Protein ligands
View Patent ↗The present invention relates to the use of an alkali-stable 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 antibodies from proteases, which method comprises
(a) contacting a liquid comprising antibodies and protease(s) with a separation matrix to adsorb antibodies;
(b) separating the liquid comprising protease(s) from the matrix; and optionally,
(c) releasing the antibodies from the matrix by adding an eluent;
the improvement being that the separation matrix comprises alkali-stable protein A-based ligands which provide increased resistance of said alkali-stable protein A-based ligands to protease degradation, wherein each of said alkali-stable protein A-based ligands is represented by SEQ ID NO: 1 or SEQ ID NO: 2, with one or two asparagine residues mutated, and the mutation is N23T; N23T, N43E; N28A; N6A; N11S; N11S, N23T; or N6A, N23T, further wherein said antibodies being separated are selected from the group consisting of IgG, IgA and IgM.
2. The method of claim 1 , wherein the alkali-stable protein A-based ligands comprise at least one B domain of Protein A (SEQ ID NO: 1) in which at least one asparagine residue has been mutated as defined in claim 1 .
3. The method of claim 1 , wherein the alkali-stable protein A-based ligands comprise at least one Protein Z domain of SEQ ID NO: 2 in which at least one asparagine residue has been mutated as defined in claim 1 .
4. The method of claim 1 , wherein the alkali-stable protein A-based ligands are multimers of two or more mutated Protein Z domains of SEQ ID NO: 2, wherein in each domain at least one asparagine residue has been mutated as defined in claim 1 .
5. The method of claim 1 , wherein the antibodies are monoclonal antibodies.