CA2024701A1 - Process for the purification of plasminogen activator inhibitor 2 (pai-2) - Google Patents
Process for the purification of plasminogen activator inhibitor 2 (pai-2)Info
- Publication number
- CA2024701A1 CA2024701A1 CA002024701A CA2024701A CA2024701A1 CA 2024701 A1 CA2024701 A1 CA 2024701A1 CA 002024701 A CA002024701 A CA 002024701A CA 2024701 A CA2024701 A CA 2024701A CA 2024701 A1 CA2024701 A1 CA 2024701A1
- Authority
- CA
- Canada
- Prior art keywords
- pai
- purification
- solution
- plasminogen activator
- buffer
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Abandoned
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
- C07K1/14—Extraction; Separation; Purification
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/81—Protease inhibitors
- C07K14/8107—Endopeptidase (E.C. 3.4.21-99) inhibitors
- C07K14/811—Serine protease (E.C. 3.4.21) inhibitors
- C07K14/8121—Serpins
- C07K14/8132—Plasminogen activator inhibitors
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- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Health & Medical Sciences (AREA)
- Medicinal Chemistry (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Biophysics (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Life Sciences & Earth Sciences (AREA)
- Molecular Biology (AREA)
- Biochemistry (AREA)
- Gastroenterology & Hepatology (AREA)
- Analytical Chemistry (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
- Medicines Containing Material From Animals Or Micro-Organisms (AREA)
- Peptides Or Proteins (AREA)
- Enzymes And Modification Thereof (AREA)
Abstract
BEHRINGWERKE ARTIENGESELLSCHAFT 89/B 038 - Ma 779 Dr. Ha/Bi Abstract of the disclosure A process for the purification of plasminogen activator inhibitor 2 (PAI-2) A process for the purification of plasminogen activator inhibitor 2 (PAI-2) is described, wherein impurities are precipitated with an acridine or quinoline base.
Description
~024701 BEHRINGWERRE AKTIENGESELLSCHAET HOE 89/B 038 - Ma 779 Dr. Ha/Bi Description A process for the purification of plasminogen activator inhibitor 2 (PAI-2) The invention relates to a process for the purification of plasminogen activator inhibitor 2 (PAI-2), wherein impurities are precipitated with an acridine or quinoline base.
Plasminogen activators (PA) are serine proteases which activate the fibrinolytic system. They convert the inactive proenzyme plasminogen into the active enzyme plasmin which degrades fibrin and fibrinogen. In the body there are two PA, the tissue plasminogen activator ~t-PA) and the PA which is identical with urokinase (u-PA) and which is produced by renal cells but also other normal or malignant cells. The t-PA possesses particular importance in the prevention of thromboses. u-PA prevents formation of fibrin clots in the urinary tract but also contributes to the healing of wounds and has been found where there is neoplastic growth.
The concentration and activity of the PA are regulated by their synthesis but also by inhibitors. Two specific PA
inhibitors (PAI) which do not inhibit plasmin or other proteases are known. One of them is produced in endothe-lial cells (PAI-l~, the other one found in placenta (PAI-2) has also been called "minactivin".
Starting from an extract from placenta a 120-fold concen-tration of PAI-2 can be achieved according to the state of the art by an ammonium sulfate fractionation, adsorp-tion of impurities on CM-Sephadex C-50, a gel filtration and hydroxyapatite chromatography. This process results in two forms of PAI-2 with molecular weights of 70 and 43 kDa. Immunoaffinity chromatography and a combination 20~7Gl of immunoaffinity chromatography and FPLC have also been used for purification. A combination of 8 process fiteps comprising a precipitation with neutral salts, chromato-graphies on CM- and DEAE- RSepharose and hydroxyapatite, and a preparative electrophoresis led, starting from placenta, to a pure PAI-2 with a molecular weight of 47 kDa. It has also been known that chromatographic purification steps can be influenced advantageously by the addition of reducing agents of the dithiothreitol type.
The object of the invention is to find a simpler process which can be used on the production scale for the isola-tion of PAI-2.
