FILLER FOR AFFINITY CHROMATOGRAPHY

• US 20130023650A1
• Filed: 03/29/2011
• Published: 01/24/2013
• Est. Priority Date: 03/31/2010
• Status: Active Grant

• Current Assignee: JSR Corporation
• Original Assignee: JSR Corporation
• Inventors: Tamori, Kouji, Abe, Takayoshi, Okano, Yusuke, Momiyama, Masaki, Kawai, Hiroshi, Hyugaji, Satoshi, Wang, Yong
• Granted Patent: US 9,090,665 B2
• Time in Patent Office: Days
• US Class Current: 530/387.1
• CPC Class Codes: B01D 15/3804 Affinity chromatography
• CPC Class Codes: B01D 15/3809 of the antigen-antibody type, e.g. protein A, G, L chromatography
• CPC Class Codes: B01J 20/286 Phases chemically bonded to a substrate, e.g. to silica or to polymers
• CPC Class Codes: C07K 1/18 Ion-exchange chromatography
• CPC Class Codes: C07K 1/22 Affinity chromatography or related techniques based upon selective absorption processes
• CPC Class Codes: C08F 216/1416 Monomers containing oxygen in addition to the ether oxygen, e.g. allyl glycidyl ether
• CPC Class Codes: C08F 220/20 of polyhydric alcohols or phenols , e.g. 2-hydroxyethyl (meth)acrylate or glycerol mono-(meth)acrylate
• CPC Class Codes: C08F 220/281 and containing only one oxygen, e.g. furfuryl (meth)acrylate or 2-methoxyethyl (meth)acrylate
• CPC Class Codes: C08F 220/325 containing glycidyl radical, e.g. glycidyl (meth)acrylate
• CPC Class Codes: C08F 220/58 containing oxygen in addition to the carbonamido oxygen , e.g. N-methylolacrylamide, N-(meth)acryloylmorpholine
• CPC Class Codes: C08F 222/1006 of polyhydric alcohols or polyhydric phenols
• CPC Class Codes: C08F 222/102 of dialcohols, e.g. ethylene glycol di(meth)acrylate or 1,4-butanediol dimethacrylate
• CPC Class Codes: C08F 222/103 of trialcohols, e.g. trimethylolpropane tri(meth)acrylate
• CPC Class Codes: C08F 222/104 of tetraalcohols, e.g. pentaerythritol tetra(meth)acrylate

Abstract

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.

Claims

1. A filler for affinity chromatography, 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 the porous particle (a), and

(c) a ring-opened epoxy group.

2. The filler for affinity chromatography according to claim 1, wherein the ligand (a) is a protein comprising at least one selected from the group consisting of a protein A, a immunoglobulin-binding domain of protein A, and a variant thereof.

3. The filler for affinity chromatography 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 for affinity chromatography according to claim 1, wherein the ring-opened epoxy group (c) is obtained by ring-opening of the epoxy group contained in the polymer with thioglycerol.

5. The filler for affinity chromatography 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).

6. The filler for affinity chromatography 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.

7. The filler for affinity chromatography according to claim 1, wherein the particle size of the porous particle (a) is 35 to 100 μ

m.

8. A method for isolating immunoglobulins, the method comprising:

absorbing immunoglobulins with the filler for affinity chromatography according to claim 1; and

eluting the immunoglobulins.

9. A packed column for affinity chromatography, which is packed with the filler for affinity chromatography according to claim 1.