Claims for Patent: 10,023,608

Summary for Patent: 10,023,608

Title:	Protein purification methods to remove impurities
Abstract:	Disclosed are methods of purifying a protein sample. More specifically, provide are methods of removing or reducing the amount of high molecular weight species and/or high mannose species from a protein sample using a mixed mode chromatography step and a hydrophobic interaction chromatography step.
Inventor(s):	Ma; Junfen (Thousand Oaks, CA), Zhao; Xiaoyang (Moorpark, CA), Williamson; Brian (Camarillo, CA)
Assignee:	AMGEN INC. (Thousand Oaks, CA)
Application Number:	14/208,043
Patent Claims:	1. A method of reducing high molecular weight (HMW) species in a protein sample comprising adalimumab or a biosimilar thereof comprising: (a) subjecting the protein sample to mixed mode chromatography (MMC) to form a first eluate using a washing buffer comprising Tris-HCl and NaCl, wherein the protein sample subjected to MMC has a pH of about 6.9 to about 7.3 and a conductivity at a temperature between about 18.degree. C. and about 22.degree. C. of about 15 to about 30 mS/cm; and (b) subjecting the first eluate to hydrophobic interaction chromatography (HIC) to form a second eluate using an elution buffer comprising 3-(N-morpholino) propanesulfonic acid (MOPS), wherein the second eluate has at least 40% fewer HMW species than the protein sample. 2. The method of claim 1, wherein the method does not comprise cation exchange chromatography or does not comprise anion exchange chromatography. 3. The method of claim 1, wherein the MMC is performed using a resin having a combination of functionalities selected from the group consisting of (i) anion exchange and hydrophobic interaction; (ii) cation exchange and hydrophilic interaction; (iii) cation exchange, anion exchange, and hydrophobic interaction; and (iv) anion exchange and hydrophobic interaction with potential for hydrogen bonding and pi-pi bonding. 4. The method of claim 1, wherein the HIC is performed using a Phenyl HP resin. 5. The method of claim 1, wherein the amount of HMW species in the second eluate is 1% by weight or molar ratio or less. 6. The method of claim 1, wherein the protein sample is from a cell supernatant or cell culture harvest. 7. The method of claim 1, wherein the method results in a decrease in the amount of high mannose species in the second eluate, compared to the amount of high mannose species in the protein sample, by about 10% or more. 8. The method of claim 1, wherein the washing buffer comprises about 175 mM Tris-HCl and about 75 mM NaCl at a pH about 7.1, and the elution buffer comprises about 20 mM MOPS and up to about 1.1M of a kosmotropic salt selected from ammonium sulfate, sodium sulfate, potassium sulfate, and sodium citrate, at a pH of about 7.0. 9. A method of decreasing high mannose species in a protein sample comprising adalimumab or a biosimilar thereof comprising: (a) subjecting the protein sample to mixed mode chromatography (MMC) to form a first eluate using a washing buffer comprising Tris-HCl and NaCl, wherein the protein sample subjected to MMC has a pH of about 6.9 to about 7.3 and a conductivity at a temperature between about 18.degree. C. and about 22.degree. C. of about 15 to about 30 mS/cm; and (b) subjecting the first eluate to hydrophobic interaction chromatography (HIC) to form a second eluate using an elution buffer comprising about 20 mM 3-(N-morpholino) propanesulfonic acid (MOPS), wherein the second eluate has at least 5% fewer high mannose species than the protein sample. 10. The method of claim 9, wherein the method does not comprise cation exchange chromatography, or does not comprise anion exchange chromatography. 11. The method of claim 9, wherein the MMC is performed using a resin having a combination of functionalities selected from the group consisting of (i) anion exchange and hydrophobic interaction; (ii) cation exchange and hydrophilic interaction; (iii) cation exchange, anion exchange, and hydrophobic interaction; and (iv) anion exchange and hydrophobic interaction with potential for hydrogen bonding and pi-pi bonding. 12. The method of claim 9, wherein the HIC is performed using a Phenyl HP resin. 13. The method of claim 9, wherein the amount of high mannose species in the second eluate is decreased by about 10% or more, compared to the amount of high mannose species in the protein sample. 14. The method of claim 9, wherein the protein sample is from a cell supernatant or cell culture harvest. 15. The method of claim 9, wherein the washing buffer comprises about 175 mM Tris-HCl and about 75 mM NaCl at a pH of about 7.1, and the elution buffer comprises about 20 mM MOPS and up to about 1.1M of a kosmotropic salt selected from ammonium sulfate, sodium sulfate, potassium sulfate, and sodium citrate, at a pH of about 7.0. 16. A method of reducing high molecular weight (HMW) species in a sample comprising adalimumab or a biosimilar thereof comprising: (a) subjecting the sample to mixed mode chromatography (MMC) in flow through mode to form a first eluate using a washing buffer comprising 100 mM to 175 mM Tris-HCl and about 75 mM to about 200 mM NaCl, wherein the sample subjected to MMC has a pH of about 6.9 to about 7.3 and a conductivity at a temperature between about 18.degree. C. and about 22.degree. C. of about 15 to about 30 mS/cm; and (b) subjecting the first eluate to hydrophobic interaction chromatography (HIC) in bind/elute mode to form a second eluate using an elution buffer comprising about 20 mM 3-(N-morpholino) propanesulfonic acid (MOPS), wherein the second eluate has at least 40% fewer HMW species than the sample. 17. The method of claim 16, wherein the second eluate has fewer high mannose species than the sample. 18. The method of claim 16, wherein the MMC step is performed using a resin having a combination of anion exchange and hydrophobic interaction functionalities. 19. The method of claim 18, wherein the resin has a protein loading of about 30 to about 120 g/L. 20. The method of claim 16, wherein the HIC step is performed using Phenyl HP resin. 21. The method of claim 16, wherein the amount of HMW species in the second eluate is 1% by weight or molar ratio or less. 22. The method of claim 16, wherein the method results in a decrease in the amount of high mannose species in the second eluate, compared to the amount of high mannose species in the sample, by about 10% or more.

Details for Patent 10,023,608

Applicant	Tradename	Biologic Ingredient	Dosage Form	BLA	Approval Date	Patent No.	Expiredate
Abbvie Inc.	HUMIRA	adalimumab	Injection	125057	December 31, 2002	10,023,608	2034-03-13
Abbvie Inc.	HUMIRA	adalimumab	Injection	125057	February 21, 2008	10,023,608	2034-03-13
Abbvie Inc.	HUMIRA	adalimumab	Injection	125057	April 24, 2013	10,023,608	2034-03-13
Abbvie Inc.	HUMIRA	adalimumab	Injection	125057	September 23, 2014	10,023,608	2034-03-13
Abbvie Inc.	HUMIRA	adalimumab	Injection	125057	November 23, 2015	10,023,608	2034-03-13
Abbvie Inc.	HUMIRA	adalimumab	Injection	125057	March 09, 2016	10,023,608	2034-03-13