Chromatography ligand
View Patent ↗The present invention relates to a chromatography ligand defined by the following formula R 1 —R 2 —N(R 3 )—R 4 —R 5 wherein R 1 is a substituted or non-substituted phenyl group; R 2 is a hydrocarbon chain comprising 0-4 carbon atoms; R 3 is a hydrocarbon chain comprising 1-3 carbon atoms; R 4 is a hydrocarbon chain comprising 1-5 carbon atoms; and R 5 is OH or H. The invention also comprises a separation matrix, comprising the described ligands coupled to a porous support, such as particles or a membrane. The ligand and matrix according to the invention is useful for purification of biomolecules or organic compounds, such as proteins, polypeptides, DNA etc. An advantageous use according to the invention is the purification of antibodies.
1. A method of separating one or more antibodies from one or more other compounds in a liquid sample, comprising contacting the liquid sample with a separation matrix to allow the one or more other compounds to be absorbed onto the separation matrix and the one or more antibodies to pass through without absorption to achieve the separation, the separation matrix comprising a chromatography ligand coupled to a support and the ligand is defined by the following formula:
R 1 —R 2 —N(R 3 )—R 4 —R 5
wherein
R 1 is a non-substituted phenyl group;
R 2 is methylene;
R 3 is a straight saturated hydrocarbon chain comprising 1-3 carbon atoms;
R 4 is a straight saturated chain hydrocarbon chain comprising 1-5 carbon atoms; and
R 5 is OH or H,
wherein said chromatography ligand is immobilized to a support via the tertiary amine nitrogen atom through a linker such that the tertiary amine of the ligand is converted to a quaternary amine, and wherein said other compounds comprise host cell proteins, protein A, or antibody aggregates.
2. The method of claim 1 , wherein the separation matrix is provided in a chromatography column, the mobile phase is passed across said column by gravity and/or pumping, and the antibodies are recovered in the flow-through of the column.
3. The method of claim 1 , wherein the liquid sample comprises a supernatant obtained from cell fermentation.
4. The method of claim 1 , wherein the contact with the separation matrix is preceded by a step of mechanical filtration and/or chromatography.
5. The method of claim 1 , wherein the liquid sample comprises a crude feed.
6. The method of claim 1 , wherein the compound(s) are host cell proteins and substantially all said proteins are adsorbed to the separation matrix.
7. The method of claim 1 , wherein R 4 is —CH 2 —CH 2 —.
8. The method of claim 1 , wherein R 3 is —CH 3 .
9. The method of claim 1 , wherein R 4 is —CH 2 —CH 2 —CH 2 —.
10. The method of claim 1 , wherein the ligand comprising N-benzyl-N-methyl ethanol amine.
11. The method of claim 1 , wherein the ligand comprising N,N-dimethyl benzyl amine.
12. The method of claim 1 , wherein the support is porous agarose.
13. The method of claim 1 , wherein the support comprises substantially spherical particles.
14. The method of claim 1 , wherein the support comprises a membranous structure.
15. The method of claim 2 , wherein the chromatography column is disposable.
16. The method of claim 2 , further comprising sterilizing the chromatography column.
17. The method of claim 2 , further comprising allowing the compound(s) to adsorb to the separation matrix before allowing the mobile phase to pass across said column.
18. The method of claim 1 , wherein the separation matrix is provided in a filter, the mobile phase is passed across said filter by gravity and/or pumping, and the antibodies are recovered in the flow-through of the filter.
19. The method of claim 18 , wherein the filter is disposable.
20. The method of claim 18 , further comprising sterilizing the filter.
21. The method of claim 18 , further comprising allowing the compound(s) to adsorb to the separation matrix before allowing the mobile phase to pass across said filter.
22. A method of separating one or more antibodies from one or more other compounds in a liquid sample, comprising contacting the liquid sample with a separation matrix to allow the one or more other compounds to be absorbed onto the separation matrix and the one or more antibodies to pass through without absorption to achieve the separation, the separation matrix comprising a chromatography ligand coupled to a support and the ligand is defined by the following formula:
R 1 —R 2 —N(R 3 )—R 4 —H
wherein
R 1 is a non-substituted phenyl group;
R 2 is a methylene;
R 3 is a straight saturated hydrocarbon chain comprising 1-3 carbon atoms; and
R 4 is a straight saturated hydrocarbon chain comprising 1-5 carbon atoms,
wherein said chromatography ligand is immobilized to a support via the tertiary amine nitrogen atom through a linker such that the tertiary amine of the ligand is converted to a quaternary amine, and wherein said other compounds comprise host cell proteins, protein A, or antibody aggregates.