Amine pegylation methods for the preparation of site-specific protein conjugates
First Claim
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, andthe 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, andthe reaction of the methoxy polyethylene glycol aldehyde with the protein yields a mono-PEGylated protein-PEG conjugate.
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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.
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Citations
33 Claims
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1. A method of making a protein-PEG conjugate, the method comprising:
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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; andreacting 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. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 26, 27, 29, 30, 33)
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9. A method of making an insulin-PEG conjugate, the method comprising:
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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; andreacting 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. - View Dependent Claims (10, 11, 12, 13, 14, 15, 16, 28, 31, 32)
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17. A method of making controlled-release microspheres containing a protein-PEG conjugate, the method comprising:
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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. - View Dependent Claims (18, 19, 20, 21, 22, 23, 24, 25)
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Specification