US4572798A
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Method for promoting disulfide bond formation in recombinant proteins
Assignee: Novartis Vaccines and Diagnostics Inc
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Filing: 1984-12-06
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Granted: 1986-02-25
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Expires: 2004-12-06
Abstract
Abstract Reduced cysteine-containing proteins consisting of recombinant IFN-β, IL-2 or muteins thereof may be oxidized selectively so that the recombinant proteins have essentially the same disulfide bridging and biological activity as their native counterparts. The oxidized product is substantially free of unwanted side products and contains a minimal amount of intermolecular oligomers. The oxidation takes place in an aqueous medium containing a solubilizing agent at a pH of about 5.5 to 9, preferably at a pH of about 7. The reaction is initiated by addition of at least an effective amount of an oxidation promoter containing a Cu +2 cation such as CuCl 2 or o-phenanthroline/Cu +2 complex in the presence of air.
Inventors
- 👤 Kirston E. Koths
- 👤 Robert F. Halenbeck
Bibliographic Details
Application Number
—
Jurisdiction
US
Data Source
google_patents
Last Synchronized
2026-11-03 20:49
CPC Classifications
C; C07; C07K; C07K1/00; C07K1/107; C07K1/113; C07K1/1133; C07K14/00; C07K14/435; C07K14/52; C07K14/54; C07K14/55; C07K14/555; C07K14/565; Y; Y10; Y10S; Y10S435/00; Y10S435/811
Claims Analysis
Total Claims: 20
Claim 1
Independent Claim
83 words
A method of oxidizing a fully reduced recombinant protein selected from the group consisting of interferon-beta, interleukin-2 and muteins thereof, whereby cysteines are oxidized preferentially to form the disulfide bridges which correspond to those present in the naturally occurring protein, which method comprises reacting an aqueous solution containing a solubilized form of the recombinant protein at a pH between about 5.5 and 9 in the presence of air with at least an effective amount of an oxidation promoter containing a Cu +2 cation.
Claim 2
Independent Claim
83 words
A method of oxidizing a fully reduced recombinant protein selected from the group consisting of interferon-beta, interleukin-2 and muteins thereof, whereby cysteines are oxidized preferentially to form the disulfide bridges which correspond to those present in the naturally occurring protein, which method comprises reacting an aqueous solution containing a solubilized form of the recombinant protein at a pH between about 5.5 and 9 in the presence of air with at least an effective amount of an oxidation promoter containing a Cu +2 cation.
Claim 3
Dependent Claim
48 words
The method of claim 1 wherein the recombinant protein is a mutein of said protein having at least one of its cysteine residues which is free to form a disulfide bond and is nonessential to the biological activity of the protein deleted or replaced by another amino acid.
Claim 4
Dependent Claim
13 words
The method of claim 2 wherein the mutein is des-ala IL-2 ser125 .
Claim 5
Dependent Claim
12 words
The method of claim 2 wherein the mutein is IFN-β ser17 .
Claim 6
Dependent Claim
15 words
The method of claim 1 wherein the pH is between about 6 and about 8.
Claim 7
Dependent Claim
11 words
The method of claim 1 wherein the pH is about 7.
Claim 8
Dependent Claim
18 words
The method of claim 1 wherein the oxidation promoter is CuCl 2 or (o-phenanthroline) 2 Cu +2 .
Claim 9
Dependent Claim
13 words
The method of claim 1 wherein the oxidation promoter is CuCl 2 .
Claim 10
Dependent Claim
15 words
The method of claim 1 wherein the oxidation promoter is (o-phenanthroline) 2 Cu +2 .
Claim 11
Dependent Claim
22 words
The method of claim 1 wherein the concentration of said protein is in the range of about 0.05 to about 2 mg/ml.
Claim 12
Dependent Claim
34 words
The method of claim 1 wherein the concentration of said oxidation promoter is approximately equivalent to the concentration of free sulfhydryl groups on the protein which are intended to be oxidized to form cystines.
Claim 13
Dependent Claim
22 words
The method of claim 10 wherein the oxidation promoter is CuCl 2 and its concentration ranges from about 1 to 400 micromolar.
Claim 14
Dependent Claim
20 words
The method of claim 1 wherein the reaction is carried out at a temperature of from 20° to 40° C.
Claim 15
Dependent Claim
14 words
The method of claim 1 wherein the protein is solubilized with sodium dodecyl sulfate.
Claim 16
Dependent Claim
25 words
The method of claim 14 wherein the concentration of sodium dodecyl sulfate prior to reaction ranges from about 0.05% to 2% by weight per volume.
Claim 17
Dependent Claim
77 words
The method of claim 1 wherein the protein is an IL-2 or IFN-β mutein, the oxidation promoter is CuCl 2 in an amount of from about 5 to 50 micromolar, the pH of the reaction is about 7, the concentration of IL-2 in the reaction mixture ranges from about 0.1 to about 1 mg/ml, and sodium dodecyl sulfate is present as a solubilizing agent at a concentration ranging from about 0.1 to 1% by weight per volume.
Claim 18
Dependent Claim
13 words
The method of claim 16 wherein the mutein is des-ala IL-2 ser125 .
Claim 19
Dependent Claim
16 words
The method of claim 16 wherein the mutein contains the three cysteines present in native IL-2.
Claim 20
Dependent Claim
12 words
The method of claim 16 wherein the mutein is IFN-β ser17 .
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) | 1989-02-24 | 1989-08-26 | 1990-02-25 | $2000.00 | Window Closed / Lapsed |
| 7.5 Years (Year 8) | 1993-02-24 | 1993-08-26 | 1994-02-25 | $3760.00 | Window Closed / Lapsed |
| 11.5 Years (Year 12) | 1997-02-24 | 1997-08-26 | 1998-02-25 | $7700.00 | Window Closed / Lapsed |