Caustic stable chromatography ligands

Patent metadata

Field	Value
Publication number	US-10072050-B2
Application number	US-65388809-A
Country	US
Kind code	B2
Filing date	Dec 18, 2009
Priority date	Dec 24, 2008
Publication date	Sep 11, 2018
Grant date	Sep 11, 2018

Abstract

The present invention relates to chromatography ligands having improved caustic stability, e.g., ligands based on immunoglobulin-binding proteins such as, Staphylococcal protein A, as well as methods of making and using such ligands.

First claim

What is claimed is:

1. An alkaline-stable chromatography ligand comprising two or more B domains of Staphylococcus protein A (SpA) attached to a chromatography resin at more than one site on the resin, wherein each B domain comprises the amino acid sequence set forth in SEQ ID NO:10 having a mutation to replace the glycine at position 29 with an amino acid other than alanine or tryptophan to reduce Fab binding, as shown in SEQ ID NO:43, wherein the ligand retains at least 95% of its binding capacity after 5 hours incubation in 0.5 M NaOH.

2. The alkaline-stable chromatography ligand of claim 1 , wherein the ligand comprises three or more B domains.

3. The alkaline-stable chromatography ligand of claim 1 , wherein the ligand comprises four or more B domains.

4. The alkaline-stable chromatography ligand of claim 1 , wherein the ligand comprises five or more B domains.

5. The alkaline-stable chromatography ligand of claim 1 , wherein the ligand comprises six or more B domains.

6. The alkaline-stable chromatography ligand of claim 1 , wherein the ligand comprises seven or more B domains.

7. A chromatography matrix comprising a ligand according to claim 1 .

8. The ligand according to claim 1 , wherein the ligand is capable of binding at least 1.5 times, or 2 times, or 3 times, or more of IgG as compared to wtSpA, following exposure to 0.5M NaOH for at least 5 hours.

9. A caustic stable affinity chromatography matrix comprising a ligand comprising three or more, or four or more, or five or more, or six or more, or seven or more B domains of Staphylococcus protein A (SpA) attached to a solid support via multipoint attachment, wherein each B domain comprises the amino acid sequence set forth in SEQ ID NO:10 having a mutation to replace the glycine at position 29 with an amino acid other than alanine or tryptophan to reduce Fab binding, as shown in SEQ ID NO:43, wherein the ligand retains at least 95% of its binding capacity after 5 hours incubation in 0.5 M NaOH.

10. The caustic stable chromatography matrix of claim 9 , wherein the solid support is selected from the group consisting of controlled pore glass, polyvinyl alcohol, zirconium oxide, agarose, polymethacrylate, polyacrylate, polyacrylamide and polystyrene.

11. A caustic stable affinity chromatography ligand comprising three B domains (B3) or four B domains (B4), or five B domains (B5), or six B domains (B6), or seven B domains (B7) of Staphylococcus protein A (SpA) attached to a solid support via multipoint attachment, wherein each B domain comprises the amino acid sequence set forth in SEQ ID NO:10 having a mutation to replace the glycine at position 29 with an amino acid other than alanine or tryptophan to reduce Fab binding, as shown in SEQ ID NO:43, and wherein the extent of caustic stability of ligand is in the order of B3<B4<B5<B6<B7, following exposure of ligand to 0.5M NaOH for at least 5 hours.

Assignees

• Bian Nanying
• Soice Neil
• Spector Shari
• Levine Kara
• Emd Millipore Corp

Inventors

• Bian Nanying
• Soice Neil
• Spector Shari
• Levine Kara

Classifications

• C07K1/22

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

• B01J20/3244

  Non-macromolecular compounds · CPC title

• C07K14/31 Primary

  from Staphylococcus (G) · CPC title

• B01J20/3274

  Proteins, nucleic acids, polysaccharides, antibodies or antigens · CPC title

• B01J20/286

  Phases chemically bonded to a substrate, e.g. to silica or to polymers · CPC title