US4259447A
Expired / Lapsed
US
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Process for the production of urokinase in pure condition
Assignee: LABORATOIRES SERONO SA A CORP OF SWITZERLAND
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Filing: 1979-06-29
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Granted: 1981-03-31
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Expires: 1999-06-29
Abstract
Abstract There is provided a process for the production of pure, concentrated urokinase starting from a crude urokinase solution. The crude urokinase solution is contacted in a medium having a pH of >/=6 with a porous solid matrix having a high specific surface area on whose surface there is immobilized aprotinin. Subsequently the urokinase adsorbed in this manner is eluted in an elution agent having a pH</=4.5. There are obtained highly concentrated and very pure solutions of urokinase. The urokinase is used in medicine as a pharmaceutical for the dissolving of fibrinous clots and coagulums.
Inventors
- 👤 Robert Hafeli
Bibliographic Details
Application Number
—
Jurisdiction
US
Data Source
google_patents
Last Synchronized
2026-10-29 13:56
CPC Classifications
C; C12; C12N; C12N9/00; C12N9/14; C12N9/48; C12N9/50; C12N9/64; C12N9/6421; C12N9/6424; C12N9/6456; C12N9/6462; C12Y; C12Y304/00; C12Y304/21; C12Y304/21073; Y; Y10; Y10S; Y10S435/00; Y10S435/814; Y10S435/815
Claims Analysis
Total Claims: 36
Claim 1
Independent Claim
74 words
A process for the production of pure urokinase of high concentration and purity starting from a preliminarily purified and concentrated urokinase crude solution comprising contacting this crude urokinase solution at a pH≧6 with a porous solid matrix having a high specific area and having immobilized on the surface aprotinin by means of covalent, chemical bonds, separating off the carrier solution and subsequently eluting the adsorbed urokinase by means of an eluting agent at pH≦4.5.
Claim 2
Dependent Claim
74 words
A process for the production of pure urokinase of high concentration and purity starting from a preliminarily purified and concentrated urokinase crude solution comprising contacting this crude urokinase solution at a pH≧6 with a porous solid matrix having a high specific area and having immobilized on the surface aprotinin by means of covalent, chemical bonds, separating off the carrier solution and subsequently eluting the adsorbed urokinase by means of an eluting agent at pH≦4.5.
Claim 3
Dependent Claim
16 words
A process according to claim 1 wherein the urokinase is separated from the crude solution affinity-chromatographically.
Claim 4
Dependent Claim
38 words
A process according to claim 2 wherein the crude urokinase solution has a concentration of 2,000 to 10,000 IU urakinase per ml of solution and a purity of 200 to 2,000 IU urokinase per ml of protein present.
Claim 5
Dependent Claim
38 words
A process according to claim 3 wherein the crude urokinase solution has a concentration of 3,000 to 5,000 IU urokinase per ml of solution and a purity of 300 to 1,000 IU urokinase per ml of protein present.
Claim 6
Dependent Claim
31 words
A process according to claim 3 wherein the porous solid matrix is a resin based on a polysaccharide, cellulose, agarose, dextran, or a copolymer addition product of a polyacrylamide with agarose.
Claim 7
Dependent Claim
33 words
A process according to claim 5 wherein the aprotinin is bound to the solid matrix either directly by covalent bonds or indirectly by a spacer which is epsilon-aminocaproic acid, divinyl sulfone or bis-oxirane.
Claim 8
Dependent Claim
25 words
A process according to claim 3 wherein the aprotinin is bound to the solid matrix either directly by covalent bonds or indirectly by a spacer.
Claim 9
Dependent Claim
16 words
A process according to claim 7 wherein the spacer is epsilon-aminocaproic acid, divinyl sulfone or bis-oxirane.
Claim 10
Dependent Claim
13 words
A process according to claim 3 wherein the solid matrix is previously activated.
Claim 11
Dependent Claim
11 words
A process according to claim 9 wherein the activator is BrCN.
Claim 12
Dependent Claim
50 words
A process according to claim 5 wherein the adsorption of the urokinase on the aprotinin takes place in a column and the column before the actual adsorption is conditioned with a liquid medium at a pH of 6 to 9 and a total salt concentration of 0.5 to 1 moles/liters.
Claim 13
Dependent Claim
17 words
A process according to claim 11 wherein the conditioning liquid contains a buffer and NaCl or KCl.
Claim 14
Dependent Claim
17 words
A process according to claim 12 wherein the buffer is a phosphate mixture or tris sodium phosphate.
