Claims for Patent: 9,556,258

Summary for Patent: 9,556,258

Title:	Purification of fusion proteins
Abstract:	The invention relates generally to methods for purifying a Fc-fusion protein produced in a eukaryotic expression system. More specifically, the invention provides a robust and scalable downstream purification process suitable for use in manufacturing TNFR:Fc for human administration which comprises an optimized Protein A affinity chromatography step and two ion exchange chromatography steps both of which are operated in the bind-and-elute mode.
Inventor(s):	Nti-Gyabaah; Joseph (West Greenwich, RI), Ikechukwu; Ijeoma (Carteret, NJ), Pettroff; Matthew (Secaucus, NJ), Lee; Christine Cho (Cohoes, NY)
Assignee:	Merck Sharp & Dohme Corp. (Rahway, NJ)
Application Number:	14/131,280
Patent Claims:	1. A method of purifying a p75 TNFR:Fc-fusion protein from one or more impurites in a sample, comprising the steps of: a) providing a sample comprising the Fc-fusion protein produced in a eukaryotic expression system; b) binding the Fc-fusion protein present in the sample to a Protein A affinity chromatography resin; c) eluting the Fc-fusion protein from the Protein A resin, by applying to the Protein A resin, an elution buffer gradient of from 50 mM Citrate pH 5.0 to 100 mM Citrate pH 4.0, wherein the eluted product provides a second sample; d) binding the second sample to a cation exchange (CEX) chromatography resin; e) eluting the second sample from the CEX resin by applying to the CEX resin, a linear elution buffer gradient of from 25 mM sodium phosphate, 25 mM arginine pH 4.0, 0.25 M NaCl to 25 mM sodium phosphate, 25 mM arginine pH 4.0, 0.65 M NaCl, wherein the eluted product provides a third sample; f) binding the third sample to an anion exchange (AEX) chromatography resin; and g) eluting the third sample from the AEX resin by applying, to the AEX resin, a linear elution buffer gradient of from 12.5 mM sodium phosphate, pH 8.0 to 12.5 mM sodium phosphate, 300 mM NaCl, pH 8.0, wherein the eluted product provides a purified p75 TNFR:Fc-fusion protein composition. 2. The method according to claim 1, wherein step b) further comprises: b') washing the bound Fc-fusion protein with a buffer having a pH ranging from 3 to 7 and conductivity range of 10 ms/cm to 50 ms/cm. 3. The method according to claim 1 wherein the Protein A chromatography resin comprises cross-linked poly(styrene-divinylbenzene). 4. The method according to claim 1 wherein the CEX chromatography step is carried out on a strong cation exchange resin and the AEX chromatography step is carried out on a strong anion exchange resin. 5. The method according to claim 4 wherein the CEX chromatography resin is surface coated with polyhydroxylated polymer functionalized with sulfopropyl and the AEX chromatography resin is surface coated with fully quaternized polyethyleneimine. 6. The method according to claim 1, wherein the sample comprising the Fc-fusion protein is mammalian cell culture broth or yeast fermentation broth. 7. The method according to claim 6, wherein the sample comprising the Fc-fusion protein is yeast fermentation broth and step a) further comprises: a') adjusting the pH of the sample comprising the Fc-fusion protein to a pH between 8 to 9 and contacting the sample with a refolding agent and a disaggregation agent. 8. The method according to claim 7, wherein the refolding agent is selected from arginine, glycerol, EDTA, TMAO, PEG-3500 or a redox reagent and the disaggregation agent is selected from urea or guanidine hydrochloride. 9. The method according to claim 7, wherein the pH of the sample comprising the Fc-fusion protein is adjusted to 8.6. 10. The method according to claim 1, wherein the purified Fc-fusion protein composition obtained in step g) provides a product with purity of >99%. 11. The method according to claim 1, wherein the purified Fc-fusion protein composition obtained in step g) is characterized by a TSA level of >18 mMol. 12. The method of claim 1 wherein the p75 TNFR:Fc fusion protein is etanercept. 13. The method of claim 1 wherein step e) comprises: eluting the second sample from the CEX resin by applying to the CEX resin, a linear elution buffer gradient of from 25 mM sodium phosphate, 25 mM arginine pH 4.0, 0.25 M NaCl to 25 mM sodium phosphate, 25 mM arginine pH 4.0, 0.65 M NaCl, and collecting product that elutes when the elution buffer ionic strength is between 37 ms/cm and 42 ms/cm; wherein the eluted product that is collected provides a third sample. 14. The method of claim 1 wherein step g) comprises: eluting the third sample from the AEX resin by applying, to the AEX resin, a linear elution buffer gradient of from 12.5 mM sodium phosphate, pH 8.0 to 12.5 mM sodium phosphate, 300 mM NaCl, pH 8.0, and collecting product that elutes when the elution buffer ionic strength is between 4 ms/cm and 24 ms/cm; wherein the eluted product provides a purified p75 TNFR:Fc-fusion protein composition. 15. The method of claim 1 comprising: a) providing a sample comprising the Fc-fusion protein produced in a Chinese hamster ovary cell expression system; b) binding the Fc-fusion protein present in the sample to a Protein A affinity chromatography resin; c) eluting the Fc-fusion protein from the Protein A resin, by applying to the Protein A resin, an elution buffer gradient of from 50 mM Citrate pH 5.0 to 100 mM Citrate pH 4.0, wherein the eluted product provides a second sample; d) binding the second sample to a cation exchange (CEX) chromatography resin; e) eluting the second sample from the CEX resin by applying to the CEX resin, a linear elution buffer gradient of from 25 mM sodium phosphate, 25 mM arginine pH 4.0, 0.25 M NaCl to 25 mM sodium phosphate, 25 mM arginine pH 4.0, 0.65 M NaCl, and collecting product that elutes when the elution buffer ionic strength is between 37 ms/cm and 42 ms/cm; wherein the eluted product that is collected provides a third sample; f) binding the third sample to an anion exchange (AEX) chromatography resin; and g) eluting the third sample from the AEX resin by applying, to the AEX resin, a linear elution buffer gradient of from 12.5 mM sodium phosphate, pH 8.0 to 12.5 mM sodium phosphate, 300 mM NaCl, pH 8.0, and collecting product that elutes when the elution buffer ionic strength is between 4 ms/cm and 24 ms/cm; wherein the eluted product provides a purified p75 TNFR:Fc-fusion protein composition. 16. The method of claim 15 wherein the TNFR:Fc-fusion protein is etanercept.

Details for Patent 9,556,258

Applicant	Tradename	Biologic Ingredient	Dosage Form	BLA	Approval Date	Patent No.	Expiredate
Immunex Corporation	ENBREL	etanercept	For Injection	103795	11/02/1998		2031-07-08
Immunex Corporation	ENBREL	etanercept	For Injection	103795	05/27/1999		2031-07-08
Immunex Corporation	ENBREL	etanercept	Injection	103795	09/27/2004		2031-07-08
Immunex Corporation	ENBREL	etanercept	Injection	103795	02/01/2007		2031-07-08
Immunex Corporation	ENBREL MINI	etanercept	Injection	103795	09/14/2017		2031-07-08