Method for purifying antibody protein

Patent metadata

Field	Value
Publication number	US-10400007-B2
Application number	US-201414785069-A
Country	US
Kind code	B2
Filing date	Apr 15, 2014
Priority date	Apr 16, 2013
Publication date	Sep 3, 2019
Grant date	Sep 3, 2019

Abstract

A method for purifying a biologically active substance from a solution mixture containing impurities and the biologically active substance, in which an ion exchange chromatography carrier comprising a matrix and a copolymer containing at least N-isopropylacrylamide as a monomer unit and immobilized to a surface of the matrix is used, and the solution mixture is allowed to flow through a container storing the carrier at a uniform temperature, thereby recovering the biologically active substance.

First claim

What is claimed is:

1. A method for purifying a biologically active substance from a solution mixture containing impurities and the biologically active substance, wherein
an ion exchange chromatography carrier having a matrix and a copolymer containing at least N-isopropylacrylamide as a monomer unit and immobilized to a surface of the matrix is used,
the mass portion of N-isopropylacrylamide in the copolymer is 0.30% to 70%, and
the solution mixture is allowed to flow through a container storing the carrier at a uniform temperature to selectively adsorb the impurities, thereby recovering the biologically active substance.

2. The method according to claim 1 , wherein the uniform temperature is a temperature at which the impurities can be adsorbed in a mass fraction of 50% or more to the carrier and the biologically active substance can be recovered in a mass fraction of 70% or more.

3. The method according to claim 1 , wherein the mass proportion of the impurities relative to the mass of a solution recovered from the container storing the carrier is 2% or less.

4. The method according to claim 1 , wherein the temperature is 5° C. or more and 60° C. or less.

5. The method according to claim 1 , wherein the temperature is 20° C. or more and 35° C. or less.

6. The method according to claim 1 , wherein the solution mixture is allowed to flow through at a flow rate of 0.1 times or more the volume of the ion exchange chromatography carrier/minute and 30 times or less the volume of the carrier/minute.

7. The method according to claim 1 , wherein the solution mixture is allowed to flow through at a flow rate of the same as or more than the volume of the ion exchange chromatography carrier/minute and 10 times or less the volume of the carrier/minute.

8. The method according to claim 1 , wherein the biologically active substance is a monomer of an antibody protein.

9. The method according to claim 8 , wherein the impurities are aggregate components of a dimer or a multimer of the antibody protein.

10. The method according to claim 1 , wherein the ion exchange chromatography carrier is a cation exchange carrier in the form of beads.

11. The method according to claim 1 , wherein the ion exchange chromatography carrier is a cation exchange carrier in the form of membrane.

12. The method according to claim 11 , wherein the cation exchange carrier is a hollow fiber membrane.

13. The method according to claim 1 , further comprising, before the solution mixture is allowed to flow through the container storing the ion exchange chromatography carrier,
purifying the solution mixture by affinity chromatography.

14. The method according to claim 13 , wherein a protein A carrier is used in the affinity chromatography.

15. The method according to claim 13 , wherein an acid elution type affinity chromatography carrier is used in the affinity chromatography.

16. The method according to claim 13 , wherein a temperature responsive affinity chromatography carrier is used in the affinity chromatography.

17. The method according to claim 1 , wherein, in the purification method, a buffer used as a mobile phase has a conductivity of 0.5 to 20 mS/cm.

18. The method according to claim 1 , wherein, in the purification method, a buffer used as a mobile phase has a hydrogen ion exponent within the range of pH 3.0 to pH 9.0.

19. The method according to claim 1 , wherein
the ion exchange chromatography carrier has a copolymer having at least a strong cation exchange group, and
the copolymer contains the strong cation exchange group in a proportion of 0.01 mol % to 500.0 mol % relative to N-isopropylacrylamide in terms of monomer.

20. The method according to claim 1 , wherein
the ion exchange chromatography carrier has a copolymer having at least a strong cation exchange group, and
the copolymer contains the strong cation exchange group in a proportion of 1 mol % to 300.0 mol % relative to N-isopropylacrylamide in terms of monomer.

21. The method according to claim 19 , wherein at least part of the monomer unit of the copolymer having the strong cation exchange group is an acrylic acid derivative or a methacrylic acid derivative and has a group represented by the following formula (1) or (2):
—CH(—OH)—CH 2 —SO 3 H  (1)

—CH(—SO 3 H)—CH 2 —OH  (2).

22. The method according to claim 19 , wherein at least part of the monomer unit of the copolymer having the strong cation exchange group is derived from a vinyl monomer having a sulfonate group.

23. The method according to claim 19 , wherein at least part of the monomer unit of the copolymer having the strong cation exchange group is represented by the following formula (3):
—CR 1 R 2 —CR 3 (—SO 3 H)—  (3)

where R 1 , R 2 and R 3 each individually represent H or Me.

24. The method according to claim 1 , wherein
the ion exchange chromatography carrier has a copolymer having at least a strong cation exchange group, and
the copolymer is formed by polymerizing a monomer composition containing a monomer having the strong cation exchange group and/or a strong cation exchange group introducing precursor monomer in a proportion of 0.01 mol % to 500.0 mol % relative to N-isopropylacrylamide, by a polymerization method.

25. The method according to claim 1 , wherein
the ion exchange chromatography carrier has a copolymer having at least a strong cation exchange group, and
the copolymer is formed by polymerizing a monomer composition containing a monomer having the strong cation exchange group and/or a strong cation exchange group introducing precursor monomer in a proportion of 1 mol % to 300.0 mol % relative to N-isopropylacrylamide, by a polymerization method.

26. The method according to claim 24 , wherein at least part of the monomer unit of the copolymer having the strong cation exchange group is an acrylic acid derivative or a methacrylic acid derivative and has a group represented by the following formula (4) or (5):
—CH(—OH)—CH 2 —SO 3 H  (4)

—CH(—SO 3 H)—CH 2 —OH  (5).

27. The method according to claim 24 , wherein at least part of the strong cation exchange group introducing precursor monomer is an acrylic acid derivative or a methacrylic acid derivative and the copolymer has a side chain represented by the following formula (6) or (7):
—CH(—OH)—CH 2 —SO 3 H  (6)

—CH(—SO 3 H)—CH 2 —OH  (7).

28. The method according to claim 24 , wherein at least part of a monomer unit of the copolymer having the strong cation exchange group is derived from a vinyl monomer having a sulfonate group.

29. The method according to claim 24 , wherein at least part of the monomer having the strong cation exchange group is a vinyl monomer having a sulfonate group.

30. The method according to claim 24 , wherein at least part of the monomer unit of the copolymer having the strong cation exchange group is represented by the following formula (8):
—CR 1 R 2 —CR 3 (—SO 3 H)—  (8)

where R 1 , R 2 and R 3 each individually represent H or Me.

31. The method according to claim 19 , wherein the strong cation exchange group is a sulfonate group.

32. The method according to claim 19 , wherein the density of the cation exchange group is 30 mmol/L or more.

33. The method according to claim 24 , wherein the polymerization method is a

Assignees

• Asahi Kasei Medical Co Ltd

Inventors

• Taniguchi Hiroki
• Goto Masako
• Koguma Ichiro
• Yokoyama Yoshiro

Classifications

• G01N2030/8813

  biological materials · CPC title

• G01N30/96

  using ion-exchange (G01N30/02, G01N30/90 take precedence) · CPC title

• G01N30/30

  of temperature · CPC title

• B01J49/06

  containing cationic exchangers · CPC title

• B01J39/26 Primary

  Cation exchangers for chromatographic processes · CPC title