Novel carbamylated EPO and method for its production

A method for producing a carbamylated erythropoietin protein having less than about 3% aggregated protein and less than about 40% by weight of over- and under-carbamylated protein as measured by ESI-mass spectrometry, comprising purifying the carbamylated erythropoietin using anion exchange chromatography.

A method for producing a carbamylated erythropoietin protein having less than about 3% aggregated protein and less than about 40% by weight of over- and under-carbamylated protein as measured by ESI mass spectrometry, which method comprises:

• (a) contacting an amount of an erythropoietin protein with an amount of cyanate at a temperature, pH, and for a time period sufficient for at least about 90% of the amine residues on the lysine and the N-terminal amino acids of the erythropoietin to become carbamylated;
• and
• (b) purifying the carbamylated erythropoietin protein using anion exchange chromatography.

(canceled)

A carbamylated erythropoietin protein having at least about 90% carbamylation of the primary amines of the lysine and amino terminal amino acids, less than about 3% aggregated protein and less than about 40% by weight of over- and under-carbamylated protein as measured by ESI-mass spectrometry.

A compound comprising a carbamylated erythropoietin protein having at least about 90% carbamylation of the primary amines of the lysine and amino terminal amino acids, less than about 40% aggregated protein, and an amount of cyanate.

A carbamylated erythropoietin protein having carbamylation of at least about 90% of the primary amines of the lysine and amino terminal amino acids, less than about 3% aggregated protein and less than about 40% by weight of over- and under-carbamylated protein as measured by ESI-mass spectrometry, which is the product of the process comprising the steps of:

• (a) contacting an amount of an erythropoietin protein with an amount of a cyanate at a temperature, pH, and for a time period sufficient for at least about 90% of the amine residues on the lysines and the N-terminal amino acid of the erythropoietin protein to become carbamylated;
• and
• (b) purifying the carbamylated erythropoietin protein using anion exchange chromatography.

A pharmaceutical composition comprising a therapeutically effective amount of a carbamylated erythropoietin protein having carbamylation of at least about 90% of the primary amines of the lysine and amino terminal amino acids, less than about 3% aggregated protein and less than about 40% by weight of over- and under-carbamylated protein, and a pharmaceutically acceptable carrier.