Claims for Patent: 9,708,365

Summary for Patent: 9,708,365

Title:	Purification process for monoclonal antibodies
Abstract:	The present invention provides an improved method for the purification of monoclonal antibody from cell culture. Process of purification of the desired monoclonal antibody comprises affinity, hydrophobic interaction and optionally ion exchange column chromatography. It provides more than 99% purity of the desired monoclonal antibody.
Inventor(s):	Mendiratta; Sanjeev Kumar (Ahmedabad, IN), Bandyopadhyay; Sanjay (Ahmedabad, IN), Singh; Avanish Kumar (Ahmedabad, IN)
Assignee:	Cadila Healthcare Limited (Ahmedabad Gujarat, IN)
Application Number:	14/895,432
Patent Claims:	1. A process for purification of a monoclonal antibody from cell culture comprising the following steps: (a) carrying out affinity chromatography (AF) on the cell culture comprising the monoclonal antibody and collecting eluate comprising the purified monoclonal antibody; and (b) carrying out hydrophobic interaction chromatography (HIC) on eluant obtained in (a), optionally followed by other suitable purification steps, wherein hydrophobic interaction chromatography is performed in bind-elute mode. 2. The process as claimed in claim 1, wherein affinity chromatography matrix is selected from protein A, protein G and protein L. 3. The process as claimed in claim 1, wherein affinity chromatography matrix is protein A. 4. The process as claimed in claim 1, wherein step (a) comprises loading of a crude mixture containing the desired antibody to the column at suitable pH and/or conductance for binding, followed by column wash prior to elution of the desired antibody in the form of a single peak. 5. The process as claimed in claim 4, wherein antibody is recovered from the column with buffer components selected from citrate, acetate, and phosphate. 6. The process as claimed in claim 4, wherein antibody is recovered from the column with additive selected from sodium chloride, arginine, and glycine. 7. The process as claimed in claim 4, wherein column wash comprises: (i) First wash with equilibration buffer at suitable pH and/or conductivity (ii) Second wash at the same pH as of the first wash buffer and/or a conductivity higher than the first wash buffer (iii) Third wash at a pH and/or a conductivity lower than the second wash buffer (iv) Elution of an antibody at lower pH and/or higher conductivity than third wash buffer. 8. The process as claimed in claim 7, wherein the column wash comprises: (i) First wash with equilibration buffer at about pH 7.4 and/or conductivity in the range of 1 mS/cm to 30 mS/cm (ii) Second wash at about pH 7.4 and/or conductivity more than 30 mS/cm (iii) Third wash at pH in the range of pH 5 to pH 6.5 and/or conductivity in the range of 1 mS/cm to 5 mS/cm (iv) Elution of an antibody at about pH 3.5 and/or conductivity higher than 5 mS/cm. 9. The process as claimed in claim 7, wherein equilibration buffer and wash buffer component is selected from iris, acetate and citrate buffer. 10. The process as claimed in claim 7, wherein elution of an antibody is performed at a pH ranging from about pH 3.5 to pH 4. 11. The process as claimed in claim 7, wherein elution is performed with additive in the buffer, wherein the additive is selected from the group consisting of sodium chloride, arginine, and glycine. 12. The process as claimed in claim 7, wherein elution of an antibody is performed at pH in the range of pH 5 to pH 7. 13. The process as claimed in claim 1, wherein an amount of aggregates in an antibody preparation after step (a) is no greater than 5%. 14. The process as claimed in claim 1, wherein step (b) is performed a pH the range of pH 5 to pH 7 and/or conductivity more than 100 mS/cm. 15. The process as claimed in claim 1, wherein in step (b) an antibody is eluted from the column with down-the-gradient salt concentration. 16. The process as claimed in claim 15, wherein salt is selected from ammonium sulphate, sodium chloride, ammonium chloride and sodium sulphate. 17. The process as claimed in claim 1, wherein hydrophobic column matrix is selected from phenyl sepharose, butyl sepharose, and octyl sepharose. 18. A process as claimed in claim 1 comprising the steps of (a) Protein A affinity chromatography (b) Hydrophobic interaction chromatography (c) Ion exchange chromatography. 19. The process as claimed in claim 18, wherein step (a) comprises loading of a crude mixture containing the desired antibody to the column at suitable pH and/or conductance for binding, followed by column wash prior to elution of the desired antibody in the form of a single peak. 20. The process as claimed in claim 18, wherein step (b) is performed at pH in the range of pH 5 to pH 7 and/or conductivity more than 100 mS/cm. 21. The process as claimed in claim 18, wherein the ion exchange chromatography is selected from cation exchange chromatography and anion exchange chromatography. 22. The process as claimed in claim 21, wherein ion exchange chromatography is anion exchange chromatography. 23. The process as claimed in claim 18, wherein the column matrix for anion exchange chromatography is selected from DEAE sepharose, Mono Q and Q sepharose XL. 24. The process as claimed in claim 23, wherein column is Q sepharose. 25. The process as claimed in claim 18, wherein elution of antibody at step (c) is performed in flow-through-and-wash mode or bind-elute mode. 26. A process as claimed in claim 9 consisting of: a) Cell separation b) Protein A chromatography c) Low-pH incubation d) Neutralization and reconditioning e) Hydrophobic interaction chromatography f) Ultrafiltration-diafiltration g) Anion exchange chromatography h) Nano-filtration i) Ultrafiltration-diafiltration j) Microfiltration. 27. The process as claimed in claim 26, wherein step (b) comprises loading of a crude mixture containing the desired antibody to the column at suitable pH and/or conductance for binding, followed by column wash prior to elution of the desired antibody in the form of a single peak. 28. The process as claimed in claim 26, wherein step (b) is performed at pH in the range of pH 5 to pH 7 and/or conductivity more than 100 mS/cm. 29. The process as claimed in claim 26, wherein diafiltration medium is selected from phosphate, acetate, citrate, succinate and combination thereof. 30. The process as claimed in claim 1, wherein the overall recovery of an antibody is in the range of not less than 50. 31. The process as claimed in claim 1, wherein purified antibody preparation contains no more than 1% aggregate. 32. The process as claimed in claim 1, wherein antibody is selected from anti-HER antibody, anti-TNF antibody, anti-VEGF antibody, anti-CD20 antibody, anti-CD52 antibody, anti-RANKL, and anti-IgE antibody. 33. The process as claimed in claim 1, wherein an antibody is selected from trastuzumab, pertuzumab, adalimumab, bevacizumab, ranibizumab, rituximab, bectumomab, and epratuzumab. 34. The process as claimed in claim 1, wherein the antibody is adalimumab, trastuzumab or rituximab.

Details for Patent 9,708,365

Applicant	Tradename	Biologic Ingredient	Dosage Form	BLA	Approval Date	Patent No.	Expiredate
Genentech, Inc.	RITUXAN	rituximab	Injection	103705	11/26/1997		2033-06-25
Idec Pharmaceuticals Corp.	RITUXAN	rituximab	Injection	103737	02/19/2002		2033-06-25
Genentech, Inc.	HERCEPTIN	trastuzumab	For Injection	103792	09/25/1998		2033-06-25
Genentech, Inc.	HERCEPTIN	trastuzumab	For Injection	103792	02/10/2017		2033-06-25
Abbvie Inc.	HUMIRA	adalimumab	Injection	125057	12/31/2002		2033-06-25
Abbvie Inc.	HUMIRA	adalimumab	Injection	125057	02/21/2008		2033-06-25