CAUSTIC STABLE CHROMATOGRAPHY LIGANDS

• US 20180362595A1
• Filed: 08/27/2018
• Published: 12/20/2018
• Est. Priority Date: 12/24/2008
• Status: Active Grant

• Current Assignee: EMD Millipore Corporation (Merck & Co., Inc.)
• Original Assignee: EMD Millipore Corporation (Merck & Co., Inc.)
• Inventors: Bian, Nanying, Soice, Neil, Spector, Shari, Levine, Kara
• Granted Patent: US 11,084,851 B2
• Time in Patent Office: Days
• CPC Class Codes: B01D 15/3809 of the antigen-antibody type, e.g. protein A, G, L chromatography
• CPC Class Codes: B01J 20/286 Phases chemically bonded to a substrate, e.g. to silica or to polymers
• CPC Class Codes: B01J 20/3244 Non-macromolecular compounds
• CPC Class Codes: B01J 20/3274 Proteins, nucleic acids, polysaccharides, antibodies or antigens
• CPC Class Codes: C07K 14/31 from Staphylococcus (G)

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.

Claims

1. An alkaline-stable chromatography ligand comprising two-or more C domains of Staphylococcus protein A (SpA) attached to a chromatography resin at more than one site on the resin, wherein each C domain comprises the amino acid sequence set forth in SEQ ID NO:

11 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;

44, wherein the ligand retains at least 95% of its binding capacity after 5 hours incubation in 0.5 M NaOH.

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 wt SpA, following exposure to 0.5M NaOH for at least 5 hours.

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

3. The alkaline-stable chromatography ligand of claim 2, wherein the ligand comprises four or more C domains.

4. The alkaline-stable chromatography ligand of claim 2, wherein the ligand comprises five or more C domains.

5. 5The alkaline-stable chromatography ligand of claim 2, wherein the ligand comprises six or more C domains.

6. The alkaline-stable chromatography ligand of claim 2, wherein the ligand comprises seven or more C domains.

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 C domains of Staphylococcus protein A (SpA) attached to a solid support via multipoint attachment, wherein each C domain comprises the amino acid sequence set forth in SEQ ID NO:

11 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;

44, 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 C domains (C3) or four C domains (C4), or five C domains (C5), or six C domains (C6), or seven C domains (C7) of Staphylococcus protein A (SpA) attached to a solid support via multipoint attachment, wherein each C domain comprises the amino acid sequence set forth in SEQ ID NO:

11 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;

44, and wherein the extent of caustic stability of ligand is in the order of C3<

C4<

C5<

C6<

C7, following exposure of ligand to 0.5M NaOH for at least 5 hours.