Amine pegylation methods for the preparation of site-specific protein conjugates

• US 10,220,075 B2
• Filed: 05/19/2016
• Issued: 03/05/2019
• Est. Priority Date: 06/04/2015
• Status: Active Grant

• Current Assignee: Rezolute, Inc.
• Original Assignee: Rezolute, Inc.
• Inventors: Rosendahl, Mary S., Mantripragada, Sankaram B.
• Primary Examiner(s): Rogers, James W
• Application Number:
  US15/158,898
• Publication Number:
  US 20160354478A1
• Time in Patent Office: 1,020 Days
• Field of Search: None
• CPC Class Codes: A61K 38/26 Glucagons
• CPC Class Codes: A61K 38/27 Growth hormone [GH], i.e. somatotropin
• CPC Class Codes: A61K 38/28 Insulins
• CPC Class Codes: A61K 38/29 Parathyroid hormone, i.e. parathormone; Parathyroid hormone-related peptides
• CPC Class Codes: A61K 47/60 the organic macromolecular compound being a polyoxyalkylene oligomer, polymer or dendrimer, e.g. PEG, PPG, PEO or polyglycerol

Abstract

Examples include a method of making a protein-PEG conjugate. The method may include providing an aqueous protein solution. The aqueous protein solution may include a protein, a pH buffer, and a chelating agent. The chelating agent may be chosen from the group consisting of an aminopolycarboxylic acid, a hydroxyaminocarboxylic acid, an N-substituted glycine, 2-(2-amino-2-oxocthyl) aminoethane sulfonic acid (BES), and deferoxamine (DEF). The method may also include introducing sodium cyanoborohydride and a methoxy polyethylene glycol aldehyde to the aqueous protein solution. The sodium cyanoborohydride in the methoxy polyethylene glycol aldehyde may have a molar ratio ranging from about 5:1 to about 1.5:1. The method may further include reacting the methoxy polyethylene glycol aldehyde with the protein to form the protein-PEG conjugate. The pH buffer may maintain a pH of the aqueous protein solution ranging from 4.0 to 4.4 during the reaction.

Claims

1. A method of making a protein-PEG conjugate, the method comprising:

providing an aqueous protein solution comprising a protein, a pH buffer, and a chelating agent, wherein;

the protein is insulin,

the pH buffer is an acetate or a citrate, and

the chelating agent is chosen from the group consisting of an aminopolycarboxylic acid, a hydroxyaminocarboxylic acid, an N-substituted glycine, 2-(2-amino-2-oxocthyl) aminoethane sulfonic acid (BES), and deferoxamine (DEF);

introducing boron-containing reducing agent and a methoxy polyethylene glycol aldehyde to the aqueous protein solution, wherein the boron-containing reducing agent and methoxy polyethylene glycol aldehyde have a molar ratio ranging from about 25;

1 to about 1.5;

1; and

reacting the methoxy polyethylene glycol aldehyde with the protein to form the protein-PEG conjugate, wherein;

the pH buffer maintains a pH of the aqueous protein solution ranging from 3.88 to 4.27 during the reaction, and

the reaction of the methoxy polyethylene glycol aldehyde with the protein yields a mono-PEGylated protein-PEG conjugate.

2. The method of claim 1, wherein the boron-containing reducing agent is selected from the group consisting of sodium cyanoborohydride, dimethylamine borane, trimethylamine borane, 2-methyl pyridine borane, sodium triacetoxyborohydride, triethylamine borane, morpholine borane, tert butylamine borane, and 5-ethyl-2-methyl-pyridine borane.

3. The method of claim 1, wherein the boron-containing reducing agent is 2-methyl pyridine borane.

4. The method of claim 1, wherein the mono-PEGylated protein-PEG conjugate comprises PEG-PheB1-insulin conjugate.

5. The method of claim 1, wherein the chelating agent is the aminopolycarboxylic acid selected from the group consisting of ethylenediaminetetraacetic acid (EDTA), diethylenetriamine pentaacetic acid (DTPA), nitrilotriacetic acid (NTA), N-2-acetamido-2-iminodiacetic acid (ADA), bis(aminoethyl)glycolether, N,N,N′

,N′

-tetraacetic acid (EGTA), trans-diaminocyclohexane tetraacetic acid (DCTA), glutamic acid, and aspartic acid.

6. The method of claim 1, wherein the chelating agent is the aminopolycarboxylic acid, and the aminopolycarboxylic acid is ethylenediaminetetraacetic acid (EDTA).

7. The method of claim 1, wherein the hydroxyaminocarboxylic acid is chosen from the group consisting of N-hydroxyethyliminodiacetic acid (HIMDA), N,N-bis-hydroxyethylglycine, and N-trishydroxymethylmethyl) glycine.

8. The method of claim 1, wherein the N-substituted glycine comprises glycylglycine.

26. The method of claim 1, wherein the boron-containing reducing agent and methoxy polyethylene glycol aldehyde have a molar ratio ranging from 22:

1 to 5.5;

1.

27. The method of claim 1, wherein the pH of the aqueous protein solution ranges from 4.0 to 4.2.

29. The method of claim 1, wherein the pH buffer is the acetate.

30. The method of claim 1, wherein the pH buffer is the citrate.

33. The method of claim 1, wherein the boron-containing reducing agent and methoxy polyethylene glycol aldehyde have a molar ratio ranging from 5:

1 to 1.5;

1.

