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US 8,993,350 B2Granted
Sensitive method to analyse free PEG-maleimide
AssigneeBristol-Myers Squibb Company
InventorsMei Lin, Anulfo Valdez
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Abstract
The present invention relates generally to a novel method using HPLC and fluorescence detection of free PEG-mal in PEGylated proteins and PEG-mal raw materials by adding a fluorescent label to the free PEG-mal.
Claims10
A method to detect free polyethylene glycol-maleimide (PEG-mal) in a solution comprising free PEG-mal and PEGylated protein said method comprising the steps of
- (a) adding the fluorescent labeled peptide probe consisting of SEQ ID NO:
- 1, to said solution;
- (b) allowing the fluorescent labeled peptide probe in the solution of step (a) to react under dark conditions with the free PEG-mal present in the solution to provide fluorescent labeled PEG-mal;
- (c) separating by fractionation the fluorescent labeled PEG-mal from the PEGylated protein using high performance liquid chromatography (HPLC) wherein the fluorescent fraction identifies the free PEG-mal.
1 more element
A method to detect free polyethylene glycol-maleimide (PEG-mal) in a solution comprising free PEG-mal and PEGylated protein said method comprising the steps of
- (a) adding a fluorescent labeled peptide probe to said solution;
- (b) allowing the fluorescent labeled peptide probe in the solution of step (a) to react under dark conditions with the free PEG-mal present in the solution to provide fluorescent labeled PEG-mal;
- (c) separating by fractionation the fluorescent labeled PEG-mal from the PEGylated protein using high performance liquid chromatography (HPLC) wherein the fluorescent fraction idetifies the free PEG-mal;
- and
- wherein the fluorescent labeled peptide probe comprises a fluorophore attached to a peptide sequence consisting of 2 to 20 amino acids wherein at least one amino acid in the peptide sequence is a cysteine at the N-terminus or C-terminus of the peptide sequence wherein the thiol group of the cysteine remains free to react with the PEG-mal in the solution;
- wherein at least one amino acid of the remainder of the peptide sequence comprises a free amine group to which the fluorophore is attached and wherein the fluorescent labeled peptide probe is soluble in aqueous solution.
3 more elements
Dependencies are inferred from the claim text.