Mutated immunoglobulin-binding polypeptides

1. An Fc-binding polypeptide with improved alkaline stability, which polypeptide comprises a mutant of a parental polypeptide, as defined by, or having at least 90% identity to, SEQ ID NO 28, SEQ ID NO 29 or SEQ ID NO 30, comprising at least the mutation wherein the glutamine residue at position 3 has been mutated to leucine, glutamic acid, valine or lysine.

2. A multimer with improved alkaline stability comprising or consisting essentially of a plurality of polypeptide units, which polypeptide comprises a mutant of a parental polypeptide, as defined by, or having at least 90% identity to, SEQ ID NO 28, SEQ ID NO 29 or SEQ ID NO 30, comprising at least the mutation wherein the glutamine residue at position 3 has been mutated to tryptophan, leucine, glutamic acid, valine or lysine.

3. The multimer of claim 2 , wherein the polypeptide units are linked by elements comprising up to 15 amino acids.

4. The multimer of claim 3 , further comprising at the C-terminal or N-terminal one or more coupling element, selected from the group consisting of a cysteine residue, a plurality of lysine residues and a plurality of histidine residues.

5. A separation matrix, comprising a plurality of polypeptides according to claim 1 coupled to a solid support.

6. A separation matrix, comprising a plurality of multimers according to claim 4 coupled to a solid support.

7. The separation matrix of claim 6 , wherein the multimers have been coupled to the solid support via thioether bonds.

8. A method of isolating an immunoglobulin, using a separation matrix according to claim 6 , the method comprising,

a) contacting a liquid sample comprising an immunoglobulin with the separation matrix according to claim 6 ,

b) washing said separation matrix with a washing liquid,

c) eluting the immunoglobulin from the separation matrix with an elution liquid, and

d) cleaning the separation matrix with a cleaning liquid.

9. The method of claim 8 , wherein the cleaning liquid is alkaline.

10. The method of claim 8 , wherein the cleaning liquid comprises 0.1-1.0 M NaOH or KOH.

11. The method of claim 8 , wherein steps a)-d) are repeated at least 50 times.

12. An Fc-binding polypeptide with improved alkaline stability, which polypeptide comprises a mutant of a B or C domain of Staphylococcus Protein A (SpA), as defined by, or having at least 90% identity to, SEQ ID NO 1 or SEQ ID NO 2, of Protein Z, as defined by, or having at least 90% identity to, SEQ ID NO 3, or of a Protein Z variant as defined by, or having at least 90% identity to, SEQ ID NO 4, comprising at least the mutation wherein the glutamine residue at position 9 has been mutated to leucine, glutamic acid, valine or lysine.

13. A multimer comprising or consisting essentially of a plurality of polypeptide units, which polypeptide comprises a mutant of a B or C domain of Staphylococcus Protein A (SpA), as defined by, or having at least 90% identity to, SEQ ID NO 1 or SEQ ID NO 2, of Protein Z, as defined by, or having at least 90% identity to, SEQ ID NO 3, or of a Protein Z variant as defined by, or having at least 90% identity to, SEQ ID NO 4, comprising at least the mutation wherein the glutamine residue at position 9 has been mutated to tryptophan, leucine, glutamic acid, valine or lysin.

14. A separation matrix, having a plurality of multimers according to claim 13 coupled to a solid support.

15. A method of isolating an immunoglobulin, comprising the steps of:

a) contacting a liquid sample comprising an immunoglobulin with a separation matrix according to claim 14 ,

b) washing said separation matrix with a washing liquid,

c) eluting the immunoglobulin from the separation matrix with an elution liquid, and

d) cleaning the separation matrix with a cleaning liquid.

16. The method of claim 15 , wherein the cleaning liquid comprises 0.1-1.0 M NaOH or KOH.

17. A polypeptide, comprising or consisting essentially of an amino acid sequence selected from the group consisting of: SEQ ID NO 31, SEQ ID NO 32, SEQ ID NO 33, SEQ ID NO 34 and SEQ ID NO 35.

18. A separation matrix, having a plurality of polypeptides according to claim 17 coupled to a solid support.

19. A method of isolating an immunoglobulin, comprising the steps of:

a) contacting a liquid sample comprising an immunoglobulin with a separation matrix according to claim 18 ,

b) washing said separation matrix with a washing liquid,

c) eluting the immunoglobulin from the separation matrix with an elution liquid, and

d) cleaning the separation matrix with a cleaning liquid.

20. A multimer with improved alkaline stability comprising or consisting essentially of a plurality of polypeptide units as defined by claim 17 .

21. The multimer of claim 20 , wherein the polypeptide units are linked by elements comprising up to 15 amino acids.

22. The multimer of claim 21 , further comprising at the C-terminal or N-terminal one or more coupling element, selected from the group consisting of a cysteine residue, a plurality of lysine residues and a plurality of histidine residues.

23. A separation matrix, comprising a plurality of multimers according to claim 20 coupled to a solid support.

24. The separation matrix of claim 23 , wherein the multimers have been coupled to the solid support via thioether bonds.

25. A method of isolating an immunoglobulin, using a separation matrix according to claim 23 , the method comprising,

a) contacting a liquid sample comprising an immunoglobulin with the separation matrix according to claim 23 ,

b) washing said separation matrix with a washing liquid,

c) eluting the immunoglobulin from the separation matrix with an elution liquid, and

d) cleaning the separation matrix with a cleaning liquid.

26. The method of claim 25 , wherein the cleaning liquid comprises 0.1-1.0 M NaOH or KOH.

27. The method of claim 23 , wherein steps a)-d) are repeated at least 50 times.