Mutant protein
View Patent ↗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 separating one or more antibodies from a liquid, which method comprises:
(a) contacting said 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ligands comprise one or more alkali-stable domain B of staphylococcal Protein A (SpA) wherein at least one glycine has been replaced by an alanine and at least the asparagine in position 23 has been replaced with a threonine.
2. The method of claim 1 , wherein the recovery of antibodies is achieved by adding an eluent having a pH in the range of 3.8-3.9.
3. The method of claim 1 , wherein at least 80% of the antibodies are recovered using an eluent having a pH in the range of 3.7-3.9.
4. The method of claim 1 , wherein at least 90% of the antibodies are recovered using an eluent having a pH in the range of 3.7-3.9.
5. The method of claim 1 , wherein at least 95% of the antibodies are recovered using an eluent having a pH in the range of 3.7-3.9.
6. The method of claim 1 , wherein the amino acid residues in position 21 and 52 are both asparagines.
7. The method of claim 3 , wherein the amino acid residues in position 21 and 52 are both asparagines.
8. The method of claim 1 , wherein the glycine at position 29 has been replaced with an alanine.