Affinity chromatography matrix

Patent metadata

Field	Value
Publication number	US-9683013-B2
Application number	US-201113996023-A
Country	US
Kind code	B2
Filing date	Dec 19, 2011
Priority date	Dec 20, 2010
Publication date	Jun 20, 2017
Grant date	Jun 20, 2017

Abstract

The present invention relates to a method of separating one or more immunoglobulin containing proteins from a liquid. The method includes first contacting the liquid with a separation matrix comprising ligands immobilized to a support; allowing the immunoglobulin containing proteins to adsorb to the matrix by interaction with the ligands; followed by an optional step of washing the matrix containing the immunoglobulin containing proteins adsorbed thereon; and recovering said immunoglobulin containing proteins by contacting the matrix with an eluent which releases the proteins. The method improves upon previous separation methods in that each of the ligands comprises one or more of a protein A domain (E, D, A, B, C), or protein Z, or a functional variant thereof, with at least one of the monomers having a substitution of the Asparagine at the position corresponding to N28 of B domain of Protein A or Protein Z, and wherein the ligand provides an increase in elution pH compared to non-substituted ligand.

First claim

What is claimed is:

1. A method of separating one or more immunoglobulin containing proteins from a liquid, the method comprising,
(a) contacting the liquid with a separation matrix comprising ligands immobilised to a support, said ligands comprises one or more monomers having the sequence of SEQ. ID. NO. 2, wherein in at least one of the monomers, the Asparagine at the position N28 of SEQ. ID. NO. 2 has been substituted with another amino acid X to give a substituted monomer having a sequence of SEQ ID No.: 9;
(b) allowing said immunoglobulin containing proteins to adsorb to the matrix by interaction with the ligands;
(c) an optional step of washing the immunoglobulin containing protein adsorbed matrix; and
(d) recovering said immunoglobulin containing proteins by contacting the matrix with an eluent which releases the proteins from the ligand at an elution pH that is higher than the elution pH when using a parental ligand that has the same sequence of said ligand except for said N28X substitution(s) under the same elution condition.

2. The method of claim 1 , wherein the ligand comprises two or more copies of SEQ. ID. NO. 9.

3. The method of claim 1 , wherein the ligand is multimeric and the other amino acid X is Glu, Leu, Gln, Trp, Lys, Asp, or Arg.

4. The method of claim 1 , wherein the ligand is multimeric and each monomer has a sequence of SEQ ID NO.: 9 where the other amino acid X is Glu, Leu, Gln, Trp, Lys, Asp, or Arg.

5. The method of claim 1 , wherein the protein is released at an elution pH that is at least 0.2 higher than when using the parental ligand under the same elution condition.

6. The method of claim 1 , wherein the protein is released at an elution pH that is at least 0.4 higher than when using the parental ligand under the same elution condition.

7. The method of claim 1 , wherein the protein is released at an elution pH of at least 4.0 with a recovery yield that is at least 80% of that of the parental ligand under the same elution condition.

8. The method of claim 1 , wherein the ligands have affinity for the Fc part of an immunoglobulin but lack affinity for the Fab part of an immunoglobulin.

9. The method of claim 1 , wherein the ligand is a dimer.

10. The method of claim 1 , wherein the ligand is a tetramer.

11. The method of claim 1 , wherein the immunoglobulin containing protein is a monoclonal antibody.

12. The method of claim 1 , wherein the immunoglobulin containing protein is polyclonal antibody.

13. The method of claim 1 , wherein the immunoglobulin containing protein is a fusion protein containing an immunoglobulin Fc portion fused with another protein.

14. The method of claim 1 , wherein a pH gradient elution is performed in the recovery step which results in a more efficient separation of aggregate and monomer species of the immunoglobulin containing protein than using the parental ligand under the same elution condition.

15. The method of claim 1 , wherein the protein is released at an elution pH of at least 4.2 with a recovery yield that is at least 80% of that of the parental ligand under the same elution condition.

16. The method of claim 1 , wherein the protein is released at an elution pH of at least 4.0 with a recovery yield that is at least >90% of that of the parental ligand under the same elution condition.

17. The method of claim 1 , wherein the protein is released at an elution pH of at least 4.2 with a recovery yield that is at least >95% of that of the parental ligand under the same elution condition.

18. The method of claim 1 , wherein the ligand is a dimeric ligand comprising dimeric monomers having the sequence of SEQ. ID. NO. 10, wherein the Asparagine at the position N28 of SEQ ID NO.: 2 has been substituted with another amino acid X.

19. The method of claim 1 , wherein the ligand is a dimeric ligand having a sequence of SEQ. ID. NO. 3.

20. A ligand comprising one or more monomers having the sequence of SEQ. ID. NO. 2, wherein in at least one of the monomers, the Asparagine at the position N28 of SEQ ID NO.: 2 has been substituted with another amino acid X to give a substituted monomer having a sequence of SEQ ID NO.: 9.

21. A matrix for affinity separation comprising multiple copies of the ligand of claim 20 coupled to a solid support.

22. The matrix of claim 21 , wherein the solid support is polysaccharide based.

23. The matrix of claim 21 , wherein the ligands are coupled via thioether bonds to the solid support.

24. The ligand of claim 20 , wherein said ligand comprises two or more copies of SEQ. ID. NO. 9.

25. The ligand of claim 20 , wherein said ligand comprises SEQ. ID. NO. 11.

26. The ligand of claim 20 , wherein the ligand is multimeric and the other amino acid X is Glu, Leu, Gln, Trp, Lys, Asp, or Arg.

27. The ligand of claim 20 , wherein the ligand is multimeric and each monomer has a sequence of SEQ ID NO.: 9 where the other amino acid X is Glu, Leu, Gln, Trp, Lys, Asp, or Arg.

28. The ligand of claim 20 , wherein the ligands has affinity for the Fc part of an immunoglobulin but lack affinity for the Fab part of an immunoglobulin.

29. The ligand of claim 20 , wherein the ligand is a dimer.

30. The ligand of claim 20 , wherein the ligand is a tetramer.

31. The ligand of claim 20 , wherein the ligand is a dimeric ligand having the sequence of SEQ. ID. NO. 10 where the other amino acid X is Ala, Glu, Leu, Gln, Trp, Lys, Asp, or Arg.

32. A dimeric ligand having the sequence of SEQ. ID. NO. 3.

Assignees

• Bjorkman Tomas
• Monie Elin
• Rodrigo Gustav
• Ge Healthcare Bioprocess R&D Ab

Inventors

• Bjorkman Tomas
• Monie Elin
• Rodrigo Gustav

Classifications

• B01J20/289

  bonded via a spacer · CPC title

• C07K14/745

  Blood coagulation or fibrinolysis factors · CPC title

• C07K14/31

  from Staphylococcus (G) · CPC title

• C07K1/22 Primary

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

• B01D15/3809

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