US4743680A
Expired / Lapsed
US
⏱️ 0.0 years remaining
Method for purifying antihemophilic factor
Assignee: New York University
•
Filing: 1985-02-01
•
Granted: 1988-05-10
•
Expires: 2005-02-01
Abstract
Abstract Disclosed are methods for purifying proteins including AHF by column chromatography in the presence of additives including sugars and polyhydric alcohols which serve to increase the electrostatic forces on the surface of said proteins while decreasing the hydrophobicity of said proteins resulting in preparations of such proteins of high purity and/or resolution and/or recovery.
Inventors
- 👤 Rita W. Mathews
- 👤 Alan J. Johnson
Bibliographic Details
Application Number
—
Jurisdiction
US
Data Source
google_patents
Last Synchronized
2026-11-04 08:56
CPC Classifications
A; A23; A23J; A23J1/00; C; C12; C12N; C12N9/00; C12N9/14; C12N9/48; C12N9/50; C12N9/64; C12N9/6421; C12N9/6424; C12N9/647; B; B01; B01J; B01J41/00; B01J41/20; C07; C07K; C07K1/00; C07K1/14; C07K1/16; C07K1/18; C07K14/00; C07K14/435; C07K14/745; C07K14/755; C07K14/76; C07K14/765; C07K16/00; C07K16/06; C07K16/065; A61; A61K; A61K38/00
Claims Analysis
Total Claims: 25
Claim 1
Independent Claim
153 words
In a process for purifying a protein that has antihemophilic factor activity by column chromatography in a column behaving predominantly as an ion-exchange chromatography column comprising the stages of: (a) equilibrating said chromatography column; (b) loading a sample containing said protein on said column, causing said protein to adsorb onto said column; (c) washing said column; (d) eluting said adsorbed protein from said column by causing it to desorb from said column; and (e) recovering said protein in purified form; the improvement comprising the step of: adding to the column a substance consisting essentially of an effective amount for selectively increasing the electrostatic forces on the surface of said protein and concomitantly decreasing the hydrophobicity of said protein of a hydration additive selected from the group consisting of sugars and polyhydric alcohols during at least one of said stages (a), (b), and (c) thereby promoting the adsorption of said protein on said column.
Claim 2
Dependent Claim
153 words
In a process for purifying a protein that has antihemophilic factor activity by column chromatography in a column behaving predominantly as an ion-exchange chromatography column comprising the stages of: (a) equilibrating said chromatography column; (b) loading a sample containing said protein on said column, causing said protein to adsorb onto said column; (c) washing said column; (d) eluting said adsorbed protein from said column by causing it to desorb from said column; and (e) recovering said protein in purified form; the improvement comprising the step of: adding to the column a substance consisting essentially of an effective amount for selectively increasing the electrostatic forces on the surface of said protein and concomitantly decreasing the hydrophobicity of said protein of a hydration additive selected from the group consisting of sugars and polyhydric alcohols during at least one of said stages (a), (b), and (c) thereby promoting the adsorption of said protein on said column.
Claim 3
Dependent Claim
36 words
The process of claim 1 wherein said hydration additive is a sugar selected from the group consisting of sucrose, maltose, lactose, glucose, ribose, arabinose, galactose, fructose, mannose, rhamnose, raffinose, melezitose, dextran, xylose, allose, 6-deoxymannose, and 6-deoxygalactose.
Claim 4
Dependent Claim
34 words
The process of claim 1 wherein said hydration additive is a polyhydric alcohol selected from the group consisting of sorbitol, mannitol, inositol, adonitol, erythritol, ethylene glycol, glycerol, propylene glycol, xylitol, 2-methyl-2,4-pentanediol, ficoll and ribitol.
Claim 5
Dependent Claim
15 words
The process of claim 1 wherein said chromatography column is an ion exchange chromatography column.
Claim 6
Dependent Claim
12 words
The process of claim 4 wherein said chromatography is anion exchange chromatography.
Claim 7
Dependent Claim
21 words
The process of claim 4 wherein said additive is incorporated into a buffer used to load said sample onto said column.
Claim 8
Dependent Claim
19 words
The process of claim 6 wherein said additive is eliminated from said column prior to elution of said protein.
