Claims for Patent: 4,530,787
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Summary for Patent: 4,530,787
Title: | Controlled oxidation of microbially produced cysteine-containing proteins |
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 pK.sub.a 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. |
Inventor(s): | Shaked; Ze\'ev (Berkeley, CA), Wolfe; Sidney N. (Richmond, CA) |
Assignee: | Cetus Corporation (Emeryville, CA) |
Application Number: | 06/661,902 |
Patent Claims: | 1. 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.sub.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.
2. 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. 3. The process of claim 1 wherein the protein is a lymphokine. 4. The process of claim 1 wherein the protein is IFN-.beta. or IL-2. 5. The process of claim 4 wherein the pH is below about 9. 6. The process of claim 4 wherein the pH is between 5.5 and 9. 7. The process of claim 1 wherein the protein is IL-2 and the pH is between 6.5 and 7.5. 8. The process of claim 1 wherein the protein is IFN-.beta. and the pH is between 6.5 and 9.0. 9. 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. 10. 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. 11. The process of claim 1 wherein the protein is IFN-.beta., 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-.beta. in the reaction mixture is in the range of about 0.3 to about 0.7 mg/ml. 12. The process of claim 1 wherein after said oxidation the oxidized product is purified using a gel filtration method. 13. The process of claim 12 wherein the filtration is carried out using a G-25 Sephadex desalting column. 14. The preparation of claim 1 wherein the preparation contains less than about 1% by weight oligomers. 15. 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. 16. 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. 17. 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. 18. The preparation of claim 16 wherein the preparation contains less than about 1% by weight oligomers. 19. A cystine-containing IFN-.beta. preparation derived from synthetic microbially produced, unglycosylated IFN-.beta. having fully reduced cysteines, which preparation consists essentially of cystine-containing IFN-.beta. which: (i) has the same disulfide bridging as native human IFN-.beta.; (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-.beta.. 20. The preparation of claim 19 wherein the preparation contains less than about 1% by weight oligomers. 21. The preparation of claim 16 wherein said IL-2 is des-ala IL-2.sub.ser125. 22. The preparation of claim 19 wherein said IFN-.beta. is IFN-.beta..sub.ser17. |
Details for Patent 4,530,787
Applicant | Tradename | Biologic Ingredient | Dosage Form | BLA | Approval Date | Patent No. | Expiredate |
---|---|---|---|---|---|---|---|
Bayer Healthcare Pharmaceuticals Inc. | BETASERON | interferon beta-1b | For Injection | 103471 | July 23, 1993 | ⤷ Sign Up | 2004-03-28 |
>Applicant | >Tradename | >Biologic Ingredient | >Dosage Form | >BLA | >Approval Date | >Patent No. | >Expiredate |
International Patent Family for US Patent 4,530,787
Country | Patent Number | Estimated Expiration |
---|---|---|
Japan | S60243021 | ⤷ Sign Up |
Japan | H0535133 | ⤷ Sign Up |
Israel | 75532 | ⤷ Sign Up |
>Country | >Patent Number | >Estimated Expiration |
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