Materials and methods for preparing protein-polymer conjugates
First Claim
1. A method for preparing a site-specific insulin-PEG conjugate comprising:
- (a) contacting insulin protein with PEG in an aqueous solution in the presence of at least one organic solvent selected from the group consisting of ethanol, methanol, DMSO, dioxane, DMF, and NMP and at least one metal chelator selected from the group consisting of metal ion chelators, EDTA, deferoxamine (DEF), diethylenetriamine pentaacetic acid (DTPA), and bis(aminoethyl)glycolether N,N,N′
,N′
-tetraacetic acid (EGTA), to form the site-specific insulin-PEG conjugate by a predominant reaction of the insulin protein at residue PheB1 without protecting the insulin protein at GlyA1 and LysB29 residues, and(b) isolating the conjugate.
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Abstract
The invention is directed to a single-step method for rapidly and efficiently preparing protein-polymer conjugates, including an insulin-polymer conjugate. According to the method of the present invention, a protein and hydrophilic polymer are contacted in the presence of at least one organic solvent and at least one metal chelator, under conditions that promote the formation of a conjugate of the protein and polymer. Thus, the invention is directed to the site-specific modification of selected proteins, such as insulin, with poly(ethylene glycol) at residue PheB1. The invention also provides a pharmaceutical formulation for encapsulating the conjugate in a biodegradable polymer.
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10 Claims
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1. A method for preparing a site-specific insulin-PEG conjugate comprising:
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(a) contacting insulin protein with PEG in an aqueous solution in the presence of at least one organic solvent selected from the group consisting of ethanol, methanol, DMSO, dioxane, DMF, and NMP and at least one metal chelator selected from the group consisting of metal ion chelators, EDTA, deferoxamine (DEF), diethylenetriamine pentaacetic acid (DTPA), and bis(aminoethyl)glycolether N,N,N′
,N′
-tetraacetic acid (EGTA), to form the site-specific insulin-PEG conjugate by a predominant reaction of the insulin protein at residue PheB1 without protecting the insulin protein at GlyA1 and LysB29 residues, and(b) isolating the conjugate. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10)
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Specification