US4677197A
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US
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Purification method for tumor necrosis factor
Assignee: Novartis Vaccines and Diagnostics Inc
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Filing: 1985-10-30
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Granted: 1987-06-30
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Expires: 2005-10-30
Abstract
Abstract An improved method for purifying TNF, especially recombinantly produced TNF in bacteria, is disclosed. The purification employs a hydrophobic support in a chromatographic column which is then developed, preferably by HPLC. Preliminary purifications using anion exchange resins are also helpful.
Inventors
- 👤 Leo S. Lin
- 👤 Ralph Yamamoto
Bibliographic Details
Application Number
—
Jurisdiction
US
Data Source
google_patents
Last Synchronized
2026-11-04 16:11
CPC Classifications
C; C07; C07K; C07K1/00; C07K1/14; C07K1/36; C07K14/00; C07K14/435; C07K14/52; C07K14/525; Y; Y10; Y10S; Y10S930/00; Y10S930/01; Y10S930/14; Y10S930/144
Claims Analysis
Total Claims: 14
Claim 1
Independent Claim
88 words
A method for purifying TNF which comprises the steps of (a) treating an aqueous mixture containing TNF with a hydrophobic interaction chromatographic support comprising hydrocarbyl substituents bound to an uncharged, polymeric, particulate matrix in the presence of an aqueous solution containing a concentration of ions that is effective to result in retention of TNF on the hydrophobic support, but ineffective to precipitate the TNF, and (b) eluting the TNF from the hydrophobic support chromatographically by decreasing the ion concentration below the concentration of ion used in step (a).
Claim 2
Independent Claim
88 words
A method for purifying TNF which comprises the steps of (a) treating an aqueous mixture containing TNF with a hydrophobic interaction chromatographic support comprising hydrocarbyl substituents bound to an uncharged, polymeric, particulate matrix in the presence of an aqueous solution containing a concentration of ions that is effective to result in retention of TNF on the hydrophobic support, but ineffective to precipitate the TNF, and (b) eluting the TNF from the hydrophobic support chromatographically by decreasing the ion concentration below the concentration of ion used in step (a).
Claim 3
Dependent Claim
11 words
The method of claim 1 wherein the TNF is recombinant TNF.
Claim 4
Dependent Claim
13 words
The method of claim 2 wherein the TNF is an N-terminal deleted mutein.
Claim 5
Dependent Claim
18 words
The method of claim 1 wherein the mixture contains bacterial proteins and is free of other mammalian proteins.
Claim 6
Dependent Claim
15 words
The method of claim 1 wherein the hydrophobic chromatographic support is phenyl TSK HPLC support.
Claim 7
Dependent Claim
20 words
The method of claim 1 wherein the the aqueous solution comprises 1.5-2.0M ammonium sulfate and 0.1M sodium phosphate, pH 7.
Claim 8
Dependent Claim
20 words
The method of claim 6 wherein the the aqueous solution comprises 1.8M ammonium sulfate and 0.1M sodium phosphate, pH 7.
Claim 9
Dependent Claim
13 words
The method of claim 1 wherein the steps are carried out as HPLC.
Claim 10
Dependent Claim
15 words
The method of claim 1 wherein the decrease in ion concentration is by continuous gradient.
Claim 11
Dependent Claim
18 words
The method of claim 1 wherein the mixture has previously been enriched in TNF by ion exchange chromatography.
Claim 12
Dependent Claim
16 words
The method of claim 1 which further includes treating the eluted TNF with a sizing gel.
Claim 13
Independent Claim
130 words
A method for purifying TNF which comprises the steps of (a) eluting unpurified TNF from an anion exchange support matrix in the presence of a concentration of ions which is effective to elute an aqueous mixture containing TNF from the support matrix but not to precipitate TNF from aqueous solution, (b) without a desalting step, treating the eluted aqueous mixture containing TNF with a hydrophobic interaction chromatographic support comprising hydrocarbyl substituents bound to an uncharged, polymeric, particulate matrix in the presence of an aqueous solution containing a concentration of ions that is effective to result in retention of TNF on the hydrophobic support, but ineffective to precipitate the TNF, and (c) eluting the TNF from the hydrophobic support chromatographically by decreasing the ion concentration below that effective for TNF retention.
Claim 14
Independent Claim
130 words
A method for purifying TNF which comprises the steps of (a) eluting unpurified TNF from an anion exchange support matrix in the presence of a concentration of ions which is effective to elute an aqueous mixture containing TNF from the support matrix but not to precipitate TNF from aqueous solution, (b) without a desalting step, treating the eluted aqueous mixture containing TNF with a hydrophobic interaction chromatographic support comprising hydrocarbyl substituents bound to an uncharged, polymeric, particulate matrix in the presence of an aqueous solution containing a concentration of ions that is effective to result in retention of TNF on the hydrophobic support, but ineffective to precipitate the TNF, and (c) eluting the TNF from the hydrophobic support chromatographically by decreasing the ion concentration below that effective for TNF retention.
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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 |