Surprisingly it has been that impurities can be precipit-ated from a solution of PAI-2 using 2-ethoxy-6,9-diamino-acridine lactate while the PAI-2 itself r0mains in solution in the supernatant.
A preincubation with dithiothreitol (DTT) proved to be advantageous.
The invention relates to a process for the purification of plasminogen act~vator inhibitor 2 (PAI-2), which comprises a PAI-2-containing solution being preincubated with an agent cleaving disulfide linkages, preferably dithiothreitol (DTT), and being mixed with ~ suitable, water-soluble acridine or quinoline base, preferably 2-ethoxy-6,9-diaminoacridine lactate, in such an amount that the precipitate formed does not contain more than a little PAI-2, this precipitate being separated off, and the PAI-2 being obtained from the supernatant and, where appropriate, further purified using known processes.
PAI-2 will also precipitate if small amounts of acridine or quinoline base in relation to the amount of proteins in solution are added. An appropriate concentration of a water-soluble salt of the base, preferably 2-ethoxy-6,9-202~7~
diaminoacridine lactate, is from 200 mg to 2 g/g ofprotein in solution (PAI-2 remains substantially in solution). The process is carried out at a pH of 5.5-8.5.
The PAI-2-containing solution may have been pasteurized beforehand, where appropriate with the addition of stabilizers, preferably glycine and/or ~ucrose.
The PAI-2-containing solution is preferably an extract of placenta or a solution containing genetically engineered PAI-2.
The disulfide-cleaving agent is used in a concentration of from 10 mmol/l to 250 mmol/l, preferably 100 mmol/l.
Preincubation is carried out for from 15 min to 3 hours, preferably for 1 hour, at 10 to 40C, preferably about 37C.
85 - 90 % of the employed PAI-2 activity, but only 5 -8 % of the employed amount of protein, are present in the supernatant when the process is carried out as described.
After the precipitation the acridine base can be salted out of the supernatant, preferably using NaCl, preferably 5 %, and the PAI-2 can be concentrated by an ammonium sulfate precipitation, for example by 80 ~ saturation of the solution with ammonium sulfate, which yields an addi-tional purification in combination with a preliminary precipitation at 30 ~ ~aturation of the solution with ammonium sulfate. ~ince about 90 ~ of the employed protein are removed by the Rivanol and ammonium sulfate precipitation, the small residual amount can then be sub-jected to purification processes which are su~ect to volume limitation: for example a chromatography on DEAE-RAffigel Blue, a chromatography gel with bifunctionalaffinity which i8 composed of diethylaminoethyl groups and RCibacron Blue F3GA dye covalently bonded to agarose, and on hydroxyapatite. Naterial of this degree of purity should already be suitable for therapeutic use. Ultra-2 ~ 2 ~ r~ o ~
purification can be carried out by hydrophobic chroma-tography on a phenylalanine column but is associated with substantial losses.
The PAI-2 which is obtained may additionally be pas-teurized, if this has not been done beforehand.
The process according to the invention is described hereinafter:
An amidolytic method was used in combination with the chromogenic substrate S-2444 (Glu-Gly-Arg-pNA) from Rabi for monitoring the isolation of PAI-2.
For the determination, 50 ~1 of urokinase (u-PA) (1000 U/ml) were incubated with 100 ~1 of PAI-2-contain-ing sample for 4 min at room temperature, and 80 ~1 of this mixture were transferred into a plastic cuvette which had been prewarmed to 37C. 50 ~1 of buffer A and 20 ~1 of S-2444 (6 mM) were then added. The increase in absorption at 405 nm was determined in a Cobas Bio centrifugal analyzer. The measurements were evaluated by comparison with a dilution plot which had been con-structed via serial dilution of an in-house PAI-2 standard.
Materials which were used for the isolation of PAI-2:
- 2-Ethoxy-6,9-diaminoacridine lactate (6,9-diamino-2-ethoxyacridine lactate): Sigma Chemie GmbH, D-6100 Darmstadt; a 2.5 % (w/v) strength solution in 12.5 mM tris, pH 6.8, was used for the fractional precipitation.
