Immobilisation of chelating groups for immobilised metal ion chromatography (IMAC)

Patent metadata

Field	Value
Publication number	US-10538552-B2
Application number	US-201715491048-A
Country	US
Kind code	B2
Filing date	Apr 19, 2017
Priority date	Aug 6, 2007
Publication date	Jan 21, 2020
Grant date	Jan 21, 2020

Abstract

The present invention refers to a method for binding a polycarboxylic acid to a solid phase. Further, the invention refers to a solid phase having a polycarboxylic acid immobilized thereto and methods of using the solid phase, e.g. for purifying His-tagged recombinant polypeptides.

First claim

The invention claimed is:

1. A method for immobilized metal ion chromatography (IMAC), comprising contacting a polypeptide sample with an immobilized chelator having 6 or more coordination groups, wherein the immobilized chelator is a solid phase having a polycarboxylic acid immobilized thereto, wherein said immobilized chelator has a structure of formula (la) or (Ib):

wherein SP is a solid phase;
R 1 is hydrogen or an organic residue, and
PCA is the residue of a polycarboxylic acid,
and wherein the immobilized chelator is complexed with a Ni 2+ ion.

2. The method according to claim 1 , wherein said coordination groups are selected from the group consisting of amino, carboxyl, carboxamide and hydroxamate groups.

3. The method according to claim 1 , wherein said PCA is the residue of an amino polycarboxylic acid or a salt thereof.

4. The method according to claim 3 , wherein the polycarboxylic acid is selected from the group consisting of ethylene diamino tetraacetic acid (EDTA), ethylene glycol-bis(2-aminoethy lether)-N, N, N′, N′-tetraacetic acid (EGTA), diethylene triamino pentaacetic acid (DPTA), triethylene tetra mine-N, N, N′, N′, N″, N″-hexa-acetic acid (TTHA), and salts thereof.

5. The method according to claim 4 , wherein said polycarboxylic acid is ethylene diamino tetraacetic acid (EDTA).

6. The method of claim 1 , wherein the solid phase has a structure of formula (II):

wherein SP is the solid phase, and
wherein one or more of the carboxylic acid groups may be deprotonated.

7. A method for immobilized metal ion chromatography (IMAC), comprising contacting a sample with an immobilized chelator comprising a solid phase having a polycarboxylic acid or salt thereof immobilized thereto, wherein the immobilized chelator has a structure of formula (la) or (Ib):

wherein SP is the solid phase;
R 1 is hydrogen or an organic residue, and
PCA is the polycarboxylic acid or a salt thereof,
wherein said polycarboxylic acid is complexed with a transition metal ion,
wherein the immobilized polycarboxylic acid amide has at least 6 or more coordination groups, which are selected from amino, carboxyl, carboxamide and hydroxamate groups,
and wherein ≥90% of the accessible primary amino groups on the surface of the solid phase are blocked.

8. The method according to claim 7 , wherein the polycarboxylic acid is selected from the group consisting of ethylene diamino tetraacetic acid (EDTA), ethylene glycol-bis(2-aminoethylether)-N,N,N′,N′-tetraacetic acid (EGTA), diethylene triamino pentaacetic acid (DPTA), triethylene tetramine-N, N, N′, N′, N″, N″-hexa-acetic acid (TTHA), and salts thereof.

9. The method according to claim 8 , wherein said polycarboxylic acid is ethylene diamino tetraacetic acid (EDTA).

10. The method of claim 7 , wherein the solid phase has a structure of formula (II):

wherein SP is the solid phase, and
wherein one or more of the carboxylic acid groups may be deprotonated.

11. The method according to claim 7 , wherein substantially all loosely bound metal ions have been removed.

12. The method according to claim 7 , wherein said transition metal is a Ni 2+ ion.

Assignees

• Max Planck Gesellschaft

Inventors

• Goerlich Dirk
• Frey Steffen

Classifications

• B01J20/22

  comprising organic material · CPC title

• B01J2220/52

  Sorbents specially adapted for preparative chromatography · CPC title

• C07K1/22 Primary

  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

• C07K1/13

  Labelling of peptides · CPC title