Stabilization of urinary erythropoietin using sodium p-aminosalicylate and extracting into phenol

Claim 1

A METHOD OF SEPARATING ERYTHROPOIETIN ACTIVITY FROM PORTESE ACTIVITY SIALIDASE ACTIVITY COMPRISONG: A. PREPARING A FEED SOLUTION OF CRUDE URINARY ERYTHROPOIETIN IN PHOSPHATE-BUFFERED-SALINE; B. ADDING SODIUM P-AMINOSALICYLATE TO THE FEED SOLUTION; C. EXTRACTING THE ERYTHROPOIETION INTO PHENOL; D. DIALYZING THE SEPARATED PHENOL FRACTION AGAINST A PHOSPHATE-BUFFERED-SALINE SOLUTION; AND E. RECOVERING ERYTHROPOIETIN FROM THE DIALYZED FRACTION, WHEREBY THE ERYTHROPOIETIN ACTIVITY IS SEPARATED FROM THE PROTEASE ACTIVIITY AND THE SIALIDASE ACTIVITY AND THE BIOLOGICAL ACTIVITY OF ERYTHROPOIETIN IS STABILIZED.

Claim 2

A METHOD OF SEPARATING ERYTHROPOIETIN ACTIVITY FROM PORTESE ACTIVITY SIALIDASE ACTIVITY COMPRISONG: A. PREPARING A FEED SOLUTION OF CRUDE URINARY ERYTHROPOIETIN IN PHOSPHATE-BUFFERED-SALINE; B. ADDING SODIUM P-AMINOSALICYLATE TO THE FEED SOLUTION; C. EXTRACTING THE ERYTHROPOIETION INTO PHENOL; D. DIALYZING THE SEPARATED PHENOL FRACTION AGAINST A PHOSPHATE-BUFFERED-SALINE SOLUTION; AND E. RECOVERING ERYTHROPOIETIN FROM THE DIALYZED FRACTION, WHEREBY THE ERYTHROPOIETIN ACTIVITY IS SEPARATED FROM THE PROTEASE ACTIVIITY AND THE SIALIDASE ACTIVITY AND THE BIOLOGICAL ACTIVITY OF ERYTHROPOIETIN IS STABILIZED.

Claim 3

A method of stabilizing urinary erythropoietin comprising: a. preparing a feed solution by dissolving crude urinary erythropoietin in a phosphate-buffered-saline solution; b. adding sodium p-aminosalicylate to said feed solution; c. equilibrating a quantity of phenol with a phosphate buffer and with sodium p-aminosalicylate; d. combining one volume of said feed solution with an equal volume of said equilibrated phenol; e. extracting said erythropoietin into the phenol fraction; f. dialyzing each of the separated fractions against a phosphate-buffered-saline solution; and g. recovering said erythropoietin from said dialyzed fractions, whereby said erythropoietin is stabilized.

Claim 4

A method of stabilizing urinary erythropoietin comprising: a. preparing a feed solution by dissolving crude urinary erythropoietin in a phosphate-buffered-saline solution; b. adding sodium p-aminosalicylate to said feed solution; c. equilibrating a quantity of phenol with a phosphate buffer and with sodium p-aminosalicylate; d. combining one volume of said feed solution with an equal volume of said equilibrated phenol; e. extracting said erythropoietin into the phenol fraction; f. dialyzing each of the separated fractions against a phosphate-buffered-saline solution; and g. recovering said erythropoietin from said dialyzed fractions, whereby said erythropoietin is stabilized.

Claim 7

A method of stabilizing urinary erythropoietin comprising: a. preparing a feed solution by dissolving crude urinary erythropoietin in a phosphate-buffered-saline solution; b. preparing a quantity of equilibrated phenol by thoroughly mixing together one volume of a phosphate-buffered-saline solution, one volume of about 0.2 molar phosphate buffer at about pH 7.0, three volumes of approximately 0.475 molar sodium p-aminosalicylate and five volumes of freshly distilled phenol, and separating the equilibrated phenol from the upper aqueous phase; c. adding to each solution of erythropoietin in phosphate-buffered-saline solution enough phosphate buffer at about pH 7.0 and sodium p-aminosalicylate to make final concentrations of about 0.4 molar and 0.285 molar, respectively; d. combining and thoroughly mixing one volume of said feed solution of erythropoietin and an equal volume of said equilibrated phenol; e. extracting said erythropoietin into the phenol fraction; f. separating the phases by centrifugation in the cold; g. dialyzing each of the separated fractions against a phosphate-buffered-saline solution in the cold; and h. recovering stabilized erythropoietin from dialyzed fractions by precipitation with cold alcohol at a final concentration of 90%.

Claim 8

A method of stabilizing urinary erythropoietin comprising: a. preparing a feed solution by dissolving crude urinary erythropoietin in a phosphate-buffered-saline solution; b. preparing a quantity of equilibrated phenol by thoroughly mixing together one volume of a phosphate-buffered-saline solution, one volume of about 0.2 molar phosphate buffer at about pH 7.0, three volumes of approximately 0.475 molar sodium p-aminosalicylate and five volumes of freshly distilled phenol, and separating the equilibrated phenol from the upper aqueous phase; c. adding to each solution of erythropoietin in phosphate-buffered-saline solution enough phosphate buffer at about pH 7.0 and sodium p-aminosalicylate to make final concentrations of about 0.4 molar and 0.285 molar, respectively; d. combining and thoroughly mixing one volume of said feed solution of erythropoietin and an equal volume of said equilibrated phenol; e. extracting said erythropoietin into the phenol fraction; f. separating the phases by centrifugation in the cold; g. dialyzing each of the separated fractions against a phosphate-buffered-saline solution in the cold; and h. recovering stabilized erythropoietin from dialyzed fractions by precipitation with cold alcohol at a final concentration of 90%.