Claims for Patent: 10,246,484

Summary for Patent: 10,246,484

Title:	Method for purifying recombinant protein
Abstract:	A method is provided for purifying a recombinant protein from a mixture comprising the recombinant protein and related proteins, comprising the steps of: A. using a first equilibrating buffer in a first conductivity and pH to make the recombinant protein bind to an ion exchange medium; B. using a second equilibration buffer in a second conductivity and pH to continually equilibrate the ion exchange medium bound to the protein; C. using a washing liquid in a third conductivity and a gradually increasing pH to wash the ion-exchange medium, and eluting the first category-related proteins; D. using a first eluent in a fourth conductivity and pH to elute the ion exchange medium, and eluting the target recombinant protein; and E. using a second eluent in a fifth conductivity and pH to continually elute the ion exchange medium, and eluting the second category-related proteins.
Inventor(s):	Hu; Hui (Shanghai, CN), Zhu; Yunbin (Shanghai, CN), Zang; Meiling (Shanghai, CN)
Assignee:	SUNSHINE GUOJIAN PHARMACEUTICAL (SHANGHAI) CO., LTD. (Shanghai, CN)
Application Number:	15/034,821
Patent Claims:	1. A method for purifying a recombinant protein from a mixture comprising the recombinant protein and its related proteins, which comprises the following steps performed sequentially: A. binding the recombinant protein to an ion exchange medium using a first equilibration buffer, wherein the first equilibration buffer is at a first conductivity and pH; the ion exchange medium is a cation exchange medium; B. equilibrating the protein-bound ion exchange medium continually using a second equilibration buffer, wherein the second equilibration buffer is at a second conductivity and pH; the conductivity of the second equilibration buffer is less than that of the first equilibration buffer, but the pH of both are the same; C. washing the ion exchange medium using a wash buffer which has different pH, so as to elute a first class of the related proteins from the ion exchange medium, wherein the wash buffer is at a third conductivity and a gradually increased pH; the conductivity of the wash buffer is less than that of the first equilibration buffer, but the pH of the wash buffer is greater than that of the first and/or second equilibration buffer; D. washing the ion exchange medium using a first elution buffer, so as to elute the target recombinant protein from the ion exchange medium, wherein the first elution buffer is at a fourth conductivity and pH; the pH of the first elution buffer is greater than that of the wash buffer, but the conductivity of both are essentially the same; and E. then washing the ion exchange medium using a second elution buffer, so as to elute a second class of related proteins from the ion exchange medium, wherein the second elution buffer is at a fifth conductivity and pH; the pH and/or conductivity of the second elution buffer are/is greater than that of the first elution buffer. 2. The method of claim 1, characterized in that, the cation exchange medium is a filler having a functional group SO.sub.3.sup.- that is bound to different substrates. 3. The method of claim 1, characterized in that, the elution of the first class of the related proteins and the target protein is achieved by changing the pH of the wash buffer and the first elution buffer. 4. The method of claim 3, characterized in that, the change of pH of the wash buffer and the first elution buffer is achieved by adjusting the mixed ratio of two salt-containing buffers that have different pH. 5. The method of claim 4, characterized in that, the change of pH of the wash buffer and the first elution buffer is achieved by changing 25% Na.sub.2HPO.sub.4 pH7.5+75% Na.sub.2HPO.sub.4 pH9.3-9.4 to 15% Na.sub.2HPO.sub.4 pH7.5+85% Na.sub.2HPO.sub.4 pH9.3-9.4. 6. The method of claim 1, characterized in that, in steps A-D, the pH is gradually increased. 7. The method of claim 1, characterized in that, the recombinant protein and its related proteins have different PI, wherein the PI of the first class of the related proteins is less than that of the recombinant protein, and the PI of the recombinant protein is less than that of the second class of related proteins. 8. The method of claim 1, characterized in that, the first class of the related proteins is an acidic variant of the recombinant protein, which is defined as a substance which has a less retention time than the target protein on CEX-HPLC; the second class of the related protein is a basic variant of the recombinant protein, which is defined as a substance which has a greater retention time than the target protein on CEX-HPLC. 9. The method of claim 1, characterized in that, the recombinant protein is an antibody. 