Claim 9
Dependent Claim
23 words
A process according to claim 2 wherein said additive is selected from the group consisting of sucrose, maltose, lactose, glucose, sorbitol and mannitol.
Claim 10
Dependent Claim
20 words
A process according to claim 8 wherein said additive is sucrose and its concentration ranges between about 0.5 and 2.0M.
Claim 11
Dependent Claim
20 words
A process according to claim 8 wherein said additive is maltose and its concentration ranges between about 0.2 and 0.4M.
Claim 12
Dependent Claim
20 words
A process according to claim 8 wherein said additive is glucose and its concentration ranges between about 0.5 and 2.0M.
Claim 13
Dependent Claim
20 words
A process according to claim 8 wherein said additive is sorbitol and its concentration ranges between about 0.5 and 2.0M.
Claim 14
Dependent Claim
20 words
A process according to claim 6 wherein said additive is also incorporated into a buffer used to equilibrate said column.
Claim 15
Dependent Claim
21 words
A process according to claim 13 wherein said column chromatography employs as a chromatographic medium a quaternary aminoethyl anion exchange resin.
Claim 16
Dependent Claim
25 words
The process of claim 13 wherein the eluting step involves use of a physiologically acceptable detergent to enhance desorption of said protein from the column.
Claim 17
Dependent Claim
20 words
The process of claim 15 wherein said additive is sorbitol used at a concentration ranging between about 0.5 and 2.0M.
Claim 18
Dependent Claim
20 words
The process of claim 16 wherein said detergent is polysorbate 80 at a concentration ranging between about 0.01 and 0.1%.
Claim 19
Dependent Claim
12 words
The process of claim 17 wherein said sorbitol concentration is about 1M.
Claim 20
Dependent Claim
26 words
The process of claim 1 further comprising using as said sample in said stage (a) an eluate containing said protein from a hydrophobic affinity column chromatography.
Claim 21
Dependent Claim
35 words
The process of claim 1 said process further comprising subjecting said eluate containing said protein from said stage (d) to a second purification using a second column behaving predominantly as a hydrophobic affinity chromatography column.
Claim 22
Dependent Claim
24 words
The process of claim 1 wherein said column is a mixed ion exchange/hydrophobic affinity chromatography column behaving predominantly as an ion exchange chromatography column.
Claim 23
Dependent Claim
14 words
The process of claim 21 wherein said column is a maleic anhydride polyelectrolyte column.
Claim 24
Dependent Claim
120 words
The process of claim 20 said second purification comprising the stages of: (i) equilibrating said second chromatography column; (ii) loading said stage (d) eluate containing said protein on said column causing said protein to absorb to said second column; (iii) washing said second column; and (iv) eluting said adsorbed protein from said second column by causing it to desorb from said second column, adding to the column during said stage (iv) a substance consisting essentially of an effective amount for selectively increasing the electrostatic forces on the surface of said protein and concomitantly decreasing the hydrophobicity of said protein of a hydration additive consisting of sugars and polyhydric alcohols, thereby promoting the desorption of said protein from said second column.
Claim 25
Dependent Claim
22 words
The process of claim 23 wherein said additive is selected from the group consisting of sucrose, maltose, lactose, glucose, sorbitol and mannitol.
References Cited (Backward Citations)
DE2635894A1
Cited
US4387092A
Cited
US4348315A
Cited
US4404132A
Cited
US4404131A
Cited
US4439357A
Cited
US4508709A
Cited
EP0173242A2
Cited
DE3432083A1
Cited
USPTO Annuity & Maintenance Windows
| Annuity Stage | Window Opens | Payment Deadline | Surcharge Period Until | Estimated Fee | Status |
|---|---|---|---|---|---|
| 3.5 Years (Year 4) | 1991-05-10 | 1991-11-09 | 1992-05-10 | $2000.00 | Window Closed / Lapsed |
| 7.5 Years (Year 8) | 1995-05-10 | 1995-11-09 | 1996-05-10 | $3760.00 | Window Closed / Lapsed |
| 11.5 Years (Year 12) | 1999-05-10 | 1999-11-09 | 2000-05-10 | $7700.00 | Window Closed / Lapsed |