Amine pegylation methods for the preparation of site-specific protein conjugates
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.
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.
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.
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).
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.
28. The method of claim 9 , 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.
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.
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.