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US4175182A
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Separation of high-activity heparin by affinity chromatography on supported protamine
Assignee: Research Corp
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Filing: 1978-07-03
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Granted: 1979-11-20
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Expires: 1998-07-03
Abstract
Abstract An affinity chromatography method for the separation of high-activity heparin from a mixture thereof with low-activity heparin. The method employs an affinity column of protamine coupled to a water-insoluble solid support material such as agarose, and a series of sodium chloride-imidazole elution buffers (pH 6.5-7.5) varying in sodium chloride molarity from about 1.3 to about 2.0. Stepwise elution of a plurality of heparin fractions from the affinity column with the elution buffers of successively increasing sodium chloride molarity results in the removal from the affinity column of first the low-activity heparin and finally the high-activity heparin.
Inventors
- 👤 Gottfried Schmer
Bibliographic Details
Application Number
—
Jurisdiction
US
Data Source
google_patents
Last Synchronized
2026-10-23 19:05
CPC Classifications
C; C08; C08B; C08B37/00; C08B37/006; C08B37/0063; C08B37/0075
Claims Analysis
Total Claims: 8
Claim 1
Independent Claim
161 words
An affinity chromatography method for the separation of heparin having a specific activity of at least about 270 IU/mg from a mixture thereof with heparin of lower specific activity, comprising the steps of: (a) preparing a solution of said heparin mixture in a solvent of about 0.6 molar sodium chloride buffered with about 0.01-0.03 molar imidazole to pH 6.5-7.5; (b) applying said heparin solution onto an affinity column composed of protamine coupled to a water-insoluble solid support material; and (c) successively eluting a plurality of heparin fractions from said affinity column with a series of elution buffers of successively increasing molarity of sodium chloride buffered with about 0.01-0.03 molar imidazole to pH 6.5-7.5, said elution buffers having a sodium chloride molarity ranging from about 1.3 to about 2.0, so as to effect a step-wise removal from said affinity column of first the heparin of lower specific activity and finally the heparin having a specific activity of at least about 270 IU/mg.
Claim 2
Dependent Claim
161 words
An affinity chromatography method for the separation of heparin having a specific activity of at least about 270 IU/mg from a mixture thereof with heparin of lower specific activity, comprising the steps of: (a) preparing a solution of said heparin mixture in a solvent of about 0.6 molar sodium chloride buffered with about 0.01-0.03 molar imidazole to pH 6.5-7.5; (b) applying said heparin solution onto an affinity column composed of protamine coupled to a water-insoluble solid support material; and (c) successively eluting a plurality of heparin fractions from said affinity column with a series of elution buffers of successively increasing molarity of sodium chloride buffered with about 0.01-0.03 molar imidazole to pH 6.5-7.5, said elution buffers having a sodium chloride molarity ranging from about 1.3 to about 2.0, so as to effect a step-wise removal from said affinity column of first the heparin of lower specific activity and finally the heparin having a specific activity of at least about 270 IU/mg.
Claim 3
Dependent Claim
38 words
The method of claim 1, wherein the elution buffer employed in eluting the final heparin fraction having a specific activity of at least about 270 IU/mg has a sodium chloride molarity of from about 1.9 to about 2.0.
Claim 4
Dependent Claim
28 words
The method of claim 2, wherein the elution buffer employed in eluting the penultimate heparin fraction has a sodium chloride molarity of from about 1.7 to about 1.9.
Claim 5
Dependent Claim
77 words
The method of claim 3, wherein said series of elution buffers comprises a first elution buffer having a sodium chloride molarity of from about 1.3 to about 1.4, a second elution buffer having a sodium chloride molarity of from about 1.4 to about 1.7, a third elution buffer having a sodium chloride molarity of from about 1.7 to about 1.9, and a fourth elution buffer having a sodium chloride molarity of from about 1.9 to about 2.0.
Claim 6
Dependent Claim
23 words
The method of claim 4, wherein said solvent and each of said elution buffers is buffered with 0.02 molar imidazole to pH 7.35.
Claim 7
Dependent Claim
25 words
The method of claim 1, wherein said affinity column is equilibrated with an additional amount of said solvent prior to application of said heparin solution.
Claim 8
Dependent Claim
11 words
The method of claim 1, wherein said support material is agarose.
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) | 1982-11-19 | 1983-05-21 | 1983-11-20 | $2000.00 | Window Closed / Lapsed |
| 7.5 Years (Year 8) | 1986-11-19 | 1987-05-21 | 1987-11-20 | $3760.00 | Window Closed / Lapsed |
| 11.5 Years (Year 12) | 1990-11-19 | 1991-05-21 | 1991-11-20 | $7700.00 | Window Closed / Lapsed |