Preparation of an activated polymer ester for protein conjugation

• US 5,281,698 A
• Filed: 07/23/1991
• Issued: 01/25/1994
• Est. Priority Date: 07/23/1991
• Status: Expired due to Term

• Current Assignee: Chiron Corporation (Novartis Ag)
• Original Assignee: Cetus Oncology Corporation (Novartis Ag)
• Inventors: Nitecki, Danute E.
• Primary Examiner(s): Russel, Jeffrey E.
• Application Number:
  US07/734,749
• Time in Patent Office: 917 Days
• Field of Search: 530/409, 530/405, 530/351, 548/520, 548/545, 548/546, 548/547
• US Class Current: 530/351
• CPC Class Codes: C07D 207/404 with only hydrogen atoms or radicals containing only hydrogen and carbon atoms directly attached to other ring carbon atoms, e.g. succinimide
• CPC Class Codes: C07K 1/1077 by covalent attachment of residues other than amino acids or peptide residues, e.g. sugars, polyols, fatty acids
• CPC Class Codes: C07K 14/53 Colony-stimulating factor [CSF]
• CPC Class Codes: C07K 14/55 IL-2
• CPC Class Codes: C07K 14/555 Interferons [IFN]

Abstract

The present invention is a process for preparing an activated ester of polyethylene glycol or a polyoxyethylated polyol. After the activated ester is prepared, it can be reacted with a protein to form a polymer/protein conjugate. Conjugation with a polymer can reduce the protein'"'"'s immunogenicity, increase its solubility, and increase its circulating in vivo half-life. Preferred proteins are IL-2, CSFs, and interferons.

Claims

1. A method for producing an activated ester of polyethylene glycol (PEG) or a polyoxyethylated polyol, comprising:

a) contacting PEG or a polyoxyethylated polyol, which has at least one hydroxyl group, with disuccinimidylcarbonate (DSC) under the appropriate reaction conditions to form a PEG or a polyoxyethylated polyol active ester.

2. A method in accordance with claim 1, wherein PEG is activated with the DSC.

3. A method in accordance with claim 2, wherein the molar ratio of DSC to PEG is 30:

1 or less.

4. A method in accordance with claim 2, wherein the molar ratio of DSC to PEG is 20:

1 or less.

5. A method in accordance with claim 2 further comprising adding a catalyst selected from the group consisting essentially of pyridine or 4-dimethylaminopyridine.

6. A method in accordance with claim 1, wherein the PEG or the polyoxyethylated polyol has a average molecular weight between 1,000 and 40,000.

7. A method in accordance with claim 1, wherein the PEG or the polyoxyethylated polyol has a average molecular weight between 2,000 and 20,000.

8. A method in accordance with claim. 1, wherein the PEG or the polyoxyethylated polyol has an average molecular weight between 3,000 and 12,000.

9. A method in accordance with claim 6, wherein the polymer is PEG.

10. A method in accordance with claim 1 further comprising:

a) contacting the PEG or the polyoxyethylated polyol active ester with a protein under the appropriate reaction conditions to form a PEG or a polyoxyethylated polyol protein conjugate.

11. A method in accordance with claim 10, wherein the PEG or the polyoxyethylated polyol active ester is contacted with the protein in the molar ratio of between 1 and 30 moles active esters to 1 mole protein.

12. A method in accordance with claim 11, wherein the active ester is contacted with the protein in the molar ratio of between 1 and 15 moles active esters to 1 mole protein.

13. A method in accordance with claim 10, wherein the PEG or the polyoxyethylated polyol has a average molecular weight between 1,000 and 40,000.

14. A method in accordance with claim 10, wherein the PEG or the polyoxyethylated polyol has a average molecular weight between 2,000 and 20,000.

15. A method in accordance with claim 10, wherein the PEG or the polyoxyethylated polyol has an average molecular weight between 3,000 and 12,000.

16. A method in accordance with claim 11, wherein the active ester is a PEG active ester.

17. A method in accordance with claim 11, wherein the protein is IL-2.

18. A method in accordance with claim 11, wherein the protein is CSF.

19. A method in accordance with claim 11, wherein the protein is interferon.

20. A method in accordance with claim 17, wherein the PEG active ester is contacted with IL-2 in the molar ratio around 5 moles of PEG esters to 1 mole IL-2.