Separation method and separation matrix
View Patent ↗The invention discloses a method of separating a biomolecule from at least one other component in a liquid, comprising a step of contacting said liquid with a separation matrix comprising a solid support and polymer chains bound to said solid support. The polymer chains comprise units derived from a first monomer of structure CH 2 ═CH-L-X, where L is a covalent bond or an alkyl ether or hydroxysubstituted alkyl ether chain comprising 2-6 carbon atoms, and X is a sulfonate or phosphonate group.
1. A method of separating a biomolecule from at least one other component in a liquid, comprising a step of contacting said liquid with a separation matrix comprising a solid support and polymer chains bound to said solid support, wherein said polymer chains comprise units derived from a first monomer of structure CH 2 ═CH-L-X, where L is a covalent bond or an alkyl ether or hydroxyl substituted alkyl ether chain comprising 2-6 carbon atoms, and X is a sulfonate or phosphonate group.
2. The method of claim 1 , wherein said biomolecule is a protein, a peptide or a nucleic acid.
3. The method of claim 1 , wherein said biomolecule is an immunoglobulin, immunoglobulin fragment or an immunoglobulin-containing protein.
4. The method of claim 1 , wherein said at least other component is a protein.
5. The method of claim 1 , wherein the liquid is an eluate from a previous chromatography step.
6. The method of claim 1 , wherein the liquid is the flow-through from a separation matrix.
7. The method of claim 1 , wherein said biomolecule is an immunoglobulin, immunoglobulin fragment or an immunoglobulin-containing protein wherein at least 1%, of said biomolecule is in the form of aggregates.
8. The method of claim 1 , further comprising a step of eluting said biomolecule from said separation matrix with an elution buffer.
9. The method of claim 1 , further comprising a step of cleaning said separation matrix with a cleaning liquid.
10. The method of claim 1 , wherein said polymer chains are copolymer chains and further comprise units derived from a second non-charged monomer.
11. The method of claim 10 , wherein the second non-charged monomer is an N-vinylamide.
12. The method of claim 10 , wherein said second non-charged monomer is selected from the group consisting of N-vinyl pyrrolidone, N-vinyl caprolactam, N-vinyl formamide and N-vinyl acetamide.
13. The method of claim 1 , wherein L is a covalent bond or —CH 2 —O-L′-, where L′ is a C 2 -C 4 or C 3 -C 4 alkylene chain, optionally substituted with at least one hydroxyl group.
14. The method of claim 1 , wherein said first monomer is selected from the group consisting of vinyl sulfonate, vinyl phosphonate and allyloxyhydroxypropyl sulfonate.
15. The method of claim 10 , wherein in said copolymer chains the molar ratio of the units derived from the first monomer to the units derived from the second monomer is 0.05 to 5.
16. A separation matrix comprising a solid support and copolymer chains bound to said solid support, wherein said copolymer chains comprise units derived from
a) a first monomer of structure CH 2 ═CH-L-X, where L is a covalent bond or an alkyl ether or hydroxysubstituted alkyl ether chain comprising 2-6 carbon atoms, and X is a sulfonate or phosphonate group and
b) a second non-charged monomer.
17. The separation matrix of claim 16 , wherein L is a covalent bond or —CH 2 —O-L′-, where L′ is a C 2 -C 4 or C 3 -C 4 alkylene chain, optionally substituted with at least one hydroxyl group.
18. The separation matrix of claim 16 , wherein said at least one charged monomer is selected from the group consisting of vinyl sulfonate, vinyl phosphonate and allyloxyhydroxypropyl sulfonate.
19. The separation matrix of claim 16 , wherein said second non-charged monomer is an N-vinyl amide.
20. The separation matrix of claim 16 , wherein said second non-charged monomer is selected from the group consisting of N-vinyl pyrrolidone, N-vinyl caprolactam, N-vinyl formamide and N-vinyl acetamide.
21. The separation matrix of claim 16 , wherein in said copolymer chains the molar ratio of the units derived from the first monomer to the units derived from the second monomer is 0.05 to 5.