Claim 15
Dependent Claim
23 words
A process according to claim 3 wherein the crude urokinase solution is adjusted to a total salt concentration of 0.5 to 1 mole/liter.
Claim 16
Dependent Claim
13 words
A process according to claim 14 wherein the salt includes NaCl or KCl.
Claim 17
Dependent Claim
23 words
A process according to claim 1 wherein the crude urokinase solution is adjusted to a total salt concentration of 0.5 to 1 mole/liter.
Claim 18
Dependent Claim
13 words
A process according to claim 16 wherein the salt includes NaCl or KCl.
Claim 19
Dependent Claim
70 words
A process according to claim 1 carried out in a separatory column and wherein after the actual adsorption of the urokinase and before the actual elution there is an intermediate rinsing of the column with a 0.5 to 1 molar NaCl solution to remove protein from the separatory column and wherein the intermediate rinsing is continued until optical purity is obtained as shown by substantial optical inactivity at 280 nm.
Claim 20
Dependent Claim
63 words
A process according to claim 3 wherein after the actual adsorption of the urokinase and before the actual elution there is an intermediate rinsing of the column with a 0.5 to 1 molar NaCl solution to remove protein from the separatory column and wherein the intermediate rinsing is continued until optical purity is obtained as shown by substantial optical inactivity at 280 nm.
Claim 21
Dependent Claim
28 words
A process according to claim 3 wherein the elution is carried out at at pH of 2 to 4.5 and a salt concentration of 0.5 to 1 molar.
Claim 22
Dependent Claim
13 words
A process according to claim 20 wherein the salt is NaCl or KCl.
Claim 23
Dependent Claim
16 words
A process according to claim 21 wherein the pH is adjusted with acetic acid or HCl.
Claim 24
Dependent Claim
28 words
A process according to claim 1 wherein the elution is carried out at a pH of 2 to 4.5 and a salt concentration of 0.5 to 1 mole/liter.
Claim 25
Dependent Claim
20 words
A process according to claim 21 wherein there is employed an aminocarboxylic acid as an elution aid in the elution.
Claim 26
Dependent Claim
14 words
A process according to claim 24 wherein the aminocarboxylic acid is lysine or arginine.
Claim 27
Dependent Claim
19 words
A process according to claim 23 wherein there is employed an aminoacid as an elution aid in the elution.
Claim 28
Dependent Claim
14 words
A process according to claim 8 wherein the spacer is divinyl sulfone or bis-oxirane.
Claim 29
Dependent Claim
12 words
A process according to claim 27 wherein the spacer is divinyl sulfone.
Claim 30
Dependent Claim
11 words
A process according to claim 27 wherein the spacer is bis-oxirane.
Claim 31
Dependent Claim
26 words
A process according to claim 19 wherein the aprotinin is bound to the solid matrix by a spacer which is epsilon-aminocaproic acid, divinyl sulfone or bis-oxirane.
Claim 32
Dependent Claim
56 words
A process according to claim 30 wherein the adsorption of the urokinase on the aprotinin takes place in a column and the column before the actual adsorption is conditioned with a liquid medium at a pH of 6 to 9 containing a buffer and NaCl or KCl, the total salt concentration being 0.5 to 1 mole/liter.
Claim 33
Dependent Claim
14 words
A process according to claim 31 wherein the spacer is divinyl sulfone or bis-oxirane.
Claim 34
Dependent Claim
14 words
A process according to claim 30 wherein the spacer is divinyl sulfone or bis-oxirane.
Claim 35
Dependent Claim
20 words
A process according to claim 31 wherein there is employed an aminocarboxylic acid as an elution aid in the elution.
Claim 36
Dependent Claim
27 words
A process according to claim 34 wherein the spacer is divinyl sulfone or bis-oxirane and the aminocarboxylic acid used as an elution aid is lysine or arginine.
References Cited (Backward Citations)
USPTO Annuity & Maintenance Windows
| Annuity Stage | Window Opens | Payment Deadline | Surcharge Period Until | Estimated Fee | Status |
|---|---|---|---|---|---|
| 3.5 Years (Year 4) | 1984-03-30 | 1984-09-29 | 1985-03-31 | $2000.00 | Window Closed / Lapsed |
| 7.5 Years (Year 8) | 1988-03-30 | 1988-09-29 | 1989-03-31 | $3760.00 | Window Closed / Lapsed |
| 11.5 Years (Year 12) | 1992-03-30 | 1992-09-29 | 1993-03-31 | $7700.00 | Window Closed / Lapsed |