Affinity purification of glycosidase-cleaving enzymes

1. 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—, CH 2 , and —S—;

Linker is selected from a straight chained, branched or cyclic aliphatic C 2 -C 20 hydrocarbon; a heteroaliphatic C 2 -C 20 hydrocarbon; an aromatic C 6 -C 20 hydrocarbon; a heteroaromatic C 2 -C 20 hydrocarbon; an aryl C 6 -C 20 hydrocarbon; a heteroaryl C 2 -C 20 hydrocarbon, a C 2 -C 12 perfluorocarbon, or a combination thereof, each of which optionally contains 1-8 heteroatoms selected from halogen, O, N, and S and combinations thereof;

R 1 , R 2 , R 3 , R 4 , and R 5 are each independently selected from H, OH, CH 2 OH, and —NH—C(O)—CH 3 , and wherein the affinity resin is capable of binding a glycoside-cleaving enzyme,

and eluting the bound glycoside-cleaving enzyme.

2. The method of claim 1 , wherein the glycoside-cleaving enzyme is purified from conditioned medium.

3. The method of claim 1 , wherein the glycoside-cleaving enzyme is purified directly from conditioned medium without manipulation of the conditioned medium.

4. The method of claim 1 , wherein the cell line is a recombinant CHO cell line.

5. The method of claim 1 , wherein the cell line is a human cell line.

6. The method of claim 1 , wherein the purity of the eluted glycoside-cleaving enzyme is at least 95%.

7. The method of claim 1 , wherein the binding capacity of the affinity resin is at least 10 mg of glycoside-cleaving enzyme per 1 mL of the affinity resin.

8. The method of claim 1 , wherein the glycoside-cleaving enzyme is selected from α-galactosidase A (α-Gal A), glucocerebrosidase (GCB), β-galactosidase, and acid alpha-glucosidase (GAA).

9. The method of claim 1 , wherein the step of eluting the bound glycoside-cleaving enzyme is performed with an elution buffer comprising tris(hydroxymethyl) aminomethane (Tris).

10. The method of claim 1 , wherein the step of eluting the bound glycoside-cleaving enzyme is performed with an elution buffer comprising the same small molecule ligand as the small molecule ligand immobilized on the affinity matrix.

11. The method of claim 1 , wherein the step of eluting the bound glycoside-cleaving enzyme is performed with an elution buffer having a pH between about pH 5 and about pH 9.

12. The method of claim 1 , wherein the step of eluting the bound glycoside-cleaving enzyme is performed with an elution buffer comprising a salt.

13. The method of claim 12 , wherein the salt is NaCl.

14. 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—, CH 2 , and —S—;

Linker is selected from a straight chained, branched or cyclic aliphatic C 2 -C 20 hydrocarbon; a heteroaliphatic C 2 -C 20 hydrocarbon; an aromatic C 6 -C 20 hydrocarbon; a heteroaromatic C 2 -C 20 hydrocarbon; an aryl C 6 -C 20 hydrocarbon; a heteroaryl C 2 -C 20 hydrocarbon, a C 2 -C 12 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—;

R 1 , R 2 , R 3 , R 4 , and R 5 are each independently selected from H, OH, and —NH—C(O)—CH 3 , and wherein the affinity resin is capable of binding a glycoside-cleaving enzyme,

and eluting the bound glycoside-cleaving enzyme.

15. The method of claim 1 , wherein R 1 is CH 2 OH, R 2 , R 3 , and R 4 are each OH, and R 5 is H.

16. The method of claim 1 , wherein R 1 is CH 2 OH, R 2 and R 3 are each OH, R 4 is —NH—C(O)—CH 3 , and R 5 is H.

17. The method of claim 1 , wherein R 1 is H, R 2 is CH 2 OH, R 3 and R 4 are each OH, and R 5 is H.

18. The method of claim 1 , wherein the functional group is —C(O)N—.

19. The method of claim 1 , wherein the functional group is —O—.

20. The method of claim 1 , wherein the linker is a straight chained, branched or cyclic aliphatic C 2 -C 20 hydrocarbon optionally containing 1-8 heteroatoms selected from halogen, O, N, and S and combinations thereof.

21. The method of claim 1 , wherein the linker is a straight chained aliphatic C 2 -C 10 hydrocarbon.

22. The method of claim 1 , wherein the linker is a straight chained aliphatic C 5 hydrocarbon.

23. The method of claim 1 , wherein the affinity resin has the formula selected from the group consisting of:

24. The method of claim 1 , wherein the affinity resin has the formula:

wherein R is a resin matrix and n is an integer from 2 to 20.

25. The method of claim 24 , wherein n is 5.

26. The method of claim 1 , wherein the affinity resin has the formula:

wherein R is a resin matrix.

27. The method of claim 14 , wherein R 1 , R 2 , R 3 , R 4 , and R 5 are each OH.

28. The method of claim 14 , wherein R 1 is CH 2 OH, R 2 and R 3 are each OH, R 4 is —NH—C(O)—CH 3 , and R 5 is H.

29. The method of claim 14 , wherein A is —NH—.

30. The method of claim 14 , wherein A is —S—.

31. The method of claim 14 , wherein the linker is a straight chained, branched or cyclic aliphatic C 2 -C 20 hydrocarbon optionally containing 1-8 heteroatoms selected from halogen, O, N, and S and combinations thereof.

32. The method of claim 14 , wherein the linker is a straight chained aliphatic C 2 -C 10 hydrocarbon.

33. The method of claim 14 , wherein the affinity resin has the formula:

34. The method of claim 14 , wherein the affinity resin has the formula selected from: