, 0.8 mol/l L-arginine, 2 mmol/l GSH, 1 mmol/1 EDTA, pH 1 1 renaturation preparation (Example 1) was then applied at a flow rate of 10 column volumes/hour. The column was rewashed with equilibrium buffer until the absorbance at 280 nm reached the blank value of the buffer.

The elution of p-t-PA was achieved by a pH jump using 0.1 mol/l Tris-HCl, 0.8 mol/l L-arginine, 2 mmol/l GSH, 1 mmol/l EDTA, pH Asp specific Volume Activity c activity (ml) (IU/ml) (mg/ml) (IU/mg) Reoxidation solution 1000 3480 0.15 23200 p-t-PA-Pool 5 682500 1.22 560000 Affinity chromatography after concentration of the renaturation solution by ultra-filtration The renaturation preparation was concentrated 1:45 using a haemodialyzer. NaCI was added to the concentrate to yield a concentration of 0.5 mol/1.

1

I

13 An ETI-Sepharose column (V 80 ml) was equilibrated with 0.1 mol/l Tris-HC1, 0.8 mol/l L-arginine, 0.5 mol/l NaCl, pH 7.5. 700 ml of concentrate was applied at a flow rate of 4 column volumes/hour. Afterwards the column was rewashed with equilibration buffer until the absorbance at 280 nm reached the blank value of the buffer.

P-t-PA was eluted with 20 mmol/l citric acid, pH 3.2.

A

sp specific Volume Activity c activity (ml) (IU/ml) (mg/ml) (IU/mg) concentrate 700 247000 25 10000 p-t-PA-Pool 1000 155000 0.18 874000 Example 3 Characterization of purified p-t-PA from E. coli Characterization of the protein SDS-PAGE and RP-HPLC (reversed-phase

HPLC)

The homogeneity of the preparation purified by affinity chromatography on ETI-Sepharose was examined using SDS-electrophoresis and RP-HPLC. Fig. 1 shows a comparison of the electrophoretic analysis of concentrated renaturation solution and purified protein.

A densitometric analysis of the gel shows that affinity 14 chromatography on ETI-Sepharose resulted in a concentration of p-t-PA to >95 A molecular weight of 2 kDa for p-t-PA from E. coli can be estimated from the relative mobilities.

Proteins differ from one another in their hydrophobic interactions with a polymeric hydrophobic matrix. This characteristic property is used in RP-HPLC as an analytical method to monitor purity. The analysis of the purified p-t-PA was carried out on a Nucleosil 300 (Knauer) separation column using a trifluoroacetic acid/acetonitrile gradient (buffer A: 1.2 ml trifluoroacetic acid in 1000 ml H 2 0; buffer B: 300 ml

H

2 0, 700 ml acetonitrile, 1 ml trifluoroacetic acid; 0 to 100 Fig. 2 shows the chromatographic analysis of purified p-t-PA from E. coli by RP-HPLC. This analysis also shows a homogeneity of >95 N-terminal amino acid sequence The N-terminal amino acid sequence was determined using an ABI 470 sequencer with a standard program and on-line PTH detection. The determined sequence S1-Y2-Q3-V4-15 corresponded with the expected amino acid sequence derived from the DNA-sequence.

Activity determination in vitro activity The in vitro activity of p-t-PA from E. coli was determined according to the test instructions of the Boehringer Mannheim Company, Order No. 1080954 for the t-PA standard. The specific activity was 0.78 0.2 x i I 15 106 IU/mg. The stimulatability of the activity in this test system by CNBr-fibrinogen fragments (activity in the presence of fibrinogen peptides divided by activity without fibrinogen peptides) was in vitro binding to fibrin The in vitro binding of p-t-PA from E. coli to fibrin was determined according to the method described by Higgins and Vehar Higgins, G.A. Vehar, Biochem.

26, (1987) 7786-91). Fig. 3 shows the binding of p-t-PA from E. coli fo fibrin at a constant fibrin concentration as a function of the p-t-PA concentration.

in vivo thrombolysis The thrombolytic activity of p-t-PA from E. coli was determined in rabbits with experimental jugular vein thrombosis in the form described by Collen et al (D.

Collen, J. M. Stassen and M. Verstraete, J. Clin.

Invest. 71, (1988) 368-376).

A dose of 800000 IU/kg body weight yielded a thrombolysis of 79 (n The spontaneous thrombolysis was 10.8 (n 7) and the maximum thrombolysis which could be achieved in the applied model was about 80 in vivo clearance The in vivo clearance (rate of degradation) of p-t-PA from E.coli was also investigated in rabbits. 200000 IU/kg body weight in a total volume of 6 ml was infused

-Y

-4 MV

.A

'-I

I,

IX i i- 16 over a period of 30 minutes into the ear vein. Plasma samples were taken from a catheter in the femoral vein.

The p-t-PA activity in the samples was determined after euglobulin precipitation according to the test instructions for the t-PA standard (Boehringer Mannheim). Fig. 4 shows the time-course of the level of activity during and after the infusion.

