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US4677194A
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Isolation of an endotoxin inactivator from human plasma
Assignee: Cambridge Biotech Corp
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Filing: 1985-08-09
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Granted: 1987-06-30
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Expires: 2005-08-09
Abstract
Abstract An endotoxin inactivator is isolated from human plasma by anion exchange (DEAE-Sephadex), dye-affinity (Cibracron Blue-Sepharose), and adsorption (on hydroxyapatite) chromatography. The endotoxin inactivator, isolated in essentially pure form, may be used to depyrogenate clinical blood products.
Inventors
- 👤 Yu-Lee Hao
Bibliographic Details
Application Number
—
Jurisdiction
US
Data Source
google_patents
Last Synchronized
2026-11-09 03:30
CPC Classifications
A; A61; A61L; A61L2/00; A61L2/16; A61L2/18; C; C07; C07K; C07K14/00; C07K14/435; C07K14/46; C07K14/47; C07K14/4701; C07K14/4702; C07K14/4703; A61L2103/00; A61L2103/05; Y; Y10; Y10S; Y10S530/00; Y10S530/827; Y10S530/829
Claims Analysis
Total Claims: 26
Claim 1
Independent Claim
59 words
A method for the isolation, from an endotoxin inactivator-containing fraction of blood, of an endotoxin inactivator in essentially pure form which comprises (a) ion-exchange chromotography of an endotoxin inactivator-containing fraction of blood; (b) dye-affinity chromatography of the product of step (a); and (c) adsorption chromatography of the product of step (b) eluting the endotoxin inactivator in essentially pure form.
Claim 2
Independent Claim
59 words
A method for the isolation, from an endotoxin inactivator-containing fraction of blood, of an endotoxin inactivator in essentially pure form which comprises (a) ion-exchange chromotography of an endotoxin inactivator-containing fraction of blood; (b) dye-affinity chromatography of the product of step (a); and (c) adsorption chromatography of the product of step (b) eluting the endotoxin inactivator in essentially pure form.
Claim 3
Dependent Claim
13 words
The method of claim 1 in which the ion-exchange chromatography is DEAE-Sephadex chromatography.
Claim 4
Dependent Claim
14 words
The method of claim 1 in which the dye-affinity chromatography is CIBACRON-BLUE Sepharose chromatography.
Claim 5
Dependent Claim
13 words
The method of claim 1 in which the adsorption chromatography is on hydroxylapatite.
Claim 6
Independent Claim
55 words
A method for the isolation from plasma or serum of an endotoxin inactivator in essentially pure form, which comprises (a) DEAE-Sephadex chromatography of plasma or serum; (b) immobilized chromophore blue chromatography of the product of step (a); and (c) hydroxylapatite chromatography of the product of step (b) eluting the endotoxin inactivator in essentially pure form.
Claim 7
Independent Claim
55 words
A method for the isolation from plasma or serum of an endotoxin inactivator in essentially pure form, which comprises (a) DEAE-Sephadex chromatography of plasma or serum; (b) immobilized chromophore blue chromatography of the product of step (a); and (c) hydroxylapatite chromatography of the product of step (b) eluting the endotoxin inactivator in essentially pure form.
Claim 8
Dependent Claim
15 words
The endotoxin inactivator isolated in essentially pure, stable form by the method of claim 5.
Claim 9
Dependent Claim
21 words
The method of claim 2, in which the endotoxin inactivator is eluted by increasing the ionic strength of an eluting buffer.
Claim 10
Dependent Claim
54 words
The method of claim 2, in which the DEAE-Sephadex chromatography comprises contacting plasma with DEAE-Sephadex in the presence of an equilibrating buffer whose ionic strength does not exceed 0.15 and whose pH is 6-9, and eluting the adsorbed material with a buffer whose ionic strength is greater than 0.15M and whose pH is 6-9.
Claim 11
Dependent Claim
29 words
The method of claim 8 in which the eluate from the DEAE-Sephadex chromatography is concentrated and diafiltered so that its ionic strength is 0.07-0.15 and its pH is 6-9.
Claim 12
Dependent Claim
31 words
The method of claim 3 in which material adsorbed on the immobilized chromophore blue adsorbent is eluted by a buffer having an ionic strength of 0.1-0.3M and a pH of 6-9.
Claim 13
Dependent Claim
16 words
The method of claim 10 in which the immobilized chromophore blue eluate is concentrated and diafiltered.
Claim 14
Dependent Claim
33 words
The method of claim 4 in which the essentially pure endotoxin inactivator is eluted by a phosphate buffer of at least 0.14M but less than 0.3M phosphate concentration, at a pH of 6-7.
Claim 15
Independent Claim
44 words
A method of separating an endotoxin inactivator from human serum albumin which comprises DEAE-Sephadex chromatography with elution of endotoxin inactivator at about 0.15-0.25M NaCl, followed by immobilized chromophore blue chromatography with elution of endotoxin inactivator at a ionic strength of less than 2.0M NaCl.
