Mutated immunoglobulin-binding polypeptides

Abstract

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 - 49 . (canceled) 
     
     
         50 . A separation matrix comprising:
 an Fc-binding polypeptide, wherein the polypeptide comprises:
 a) a mutant of an Fc-binding domain of  Staphylococcus  Protein A (SpA) having the amino acid sequence of SEQ ID NO: 52, wherein the amino acid residue at the position corresponding to position 5 in SEQ ID NO: 52 is alanine, or a variant of the mutant having at least 90% identity thereto; and 
 b) a linker comprising 5-10 amino acids coupled to the mutant, 
 wherein linker is free of proline, asparagine, and glutamine; and 
   a solid support comprising crosslinked agarose, wherein the Fc-binding polypeptide is coupled to the solid support.   
     
     
         51 . The separation matrix of  claim 50 , wherein the IgG capacity of the matrix after 24 h incubation in 0.5 M NaOH at 22+/−2° C. is at least 95% of the IgG capacity before the incubation. 
     
     
         52 . The separation matrix of  claim 50 , wherein the polypeptide coupling to the solid support comprises a bridge coupling between at least one coupling amino acid on the polypeptide and an electrophilic group on the support. 
     
     
         53 . The separation matrix of  claim 52 , wherein the bridge coupling comprises a thioether bond. 
     
     
         54 . The separation matrix of  claim 52 , wherein the coupling amino acid is selected from one or more of cysteine, lysine, or histidine. 
     
     
         55 . The separation matrix of  claim 52 , wherein the coupling is multipoint coupling through two lysines. 
     
     
         56 . The separation matrix of  claim 50 , wherein a plurality of Fc binding polypeptides have been coupled to the crosslinked agarose. 
     
     
         57 . A separation matrix comprising:
 a multimer comprising a plurality of Fc-binding polypeptides, wherein each polypeptide comprises:
 a) a mutant of an Fc-binding domain of  Staphylococcus  Protein A (SpA) having the amino acid sequence of SEQ ID NO: 52, wherein the amino acid residue at the position corresponding to position 5 in SEQ ID NO: 52 is alanine, or a variant of the mutant having at least 90% identity thereto; and 
 b) a linker comprising 5-10 amino acids coupled to the mutant, 
 wherein linker is free of proline, asparagine, and glutamine; and 
   a solid support comprising crosslinked agarose, wherein the multimer is coupled to the solid support.   
     
     
         58 . The separation matrix of  claim 57 , which is a dimer, trimer, tetramer, pentamer, hexamer, heptamer, octamer, or nonamer. 
     
     
         59 . The separation matrix of  claim 57 , which is a pentamer. 
     
     
         60 . The separation matrix of  claim 57 , wherein the IgG capacity of the matrix after 24 h incubation in 0.5 M NaOH at 22+/−2° C. is at least 95% of the IgG capacity before the incubation. 
     
     
         61 . The separation matrix of  claim 57 , wherein the multimer coupling to the solid support comprises a bridge coupling between at least one coupling amino acid on a polypeptide of the multimer and an electrophilic group on the support. 
     
     
         62 . The separation matrix of  claim 61 , wherein the bridge coupling comprises a thioether bond. 
     
     
         63 . The separation matrix of  claim 61 , wherein the coupling amino acid is selected from one or more of cysteine, lysine, or histidine. 
     
     
         64 . The separation matrix of  claim 63 , wherein the coupling is multipoint coupling through two lysines. 
     
     
         65 . The separation matrix of  claim 57 , wherein a plurality of multimers have been coupled to the crosslinked agarose.