- Dithiothreitol (DTT): Serva Feinbiochemika GmbH
Co./ D-6900 Heidelberg - Hydroxyapatite: RBioRad, Richmond, CA, USA
- Phenylalanine-RSepharose and CNBr-activated 2~2~701 RSepharose 4B: Deutsche Pharmacia GmbH, D-69~0 Freiburg - S-2444: Rabi Vitrum, S-11287 Stockholm, Sweden - Tris(hydroxymethyl)aminomethane (tris): E. Merck, D-6100 Darmstadt - Urokinase (RActosolv): Behringwerke AG, D-3550 Marburg - Buffer A: 50 mM tris, pH 8.4, 1 % polygeline, 100 mM
NaCl, 0.01 % ~Triton X 100 and 0.01 ~ NaN3 - Buffer B: 20 mM tris, p~ 7.5, 20 mM DTT
- Buffer C: 20 mM Na2HPO4, pH 6.8, 20 mM DTT
- Buffer D: 20 mM tris, pH 7.5, 20 mM DTT, 30 %
saturation with ammonium sulfate - Buffer E: 20 mM tris, pH 7.5, 100 mM NaCl - Buffer F: 20 mM tris, pH 6.8 Example Frozen human placenta which had been washed free from blood was used as raw material for the preparation of PAI-2; the placenta had been chopped up in a cutter and then extracted with 0.5 % NaCl and 3 mM EDTA. The rem-nants of cells were removed by centrifugation and the supernatant was precipitated with 8 % Rivanol, and the precipitate was dissolved and sub~ected to a fractional precipitation with ammonium sulfate. The precipitate which contained the PAI-2 was dissolved in buffer F and dialyzed against it. The concentrated extract of placenta contained 4,615 U of P~I-2Jml with a specific activity of 120 U/mg. This material was used for the ultrapurification.
202~
- Rivanol and ammonium sulfate precipitation 500 ml of the starting material described above (Tab. 1) were incubated with 7.7 g of DTT (final concentration 100 mM) at 37C for 1 hour. 1,520 ml of a 2.5 % strength Rivanol solution were added dropwise to this solution while continuously stirring cautiously, thus reaching a final concentration corresponding to 200 %. The precipi-tate resulting from the precipitation was removed by centrifugation, and excess Rivanol was removed from the supernatant by addition of 100 g of solid NaCl to final concentration of 5 %. 343 g of solid ammonium sulfate were then added to the supernatant until a final con-centration corresponding to 30 ~ saturation was reached.
The precipitate was removed by centrifugation (20 min, 3000 rpm), and 694 g of solid ammonium sulfate were then added to a final concentration of 80 % saturation. The precipitate resulting from this was dissolved buffer B in high concentration and dialyzed. This resulted in 80 ml of dissolved and dialyzed ammonium sulfate residue.
- DEAE-~Affigel Blue chromatography 80 ml of the ammonium sulfate precipitate were applied to a DEAE-Affigel Blue column (17.5 x 4.4 cm, 260 ml gel bed) which had been equilibrated with buffer B. The column was eluted using an NaCl gradient in buffer B
(2 x 10~0 ml) which covered a range from 0 to 200 mM NaCl at a flow rate of 90 ml/h. The protein concentration was continuously measured via the O~ at 280 nm and the PAI-2 activity was determined as described.
- Hydroxyapatite chromatography 472 ml of the PAI-2-containing fractions from the previ-ous step were collected, dialyzed against buffer C and applied to a hydroxyapatite column (15 x 3.2 cm, 56 ml gel bed) (Table 1) which had been equilibrated with the same buffer. The column was eluted at a flow rate of 202~7Gl same buffer. The column was elu~ed at a flow rate of 50 ml/h and using a salt gradient of sodium phosphate (0.02 to 0.3 M; 2 x 250 ml) in buffer C. The protein concentration and PAI-2 activity of the eluate were determined continuously.
