Method for preparation of a separation matrix
Methods that include providing and reacting a solid support and an alkali-stable ligand derived from an immunoglobulin-binding bacterial protein to form a separation matrix having covalently coupled alkali-stable ligands; and washing with a wash solution comprising at least 10 mM of an alkali metal hydroxide.
1. A method for preparation of a separation matrix, comprising the steps of:
a) providing a solid support and an alkali-stable ligand derived from a bacterial immunoglobulin-binding protein;
b) reacting said alkali-stable ligand with said solid support to form a separation matrix having covalently coupled alkali-stable ligands; and
c) washing said separation matrix having covalently coupled alkali-stable ligands with a wash solution comprising at least 10 mM of an alkali metal hydroxide;
wherein the separation matrix is substantially free from non-covalently coupled ligands derived from said bacterial immunoglobulin-binding protein; and
wherein said alkali-stable ligand derived from a bacterial immunoglobulin-binding protein is capable of withstanding incubation with 0.1 M NaOH for 100×10 min at 22+/−2° C. with less than a 45% reduction in binding capacity towards IgG.
2. The method of claim 1 , wherein said alkali-stable ligand derived from a bacterial immunoglobulin-binding protein is an alkali-stable Protein A ligand.
3. The method of claim 1 , wherein said alkali-stable ligand derived from a bacterial immunoglobulin-binding protein is an alkali-stable Protein L ligand.
4. The method of claim 1 , wherein said alkali-stable ligand derived from a bacterial immunoglobulin-binding protein is an alkali-stable Protein G ligand.
5. The method of claim 1 , wherein said solid support comprises a plurality of support particles.
6. The method of claim 1 , wherein step c) is repeated at least once.
7. The method of claim 1 , wherein step c) is repeated at least 5 times.
8. The method of claim 1 , further comprising, after step c), a step d) of transferring said separation matrix to a storage solution.
9. The method of claim 8 , further comprising, after step d), a step of dispensing said separation matrix into transport containers.
10. The method of claim 1 , wherein step c) is performed within 24 h after step b).
11. The method of claim 1 , wherein in step c) the wash solution comprises 25 mM-1 M of an alkali metal hydroxide.
12. The method of claim 1 , wherein in step c) the wash solution comprises 40 mM-1 M of an alkali metal hydroxide.
13. The method of claim 1 , wherein in step c) the wash solution comprises 40 mM-1 M NaOH or KOH.
14. The method of claim 1 , wherein in step c) the separation matrix is incubated with the wash solution during 2-60 min.
15. The method of claim 14 , wherein, after incubation, the wash solution is removed by filtration.
16. The method of claim 1 , wherein in step c) the temperature is 2-40° C.
17. The method of claim 1 , further comprising, before step b), a step a′) of activating said solid support.
18. The method of claim 17 , wherein step a′) comprises the formation of aldehyde groups on said solid support.
19. The method of claim 17 , wherein step a′) comprises the formation of epoxide groups on said solid support.
20. The method of claim 19 , wherein after step b), said separation matrix comprises residual epoxide groups.
21. The method of claim 15 , wherein the alkali-stable ligand derived from a bacterial immunoglobulin-binding protein comprises lysine residues.
22. The method of claim 19 , wherein the alkali-stable ligand derived from a bacterial immunoglobulin-binding protein comprises a cysteine residue with a thiol capable of reacting with said epoxide groups.
23. The method of claim 22 , wherein said cysteine residue is proximal to a C-terminus or an N-terminus of said alkali-stable ligand derived from a bacterial immunoglobulin-binding protein.
24. The method of claim 1 , wherein said alkali-stable ligand derived from a bacterial immunoglobulin-binding protein is capable of withstanding incubation with 0.5 M NaOH for 100×10 min at 22+/−2° C. with less than 45% reduction in binding capacity towards IgG.
25. The method of claim 1 , wherein said alkali-stable ligand derived from a bacterial immunoglobulin-binding protein comprises at least one alkali-stable Protein A domain.
26. The method of claim 1 , wherein said alkali-stable ligand derived from a bacterial immunoglobulin-binding protein comprises a multimer of alkali-stable Protein A domains.
27. The method of claim 26 , wherein said multimer comprises at least four alkali-stable Protein A domains.
28. The method of claim 26 , wherein said multimer comprises at least six alkali-stable Protein A domains.
29. The method of claim 1 , wherein said alkali-stable ligand derived from a bacterial immunoglobulin-binding protein comprises at least one alkali-stable Protein L domain.
30. The method of claim 1 , wherein said alkali-stable ligand derived from a bacterial immunoglobulin-binding protein comprises a multimer of alkali-stable Protein L domains.
31. The method of claim 1 , wherein said alkali-stable ligand derived from a bacterial immunoglobulin-binding protein comprises at least one alkali-stable Protein G domain.
32. The method of claim 1 , wherein said alkali-stable ligand derived from a bacterial immunoglobulin-binding protein comprises a multimer of alkali-stable Protein G domains.
33. The method of claim 1 , wherein said solid support comprises a crosslinked polysaccharide.
34. The method of claim 1 , wherein said solid support comprises crosslinked agar or agarose.
35. The method of claim 1 , wherein said separation matrix comprises at least 11 mg covalently bound ligand per mL matrix.
36. The method of claim 1 , wherein after step c), said separation matrix produces a ligand leakage of less than 40 ng ligand per mg IgG, as measured by an ELISA assay in an IgG eluate pool.
37. The method of claim 1 , which is a method of removing non-covalently bound Protein A ligand from the separation matrix.
38. The method of claim 37 , which is a method of removing non-covalently bound Protein A ligand from the separation matrix during preparation of the separation matrix.
39. The method of claim 1 , which is a method of preparing a separation matrix comprising covalently coupled alkali-stable Protein A ligands and being substantially free from non-covalently coupled Protein A ligands.
40. The method of claim 1 , which is a method of preparing a separation matrix comprising covalently coupled alkali-stable Protein L ligands and being substantially free from non-covalently coupled Protein L ligands.
41. The method of claim 1 , which is a method of preparing a separation matrix comprising covalently coupled alkali-stable Protein G ligands and being substantially free from non-covalently coupled Protein G ligands.
42. The method of claim 1 , wherein said alkali metal hydroxide comprises NaOH, KOH, or any mixture thereof.
43. The method of claim 1 , wherein said solid support comprises a plurality of porous support particles.
44. The method of claim 1 , wherein in step c) the wash solution comprises 50-200 mM of an alkali metal hydroxide.
45. The method of claim 1 , wherein in step c) the wash solution comprises 50-200 mM NaOH or KOH.
46. The method of claim 1 , wherein in step c) the separation matrix is incubated with the wash solution during 5-30 min.
47. The method of claim 1 , wherein in step c) the temperature is 20-25° C.
48. The method of claim 1 , wherein said separation matrix comprises at least 15 mg covalently bound ligand per mL matrix.