9. A method of making an insulin-PEG conjugate, the method comprising:

providing an aqueous insulin solution comprising an insulin, a pH buffer, an organic solvent, and a chelating agent comprising ethylenediaminetetraacetic acid (EDTA);

introducing a boron-containing reducing agent and a methoxy polyethylene glycol aldehyde to the aqueous insulin solution, wherein the boron-containing reducing agent and methoxy polyethylene glycol aldehyde have a molar ratio ranging from about 25;

1 to about 1.5;

1; and

reacting the methoxy polyethylene glycol aldehyde with the insulin to form the insulin-PEG conjugate, wherein;

the pH buffer maintains a pH of the aqueous insulin solution in a range from 3.88 to 4.27 during the reaction,

the pH buffer is an acetate or a citrate, and

the reaction of the methoxy polyethylene glycol aldehyde with the insulin yields a mono-PEGylated insulin-PEG conjugate.

10. The method of claim 9, wherein the boron-containing reducing agent is selected from the group consisting of sodium cyanoborohydride, dimethylamine borane, trimethylamine borane, 2-methyl pyridine borane, sodium triacetoxyborohydride, triethylamine borane, morpholine borane, tert butylamine borane, and 5-ethyl-2-methyl-pyridine borane.

11. The method of claim 9, wherein the boron-containing reducing agent is 2-methyl pyridine borane.

12. The method of claim 9, wherein the methoxy polyethylene glycol aldehyde and the insulin have a molar ratio of about 10:

1 to about 1;

1 when the reaction starts.

13. The method of claim 9, wherein the boron-containing reducing agent and methoxy polyethylene glycol aldehyde have a molar ratio ranging from about 22:

1 to about 5.5;

1.

14. The method of claim 9, wherein the organic solvent is chosen from the group consisting of ethanol, methanol, dimethyl sulfoxide (DMSO), dioxane, acetonitrile, dimethylformamide (DMF), and N-methylpyrrolidone (NMP).

15. The method of claim 9, wherein the organic solvent is dioxane.

16. The method of claim 9, wherein the insulin comprises human insulin.

28. The method of claim 9, wherein the pH of the aqueous protein solution ranges from 4.0 to 4.2.

31. The method of claim 9, wherein the pH buffer is the acetate.

32. The method of claim 9, wherein the pH buffer is the citrate.

17. A method of making controlled-release microspheres containing a protein-PEG conjugate, the method comprising:

providing an aqueous protein solution comprising a protein, a pH buffer, and a chelating agent, wherein;

the protein is insulin,

the pH buffer is an acetate or a citrate, and

the chelating agent is chosen from the group consisting of an aminopolycarboxylic acid, a hydroxyaminocarboxylic acid, an N-substituted glycine, 2-(2-amino-2-oxocthyl) aminoethane sulfonic acid (BES), and deferoxamine (DEF);

introducing a boron-containing reducing agent and a methoxy polyethylene glycol aldehyde to the aqueous protein solution, wherein the boron-containing reducing agent and methoxy polyethylene glycol have a molar ratio ranging from about 25;

1 to about 1.5;

1;

reacting the methoxy polyethylene glycol aldehyde with the protein to form the protein-PEG conjugate, wherein the pH buffer maintains a pH of the aqueous protein solution ranging from 3.88 to 4.27 during the reaction;

mixing the protein-PEG conjugate in an organic solvent with a biodegradable polymer to form a mixture;

emulsifying the mixture of the protein-PEG conjugate and the biodegradable polymer in an aqueous solution to form an emulsified mixture; and

hardening the emulsified mixture of the protein-PEG conjugate and the biodegradable polymer into the controlled-release microspheres, and

the reaction of the methoxy polyethylene glycol aldehyde with the protein yields a mono-PEGylated protein-PEG conjugate.

18. The method of claim 17, wherein the boron-containing reducing agent is selected from the group consisting of sodium cyanoborohydride, dimethylamine borane, trimethylamine borane, 2-methyl pyridine borane, sodium triacetoxyborohydride, triethylamine borane, morpholine borane, tert butylamine borane, and 5-ethyl-2-methyl-pyridine borane.

19. The method of claim 17, wherein the boron-containing reducing agent is 2-methyl pyridine borane.

20. The method of claim 18, wherein the mono-PEGylated protein-PEG conjugate comprises PEG-PheB1-insulin conjugate.

21. The method of claim 17, wherein the chelating agent comprises ethylenediaminetetraacetic acid (EDTA).

22. The method of claim 17, wherein the boron-containing reducing agent and methoxy polyethylene glycol aldehyde have a molar ratio ranging from about 25:

1 to about 5;

1.

23. The method of claim 17, wherein the organic solvent comprises methylene chloride.

24. The method of claim 17, wherein the biodegradable polymer is chosen from the group consisting of a polylactide;

a polyglycolide;

a poly(d,1-lactide-co-glycolide);

a polycaprolactone;

a polyorthoester;

a copolymer of a polyester and a polyether; and

a copolymer of polylactide and polyethylene glycol.

25. The method of claim 17, wherein the biodegradable polymer comprises poly(d,1-lactide-co-glycolide).