US4530787A
Expired / Lapsed
US
⏱️ 0.0 years remaining
Controlled oxidation of microbially produced cysteine-containing proteins
Assignee: Novartis Vaccines and Diagnostics Inc
•
Filing: 1984-10-17
•
Granted: 1985-07-23
•
Expires: 2004-10-17
Abstract
Abstract Method of oxidizing reduced cysteine-containing microbially produced synthetic proteins, such as synthetic IFN- beta or synthetic IL-2, in a controlled manner so that the synthetic proteins have the same disulfide bridging as their native counterparts. The oxidation employs o-iodosobenzoate as oxidizing agent and is carried out in an aqueous medium at a pH at least about one-half pH unit less than the pKa of the cysteines to be oxidized, a synthetic protein concentration of less than about 5 mg/ml, and an oxidizing agent:protein mol ratio that is at least stoichiometric, provided that the oxidizing agent is in excess in the terminal portion of the reaction.
Inventors
- 👤 Ze'ev Shaked
- 👤 Sidney N. Wolfe
Bibliographic Details
Application Number
—
Jurisdiction
US
Data Source
google_patents
Last Synchronized
2026-10-23 05:47
CPC Classifications
C; C07; C07K; C07K14/00; C07K14/435; C07K14/52; C07K14/54; C07K14/55; C07K1/00; C07K1/107; C07K1/113; C07K1/1133; C07K1/14; C07K1/36; C07K14/555; C07K14/565; Y; Y10; Y10S; Y10S435/00; Y10S435/811; Y10S530/00; Y10S530/808; Y10S530/82; Y10S530/825; Y10S930/00; Y10S930/01; Y10S930/14; Y10S930/141; Y10S930/142
Claims Analysis
Total Claims: 26
Claim 1
Independent Claim
143 words
A preparative process for oxidizing a microbially produced synthetic protein having fully reduced cysteines and having an amino acid sequence substantially identical to a useful protein which sequence includes cysteines which in the useful protein are linked intramolecularly to form a cystine in a controlled manner whereby said cysteines are oxidized selectively to form said cystine with minimal overoxidation and formation of nonconforming cysteine groups or oligomers comprising reacting the fully reduced microbially produced synthetic protein with o-iodosobenzoate in an aqueous medium at a pH at least about one-half pH unit below the pK a of said cysteines and wherein the concentration of synthetic protein in the reaction mixture is less than about 5 mg/ml and the mol ratio of o-iodosobenzoate to protein is at least stoichiometric, with the proviso that the o-iodosobenzoate is in excess in the terminal portion of the reaction.
Claim 2
Dependent Claim
143 words
A preparative process for oxidizing a microbially produced synthetic protein having fully reduced cysteines and having an amino acid sequence substantially identical to a useful protein which sequence includes cysteines which in the useful protein are linked intramolecularly to form a cystine in a controlled manner whereby said cysteines are oxidized selectively to form said cystine with minimal overoxidation and formation of nonconforming cysteine groups or oligomers comprising reacting the fully reduced microbially produced synthetic protein with o-iodosobenzoate in an aqueous medium at a pH at least about one-half pH unit below the pK a of said cysteines and wherein the concentration of synthetic protein in the reaction mixture is less than about 5 mg/ml and the mol ratio of o-iodosobenzoate to protein is at least stoichiometric, with the proviso that the o-iodosobenzoate is in excess in the terminal portion of the reaction.
Claim 3
Dependent Claim
32 words
The process of claim 1 wherein the useful protein is a native protein having useful biological activity and the intramolecular linking is essential to the biological activity or enhances the biological activity.
Claim 4
Dependent Claim
11 words
The process of claim 1 wherein the protein is a lymphokine.
Claim 5
Dependent Claim
12 words
The process of claim 1 wherein the protein is IFN-β or IL-2.
Claim 6
Dependent Claim
12 words
The process of claim 4 wherein the pH is below about 9.
Claim 7
Dependent Claim
13 words
The process of claim 4 wherein the pH is between 5.5 and 9.
Claim 8
Dependent Claim
18 words
The process of claim 1 wherein the protein is IL-2 and the pH is between 6.5 and 7.5.
Claim 9
Dependent Claim
18 words
The process of claim 1 wherein the protein is IFN-β and the pH is between 6.5 and 9.0.
Claim 10
Dependent Claim
22 words
The process of claim 1 wherein said concentration of synthetic protein is in the range of about 0.3 to about 0.7 mg/ml.
Claim 11
Dependent Claim
52 words
The process of claim 1 wherein the protein is an IL-2, said mol ratio is in the range of about 1:1 and about 5:1, the pH is between 6.5 and 7.5, and the concentration of synthetic IL-2 in the reaction mixture is in the range of about 0.3 to about 0.7 mg/ml.