- Phenylalanine-RSepharose The PAI-2-containing fractions after hydroxyapatite chromatography were collected, and 13.2 g of solid ammo-nium sulfate were added to 120 ml of thi6 material to reach 30 ~ saturation. Thi6 solution was applied to a phenylalanine-RSepharose column which had been equili-brated with buffer D. The resin was eluted at a flow rate of 20 ml/h and using a gradient formed from buffer D and B (2 x 100 ml). The PAI-2-containing fractions were col-lected and characterized. The purest material had aspecific activity of 60,000 U (= units)/mg and migrated in SDS polyacrylamide gel electrophoresis as one band which had a molecular weight of 43 kDa and could be demonstrated in an immunoblot with a specific monoclonal anti~ody.
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Plasminogen activators (PA) are serine proteases which activate the fibrinolytic system. They convert the inactive proenzyme plasminogen into the active enzyme plasmin which degrades fibrin and fibrinogen. In the body there are two PA, the tissue plasminogen activator ~t-PA) and the PA which is identical with urokinase (u-PA) and which is produced by renal cells but also other normal or malignant cells. The t-PA possesses particular importance in the prevention of thromboses. u-PA prevents formation of fibrin clots in the urinary tract but also contributes to the healing of wounds and has been found where there is neoplastic growth.
The concentration and activity of the PA are regulated by their synthesis but also by inhibitors. Two specific PA
inhibitors (PAI) which do not inhibit plasmin or other proteases are known. One of them is produced in endothe-lial cells (PAI-l~, the other one found in placenta (PAI-2) has also been called "minactivin".
Starting from an extract from placenta a 120-fold concen-tration of PAI-2 can be achieved according to the state of the art by an ammonium sulfate fractionation, adsorp-tion of impurities on CM-Sephadex C-50, a gel filtration and hydroxyapatite chromatography. This process results in two forms of PAI-2 with molecular weights of 70 and 43 kDa. Immunoaffinity chromatography and a combination 20~7Gl of immunoaffinity chromatography and FPLC have also been used for purification. A combination of 8 process fiteps comprising a precipitation with neutral salts, chromato-graphies on CM- and DEAE- RSepharose and hydroxyapatite, and a preparative electrophoresis led, starting from placenta, to a pure PAI-2 with a molecular weight of 47 kDa. It has also been known that chromatographic purification steps can be influenced advantageously by the addition of reducing agents of the dithiothreitol type.
The object of the invention is to find a simpler process which can be used on the production scale for the isola-tion of PAI-2.
Surprisingly it has been that impurities can be precipit-ated from a solution of PAI-2 using 2-ethoxy-6,9-diamino-acridine lactate while the PAI-2 itself r0mains in solution in the supernatant.
A preincubation with dithiothreitol (DTT) proved to be advantageous.
The invention relates to a process for the purification of plasminogen act~vator inhibitor 2 (PAI-2), which comprises a PAI-2-containing solution being preincubated with an agent cleaving disulfide linkages, preferably dithiothreitol (DTT), and being mixed with ~ suitable, water-soluble acridine or quinoline base, preferably 2-ethoxy-6,9-diaminoacridine lactate, in such an amount that the precipitate formed does not contain more than a little PAI-2, this precipitate being separated off, and the PAI-2 being obtained from the supernatant and, where appropriate, further purified using known processes.
PAI-2 will also precipitate if small amounts of acridine or quinoline base in relation to the amount of proteins in solution are added. An appropriate concentration of a water-soluble salt of the base, preferably 2-ethoxy-6,9-202~7~
diaminoacridine lactate, is from 200 mg to 2 g/g ofprotein in solution (PAI-2 remains substantially in solution). The process is carried out at a pH of 5.5-8.5.
The PAI-2-containing solution may have been pasteurized beforehand, where appropriate with the addition of stabilizers, preferably glycine and/or ~ucrose.
The PAI-2-containing solution is preferably an extract of placenta or a solution containing genetically engineered PAI-2.
The disulfide-cleaving agent is used in a concentration of from 10 mmol/l to 250 mmol/l, preferably 100 mmol/l.
Preincubation is carried out for from 15 min to 3 hours, preferably for 1 hour, at 10 to 40C, preferably about 37C.
