Filler for affinity chromatography
Provided is a filler for affinity chromatography which is useful for protein purification and contains porous particles that have a high dynamic binding capacity for proteins and excellent pressure characteristics. The filler for affinity chromatography of the present invention is characterized in that it includes a porous particle consisting of a polymer of vinyl monomer including a cross-linkable vinyl monomer that contains hydroxyl group but does not contain epoxy group and an epoxy group-containing non-cross-linkable vinyl monomer, or a cross-linkable vinyl monomer that contains hydroxyl group and epoxy group, ligands bound to the porous particle, and ring-opened epoxy groups.
1. A filler comprising:
(a) a porous particle consisting of a polymer of vinyl monomer comprising
a cross-linkable vinyl monomer comprising a hydroxyl group but not an epoxy group and an epoxy group-containing non-cross-linkable vinyl monomer,
or a cross-linkable vinyl monomer comprising a hydroxyl group and epoxy group,
(b) a ligand bound to an epoxy group of the porous particle (a), and
(c) a ring-opened epoxy group, obtained by ring-opening of the epoxy group contained in the polymer with thioglycerol,
wherein the filler is suitable for affinity chromatography.
2. The filler according to claim 1 , wherein the ligand (b) is a protein comprising at least one selected from the group consisting of a protein A, an immunoglobulin-binding domain of protein A, and a variant thereof.
3. The filler according to claim 1 , wherein the vinyl monomer consists of:
20 to 90 parts by mass of (X-1) a cross-linkable vinyl monomer comprising a hydroxyl group but not an epoxy group,
0 to 40 parts by mass of (X-2) a cross-linkable vinyl monomer comprising neither a hydroxyl group nor an epoxy group,
1 to 40 parts by mass of (M-1) an epoxy group-containing non-cross-linkable vinyl monomer, and
0 to 70 parts by mass of (M-2) a non-cross-linkable vinyl monomer that does not contain epoxy group,
with the proviso that the total amount of (X-1), (X-2), (M-1) and (M-2) is 100 parts by mass.
4. The filler according to claim 1 , wherein the porous particle (a) is obtained by performing suspension polymerization of:
100 parts by mass of the vinyl monomer, and
a water-based mixture comprising (P-1) as an essential component, wherein the (P-1) is at least one porogen selected from the group consisting of a linear, branched or cyclic, C7 to C14 alcohol, an ether, an aldehyde, a ketone and an ester, and C8 to C10 alkylbenzene,
with the proviso that the total amount of porogens is 100 to 400 parts by mass, and the content of (P-1) is 10% by mass or more in 100% by mass of the total amount of porogens.
5. The filler according to claim 1 , wherein the pore volume of the porous particle (a) obtainable by measuring micropores having pore diameter in the range of 10 to 5,000 nm with a mercury porosimeter, is 1.00 to 1.90 ml/g.
6. The filler according to claim 1 , wherein the particle size of the porous particle (a) is 35 to 100 μm.
7. A method for isolating immunoglobulins, the method comprising:
absorbing immunoglobulins with the filler according to claim 1 , wherein the ligand is protein A, protein G or an immunoglobulin binding protein; and
eluting the immunoglobulins.
8. A packed column, which is packed with the filler according to claim 1 .