Claims for Patent: 10,723,775

Summary for Patent: 10,723,775

Title:	Method for purifying darbepoetin alfa
Abstract:	The present invention relates to a method for purifying darbepoetin alfa by selectively separating only a structural isoform having a high content of sialic acid from a mixture of structural isoforms of darbepoetin alfa having various contents of sialic acid. Since the method of the present invention is a novel method for purifying darbepoetin alfa which can be conveniently and simply produced, it is possible to remarkably increase productivity due to process efficiency improvement, as well as to yield high purity darbepoetin alfa when mass-producing darbepoetin alfa according to the present invention.
Inventor(s):	Lee; Yoon Jung (Yongin-si, KR), Kim; Kyung Hwa (Busan, KR), Yang; Yoo Hee (Yongin-si, KR), Yoo; Jung Min (Seoul, KR), Kim; Se Jun (Gwacheon-si, KR), Moon; Ji Hyun (Seoul, KR), Oh; Hoo Keun (Suwon-si, KR), Lee; Dong Eok (Seoul, KR), Lee; Won Jeong (Seoul, KR), Lee; Jung Rok (Busan, KR), Lee; Chung Min (Yongin-si, KR), Choi; Eun Young (Seoul, KR), Ha; Gyong Sik (Suwon-si, KR)
Assignee:	CJ Healthcare Corporation (Seoul, KR)
Application Number:	15/039,569
Patent Claims:	1. A method for purifying darbepoetin alfa, comprising: (a) binding darbepoetin alfa to an anion exchange chromatography column by loading a mixture comprising darbepoetin alfa having various contents of sialic acid into the anion exchange chromatography column; (b) washing the chromatography column with a primary washing buffer solution having a pH ranging 6 to 8, wherein the primary washing buffer solution is a sodium phosphate solution; (c) removing darbepoetin alfa having an isoelectric point (pl) less than 2.0 and above 4.0 by washing the chromatography column with a secondary washing buffer solution containing arginine and having a pH ranging 3 to 5, wherein the secondary washing buffer solution is a glycine-HCI solution; and (d) eluting the darbepoetin alfa, which remains bound to the chromatography column, from the column, wherein the darbepoetin alfa eluted in step (d) has an isoeletric point ranging from pl 2.0 to 4.0. 2. The method of claim 1, wherein the washing buffer solution containing arginine further comprises at least one selected from the group consisting of NaCI and urea. 3. The method of claim 2, wherein the washing buffer solution comprises NaCI at a concentration ranging from 5 mM to 90 mM. 4. The method of claim 2, wherein the washing buffer solution comprises urea at a concentration ranging from 3 M to 8 M. 5. The method of claim 1, further comprising washing with a washing buffer solution with or without arginine, before or after step (c). 6. The method of claim 1, wherein the biological fluid is an yeast culture, a plant cell culture, or an animal cell culture. 7. The method of claim 1, wherein the anion exchange chromatography column comprises a resin having a functional group which is selected from the group consisting of a quaternary amine (Q), diethylaminoethyl (DEAE), and quaternary aminoethyl (QAE). 8. The method of claim 1, further comprising (e) fractionating the anion exchange chromatography eluate obtained in step (d) by applying it to gel filtration chromatography. 9. A method for purifying darbepoetin alfa, comprising: (a) eluting a darbepoetin alfa-containing fraction by applying a biological fluid comprising darbepoetin alfa to anion exchange chromatography; (b) eluting a darbepoetin alfa-containing fraction by applying the eluate produced in step (a) to adsorption chromatography; (c) binding darbepoetin alfa to an anion exchange chromatography column by loading the eluate produced in step (b) into the anion exchange chromatography column; (d) washing the chromatography column with a primary washing buffer solution having a pH ranging 6 to 8, wherein the primary washing buffer is a sodium phosphate solution; (e) removing darbepoetin alfa having an isoelectric point (pl) less than 2.0 and above 4.0 by washing the column treated in step (d) with a secondary washing buffer solution containing arginine and having a pH ranging 3 to 5, wherein the secondary washing buffer solution is a glycine-HCI solution; and (f) eluting the darbepoetin alfa, which remains bound to the chromatography column by washing in step (e), from the column, wherein the darbepoetin alfa eluted in step (f) has an isoeletric point ranging from pl 2.0 to 4.0. 10. The method of claim 1, wherein the stationary phase of the adsorption chromatography is a hydroxyapatite resin. 11. The method of claim 9, further comprising (g) fractionating the anion exchange chromatography eluate obtained in step (f) by applying it to gel filtration chromatography. 12. The method of claim 9, wherein step (a) comprises adsorbing a darbepoetin alfa-containing biological fluid to an equilibrated anion exchange resin by adding it thereto, washing the resulting resin with a washing buffer solution which has a pH from 6 to 8 and contains 0 mM to 100 mM NaCI, and eluting a darbepoetin alfa-containing fraction with an elution buffer solution which has a pH from 6 to 8 and contains 100 mM to 300 mM NaCI. 13. The method of claim 9, wherein step (b) comprises loading the eluate recovered in step (a) into an equilibrated hydroxyapatite resin, washing the resulting resin with a washing buffer solution which has a pH from 6 to 8 and contains 0 mM to 100 mM sodium phosphate, and obtaining a darbepoetin alfa-containing fraction from the liquid coming out of the resin without being adsorbed thereto during loading and washing. 14. A method for purifying darbepoetin alfa, comprising: (a) removing biological impurities from a darbepoetin alfa solution. (b) binding darbepoetin alfa in the darbepoetin alfa solution to an anion exchange chromatography column by loading a mixture comprising darbepoetin alfa having various contents of sialic acid into the anion exchange chromatography column; (c) washing the chromatography column with a primary washing buffer solution having a pH ranging 6 to 8, wherein the primary washing buffer solution is a sodium phosphate solution; (d) removing darbepoetin alfa having an isoelectric point (pl) less than 2.0 and above 4.0 by washing the chromatography column with a secondary washing buffer solution containing arginine and having a pH ranging 3 to 5, wherein the secondary washing buffer solution is a glycine-HCI solution; and e) eluting the darbepoetin alfa, which remains bound to the chromatography column, from the column, wherein the darbepoetin alfa eluted has an isoeletric point ranging from pl 2.0 to 4.0.

Details for Patent 10,723,775

Applicant	Tradename	Biologic Ingredient	Dosage Form	BLA	Approval Date	Patent No.	Expiredate
Amgen Inc.	ARANESP	darbepoetin alpha	Injection	103951	September 17, 2001	10,723,775	2034-11-28
Amgen Inc.	ARANESP	darbepoetin alpha	Injection	103951	July 19, 2002	10,723,775	2034-11-28
Amgen Inc.	ARANESP	darbepoetin alpha	Injection	103951	December 17, 2002	10,723,775	2034-11-28
Amgen Inc.	ARANESP	darbepoetin alpha	Injection	103951	December 12, 2003	10,723,775	2034-11-28
Amgen Inc.	ARANESP	darbepoetin alpha	Injection	103951	June 29, 2006	10,723,775	2034-11-28
Amgen Inc.	ARANESP	darbepoetin alpha	Injection	103951		10,723,775	2034-11-28

International Patent Family for US Patent 10,723,775

Country	Patent Number	Estimated Expiration
Brazil	112016009421
China	105764915
European Patent Office	3075740
Hong Kong	1223633
Japan	2016538261
Japan	6232130