Affinity purification of glycoside-cleaving enzymes

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.

1 . An affinity resin comprising a small molecule ligand having the formula: 
       
         
           
           
               
               
           
         
         wherein R is a resin matrix; 
         FG is absent or is a functional group selected from —O—, —NH—, —C(O)N—, —C(O)O—, CH 2 , and —S—; Linker is absent or 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. 
       
     
     
         2 . The affinity resin 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. 
     
     
         3 . The affinity resin 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. 
     
     
         4 . The affinity resin 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. 
     
     
         5 . The affinity resin of  claim 1 , wherein the functional group is —C(O)N—. 
     
     
         6 . The affinity resin of  claim 1 , wherein the functional group is —O—. 
     
     
         7 . The affinity resin 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. 
     
     
         8 . The affinity resin of  claim 1 , wherein the linker is a straight chained aliphatic C 2 -C 10  hydrocarbon. 
     
     
         9 . The affinity resin of  claim 1 , wherein the linker is a straight chained aliphatic C 5  hydrocarbon. 
     
     
         10 . The affinity resin of  claim 1 , having the formula selected from the group consisting of: 
       
         
           
           
               
               
           
         
       
     
     
         11 .- 13 . (canceled) 
     
     
         14 . The affinity resin of  claim 1 , having the formula: 
       
         
           
           
               
               
           
         
         wherein R is a resin matrix and n is an integer from 2 to 20. 
       
     
     
         15 . The affinity resin of  claim 14 , wherein n is 5. 
     
     
         16 . The affinity resin of  claim 1  having the formula: 
       
         
           
           
               
               
           
         
         wherein R is a resin matrix. 
       
     
     
         17 . An affinity resin comprising a small molecule ligand having the formula: 
       
         
           
           
               
               
           
         
         wherein R is a resin matrix; 
         FG is absent or is a functional group selected from —O—, —NH—, —C(O)N—, —C(O)O—, CH 2 , and —S—; 
         Linker is absent or 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. 
       
     
     
         18 . The affinity resin of  claim 17 , wherein R 1 , R 2 , R 3 , R 4 , and R 5  are each OH. 
     
     
         19 . The affinity resin of  claim 17 , 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. 
     
     
         20 . The affinity resin of  claim 17 , wherein A is —NH—. 
     
     
         21 . The affinity resin of  claim 17 , wherein A is —S—. 
     
     
         22 . The affinity resin of  claim 17 , 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. 
     
     
         23 . The affinity resin of  claim 17 , wherein the linker is a straight chained aliphatic C 2 -C 10  hydrocarbon. 
     
     
         24 . The affinity resin of  claim 17 , having the formula: 
       
         
           
           
               
               
           
         
       
     
     
         25 . The affinity resin of  claim 17 , having the formula selected from: 
       
         
           
           
               
               
           
         
       
     
     
         26 . The affinity resin of  claim 1 , wherein the affinity resin is capable of purifying the glycoside-cleaving enzyme directly from conditioned medium of a cell line. 
     
     
         27 . The affinity resin of  claim 1 , wherein the affinity resin is capable of purifying the glycoside-cleaving enzyme directly from conditioned medium of a cell line without manipulation of the conditioned medium. 
     
     
         28 . The affinity resin of  claim 1  having the binding capacity of at least 10 mg of glycoside-cleaving enzyme per 1 mL of the affinity resin. 
     
     
         29 . The affinity resin of  claim 1  having the binding capacity of about 50 mL to about 100 mL of the conditioned medium per 1 mL of the affinity resin. 
     
     
         30 . The affinity resin of  claim 1 , wherein the affinity resin maintains purification performance after exposure to NaOH. 
     
     
         31 .- 54 . (canceled) 
     
     
         55 . The affinity resin of  claim 17 , wherein the affinity resin is capable of purifying the glycoside-cleaving enzyme directly from conditioned medium of a cell line. 
     
     
         56 . The affinity resin of  claim 17 , wherein the affinity resin is capable of purifying the glycoside-cleaving enzyme directly from conditioned medium of a cell line without manipulation of the conditioned medium. 
     
     
         57 . The affinity resin of  claim 17  having the binding capacity of at least 10 mg of glycoside-cleaving enzyme per 1 mL of the affinity resin. 
     
     
         58 . The affinity resin of  claim 17  having the binding capacity of about 50 mL to about 100 mL of the conditioned medium per 1 mL of the affinity resin. 
     
     
         59 . The affinity resin of  claim 17 , wherein the affinity resin maintains purification performance after exposure to NaOH. 
     
     
         60 . 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 the affinity resin of  claim 17  and eluting the bound glycoside-cleaving enzyme.