Chromatography ligand comprising domain C from
The present invention relates to a chromatography ligand, which comprises Domain C from Staphylococcus protein A (SpA), or a functional fragment or variant thereof. The chromatography ligand presents an advantageous capability of withstanding harsh cleaning in place (CIP) conditions, and is capable of binding Fab fragments of antibodies. The ligand may be provided with a terminal coupling group, such as arginine or cysteine, to facilitate its coupling to an insoluble carrier such as beads or a membrane. The invention also relates to a process of using the ligand in isolation of antibodies, and to a purification protocol which may include washing steps and/or regeneration with alkali.
1. A process for isolating one or more target compound(s), the process comprising:
(a) contacting a first liquid with a chromatography matrix, the first liquid comprising the target compound(s) and the chromatography matrix comprising:
(i) a solid support; and
(ii) at least one ligand coupled to the solid support, the ligand capable of binding the one or more target compound(s) and comprising at least two polypeptides, wherein the amino acid sequence of each polypeptide comprises at least 52 contiguous amino acids of a modified SEQ ID NO. 1, and wherein the modified SEQ ID NO. 1 has an alanine (A) instead of glycine (G) at a position corresponding to position 29 of SEQ ID NO. 1; and
(b) adsorbing the target compound(s) to the ligand;
(c) eluting the compound(s) by passing a second liquid through the chromatography matrix that releases the compound(s) from the ligand; and,
(d) performing a cleaning in place (CIP) process involving exposing the chromatography matrix to a CIP solution with a NaOH concentration of at least 0.1 M.
2. The process of claim 1 , wherein the amino acid sequence of each polypeptide comprises at least 55 contiguous amino acids of a modified SEQ ID NO. 1.
3. The process of claim 1 , wherein the CIP process involves exposing the chromatography matrix to a CIP solution with a NaOH concentration of at least 0.5 M.
4. The process of claim 1 , wherein the process for isolating one or more target compound(s) is repeated multiple times such that the total contact time between the CIP solution and the chromatography matrix is at least 5 hours, and wherein the chromatography matrix retains at least 80% of its original binding capacity after the repetitions.
5. The process of claim 3 , wherein the process for isolating one or more target compound(s) is repeated multiple times such that the total contact time between the CIP solution and the chromatography matrix is at least 5 hours, and wherein the chromatography matrix retains at least 80% of its original binding capacity after the repetitions.
6. The process of claim 1 , wherein the chromatography matrix is capable of retaining at least 95% of its original binding capacity after 5 hours incubation in 0.5 M NaOH.
7. The process of claim 1 , wherein the chromatography matrix is capable of retaining at least 90% of its original binding capacity after 10 hours incubation in 0.5 M NaOH.
8. The process of claim 1 , wherein the chromatography matrix is capable of retaining at least 80% of its original binding capacity after 20 hours incubation in 0.5 M NaOH.
9. The process of claim 1 , wherein the target compound is an antibody.
10. The process of claim 1 , wherein the ligand comprises 2-8 of the polypeptides, optionally coupled via linker segments.
11. The process of claim 1 , wherein the ligand binds to the Fab part of an antibody.
12. The process of claim 1 , wherein the ligand comprises a terminal coupling group comprising at least one nitrogen and/or sulfur atom(s).
13. The process of claim 12 , wherein the terminal group comprises arginine or cysteine.
14. The process of claim 1 , wherein the ligand is coupled to the solid support via thioether bonds.
15. The process of claim 1 , wherein the ligand further comprises one or more other alkaline-stable protein-based units.
16. The process of claim 1 , wherein the solid support is selected from a polysaccharide, a crosslinked synthetic polymer or an inorganic polymer.
17. The process of claim 1 , wherein the solid support is comprised of substantially spherical particles.
18. The process of claim 1 , wherein the solid support is porous.
19. The process of claim 1 , wherein the solid support is a comprised of substantially spherical particles with a particle size in the range of 20-80 μm and made of a crosslinked synthetic polymer.
20. A process for isolating one or more target compound(s), the process comprising:
(a) contacting a first liquid with a chromatography matrix, the first liquid comprising the target compound(s) and the chromatography matrix comprising:
(i) a solid support; and
(ii) at least one ligand coupled to the solid support, the ligand capable of binding the one or more target compound(s) and comprising at least two polypeptides, wherein the amino acid sequence of each polypeptide comprises at least 55 amino acids in alignment with SEQ ID NO. 1, and wherein each polypeptide has an alanine (A) instead of glycine (G) at a position corresponding to position 29 of SEQ ID NO. 1;
(b) adsorbing the target compound(s) to the ligand; and,
(d) performing a clean in place (CIP) process involving exposing the chromatography matrix to a CIP solution with a NaOH concentration of at least 0.1 M.
21. The process of claim 20 , wherein the CIP process involves exposing the chromatography matrix to a CIP solution with a NaOH concentration of at least 0.5 M.
22. The process of claim 20 , wherein the process for isolating one or more target compound(s) is repeated multiple times such that the total contact time between the CIP solution and the chromatography matrix is at least 5 hours, and wherein the chromatography matrix retains at least 80% of its original binding capacity after the repetitions.
23. The process of claim 21 , wherein the process for isolating one or more target compound(s) is repeated multiple times such that the total contact time between the CIP solution and the chromatography matrix is at least 5 hours, and wherein the chromatography matrix retains at least 80% of its original binding capacity after the repetitions.
24. The process of claim 20 , wherein the chromatography matrix is capable of retaining at least 95% of its original binding capacity after 5 hours incubation in 0.5 M NaOH.
25. The process of claim 20 , wherein the chromatography matrix is capable of retaining at least 90% of its original binding capacity after 10 hours incubation in 0.5 M NaOH.
26. The process of claim 20 , wherein the chromatography matrix is capable of retaining at least 80% of its original binding capacity after 20 hours incubation in 0.5 M NaOH.
27. The process of claim 20 , wherein the target compound is an antibody.
28. The process of claim 20 , wherein the ligand comprises 2-8 of the polypeptides, optionally coupled via linker segments.
29. The process of claim 20 , wherein the ligand binds to the Fab part of an antibody.
30. The process of claim 20 , wherein the ligand comprises a terminal coupling group comprising at least one nitrogen and/or sulfur atom(s).
31. The process of claim 30 , wherein the terminal group comprises arginine or cysteine.
32. The process of claim 20 , wherein the ligand is coupled to the solid support via thioether bonds.
33. The process of claim 20 , wherein the ligand further comprises one or more other alkaline-stable protein-based units.
34. The process of claim 20 , wherein the solid support selected from the group of a polysaccharide, a crosslinked synthetic polymer and an inorganic polymer.
35. The process of claim 20 , wherein the solid support is comprised of substantially spherical particles.
36. The process of claim 20 , wherein the solid support is porous.
37. The process of claim 20 , wherein the solid support is a comprised of substantially spherical particles with a particle size in the range of 20-80 μm and made of a crosslinked synthetic polymer.