Claim 12
Dependent Claim
51 words
The process of claim 1 wherein the protein is IFN-β, said mol ratio is in the range of about 1:1 and about 5:1, the pH is between 6.5 and 9.0 and the concentration of synthetic IFN-β in the reaction mixture is in the range of about 0.3 to about 0.7 mg/ml.
Claim 13
Dependent Claim
19 words
The process of claim 1 wherein after said oxidation the oxidized product is purified using a gel filtration method.
Claim 14
Dependent Claim
17 words
The process of claim 12 wherein the filtration is carried out using a G-25 Sephadex desalting column.
Claim 15
Dependent Claim
16 words
The preparation of claim 1 wherein the preparation contains less than about 1% by weight oligomers.
Claim 16
Dependent Claim
58 words
The preparation of claim 1 wherein the synthetic protein is a synthetic mutein of a biologically active protein which protein has at least one cysteine residue that is free to form a disulfide link and is nonessential to said biological activity, said mutein having at least one of said cysteine residues deleted or replaced by another amino acid.
Claim 17
Dependent Claim
53 words
A cystine-containing IL-2 preparation derived from synthetic microbially produced IL-2 having fully reduced cysteines comprising cystine-containing IL-2 which: (i) has the same disulfide bridging as native human IL-2; (ii) is substantially free of oligomers; and (iii) contains less than about 15% by weight of isomers having disulfide bridging different from native human IL-2.
Claim 18
Dependent Claim
53 words
A cystine-containing IL-2 preparation derived from synthetic microbially produced IL-2 having fully reduced cysteines comprising cystine-containing IL-2 which: (i) has the same disulfide bridging as native human IL-2; (ii) is substantially free of oligomers; and (iii) contains less than about 15% by weight of isomers having disulfide bridging different from native human IL-2.
Claim 19
Dependent Claim
90 words
A cystine-containing protein preparation derived from a synthetic microbially produced, unglycosylated protein having fully reduced cysteines and having an amino acid sequence substantially identical to a useful protein which sequence includes cysteines which is the useful protein are linked intramolecularly to form a cystine, which preparation consists essentially of a cystine-containing protein which: (i) has the same disulfide bridging as the native useful protein; (ii) is substantially free of oligomers; and (iii) contains less than about 15% by weight of isomers having disulfide bridging different from the native useful protein.
Claim 20
Dependent Claim
90 words
A cystine-containing protein preparation derived from a synthetic microbially produced, unglycosylated protein having fully reduced cysteines and having an amino acid sequence substantially identical to a useful protein which sequence includes cysteines which is the useful protein are linked intramolecularly to form a cystine, which preparation consists essentially of a cystine-containing protein which: (i) has the same disulfide bridging as the native useful protein; (ii) is substantially free of oligomers; and (iii) contains less than about 15% by weight of isomers having disulfide bridging different from the native useful protein.
Claim 21
Dependent Claim
16 words
The preparation of claim 16 wherein the preparation contains less than about 1% by weight oligomers.
Claim 22
Dependent Claim
58 words
A cystine-containing IFN-β preparation derived from synthetic microbially produced, unglycosylated IFN-β having fully reduced cysteines, which preparation consists essentially of cystine-containing IFN-β which: (i) has the same disulfide bridging as native human IFN-β; (ii) is substantially free of oligomers; and (iii) contains less than about 15% by weight of isomers having disulfide bridging different from native human IFN-β.
Claim 23
Dependent Claim
58 words
A cystine-containing IFN-β preparation derived from synthetic microbially produced, unglycosylated IFN-β having fully reduced cysteines, which preparation consists essentially of cystine-containing IFN-β which: (i) has the same disulfide bridging as native human IFN-β; (ii) is substantially free of oligomers; and (iii) contains less than about 15% by weight of isomers having disulfide bridging different from native human IFN-β.
Claim 24
Dependent Claim
16 words
The preparation of claim 19 wherein the preparation contains less than about 1% by weight oligomers.
Claim 25
Dependent Claim
13 words
The preparation of claim 16 wherein said IL-2 is des-ala IL-2 ser125 .
Claim 26
Dependent Claim
12 words
The preparation of claim 19 wherein said IFN-β is IFN-β ser17 .
References Cited (Backward Citations)
US4061538A
Cited
US4414147A
Cited
US4411993A
Cited
US4478744A
Cited
US4450103A
Cited
US4462940A
Cited
US4432895A
Cited
USPTO Annuity & Maintenance Windows
| Annuity Stage | Window Opens | Payment Deadline | Surcharge Period Until | Estimated Fee | Status |
|---|---|---|---|---|---|
| 3.5 Years (Year 4) | 1988-07-22 | 1989-01-21 | 1989-07-23 | $2000.00 | Window Closed / Lapsed |
| 7.5 Years (Year 8) | 1992-07-22 | 1993-01-21 | 1993-07-23 | $3760.00 | Window Closed / Lapsed |
| 11.5 Years (Year 12) | 1996-07-22 | 1997-01-21 | 1997-07-23 | $7700.00 | Window Closed / Lapsed |