Claim 16
Independent Claim
44 words
A method of separating an endotoxin inactivator from human serum albumin which comprises DEAE-Sephadex chromatography with elution of endotoxin inactivator at about 0.15-0.25M NaCl, followed by immobilized chromophore blue chromatography with elution of endotoxin inactivator at a ionic strength of less than 2.0M NaCl.
Claim 17
Dependent Claim
38 words
A method of obtaining an endotoxin inactivator in essentially pure form which comprises separating the endotoxin inactivator from albumin by the method of claim 13, and then isolating the endotoxin inactivator in essentially pure form by hydroxyapatite chromatography.
Claim 18
Independent Claim
121 words
A method of obtaining an endotoxin inactivator in essentially pure form which consists essentially of (a) contacting an endotoxin inactivator containing blood or blood fraction with DEAE-Sephadex in the presence of an equilibrating buffer whose ionic strength does not exceed 0.15M and eluting the adsorbed endotoxin inactivator in a state of enhanced purity with a buffer whose ionic strength is greater than 0.15M, (b) contacting the eluate of step (a) with immobilized chromophore blue, and eluting the adsorbed endotoxin inactivator in a state of enhanced purity by a buffer having an ionic strength of 0.1-0.3M, and (c) contacting the eluate of step (b) with hydroxyapatite, and eluting the adsorbed endotoxin inactivator in essentially pure form by a phosphate buffer of 0.14-0.3M.
Claim 19
Independent Claim
121 words
A method of obtaining an endotoxin inactivator in essentially pure form which consists essentially of (a) contacting an endotoxin inactivator containing blood or blood fraction with DEAE-Sephadex in the presence of an equilibrating buffer whose ionic strength does not exceed 0.15M and eluting the adsorbed endotoxin inactivator in a state of enhanced purity with a buffer whose ionic strength is greater than 0.15M, (b) contacting the eluate of step (a) with immobilized chromophore blue, and eluting the adsorbed endotoxin inactivator in a state of enhanced purity by a buffer having an ionic strength of 0.1-0.3M, and (c) contacting the eluate of step (b) with hydroxyapatite, and eluting the adsorbed endotoxin inactivator in essentially pure form by a phosphate buffer of 0.14-0.3M.
Claim 20
Independent Claim
57 words
A method for the isolation in essentially pure form of an endotoxin inactivator which consists essentially of: (a) DEAE-Sephadex chromatography of an endotoxin-inactivator containing fraction of blood; (b) immobilized chromophore blue chromatography of the product of step (a); and (c) hydroxyapatite chromatography of the product of step (b), the endotoxin inactivator being eluted in essentially pure form.
Claim 21
Independent Claim
57 words
A method for the isolation in essentially pure form of an endotoxin inactivator which consists essentially of: (a) DEAE-Sephadex chromatography of an endotoxin-inactivator containing fraction of blood; (b) immobilized chromophore blue chromatography of the product of step (a); and (c) hydroxyapatite chromatography of the product of step (b), the endotoxin inactivator being eluted in essentially pure form.
Claim 22
Independent Claim
35 words
A method of separating an endotoxin inactivator from human serum albumin which consists essentially of DEAE-Sephadex chromatography with elution at about 0.15-0.25M NaCl, and immobilized chromophore blue chromatography with elution at less than 2.0M NaCl.
Claim 23
Independent Claim
35 words
A method of separating an endotoxin inactivator from human serum albumin which consists essentially of DEAE-Sephadex chromatography with elution at about 0.15-0.25M NaCl, and immobilized chromophore blue chromatography with elution at less than 2.0M NaCl.
Claim 24
Dependent Claim
14 words
The method of claim 3 in which the immobilized chromophore blue is CIBACRON-BLUE SEPHAROSE.
Claim 25
Dependent Claim
29 words
The method of claim 3 in which the endotoxin inactivator is eluted from the chromophore blue in an eluting buffer having an ionic strength of less than 2.0M NaCl.
Claim 26
Dependent Claim
27 words
The product of claim 6, said product retaining endotoxin inactivating activity for at least 48 hours at 5° C. and at least one month at -20° C.
References Cited (Backward Citations)
US4382028A
Cited
USPTO Annuity & Maintenance Windows
| Annuity Stage | Window Opens | Payment Deadline | Surcharge Period Until | Estimated Fee | Status |
|---|---|---|---|---|---|
| 3.5 Years (Year 4) | 1990-06-29 | 1990-12-29 | 1991-06-30 | $2000.00 | Window Closed / Lapsed |
| 7.5 Years (Year 8) | 1994-06-29 | 1994-12-29 | 1995-06-30 | $3760.00 | Window Closed / Lapsed |
| 11.5 Years (Year 12) | 1998-06-29 | 1998-12-29 | 1999-06-30 | $7700.00 | Window Closed / Lapsed |