Packing material for affinity chromatography
An affinity chromatography packing material includes porous mother particles that include a copolymer of a monomer mixture including a crosslinkable vinyl monomer and an epoxy group-containing vinyl monomer, a ligand being bound to the porous mother particles, and the porous mother particles including a ring-opening epoxy group produced by ring-opening of the epoxy group included in the porous mother particles.
1. An affinity chromatography packing material, comprising:
at least one porous mother particle that comprises a copolymer of a monomer mixture comprising a crosslinkable vinyl monomer and an epoxy group-comprising vinyl monomer,
wherein:
a ligand is bound to the porous mother particle;
the porous mother particle comprises a ring-opening epoxy group produced by ring-opening of the epoxy group comprised in the porous mother particles;
the ligand is bound to the porous mother particle by (i) utilizing the epoxy group comprised in the porous mother particles as a ligand binding site, (ii) activating an alcoholic hydroxyl group produced by ring-opening of the epoxy group comprised in the porous mother particles, and binding the ligand to the porous mother particles, or (iii) forming a linker that extends from the epoxy group comprised in the porous mother particles or a group produced by ring-opening of the epoxy group, and binding the ligand to the porous mother particles via the linker; and
the ligand is an immunoglobulin-binding protein of formula (1),
R-R 2 (1)
wherein
R represents an amino acid sequence that comprises 4 to 300 amino acids and a site including 4 to 20 consecutive histidine residues, and
R 2 represents an amino acid sequence that comprises 50 to 500 amino acids and at least one immunoglobulin-binding domain of protein A, provided that a terminal of the immunoglobulin-binding domain binds to R.
2. The affinity chromatography packing material of claim 1 , wherein the ring-opening epoxy group is a substituted or unsubstituted 2,3-dihydroxypropyl group.
3. The affinity chromatography packing material of claim 1 , wherein the immunoglobulin-binding domain of protein A is at least one immunoglobulin-binding domain selected from the group consisting of an A-domain, a B-domain, a C-domain, a D-domain, an E-domain, and a Z-domain.
4. The affinity chromatography packing material of claim 1 , wherein R- in formula (1) is a group of formula (2),
R 1 -r- (2)
wherein
R 1 represents an amino acid sequence that comprises 4 to 100 amino acids and a site comprising 4 to 20 consecutive histidine residues, provided that a terminal of the site comprising 4 to 20 consecutive histidine residues binds to r, and
r represents an arbitrary amino acid sequence that comprises 7 to 200 amino acids and a TEV domain.
5. The affinity chromatography packing material of claim 1 , wherein at least one selected from the group consisting of the amino acid sequence represented by R and the amino acid sequence represented by R 2 in formula (1), comprises a domain t that comprises 1 to 50 amino acids and one amino acid selected from the group consisting of lysine, arginine, and cysteine.
6. The affinity chromatography packing material of claim 1 , wherein the monomer mixture comprises:
20 to 50 wt% of the crosslinkable vinyl monomer; and
50 to 80 wt% of the epoxy group-comprising vinyl monomer.
7. The affinity chromatography packing material of claim 1 , wherein the at least one porous mother particle has a volume average particle size of 20 to 80 micrometers.
8. The affinity chromatography packing material of claim 1 , wherein the at least one porous mother particle has a volume average particle size of 30 to 60 micrometers.
9. The affinity chromatography packing material of claim 1 , wherein the at least one porous mother particle has a specific surface area of 50 to 150 m 2 /g.
10. The affinity chromatography packing material of claim 1 , wherein the at least one porous mother particle has a specific surface area of 80 to 120 m 2 /g.
11. The affinity chromatography packing material of claim 1 , wherein the at least one porous mother particle has a volume average pore size of 100 to 400 nm.
12. The affinity chromatography packing material of claim 1 , wherein the ring-opening epoxy group comprises a substituted 2,3-dihydroxypropyl group, wherein the substituted 2,3-dihydroxypropyl group is produced by ring-opening of the epoxy group comprised in the at least one porous mother particle with a mercapto group-comprising blocking agent.
13. The affinity chromatography packing material of claim 10 , wherein the substituted 2,3-dihydroxypropyl group is produced by ring-opening of the epoxy group comprised in the at least one porous mother particle with mercaptoethanol.
14. The affinity chromatography packing material of claim 1 , wherein the ring-opening epoxy group comprises a substituted 2,3-dihydroxypropyl group, wherein the substituted 2,3-dihydroxypropyl group is produced by ring-opening of the epoxy group comprised in the at least one porous mother particle with an amino group-comprising blocking agent.
15. The affinity chromatography packing material of claim 10 , wherein the substituted 2,3-dihydroxypropyl group is produced by ring-opening of the epoxy group comprised in the at least one porous mother particle with monoethanolamine.