Mono- and di-peg IL-10 production; and uses

• US 9,566,345 B2
• Filed: 01/06/2016
• Issued: 02/14/2017
• Est. Priority Date: 12/17/2008
• Status: Active Grant

• Current Assignee: Merck Sharp & Dohme Corporation (Merck & Co., Inc.)
• Original Assignee: Merck Sharp & Dohme Corporation (Merck & Co., Inc.)
• Inventors: Blaisdell, Steven J., Cutler, Collette M., Paporello, Brittany C., Ambrogelly, Alexandre
• Primary Examiner(s): Saoud, Christine J
• Assistant Examiner(s): Seharaseyon, Jegatheesan
• Application Number:
  US14/989,068
• Publication Number:
  US 20160193352A1
• Time in Patent Office: 405 Days
• Field of Search: None
• US Class Current: 1/1
• CPC Class Codes: A61K 31/506 not condensed and containing further heterocyclic rings
• CPC Class Codes: A61K 31/513 having oxo groups directly attached to the heterocyclic ring, e.g. cytosine
• CPC Class Codes: A61K 33/243 Platinum; Compounds thereof
• CPC Class Codes: A61K 38/2066 IL-10
• CPC Class Codes: A61K 45/06 Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
• CPC Class Codes: A61K 47/60 the organic macromolecular compound being a polyoxyalkylene oligomer, polymer or dendrimer, e.g. PEG, PPG, PEO or polyglycerol
• CPC Class Codes: A61P 35/00 Antineoplastic agents

Abstract

Provided are methods of producing mono- and di-pegylated IL-10.

Claims

1. A method of producing a mixture of mono- and di-pegylated interleukin-10 (IL-10), wherein at least one PEG molecule is covalently attached to at least one amino acid residue of at least one subunit of IL-10, comprising:

a) reacting 1 mg/ml to 12 mg/ml of IL-10 with an activated PEG-linker in the presence of 25 mM to 35 mM of a reducing agent, wherein the PEG-linker is selected from the group consisting of succinimidylcarbonate-PEG, PEG-butyraldehyde, PEG-pentaldehyde, PEG-amido-propionaldehyde, PEG-urethano-propioaldehyde, and PEG-propylaldehyde; and

b) purifying the mixture of mono- and di-pegylated IL-10.

2. The method of claim 1, wherein the PEG-linker is PEG-propylaldehyde.

3. The method of claim 1, wherein the molecular mass of PEG comprising the PEG-linker is from 5,000 daltons to 20,000 daltons.

4. The method of claim 1, wherein the molecular mass of PEG comprising the PEG linker is 5,000 daltons.

5. The method of claim 1, wherein the reducing agent is selected from the group consisting of borohydride, sodium cyanoborohydride, amine borane, and picoline borane.

6. The method of claim 5, wherein the reducing agent is selected from the group consisting of sodium cyanoborohydride and picoline borane.

7. The method of claim 1, wherein the mixture of mono- and di-PEG is purified by chromatography selected from the group consisting of cation exchange, anion exchange, size exclusion, and hydrophobic interaction.

8. The method of claim 7, wherein the mixture of mono- and di-PEG is purified by size exclusion chromatography.

9. The method of claim 1, wherein the ratio of IL-10 to PEG linker is 1:

1-1;

7.7.

10. The method of claim 9, wherein the ratio is 1:

3.5.

11. The method of claim 1, wherein the reacting is at a pH of about 5.0-7.4.

12. The method of claim 1, wherein the reacting is at a temperature of 5°

C. to 30°

C.

13. The method of claim 1, wherein the reacting is for 3-24 hours.

14. The method of claim 13, wherein the reacting is for 15-18 hours.

15. The method of claim 1, wherein the method produces a 1:

1 ratio of mono-pegylated IL-10 to di-pegylated IL-10.

16. The method of claim 1, wherein the reacting is in the presence of 25 mM of a reducing agent.