Mutated immunoglobulin-binding polypeptides
An Fc-binding polypeptide of improved alkali stability, comprising a mutant of an Fc-binding domain of Staphylococcus Protein A (SpA), as defined by SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO:3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:22, SEQ ID NO 51 or SEQ ID NO 52 wherein at least the asparagine or serine residue at the position corresponding to position 11 in SEQ ID NO:4-7 has been mutated to an amino acid selected from the group consisting of glutamic acid, lysine, tyrosine, threonine, phenylalanine, leucine, isoleucine, tryptophan, methionine, valine, alanine, histidine and arginine.
1. An Fc-binding polypeptide comprising a mutant of an Fc-binding domain of Staphylococcus Protein A (SpA) wherein the polypeptide is selected from the group consisting of SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:16, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:44, SEQ ID NO:46, and SEQ ID NO:48, and wherein the mutant binds the Fc domain of an antibody and has improved alkaline stability compared to SEQ ID NO: 47.
2. The polypeptide of claim 1 , wherein the alkaline stability is improved as measured by the remaining IgG-binding capacity after 24 or 25 h incubation in 0.5 M or 1.0 M aqueous NaOH at 22+/−2° C.
3. A multimer comprising a plurality of polypeptides as defined by claim 1 .
4. The multimer according to claim 3 , which is a dimer, trimer, tetramer, pentamer, hexamer, heptamer, octamer or nonamer.
5. A nucleic acid or a vector encoding a polypeptide or multimer according to claim 3 .
6. An expression system, which comprises a nucleic acid or vector according to claim 5 .
7. A separation matrix, wherein a plurality of polypeptides or multimers according to claim 3 have been coupled to a solid support.
8. The separation matrix according to claim 7 , wherein the polypeptides or multimers have been coupled to the solid support via thioether bonds.
9. The separation matrix of claim 7 , wherein the solid support is a polysaccharide.
10. The separation matrix of claim 6 , wherein the IgG capacity of the matrix after 24 incubation in 0.5 M NaOH at 22+/−2° C. is at least 80% of the IgG capacity before the incubation.
11. The separation matrix of claim 7 , wherein the IgG capacity of the matrix after 24 incubation in 1.0 M NaOH at 22+/−2° C. is at least 70% of the IgG capacity before the incubation.
12. A method of isolating an immunoglobulin, wherein a separation matrix according to claim 7 is used.
13. The method of claim 12 , comprising the steps of:
a) contacting a liquid sample comprising an immunoglobulin with the separation matrix,
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.
14. The method of claim 13 , wherein the cleaning liquid is alkaline with a pH of 13-14.
15. The method of claim 13 , wherein the cleaning liquid comprises 0.1-1.0 M NaOH or KOH.
16. The method of claim 13 , wherein steps a)-d) are repeated at least 10 times.
17. The method of claim 13 , wherein steps a)-c) are repeated at least 10 times and wherein step d) is performed after a plurality of instances of step c).
18. The polypeptide of claim 1 , comprising the sequence of SEQ ID NO:8.
19. The polypeptide of claim 1 , comprising the sequence of SEQ ID NO:9.
20. The polypeptide of claim 1 , comprising the sequence of SEQ ID NO:10.
21. The polypeptide of claim 1 , comprising the sequence of SEQ ID NO:11.
22. The polypeptide of claim 1 , comprising the sequence of SEQ ID NO:16.
23. The polypeptide of claim 1 , comprising the sequence of SEQ ID NO:23.
24. The polypeptide of claim 1 , comprising the sequence of SEQ ID NO:24.
25. The polypeptide of claim 1 , comprising the sequence of SEQ ID NO:25.
26. The polypeptide of claim 1 , comprising the sequence of SEQ ID NO:26.
27. The polypeptide of claim 1 , comprising the sequence of SEQ ID NO:27.
28. The polypeptide of claim 1 , comprising the sequence of SEQ ID NO:28.
29. The polypeptide of claim 1 , comprising the sequence of SEQ ID NO:44.
30. The polypeptide of claim 1 , comprising the sequence of SEQ ID NO:46.
31. The polypeptide of claim 1 , comprising the sequence of SEQ ID NO:48.