Mutant protein

Abstract

The present invention relates to an immunoglobulin-binding protein, wherein at least one asparagine residue has been mutated to an amino acid other than glutamine or aspartic acid, which mutation confers an increased chemical stability at pH-values of up to about 13-14 compared to the parental molecule. The protein can for example be derived from a protein capable of binding to other regions of the immunoglobulin molecule than the complementarity determining regions (CDR), such as protein A, and preferably the B-domain of Staphylococcal protein A. The invention also relates to a matrix for affinity separation, which comprises an immunoglobulin-binding protein as ligand coupled to a solid support, in which protein ligand at least one asparagine residue has been mutated to an amino acid other than glutamine.

1 . In a method of preparing a separation matrix comprising at least one ligand with affinity for the Fc part of an antibody, which method comprises 
 (a) providing a nucleic acid sequence encoding at least one alkali-stable domain B of staphylococcal Protein A (SpA);    (b) mutating said nucleic acid sequence to encode for a recombinant protein wherein at least one glycine has been replaced by an alanine;    (c) expressing the protein encoded by the nucleic acid sequence resulting from (b) in a host cell; and    (d) coupling the expressed protein to a support, the improvement being that the ligand(s) so prepared lack affinity for the Fab part of an antibody.    
     
     
         2 . The method of  claim 1 , wherein in the alkali-stable domain B, the alkali-stability has been achieved by mutating at least one asparagine residue to an amino acid other than glutamine.  
     
     
         3 . The method of  claim 1 , wherein in the alkali-stable domain B, the alkali-stability has been achieved by mutating at least one asparagine residue to an amino acid other than glutamine; and wherein glycine at position 29 of the alkali-stable domain B has been replaced by an alanine.  
     
     
         4 . The method of  claim 1 , wherein the recombinant protein expressed in (c) is Protein Z in which the alkali-stability has been achieved by mutating at least one asparagine residue to an amino acid other than glutamine.  
     
     
         5 . The method of  claim 1 , wherein the recombinant protein expressed in (c) is Protein Z in which the alkali-stability has been achieved by mutating at least the asparagine residue at position 23 to an amino acid other than glutamine.  
     
     
         6 . The method of  claim 1 , wherein the nucleic acid provided encodes a multimer of two or more domains wherein at least one is Protein Z in which the alkali-stability has been achieved by mutating at least one asparagine residue to an amino acid other than glutamine.  
     
     
         7 . In a method of separating one or more antibodies from a liquid, which method comprises 
 (a) contacting liquid with a separation matrix comprising ligands immobilised to a support;    (b) allowing antibodies to adsorb to said matrix by interaction with said ligands;    (c) optionally washing the adsorbed antibodies; and    (d) recovering antibodies by contacting said matrix with an eluent which releases the antibodies;    the improvement being that said ligands comprise one or more alkali-stable domain B of staphylococcal Protein A (SpA) wherein at least one glycine has been replaced by an alanine.    
     
     
         8 . The method of  claim 7 , wherein the recovery of antibodies is achieved by adding an eluent having a pH in the range of 3.8-3.9.  
     
     
         9 . The method of  claim 7 , wherein at least 80% of the antibodies are recovered using an eluent having a pH in the range of 3.7-3.9.  
     
     
         10 . The method of  claim 7 , wherein at least 90% of the antibodies are recovered using an eluent having a pH in the range of 3.7-3.9.  
     
     
         11 . The method of  claim 7 , wherein at least 95% of the antibodies are recovered using an eluent having a pH in the range of 3.7-3.9.