Chromatography method
The present invention relates to a method for removal of large contaminants, such as virus, in a chromatographic process for purification of a target molecule, preferably monoclonal antibodies, mAbs, by using a specifically designed chromatographic bead having a thin outer layer and a core functionalized with a ligand adsorbing the mAbs or parts thereof.
1. A method of making affinity chromatography beads suitable for purification of immunoglobulin containing proteins or parts thereof, from contaminants that are larger than immunoglobulin containing proteins, the method comprising:
providing allylated chromatography beads having an allylated outer layer and an allylated inner core;
inactivating the allylated outer layer of the allylated chromatography beads to form a lid consisting of agarose modified with hydrolyzed allyl groups and having a thickness of 1-8 μm;
immobilizing one or more affinity ligands to the allylated inner core of the allylated chromatography beads to form the affinity chromatography beads;
wherein the lid and the allylated inner core of the allylated chromatography beads have a defined pore size corresponding to K D of 0.1 to 1.
2. Affinity chromatography beads made by the method of claim 1 .
3. The affinity chromatography beads of claim 2 , having average diameter size of 10-500 μm.
4. The affinity chromatography beads of claim 2 , wherein the thickness of the lid is 3-5 μm.
5. The affinity chromatography beads of claim 2 , wherein the porosity of the lid and core is 0.30 to 0.95.
6. The affinity chromatography beads of claim 2 , wherein the affinity ligand is a proteinaceus affinity ligand.
7. The affinity chromatography beads of claim 2 , wherein the affinity ligand is Protein A or affinity ligand derived from Protein A.
8. The affinity chromatography beads of claim 2 , wherein the affinity ligand is Protein L or an affinity ligand derived from Protein L.
9. The affinity chromatography beads of claim 2 , wherein the affinity ligand is Protein G or an affinity ligand derived from Protein G.
10. The affinity chromatography beads of claim 2 , wherein the affinity chromatography beads are magnetic.
11. The affinity chromatography beads of claim 2 , wherein the affinity ligand is Z4.
12. A method of making affinity chromatography beads suitable for purification of immunoglobulin containing proteins or parts thereof, from contaminants that are larger than immunoglobulin containing proteins, the method comprising:
providing allylated chromatography beads having an allylated outer layer and an allylated inner core;
inactivating the allylated outer layer of the allylated chromatography beads to form a lid consisting of agarose modified with hydrolyzed allyl groups having a thickness of 3-5 μm;
immobilizing one or more affinity ligands to the allylated inner core of the allylated chromatography beads while leaving the lid intact to form the affinity chromatography beads;
wherein the average diameter size of the affinity chromatography beads is 250-450 μm,
wherein the lid and the allylated inner core of the allylated chromatography beads have a defined pore size corresponding to K D of about 0.3 to 0.7,
wherein the porosity of the lid and core is 0.60 to 0.85, and
wherein the lid and core are made of agarose.
13. Affinity chromatography beads made by the method of claim 12 .
14. The affinity chromatography beads of claim 13 , wherein the affinity ligand is a proteinaceus affinity ligand.
15. The affinity chromatography beads of claim 13 , wherein the affinity ligand is Protein A or affinity ligand derived from Protein A.
16. The affinity chromatography beads of claim 13 , wherein the affinity ligand is Protein L or an affinity ligand derived from Protein L.
17. The affinity chromatography beads of claim 13 , wherein the affinity ligand is Protein G or an affinity ligand derived from Protein G.
18. The affinity chromatography beads of claim 13 , wherein the affinity chromatography beads are magnetic.
19. Affinity chromatography beads suitable for purification of immunoglobulin containing proteins or parts thereof, from contaminants that are larger than immunoglobulin containing proteins, manufactured by:
providing allylated chromatography beads having an allylated outer layer and an allylated inner core;
inactivating the allylated outer layer of the allylated chromatography beads to form a lid consisting of agarose modified with hydrolyzed allyl groups and having a thickness of 3-5 μm;
immobilizing one or more Z4 affinity ligands to the allylated inner core of the allylated chromatography beads to form the affinity chromatography beads;
wherein the average diameter size of the affinity chromatography beads is 250-450 μm,
wherein the lid and the allylated inner core of the allylated chromatography beads leg have a defined pore size corresponding to K D of about 0.3 to 0.7,
wherein the porosity of the lid and core is 0.60 to 0.85, and
wherein the lid and core are made of agarose.