Methods and compositions for treatment of hunter syndrome

Abstract

The present invention provides, among other things, improved methods for purifying 12S protein produced recombinantly for enzyme replacement therapy. The present invention is, in part, based on the surprising discovery that recombinant 12S protein can be purified from unprocessed biological materials, such as, 12S-containing cell culture medium, using a process involving as few as four chromatography columns.

1 . A composition comprising purified recombinant iduronate-sulfatase (I2S) having an amino acid sequence at least 70% identical to SEQ ID NO: 1,
 wherein the purified recombinant I2S comprises at least about 70% conversion of the cysteine residue corresponding to Cys59 of SEQ ID NO: 1 to Cα-formylglycine (FGly) and   further wherein the purified recombinant I2S contains less than 150 ng/mg Host Cell Protein (HCP).   
     
     
         2 - 3 . (canceled) 
     
     
         4 . The composition of  claim 1 , wherein the purified recombinant I2S contains less than 60 ng/mg HCP. 
     
     
         5 . A composition of  claim 1 ,
 wherein the purified recombinant I2S contains, on average, at least 16 sialic acids per molecule.   
     
     
         6 . (canceled) 
     
     
         7 . The composition of  claim 5 , wherein the purified recombinant I2S contains, on average, at least 20 sialic acids per molecule. 
     
     
         8 . A composition of  claim 1 ,
 wherein the purified recombinant I2S contains at least 10% bis-phosphorylated oligosaccharides per enzyme.   
     
     
         9 . (canceled) 
     
     
         10 . A composition of  claim 1 ,
 wherein the purified I2S is characterized with a glycan map comprising seven or fewer peak groups selected from the peak groups indicative of neutral (peak group 1), mono-sialylated (peak group 2), di-sialylated (peak group 3), monophosphorylated (peak group 4), tri-sialylated (peak group 5), tetra-sialylated (peak group 6), or diphosphorylated (peak group 7) I2S protein.   
     
     
         11 - 20 . (canceled) 
     
     
         21 . A formulation comprising the composition of  claim 1  and a physiologically acceptable carrier. 
     
     
         22 . The formulation of  claim 21 , wherein the formulation is suitable for intravenous administration. 
     
     
         23 . The formulation of  claim 21 , wherein the formulation is suitable for intrathecal administration. 
     
     
         24 . The formulation of  claim 21 , wherein the formulation is suitable for subcutaneous administration. 
     
     
         25 . The formulation of  claim 21 , wherein the formulation is for treating Hunter syndrome. 
     
     
         26 . A method comprising
 purifying recombinant iduronate-2-sulfatase (I2S) protein from an impure preparation by conducting one or more of anion-exchange chromatography, cation-exchange chromatography, mixed-mode chromatography, and hydrophobic interaction chromatography, and   wherein the method involves less than 6 chromatography steps and wherein the purified recombinant I2S protein contains less than 100 ng/mg Host Cell Protein (HCP).   
     
     
         27 - 30 . (canceled) 
     
     
         31 . The method of  claim 26 , wherein the method involves 5 chromatography steps or less. 
     
     
         32 - 45 . (canceled) 
     
     
         46 . The method of  claim 26 , wherein the method further comprises a step of viral inactivation. 
     
     
         47 - 52 . (canceled) 
     
     
         53 . The method of  claim 26 , wherein the recombinant I2S protein has an amino acid sequence identical to SEQ ID NO: 1. 
     
     
         54 - 61 . (canceled) 
     
     
         62 . The method of  claim 26 , wherein the purified recombinant I2S protein contains less than 80 ng/mg HCP. 
     
     
         63 . (canceled) 
     
     
         64 . The method of  claim 26 , wherein the purified recombinant I2S protein contains, on average, 16-22 sialic acids per molecule. 
     
     
         65 . The method  claim 26 , wherein the purified recombinant I2S protein has specific activity of at least 60 U/mg as determined by an in vitro sulfate release activity assay using heparin disaccharide as substrate. 
     
     
         66 . A pharmaceutical composition comprising a recombinant I2S protein purified according to a method of  claim 26 . 
     
     
         67 . A method of treating Hunter syndrome comprising administering into a subject in need of treatment a pharmaceutical composition of  claim 66 .