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:8, SEQ ID NO 26 or SEQ ID NO 27, wherein at least the alanine residue at the position corresponding to position 42 in SEQ ID NO:4-7 has been mutated to arginine and/or wherein at least the aspartic acid residue at the position corresponding to position 37 in SEQ ID NO:4-7 has been mutated to glutamic acid.
1. An Fc-binding polypeptide comprising a mutant of an Fc-binding domain of Staphylococcus Protein A (SpA) wherein the mutant is selected from the group consisting of SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, and SEQ ID NO:17, and wherein the mutant binds the Fc domain of an antibody and has improved alkaline stability compared to SEQ ID NO:7.
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. The polypeptide of claim 1 , comprising the sequence of SEQ ID NO:9.
4. The polypeptide of claim 1 , comprising the sequence of SEQ ID NO:10.
5. The polypeptide of claim 1 , comprising the sequence of SEQ ID NO:11.
6. The polypeptide of claim 1 , comprising the sequence of SEQ ID NO:12.
7. The polypeptide of claim 1 , comprising the sequence of SEQ ID NO:13.
8. The polypeptide of claim 1 , comprising the sequence of SEQ ID NO:14.
9. The polypeptide of claim 1 , comprising the sequence of SEQ ID NO:15.
10. The polypeptide of claim 1 , comprising the sequence of SEQ ID NO:16.
11. The polypeptide of claim 1 , comprising the sequence of SEQ ID NO:17.
12. The polypeptide according to claim 1 , further comprising at, or within 1-5 amino acid residues from, the C-terminal or N-terminal one or more coupling element, selected from the group consisting of one or more cysteine residues, a plurality of lysine residues and a plurality of histidine residues.
13. A multimer comprising a plurality of polypeptides as defined by claim 1 .
14. The multimer of claim 3 , which is a dimer, trimer, tetramer, pentamer, hexamer, heptamer, octamer or nonamer.
15. A nucleic acid or a vector encoding a polypeptide or multimer according to claim 1 .
16. An expression system, which comprises a nucleic acid or vector of claim 6 .
17. A separation matrix, wherein a plurality of polypeptides or multimers of claim 1 have been coupled to a solid support.
18. The separation matrix of claim 17 , wherein the polypeptides or multimers have been coupled to the solid support via thioether bonds.
19. The separation matrix of claim 17 , wherein the solid support is a polysaccharide.
20. The separation matrix of claim 17 , 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.
21. The separation matrix of claim 17 , 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.
22. The separation matrix of claim 17 , 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.
23. A method of isolating an immunoglobulin, wherein a separation matrix of claim 17 is used.
24. 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 17 ,
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.
25. The method of claim 24 , wherein the cleaning liquid is alkaline, such as with a pH of 13-14.
26. The method of claim 24 , wherein the cleaning liquid comprises 0.1-1.0 M NaOH or KOH.
27. The method of claim 24 , wherein steps a)-d) are repeated at least 10 times.
28. The method of claim 24 , wherein steps a)-c) are repeated at least 10 times, and wherein step d) is performed after a plurality of instances of step c).