22. The separation matrix according to claim 16 , wherein the ion capacity of said matrix is 20-300 micromol/mL.
23. The separation matrix according to claim 16 , wherein the solid support comprises a polyhydroxy polymer.
24. The separation matrix according to claim 16 , wherein the solid support comprises agar or agarose.
25. The separation matrix according to claim 16 , wherein the solid support is crosslinked.
26. The separation matrix according to claim 16 , wherein the solid support is porous.
27. The separation matrix according to claim 16 , wherein the solid support has a pore size corresponding to a K D value of 0.5-0.9, measured with dextran of Mw 110 kDa as the probe molecule.
28. The separation matrix according to claim 16 , wherein the matrix has a pore size corresponding to a K D value of 0.1-0.8, measured with dextran of Mw 110 kDa as the probe molecule.
29. A method of manufacturing a separation matrix according to claim 16 , comprising the steps of:
a) providing a solid support comprising moieties with copolymerizable C═C double bonds or moieties susceptible to formation of free radicals;
b) contacting the solid support with a mixture comprising the first and second monomer and;
c) initiating radical polymerization.
30. The method of claim 29 , wherein step c) is performed under conditions such that copolymer chains comprising units derived from said first and second monomer are formed and covalently linked to the solid support either by copolymerization with said C═C double bonds or by initiation or chain transfer from said moieties susceptible to free radical formation.
31. The method of claim 29 , further comprising a step, before step a), of derivatizing said solid support with moieties comprising copolymerizable C═C double bonds or moieties susceptible to formation of free radicals.
32. The method of claim 29 , wherein said moieties comprising copolymerizable C═C double bonds are allyl groups.
33. The method of claim 29 , wherein said moieties susceptible to formation of free radicals comprise i) chain transfer groups or ii) initiating groups.
34. The method of claim 1 , wherein said biomolecule is an antibody, an antibody fragment, an antibody conjugate, or an antibody fusion protein.
35. The method of claim 1 , wherein said at least other component is a host cell protein, protein A or an aggregate of immunoglobulins, immunoglobulin fragments, or immunoglobulin-containing proteins.
36. The method of claim 1 , wherein the liquid is an eluate from a previous affinity chromatography step, ion exchange chromatography step, multimodal chromatography step, or hydrophobic interaction chromatography step.
37. The method of claim 1 , wherein the liquid is the flow-through from an ion exchange separation matrix, a multimodal separation matrix, or a hydrophobic interaction separation matrix.
38. The method of claim 1 , wherein said biomolecule is an antibody, an antibody fragment, an antibody conjugate or an antibody fusion protein and wherein at least 1% of said biomolecule is in the form of aggregates.
39. The method of claim 7 , wherein at least 5% or 10% of said biomolecule is in the form of aggregates.
40. The method of claim 38 , wherein at least 5% or 10% of said biomolecule is in the form of aggregates.
41. The method of claim 9 , wherein the cleaning liquid is an alkaline cleaning liquid comprising at least 0.1 mol/L NaOH or 0.5-2 mol/L NaOH.
42. The method of claim 10 , wherein in said copolymer chains the molar ratio of the units derived from the first monomer to the units derived from the second monomer is 0.10 to 2 or 0.5 to 2.
43. The separation matrix of claim 16 , wherein in said copolymer chains the molar ratio of the units derived from the first monomer to the units derived from the second monomer is 0.10 to 2 or 0.5 to 2.
44. The separation matrix according to claim 16 , wherein the ion capacity of said matrix is 20-200 or 20-80 micromol/mL.
45. The separation matrix according to claim 16 , wherein the solid support comprises a polysaccharide.
46. The separation matrix according to claim 16 , wherein the solid support is crosslinked with hydroxyalkyl ether crosslinks.
47. The separation matrix according to claim 16 , wherein the solid support is porous in the form of porous beads or a porous membrane.
48. The separation matrix according to claim 16 , wherein the solid support has a pore size corresponding to a K D value of 0.6-0.8, measured with dextran of Mw 110 kDa as the probe molecule.
49. The separation matrix according to claim 16 , wherein the matrix has a pore size corresponding to a K D value of 0.2-0.6, measured with dextran of Mw 110 kDa as the probe molecule.
50. The method of claim 29 , wherein said moieties comprising copolymerizable C═C double bonds are allyl ether groups or allyl hydroxypropyl ether groups.
51. The method of claim 29 , wherein said moieties susceptible to formation of free radicals comprise i) thiols or hydrogens in alpha position to hydroxyl groups or ii) peroxides, hydroperoxides, persulfates or azo compounds.