Mutated immunoglobulin-binding polypeptides

Patent metadata

Field	Value
Publication number	US-11136359-B2
Application number	US-201815900419-A
Country	US
Kind code	B2
Filing date	Feb 20, 2018
Priority date	Jul 10, 2013
Publication date	Oct 5, 2021
Grant date	Oct 5, 2021

Abstract

The invention discloses a polypeptide with improved alkaline stability, which polypeptide comprises a mutant of a B or C domain of Staphylococcus Protein A (SpA), as specified by SEQ ID NO 1 or SEQ ID NO 2, or of Protein Z, as specified by SEQ ID NO 3, wherein at least the glutamine residue at position 9 has been mutated to an amino acid other than asparagine. The invention also discloses multimers of said polypeptide, as well as separation matrices comprising the multimers or polypeptides.

First claim

What is claimed is:

1. An immunoglobulin-binding polypeptide comprising a mutant B-domain or C-domain of Staphylococcus Protein A (SpA), as specified by SEQ ID NO:4, wherein the glutamine residue at position 9 has been mutated to an alanine residue or a threonine residue, or wherein the glutamine residue at position 9 has been mutated to an alanine residue or a threonine residue with further mutations in at least one of E15 mutated to a lysine residue, D36 mutated to a threonine residue or an alanine residue, and E47 mutated to a threonine residue; and wherein the polypeptide binds the kappa light chain of an antibody or fragment thereof and has improved alkaline stability compared to a non-mutated B-domain or C-domain of SpA.

2. The polypeptide of claim 1 , wherein in the further mutations the original amino acid residue is substituted at E15 with lysine.

3. The polypeptide of claim 1 , wherein in the further mutations the original amino acid residue is substituted at D36 with threonine.

4. The polypeptide of claim 1 , wherein in the further mutations the original amino acid residue is substituted at D36 with alanine.

5. The polypeptide of claim 1 , wherein in the further mutations the original amino acid residue is substituted at E47 with threonine.

6. The polypeptide of claim 1 , wherein the amino acid residue at position 9 is alanine.

7. The polypeptide of claim 1 , wherein the amino acid residue at position 9 is threonine.

8. The polypeptide or multimer according to claim 1 , further comprising at the C-terminal or N-terminal one or more coupling elements, selected from the group consisting of a cysteine residue, a plurality of lysine residues and a plurality of histidine residues.

9. A nucleic acid or a vector encoding a polypeptide or multimer according to claim 1 .

10. A separation matrix, wherein a plurality of polypeptides or multimers according to claim 1 have been coupled to a solid support.

11. A method of isolating an immunoglobulin, wherein a separation matrix according to claim 10 is used.

12. The method of claim 11 , comprising the steps of:
contacting a liquid sample comprising an immunoglobulin with the separation matrix,
washing said separation matrix with a washing liquid,
eluting the immunoglobulin from the separation matrix with an elution liquid, and cleaning the separation matrix with a cleaning liquid.

Assignees

• Cytiva Bioprocess R & D Ab

Inventors

• Rodrigo Gustav
• Ander Mats
• Bjorkman Tomas
• Bauren Goran

Classifications

• B01J20/3212

  consisting of a polymer obtained by reactions otherwise than involving only carbon to carbon unsaturated bonds · CPC title

• B01D15/3809

  of the antigen-antibody type, e.g. protein A, G or L chromatography · CPC title

• C07K14/31 Primary

  from Staphylococcus (G) · CPC title

• C07K1/22

  Affinity chromatography or related techniques based upon selective absorption processes · CPC title

• B01J20/3219

  involving a particular spacer or linking group, e.g. for attaching an active group · CPC title