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 chromatography ligand, which ligand comprises one or more Domain C units from  Staphylococcus  protein A (SpA), wherein the Domain C sequence has a sequence that is at least 94% identical to the amino acid sequence shown in SEQ ID NO:2 with mutation G25A. 
     
     
         51 . The chromatography ligand according to  claim 50 , wherein after 15 hours incubation in 0.5 M NaOH the ligand retains at least 80% of its binding capacity compared to the untreated ligand. 
     
     
         52 . The chromatography ligand according to  claim 50 , wherein after 15 hours incubation in 0.5 M NaOH the ligand retains at least 90% of its binding capacity compared to the untreated ligand. 
     
     
         53 . The chromatography ligand according to  claim 51 , wherein after 15 hours incubation in 0.5 M NaOH the ligand retains at least 90% of its binding capacity compared to the untreated ligand. 
     
     
         54 . The chromatography ligand according to  claim 50 , wherein at least one asparagine in the sequence of the ligand is replaced with an amino acid other than glutamine or aspartic acid. 
     
     
         55 . The chromatography ligand according to  claim 51 , wherein at least one asparagine in the sequence of the ligand is replaced with an amino acid other than glutamine or aspartic acid. 
     
     
         56 . The chromatography ligand according to  claim 52 , wherein at least one asparagine in the sequence of the ligand is replaced with an amino acid other than glutamine or aspartic acid. 
     
     
         57 . The chromatography ligand according to  claim 50 , wherein the ligand is multimeric and comprises at least two Domain C mutants. 
     
     
         58 . The chromatography ligand according to  claim 51 , wherein the ligand is multimeric and comprises at least two Domain C mutants. 
     
     
         59 . The chromatography ligand according to  claim 50 , wherein the multimeric ligand comprises 2-8 Domain C units, optionally coupled via linker segments. 
     
     
         60 . The chromatography ligand according to  claim 51 , wherein the multimeric ligand comprises 2-8 Domain C units, optionally coupled via linker segments. 
     
     
         61 . The chromatography ligand according to  claim 50 , wherein the ligand comprises a terminal coupling group comprising at least one nitrogen and/or sulphur atom(s). 
     
     
         62 . The chromatography ligand according to  claim 51 , wherein the ligand comprises a terminal coupling group comprising at least one nitrogen and/or sulphur atom(s). 
     
     
         63 . The chromatography ligand according to  claim 61 , wherein the terminal group comprises arginine or cysteine. 
     
     
         64 . The chromatography ligand according to  claim 62 , wherein the terminal group comprises arginine or cysteine. 
     
     
         65 . A separation matrix comprising the ligand of  claim 50 . 
     
     
         66 . A separation matrix comprising the ligand of  claim 51 .