Affinity purification of glycosidase-cleaving enzymes
AssigneeTakeda Pharmaceutical Company Limited
InventorsBrian Dwyer, Bohong Zhang, Jun Hu, Muthuraman Meiyappan, Thomas Miller, Paul McLean, Clark Pan
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Abstract
The invention relates to an affinity resin functionalized with small molecule inhibitors of glycoside-cleaving enzymes, e.g., α-galactosidase A (α-Gal A), glucocerebrosidase (GCB), β-galactosidase, and acid alpha-glucosidase (GAA), and a method for purifying glycoside-cleaving enzymes produced in a cell line using the small molecule inhibitor-functionalized affinity resin.
Claims34
A method of purifying a glycoside-cleaving enzyme produced in a cell line, said method comprising the steps of binding the glycoside-cleaving enzyme to an affinity resin comprising a small molecule ligand having the formula:
- wherein R is a resin matrix;
- FG is a functional group selected from —O—, —NH—, —C(O)N—, —C(O)O—, CH2, and —S—;
- Linker is selected from a straight chained, branched or cyclic aliphatic C2-C20 hydrocarbon;
- a heteroaliphatic C2-C20 hydrocarbon;
- an aromatic C6-C20 hydrocarbon;
- a heteroaromatic C2-C20 hydrocarbon;
- an aryl C6-C20 hydrocarbon;
- a heteroaryl C2-C20 hydrocarbon, a C2-C12 perfluorocarbon, or a combination thereof, each of which optionally contains 1-8 heteroatoms selected from halogen, O, N, and S and combinations thereof;
- R1, R2, R3, R4, and R5 are each independently selected from H, OH, CH2OH, and —NH—C(O)—CH3, and wherein the affinity resin is capable of binding a glycoside-cleaving enzyme,
- and eluting the bound glycoside-cleaving enzyme.
A method of purifying a glycoside-cleaving enzyme produced in a cell line, said method comprising the steps of binding the glycoside-cleaving enzyme to an affinity resin comprising a small molecule ligand having the formula:
- wherein R is a resin matrix;
- FG is a functional group selected from —O—, —NH—, —C(O)N—, —C(O)O—, CH2, and —S—;
- Linker is selected from a straight chained, branched or cyclic aliphatic C2-C20 hydrocarbon;
- a heteroaliphatic C2-C20 hydrocarbon;
- an aromatic C6-C20 hydrocarbon;
- a heteroaromatic C2-C20 hydrocarbon;
- an aryl C6-C20 hydrocarbon;
- a heteroaryl C2-C20 hydrocarbon, a C2-C12 perfluorocarbon, or a combination thereof, each of which optionally contains 1-8 heteroatoms selected from halogen, O, N, and S and combinations thereof;
- A is selected from —NH— and —S—;
- R1, R2, R3, R4, and R5 are each independently selected from H, OH, and —NH—C(O)—CH3, and wherein the affinity resin is capable of binding a glycoside-cleaving enzyme,
- and eluting the bound glycoside-cleaving enzyme.
Dependencies are inferred from the claim text.