85 - 90 % of the employed PAI-2 activity, but only 5 -8 % of the employed amount of protein, are present in the supernatant when the process is carried out as described.
After the precipitation the acridine base can be salted out of the supernatant, preferably using NaCl, preferably 5 %, and the PAI-2 can be concentrated by an ammonium sulfate precipitation, for example by 80 ~ saturation of the solution with ammonium sulfate, which yields an addi-tional purification in combination with a preliminary precipitation at 30 ~ ~aturation of the solution with ammonium sulfate. ~ince about 90 ~ of the employed protein are removed by the Rivanol and ammonium sulfate precipitation, the small residual amount can then be sub-jected to purification processes which are su~ect to volume limitation: for example a chromatography on DEAE-RAffigel Blue, a chromatography gel with bifunctionalaffinity which i8 composed of diethylaminoethyl groups and RCibacron Blue F3GA dye covalently bonded to agarose, and on hydroxyapatite. Naterial of this degree of purity should already be suitable for therapeutic use. Ultra-2 ~ 2 ~ r~ o ~
purification can be carried out by hydrophobic chroma-tography on a phenylalanine column but is associated with substantial losses.
The PAI-2 which is obtained may additionally be pas-teurized, if this has not been done beforehand.
The process according to the invention is described hereinafter:
An amidolytic method was used in combination with the chromogenic substrate S-2444 (Glu-Gly-Arg-pNA) from Rabi for monitoring the isolation of PAI-2.
For the determination, 50 ~1 of urokinase (u-PA) (1000 U/ml) were incubated with 100 ~1 of PAI-2-contain-ing sample for 4 min at room temperature, and 80 ~1 of this mixture were transferred into a plastic cuvette which had been prewarmed to 37C. 50 ~1 of buffer A and 20 ~1 of S-2444 (6 mM) were then added. The increase in absorption at 405 nm was determined in a Cobas Bio centrifugal analyzer. The measurements were evaluated by comparison with a dilution plot which had been con-structed via serial dilution of an in-house PAI-2 standard.
Materials which were used for the isolation of PAI-2:
- 2-Ethoxy-6,9-diaminoacridine lactate (6,9-diamino-2-ethoxyacridine lactate): Sigma Chemie GmbH, D-6100 Darmstadt; a 2.5 % (w/v) strength solution in 12.5 mM tris, pH 6.8, was used for the fractional precipitation.
- Dithiothreitol (DTT): Serva Feinbiochemika GmbH
Co./ D-6900 Heidelberg - Hydroxyapatite: RBioRad, Richmond, CA, USA
- Phenylalanine-RSepharose and CNBr-activated 2~2~701 RSepharose 4B: Deutsche Pharmacia GmbH, D-69~0 Freiburg - S-2444: Rabi Vitrum, S-11287 Stockholm, Sweden - Tris(hydroxymethyl)aminomethane (tris): E. Merck, D-6100 Darmstadt - Urokinase (RActosolv): Behringwerke AG, D-3550 Marburg - Buffer A: 50 mM tris, pH 8.4, 1 % polygeline, 100 mM
NaCl, 0.01 % ~Triton X 100 and 0.01 ~ NaN3 - Buffer B: 20 mM tris, p~ 7.5, 20 mM DTT
- Buffer C: 20 mM Na2HPO4, pH 6.8, 20 mM DTT
- Buffer D: 20 mM tris, pH 7.5, 20 mM DTT, 30 %
saturation with ammonium sulfate - Buffer E: 20 mM tris, pH 7.5, 100 mM NaCl - Buffer F: 20 mM tris, pH 6.8 Example Frozen human placenta which had been washed free from blood was used as raw material for the preparation of PAI-2; the placenta had been chopped up in a cutter and then extracted with 0.5 % NaCl and 3 mM EDTA. The rem-nants of cells were removed by centrifugation and the supernatant was precipitated with 8 % Rivanol, and the precipitate was dissolved and sub~ected to a fractional precipitation with ammonium sulfate. The precipitate which contained the PAI-2 was dissolved in buffer F and dialyzed against it. The concentrated extract of placenta contained 4,615 U of P~I-2Jml with a specific activity of 120 U/mg. This material was used for the ultrapurification.
