Purification of immunoglobulins
View Patent ↗The present invention relates to a separation matrix comprised of a porous or non-porous support to which ligands have been immobilized, wherein said ligands comprise at least one aliphatic sulfamide. The invention also relates to a chromatography column that contains the described separation matrix, as well as to a method of isolating immunoglobulins, such as IgG, Fab fragments, fusion proteins containing immunoglobulins etc, by adsorption to a separation matrix that comprises the aliphatic sulfamide ligands of the invention.
1. A separation matrix comprised of a support to which at least one ligand has been covalently immobilized, optionally via a spacer arm, wherein said ligand comprises one aliphatic sulfamide group described by the following formula:
—N(R 1 )—S(O) 2 —N(R 2 )(R 3 ) (I)
wherein R 1 , R 2 and R 3 are, independently from each other, either hydrogen or linear, optionally branched carbon chains, and wherein the content of aliphatic sulfamide groups is 113-337 micromol/ml.
2. The matrix of claim 1 , wherein the support is porous.
3. The matrix of claim 1 , wherein R 1 is hydrogen.
4. The matrix of claim 1 , wherein R 2 and/or R 3 are C1-C3 carbon chains.
5. The matrix of claim 1 , wherein R 1 ═H and R 2 ═R 3 ═CH 3 having the following structure
6. The matrix of claim 1 , wherein the sulfamide groups are present as repetitive units of a polymer covalently immobilized to the support.
7. The matrix of claim 1 , which comprises sulfamide ligands coupled to a support via spacer arms, which optionally comprises one or more nitrogen atoms.
8. The matrix of claim 1 , further comprising ion exchange ligands or hydrophobic interaction chromatography (HIC) ligands.
9. The matrix of claim 1 , wherein the support is a cross-linked polysaccharide.
10. The separation matrix of claim 1 in a solution phase.
11. A chromatography column packed with the separation matrix of claim 1 .
12. The chromatography column of claim 11 , which is substantially sterile.
13. The chromatography column of claim 11 , which is a disposable product.
14. A process of preparing the separation matrix of claim 1 , said method comprises a first step of covalently immobilizing amines to a support and a subsequent step of sulfamoylating said amines to provide aliphatic sulphamide ligands.
15. The process of claim 14 , wherein the amines and/or polyamines are covalently immobilized to the support via spacer arms.
16. A method of isolating at least one immunoglobulin from a liquid, which method comprises the steps of:
(a) providing a liquid that comprises at least one immunoglobulin:
(b) contacting said liquid with aliphatic sulfamide ligands to adsorb one or more immunoglobulins to the matrix of claim 1 containing said aliphatic sulfamide ligands; and, optionally,
(c) contacting said matrix with an eluent to release one or more immunoglobulins; and
(d) recovering at least one immunoglobulin.
17. The method of claim 16 , wherein the liquid provided in step (a) also comprises one or more other proteins.
18. The method of claim 16 , wherein the separation matrix of step (b) is provided in a chromatography column.
19. The method of claim 16 , wherein step (b) is performed at a neutral or close to neutral pH.
20. The method of claim 16 , wherein step (c) is a gradient elution performed by passing an eluent of decreasing pH across the matrix.
21. The method of claim 16 , wherein the immunoglobulins recovered in step (d) are human or humanised immunoglobulins.
22. The method of claim 21 , wherein the immunoglobulins recovered in step (d) are immunoglobulin G (IgG).
23. A method of determining the quantity of an immunoglobulin, which method encompass the method of claim 16 and in addition a step (e) of determining the amount of immunoglobulin spectrophotometrically.