Affinity chromatography matrix
View Patent ↗The present invention relates to a method of separating one or more immunoglobulin containing proteins from a liquid. The method includes first contacting the liquid with a separation matrix comprising ligands immobilised to a support; allowing the immunoglobulin containing proteins to adsorb to the matrix by interaction with the ligands; followed by an optional step of washing the adsorbed immunoglobulin containing proteins; and recovering said immunoglobulin containing proteins by contacting the matrix with an eluent which releases the proteins. The method improves upon previous separation methods in that each of the ligands comprises one or more of a protein A domain (E, D, A, B, C), or protein Z, or a functional variant thereof, with at least one of the monomers having a substitution of the C-terminal most proline residue after the third alpha-helix.
1. A ligand for separation of immunoglobulin containing proteins, which ligand comprises one or more monomeric domains of staphylococcal protein A (SpA) (E, D, A, B, C) or protein Z, wherein the proline nearest the C terminus in at least one of the domains has been substituted with any other amino acid.
2. The ligand of claim 1 , wherein said one or more domains of protein A or protein Z are two or more copies from the same domain of SpA (E, D, A, B, C) or protein Z respectively.
3. The ligand of claim 1 , wherein the proline nearest the C terminus in at least one of the monomers in a multimeric ligand has been substituted with another amino acid.
4. The ligand of claim 1 , wherein the proline nearest the C terminus in all the monomers in a multimeric ligand have been substituted with another amino acid.
5. The ligand of claim 1 , wherein the proline nearest the C terminus in at least one monomer is substituted with an amino acid selected from Y, W, F, M, I, V, T.
6. The ligand of claim 1 , wherein the proline nearest the C terminus in at least one monomer is substituted with isoleucine.
7. The ligand of claim 1 , wherein the proline nearest the C terminus in at least one monomer is substituted with an amino acid selected from E, A, L, H, M, Q, K.
8. The ligand of claim 1 , wherein the ligand achieves alkali stability by mutating at least one asparagine residue to an amino acid other than glutamine.
9. The ligand of claim 1 , wherein the ligand has affinity for the Fc part of an immunoglobulin but lacks affinity for the Fab part of an immunoglobulin.
10. The ligand of claim 1 , wherein at least one glycine of said ligands has been replaced by an alanine.
11. The ligand of claim 1 , wherein the ligand 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.
12. The ligand of claim 11 , wherein the alkali-stability of protein Z has been achieved by mutating at least the asparagine residue at position 23 to an amino acid other than glutamine.
13. The ligand of claim 1 , wherein said multimer is a dimer or a tetramer.