202~
- Rivanol and ammonium sulfate precipitation 500 ml of the starting material described above (Tab. 1) were incubated with 7.7 g of DTT (final concentration 100 mM) at 37C for 1 hour. 1,520 ml of a 2.5 % strength Rivanol solution were added dropwise to this solution while continuously stirring cautiously, thus reaching a final concentration corresponding to 200 %. The precipi-tate resulting from the precipitation was removed by centrifugation, and excess Rivanol was removed from the supernatant by addition of 100 g of solid NaCl to final concentration of 5 %. 343 g of solid ammonium sulfate were then added to the supernatant until a final con-centration corresponding to 30 ~ saturation was reached.
The precipitate was removed by centrifugation (20 min, 3000 rpm), and 694 g of solid ammonium sulfate were then added to a final concentration of 80 % saturation. The precipitate resulting from this was dissolved buffer B in high concentration and dialyzed. This resulted in 80 ml of dissolved and dialyzed ammonium sulfate residue.
- DEAE-~Affigel Blue chromatography 80 ml of the ammonium sulfate precipitate were applied to a DEAE-Affigel Blue column (17.5 x 4.4 cm, 260 ml gel bed) which had been equilibrated with buffer B. The column was eluted using an NaCl gradient in buffer B
(2 x 10~0 ml) which covered a range from 0 to 200 mM NaCl at a flow rate of 90 ml/h. The protein concentration was continuously measured via the O~ at 280 nm and the PAI-2 activity was determined as described.
- Hydroxyapatite chromatography 472 ml of the PAI-2-containing fractions from the previ-ous step were collected, dialyzed against buffer C and applied to a hydroxyapatite column (15 x 3.2 cm, 56 ml gel bed) (Table 1) which had been equilibrated with the same buffer. The column was eluted at a flow rate of 202~7Gl same buffer. The column was elu~ed at a flow rate of 50 ml/h and using a salt gradient of sodium phosphate (0.02 to 0.3 M; 2 x 250 ml) in buffer C. The protein concentration and PAI-2 activity of the eluate were determined continuously.
- Phenylalanine-RSepharose The PAI-2-containing fractions after hydroxyapatite chromatography were collected, and 13.2 g of solid ammo-nium sulfate were added to 120 ml of thi6 material to reach 30 ~ saturation. Thi6 solution was applied to a phenylalanine-RSepharose column which had been equili-brated with buffer D. The resin was eluted at a flow rate of 20 ml/h and using a gradient formed from buffer D and B (2 x 100 ml). The PAI-2-containing fractions were col-lected and characterized. The purest material had aspecific activity of 60,000 U (= units)/mg and migrated in SDS polyacrylamide gel electrophoresis as one band which had a molecular weight of 43 kDa and could be demonstrated in an immunoblot with a specific monoclonal anti~ody.
202470 ~
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a~ o OD U~
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~ , ~, .,, 4 ~1 C
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P~
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Claims (9)
1. A process for the purification of plasminogen activator inhibitor 2 (PAI-2), which comprises a PAI-2-containing solution being preincubated with an agent cleaving disulfide linkages, and being mixed with a suitable water-soluble acridine or quinoline base, preferably 2-ethoxy-6,9-diaminoacridine lactate, in such an amount that the precipitate formed does not contain more than a little PAI-2, this precipitate being separated off and the PAI-2 being obtained from the supernatant and, where ap-propriate, further purified using known processes.
2. The process as claimed in claim 1, wherein the PAI-2-containing solution is an extract of placenta or a solution of genetically engineered PAI-2.
3. The process as claimed in claim 1, wherein the disulfide-cleaving agent is dithiothreitol.
4. The process as claimed in claim 1, wherein the disulfide-cleaving agent is used in a concentration of from 10 mmol/l to 250 mmol/l, preferably 100 mmol/l.
5. The process as claimed in claim 1, wherein pre-incubation is carried out for 15 min to 3 hours, preferably for 1 hour.
