Claims for Patent: 9,683,012

Summary for Patent: 9,683,012

Title:	Method of antibody purification
Abstract:	The present invention relates to a method for preparing a population of antibodies to have high purity and high quality by removing antibody isoforms and impurities through the use of a cation exchange column, a hydrophobic interaction column, and an anion exchange column successively, without using a protein A column; and to a population of antibodies prepared by the above method.
Inventor(s):	Yoon; Ji Yong (Yuseong-gu, KR), Lee; Dong Eun (Yuseong-gu, KR), Kim; Won Kyum (Yuseong-gu, KR), Youn; Jeong Won (Sejong, KR), Baek; Jung Eun (Daejeon, KR)
Assignee:	PRESTIGE BIOPHARMA PTE. LTD. (Singapore, SG)
Application Number:	14/365,027
Patent Claims:	1. A method for preparing a population of antibodies wherein over 65% of the population are active antibodies, comprising: (a) loading a sample comprising a mixture of antibodies to a pre-equilibrated cation exchange column, then optionally washing the column with a wash buffer, and eluting antibodies bound to the column with an elution buffer, thereby removing host cell proteins (HCPs) and antibody isoforms from the sample, wherein the sample of step (a) has a conductivity of 5 to 7 mS/cm; (b) loading a sample prepared by mixing salt with the eluate of step (a) to a hydrophobic interaction column (HIC), and eluting the antibodies bound to the column with an elution buffer, thereby removing the host cell proteins (HCPs) from the eluate of step (a); and (c) loading the eluate of step (b) to an anion exchange column and collecting the flow-through. 2. The method according to claim 1, wherein the sample of step (a) is prepared by adjusting the pH of a culture supernatant to be in a range from pH 4 to 6 in order to remove precipitates. 3. The method according to claim 1, wherein the antibody has an isoelectric point of 8 to 10. 4. The method according to claim 1, wherein the antibody is Trastuzumab. 5. The method according to claim 1, wherein the eluate of step (a) comprises 65 to 80% active antibodies, 15 to 30% acidic antibody isoforms, and 5 to 20% basic antibody isoforms. 6. The method according to claim 1, wherein the antibody isoform of step (a) is an acidic antibody isoform. 7. The method according to claim 6, wherein the cation exchange column of step (a) comprises a cross-linked agarose resin having a --OCH.sub.2COO-- functional group. 8. The method according to claim 1, wherein the step (a) comprises: (i) loading the sample to the cation exchange column pre-equilibrated with an equilibration buffer comprising 20 to 30 mM sodium acetate (pH 4.5 to 5.5) and 35 to 45 mM sodium chloride; (ii) washing the column with a buffer comprising 20 to 30 mM sodium acetate (pH 4.5 to 5.5) and 35 to 45 mM sodium chloride; (iii) washing the column with a buffer comprising 20 to 30 mM Tris-hydrogen chloride (Tris-HCl) (pH 7.0 to 7.5); (iv) washing the column with a buffer comprising 20 to 30 mM Tris-hydrogen chloride (pH 7.0 to 7.5) and 20 to 30 mM sodium chloride; (v) washing the column with a buffer comprising 20 to 30 mM Tris-hydrogen chloride (pH 7.0 to 7.5); and (vi) eluting antibodies from the column with an elution buffer comprising 20 to 30 mM Tris-hydrogen chloride (pH 7.0 to 7.5) and 80 to 100 mM sodium chloride. 9. The method according to claim 1, wherein the antibody isoform of step (a) is an acidic antibody isoform and a basic antibody isoform. 10. The method according to claim 9, wherein the cation exchange column of step (a) comprises a methacrylate based polymer resin having a COO-- functional group. 11. The method according to claim 10, wherein the step (a) comprises: (i) loading the sample to the cation exchange column pre-equilibrated with an equilibration buffer comprising 20 to 30 mM sodium acetate (pH 4.5 to 5.5) and 35 to 45 mM sodium chloride (NaCl); (ii) washing the column with a buffer comprising 20 to 30 mM sodium acetate (pH 4.5 to 5.5) and 35 to 45 mM sodium chloride; (iii) washing the column with a buffer comprising 25 to 35 mM sodium acetate (pH 5.5 to 6.5); (iv) washing the column with a buffer comprising 25 to 35 mM sodium acetate (pH 5.5 to 6.5) and 45 to 55 mM sodium chloride; (v) washing the column with a buffer comprising 25 to 35 mM sodium acetate (pH 5.5 to 6.5); and (vi) eluting antibodies from the column with an elution buffer comprising 25 to 35 mM sodium acetate and 70 to 90 mM sodium chloride. 12. The method according to claim 1, wherein the antibodies bound to the column of step (b) are eluted with a linear gradient in salt component of the elution buffer. 13. The method according to claim 12, wherein the step (b) comprises (i) loading a sample prepared by adjusting the citrate concentration of the eluate of step (a) to be the same as in an equilibration buffer comprising 25 to 35 mM acetate (pH 5.5 to 6.5) and 0.3 to 1.0 M sodium citrate, to hydrophobic interaction column (HIC) which is pre-equilibrated with the equilibration buffer; and (ii) eluting the antibodies with an elution buffer comprising 25 to 35 mM acetate (pH 5.5 to 6.5) in a linear gradient. 14. The method according to claim 1, wherein the hydrophobic interaction column of step (b) comprises a cross-linked agarose resin having a phenyl functional group. 15. The method according to claim 1, wherein the anion exchange column is equilibrated with an equilibration buffer in a range from pH 7.0 to 8.0, before loading. 16. The method according to claim 15, wherein the equilibration buffer comprises Tris-HCl (pH 7.0 to 8.0). 17. The method according to claim 1, wherein the anion exchange column of step (c) comprises a cross-linked agarose resin having a quaternary amine functional group. 18. The method according to claim 1, wherein the flow-through of the anion exchange column obtained from step (c) has a host cell protein (HCP) concentration of 0.001 to 5 ppm.

Details for Patent 9,683,012

Applicant	Tradename	Biologic Ingredient	Dosage Form	BLA	Approval Date	Patent No.	Expiredate
Genentech, Inc.	HERCEPTIN	trastuzumab	For Injection	103792	09/25/1998		2031-12-15
Genentech, Inc.	HERCEPTIN	trastuzumab	For Injection	103792	02/10/2017		2031-12-15
Genentech, Inc.	HERCEPTIN HYLECTA	trastuzumab and hyaluronidase-oysk	Injection	761106	02/28/2019		2031-12-15