Methods of increasing protein purity using protein A based chromatography

Patent metadata

Field	Value
Publication number	US-10472389-B2
Application number	US-201414768254-A
Country	US
Kind code	B2
Filing date	Mar 7, 2014
Priority date	Mar 14, 2013
Publication date	Nov 12, 2019
Grant date	Nov 12, 2019

Abstract

The present invention provides methods for increasing purity of an Fc-containing protein by removing protein aggregates during the Protein A chromatography step used during the purification of the Fc-containing protein.

First claim

What is claimed is:

1. A method of reducing the level of protein aggregates in an elution pool containing an Fc-containing protein, the method comprising the steps of:
(a) providing a sample comprising an Fc-containing protein and protein aggregates;
(b) contacting the sample with a protein A ligand immobilized onto a solid support, wherein the protein A ligand consists of one or more C-based domains of Protein A, such that the Fc-region containing protein binds to the protein A ligand;
(c) obtaining an elution pool containing the Fc-containing protein using a pH gradient method employing a high pH buffer and a low pH buffer;
wherein at least 30% of the protein aggregates are removed prior to the elution of the Fc-containing protein in addition to protein aggregates that are removed after the elution of the Fc-containing protein, thereby reducing the level of protein aggregates in the elution pool.

2. A method of reducing the level of protein aggregates in an elution pool containing an Fc-containing protein, the method comprising the steps of:
(d) providing a sample comprising an Fc-containing protein and protein aggregates;
(e) contacting the sample with a protein A ligand immobilized onto a solid support, wherein the protein A ligand consists of one or more C-based domains of Protein A, such that the Fc-region containing protein binds to the protein A ligand;
(f) obtaining an elution pool containing the Fc-containing protein using a pH step method employing two or more buffers used sequentially in the order of descending pH values,
wherein at least 30% of the protein aggregates are removed prior to the elution of the Fe-containing protein in addition to protein aggregates that are removed after the elution of the Fc-containing protein, thereby reducing the level of protein aggregates in the elution pool.

3. The method of claim 2 , wherein step (f) comprises two or more, or three or more, or four or more, or five or more, or six or more, or seven or more, or eight or more, or nine or more, or ten or more small pH change steps, with pH ranging from about 5.0 to about 3.0.

4. The method of claim 1 , wherein high pH buffer has a pH of about 6.0 and the low pH buffer has a pH of about 3.0.

5. The method of claim 2 , wherein at least one of the buffers used in the step elution method has a pH ranging from 3.6 to 4.4.

6. The method of claim 1 , wherein the Fc-containing protein is selected from the group consisting of an antibody and an Fc-fusion protein.

7. The method of claim 2 , wherein the Fc-containing protein is an antibody.

8. The method of claim 7 , wherein the antibody is a monoclonal antibody.

9. The method of claim 2 , wherein the Fc-containing protein is selected from the group consisting of an antibody and an Fc-fusion protein.

10. The method of claim 9 , wherein the antibody is a monoclonal antibody.

11. The method of claim 1 , wherein the pH gradient spans 5 column volumes to 30 column volumes.

12. The method of claim 1 , wherein the solid support is selected from the group consisting of solid support is selected from the group consisting of controlled pore glass, silica, zirconium oxide, titanium oxide, agarose, polymethacrylate, polyacrylate, polyacrylamide, polyvinylether, polyvinyl alcohol and polystyrene and derivatives thereof.

13. The method of claim 2 , wherein the solid support is selected from the group consisting of solid support is selected from the group consisting of controlled pore glass, silica, zirconium oxide, titanium oxide, agarose, polymethacrylate, polyacrylate, polyacrylamide, polyvinylether, polyvinyl alcohol and polystyrene and derivatives thereof.

14. The method of claim 1 , wherein the Protein A ligand comprises the amino acid sequence set forth in SEQ ID NO:3 or SEQ ID NO:4.

15. The method of claim 2 , wherein the Protein A ligand comprises the amino acid sequence set forth in SEQ ID NO:3 or SEQ ID NO:4.

16. The method of claim 1 , wherein the solid support is polyvinyl alcohol or polyvinylether.

17. The method of claim 2 , wherein the solid support is polyvinyl alcohol or polyvinylether.

18. A method of eluting Fc-containing protein aggregates before elution of Fc-containing monomer protein from an affinity ligand, the method comprising:
(a) contacting a sample comprising a mixture of Fc-containing protein aggregates and an Fc-containing protein with an affinity ligand immobilized on a solid support, the affinity ligand consisting of one or more C-based domains of Protein A and which binds Fc-containing protein and Fc-containing protein aggregates;
(b) applying a pH gradient elution buffer from a high pH to a low pH, thereby eluting at least 20% of aggregates from the mixture of aggregates and monomer bound to the affinity ligand on the solid support before elution of Fc-containing monomer protein from the affinity ligand.

19. The method of claim 18 , wherein the high pH is about 6.0 and the low pH is about 3.0.

20. A method of eluting Fc-containing protein aggregates before elution of Fc-containing monomer protein from an affinity ligand, the method comprising:
(a) contacting a sample comprising a mixture of Fc-containing protein aggregates and an Fc-containing protein with an affinity ligand immobilized on a solid support, the affinity ligand consisting of one or more C-based domains of Protein A and which binds Fc-containing protein and Fc-containing protein aggregates;
(b) applying a pH step elution using two or more buffers with sequentially descending pH values from a high pH to a low pH, thereby eluting at least 20% of aggregates from the mixture of aggregates and monomer bound to the affinity ligand on the solid support before elution of Fc-containing monomer protein from the affinity ligand.

21. The method of claim 20 , wherein the pH step elution comprises two, three, four, five, six, seven, eight, nine, ten or more step changes, wherein the pH step elution results in a pH change from between about pH 5.0 to about 3.0.

Assignees

• Emd Millipore Corp

Inventors

• Bian Nanying
• Holstein Melissa

Classifications

• C07K16/065

  Purification, fragmentation · CPC title

• C07K1/36

  by a combination of two or more processes of different types · CPC title

• C07K1/22 Primary

  Affinity chromatography or related techniques based upon selective absorption processes · CPC title

• B01D15/168

  pH gradient or chromatofocusing, i.e. separation according to the isoelectric point pI · CPC title

• B01D15/3809

  of the antigen-antibody type, e.g. protein A, G or L chromatography · CPC title