10. The method of claim 9, characterized in that, the antibody is selected from the group consisting of trastuzumab, pertuzumab and other HER2 antigen-binding recombinant antibodies. 11. The method of claim 1, characterized in that, the method further comprises subjecting the mixture comprising the recombinant protein and its related proteins to one or more purification steps, either before, during, or after the ion exchange chromatography, so as to obtain a homogeneous preparation of the recombinant protein. 12. A method for purifying a recombinant anti-HER2 antibody from a mixture comprising the recombinant anti-HER2 antibody and its related proteins, which comprises the following steps performed sequentially: a). binding the recombinant protein to a cation exchange medium using a first equilibration buffer, wherein the first equilibration buffer is at a first conductivity and pH; b). then equilibrating the protein-bound cation exchange medium using a second equilibration buffer, wherein the second equilibration buffer is at a second conductivity and pH; c). washing the ion exchange medium using a wash buffer which has different pH, so as to elute the first class of the related proteins from the cation exchange medium, wherein the wash buffer is at a third conductivity and a gradually increased pH; d). washing the ion exchange medium using a first elution buffer, so as to elute the target recombinant protein from the cation exchange medium, wherein the first elution buffer is at a fourth conductivity and pH; and e). washing the ion exchange medium using a second elution buffer, so as to elute the second class of the related proteins from the cation exchange medium, wherein the second elution buffer is at a fifth conductivity and pH; the conductivity of the wash buffer is less than that of the first equilibration buffer, but the pH of the wash buffer is greater than that of the first and/or second equilibration buffer; the pH of the first elution buffer is greater than that of the wash buffer, and the conductivity of the first elution buffer is basically the same as that of the wash buffer; the pH and conductivity of the second elution buffer are/is greater than that of the first elution buffer. 13. The method of claim 12, characterized in that, the cation exchange medium is a filler having a functional group SO.sub.3.sup.- which is bound to different substrates. 14. The method of claim 13, characterized in that, the cation exchange medium is at least one selected from the group consisting of carboxy-methyl-cellulose, sulphopropyl immobilized on agarose, sulphopropyl immobilized on polystyrene/divinyl benzene, sulfonyl immobilized on agarose, and sulphopropyl immobilized on hydrophilic polyacrylamides. 15. The method of claim 12, characterized in that, the first equilibration buffer is a salt-containing buffer, of which the buffer is 10-50 mmol/L acetic acid buffer, the salt is 40-60 mmol/L sodium chloride or ammonium sulfate, the pH is 4-6, and the conductivity is 8-13 ms/cm; the second equilibration buffer is a salt-free buffer, of which the buffer is 10-50 mmol/L acetic acid buffer, the pH is the same as that of the first equilibration buffer, and the conductivity is 1-2 ms/cm. 16. The method of claim 12, characterized in that, the change of pH of the wash buffer and the first elution buffer is achieved by adjusting the mixed ratio of two salt-containing buffers that have different pH, and achieved by changing 25% Na.sub.2HPO.sub.4 pH7.5+75% Na.sub.2HPO.sub.4 pH9.3-9.4 to 15% Na.sub.2HPO.sub.4 pH7.5+85% Na.sub.2HPO.sub.4 pH9.3-9.4. 17. The method of claim 12, characterized in that, the recombinant anti-HER2 antibody is selected from the group consisting of trastuzumab, pertuzumab and other HER2 antigen-binding recombinant antibodies. 18. The method of claim 12, characterized in that, the method further comprises subjecting the mixture comprising the recombinant anti-HER2 antibody and its related proteins to one or more purification steps, either before, during, or after the cation exchange chromatography, so as to obtain a homogeneous preparation of the recombinant anti-HER2 antibody.

Details for Patent 10,246,484

Applicant	Tradename	Biologic Ingredient	Dosage Form	BLA	Approval Date	Patent No.	Expiredate
Genentech, Inc.	HERCEPTIN	trastuzumab	For Injection	103792	09/25/1998		2033-11-06
Genentech, Inc.	HERCEPTIN	trastuzumab	For Injection	103792	02/10/2017		2033-11-06
Genentech, Inc.	PERJETA	pertuzumab	Injection	125409	06/08/2012		2033-11-06
Genentech, Inc.	HERCEPTIN HYLECTA	trastuzumab and hyaluronidase-oysk	Injection	761106	02/28/2019		2033-11-06