6. The process as claimed in claim 1, wherein pre-incubation is carried out at 10 to 40°C, preferably about 37°C.
7. The process as claimed in claim 1, wherein the base is used in dissolved form at a pH of 5.5 - 8.5.
8. The process as claimed in claim 1, wherein a PAI-2-containing solution is pasteurized.
9. The process as claimed in claim 1 and substantially as described herein.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| DE3929504A DE3929504A1 (en) | 1989-09-06 | 1989-09-06 | METHOD FOR PURIFYING PLASMINOGEN ACTIVATOR INHIBITOR 2 (PAI-2) |
| DEP3929504.4 | 1989-09-06 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA2024701A1 true CA2024701A1 (en) | 1991-03-07 |
Family
ID=6388683
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA002024701A Abandoned CA2024701A1 (en) | 1989-09-06 | 1990-09-05 | Process for the purification of plasminogen activator inhibitor 2 (pai-2) |
Country Status (11)
| Country | Link |
|---|---|
| EP (1) | EP0418647B1 (en) |
| JP (1) | JPH0393798A (en) |
| KR (1) | KR910006477A (en) |
| AT (1) | ATE115589T1 (en) |
| AU (1) | AU642661B2 (en) |
| CA (1) | CA2024701A1 (en) |
| DE (2) | DE3929504A1 (en) |
| DK (1) | DK0418647T3 (en) |
| ES (1) | ES2066066T3 (en) |
| IE (1) | IE65590B1 (en) |
| PT (1) | PT95205A (en) |
Families Citing this family (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| DE4214999C2 (en) * | 1992-05-06 | 1994-07-14 | Behringwerke Ag | Purification of plasminogen activator inhibitor 2 by immunoaffinity chromatography |
| ES2287687T3 (en) * | 2003-01-09 | 2007-12-16 | Genentech, Inc. | PURIFICATION OF POLYPEPTIDES. |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| DE3713272A1 (en) * | 1987-04-18 | 1988-11-03 | Behringwerke Ag | Plasminogen activator inhibitor type 2 (PAI-2) |
-
1989
- 1989-09-06 DE DE3929504A patent/DE3929504A1/en not_active Withdrawn
-
1990
- 1990-09-04 KR KR1019900013890A patent/KR910006477A/en active IP Right Grant
- 1990-09-05 CA CA002024701A patent/CA2024701A1/en not_active Abandoned
- 1990-09-05 AU AU62175/90A patent/AU642661B2/en not_active Ceased
- 1990-09-05 DK DK90117038.1T patent/DK0418647T3/en active
- 1990-09-05 ES ES90117038T patent/ES2066066T3/en not_active Expired - Lifetime
- 1990-09-05 IE IE323090A patent/IE65590B1/en not_active IP Right Cessation
- 1990-09-05 JP JP2233445A patent/JPH0393798A/en active Pending
- 1990-09-05 PT PT95205A patent/PT95205A/en not_active Application Discontinuation
- 1990-09-05 DE DE59008002T patent/DE59008002D1/en not_active Expired - Lifetime
- 1990-09-05 AT AT90117038T patent/ATE115589T1/en not_active IP Right Cessation
- 1990-09-05 EP EP90117038A patent/EP0418647B1/en not_active Expired - Lifetime
Also Published As
| Publication number | Publication date |
|---|---|
| DE59008002D1 (en) | 1995-01-26 |
| AU642661B2 (en) | 1993-10-28 |
| EP0418647B1 (en) | 1994-12-14 |
| DE3929504A1 (en) | 1991-03-07 |
| KR910006477A (en) | 1991-04-29 |
| PT95205A (en) | 1991-05-22 |
| ES2066066T3 (en) | 1995-03-01 |
| IE903230A1 (en) | 1991-03-13 |
| AU6217590A (en) | 1991-03-14 |
| IE65590B1 (en) | 1995-11-01 |
| JPH0393798A (en) | 1991-04-18 |
| ATE115589T1 (en) | 1994-12-15 |
| EP0418647A1 (en) | 1991-03-27 |
| DK0418647T3 (en) | 1995-05-15 |
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