Examp e 4 Solubility properties of p-t-PA from E. coli p-t-PA from E. coli exhibits only a slight solubility in dilute aqueous buffer solutions at physiological pH-values in 0.1 mol/l Tris-HCl, pH 7.5 or 0.1 mol/l sodium phosphate, pH For an efficient purification by conventional chromatographic separation methods, it is essential to define conditions under which p-t-PA from E. coli is readily soluble. The same applies to the development of a pharmaceutically tolerable galenic formulation. In the following some data on the solubility of p-t-PA in different buffer solutions are summarized.

Solubility experiments with concentrated renaturation solution (cf. Example 1) The concentrate of the renaturation solution was dialyzed against 20 mmol/l citric acid, pH 2.5 at 4°C and centrifuged. The clear supernatant was then dialyzed at 4°C in 0.5 ml portions against 300 ml buffer solution and centrifuged. The solubility was determined by measurement of the activity in the supernatant.

sf

"I.

c9 17 Dependency of solubility on pH in 20 mmrol/l citric acid/NaOH or 20 mmol/l sodium phosphate.

pH p-t-PA (g.g/ml) 403 420 38 3 2 4 6 Dependency of solubility on pH in the presence of 0.3 rnol/l arginine HCl plus 20 mmol/l citric acid mmol/l Tris-HC1.

PH p-t-PA (gg/ml) or 409 504 607 865 696 698 725

ON

L

[I

18 Dependency of solubility on pH in the presence of 0.3 mol/1 guanidinium HC1 plus 20 mmol/1 citric acid or mmol/1 Tris-HC1.

pH p-t-PA (Ag/ml) 211 220 432 509 243 112 104 Influence of urea, urea derivatives or amides of carboxylic acid on the solubility in 20 mmol/1 citric acid /NaOH, pH Comnound n---PA Itnn/m11 n-t-PA ("q1M1% 0.3 mol/1 urea 0.3 mol/l dimethylurea 0.3 mol/1 tetramethylurea 1 mol/1 dimethylformamide 512 455 539 451 Solubility experiments with p-t-PA from E. coli after purification by affinity chromatography (cf. Example 2) Purified p-t-PA from E. coli in 0.5 mol/1 L-arginine/H 3

PO

4 pH 7.2, 0.01 Tween 80 was dialyzed in 0.5 ml portions against 200 ml buffer solution (L-arginine/H 3

PO

4 pH 7.2, 0.01 Tween 80) and centrifuged. The solubility was determined by measurement of the activity in the supernatant.

:I

19 L-Arainine (mol/11 D-t-PA (gqlml)l 0.05 0.1 0.2 0.3 0.4 89 264 389 400 440 The reduction in solubility at low arginine concentrations can be partially compensated by addition of tetramethylurea.

It can thus be seen that purified p-t-PA has a good solubility at a pH of less than 4, especially in the presence of L-arginine or guanidinium hydrochloride.

2r~~ ILbm i N;t

Claims (9)

1. A preparation of recombinant plasminogen activator (p-t-PA) expressed in prokaryotes, wherein it has a content of active p-t-PA of more than 90 with respect to total protein and a specific activity of more than 0.4 x 106 IU/mg.

2. Preparation as claimed in claim 1, wherein it has a content of active p-t-PA of more than 95 with respect to total protein and in particular of more than 98

3. Process for the production of a preparation of recombinant plasminogen activator (p-t-FA) expressed in prokaryotes with a content of active p-t-PA of more than 90 with respect to total protein and a specific activity of more than 0.4 x 106 IU/mg by lysis of the prokaryote cells which express the p-t-PA, isolation of inclusion bodies solubilization of the IB's under denaturing and reducing conditions, derivatization to mixed disulphides and reactivation under controlled redox conditions, wherein after reactivation p-t-PA is concentrated in the reactivation mixture and this is followed by a chromatographic purification by affinity chromatography.

4. Process as claimed in claim 3, wherein the concentration of p-t-PA in the reactivation mixture is carried out, especially for small volumes, by ammonium sulphate precipitation. 21 Process as claimed in claim 3, wherein the concentration of p-t-PA in the reactivation mixture is carried out, especially for larger volumes, in a haemodialyzer.

6. Process as claimed in one of the claims 3 to wherein an ETI adsorber column is used for the chromatographic purification.

7. Process as claimed in one of the claims 3 to 6, wherein the process is carried out in the presence of 25 to 1000 mmol/l L-arginine.

8. Process as claimed in one of the claims 3 to 7, wherein the chromatographic purification is carried out with the concentrate of the reactivation mixture over an ETI adsorber column which contains to 1000 mmol/l, preferably 500 to 1000 mmol/l L-arginine.

9. Process as claimed in claim 8, wherein the concentrate used for the chromatographic purification is adjusted to a pH value of more than Process as claimed in one of the claims 3 to 9, wherein the elution is carried out at a pH value of to

11. Use of a preparation as claimed in claims 1 or 2 in an agent to dissolve blood clots. DATED this FOURTH day of MAY 1990 Boehringer Mannheim GmbH by DAVIES COLLISON Patent Attorneys for the applicant(s) MVS W a L i