Cells for producing recombinant iduronate-2-sulfatase

Abstract

The present invention provides, among other things, methods and compositions for production of recombinant I2S protein with improved potency and activity using cells co-express I2S and FGE protein. In some embodiments, cells according to the present invention are engineered to simultaneously over-express recombinant I2S and FGE proteins. Cells according to the invention are adaptable to various cell culture conditions. In some embodiments, cells of the present invention adaptable to a large-scale suspension serum-free culture.

1 . A cell comprising
 a first nucleic acid encoding an iduronate-2-sulfatase (I2S) protein comprising an amino acid sequence at least 70% identical to SEQ ID NO:1; and   a second nucleic acid encoding a formylglycine generating enzyme (FGE) protein comprising an amino acid sequence at least 70% identical to SEQ ID NO:5,   wherein the first and/or the second nucleic acid are exogenous and wherein the cell, once cultivated under a cell culture condition, produces I2S protein comprising at least about 70% conversion of the cysteine residue corresponding to Cys59 of SEQ ID NO:1 to Cα-formylglycine (FGly).   
     
     
         2 . A cell comprising
 a first nucleic acid encoding an iduronate-2-sulfatase (I2S) protein comprising an amino acid sequence at least 70% identical to SEQ ID NO:1; and   a second nucleic acid encoding a formylglycine generating enzyme (FGE) protein comprising an amino acid sequence at least 70% identical to SEQ ID NO:5,   wherein the first and/or the second nucleic acid are exogenous and wherein the cell, once cultivated under a cell culture condition, produces I2S protein comprising at least about 60% conversion of the cysteine residue corresponding to Cys59 of SEQ ID NO:1 to Cα-formylglycine (FGly) and at a specific productivity rate of great than about 30 picogram/cell/day.   
     
     
         3 . The cell of  claim 1 , wherein the cell, once cultivated under a cell culture condition, produces the I2S protein comprising at least about 80% conversion of the cysteine residue corresponding to Cys59 of SEQ ID NO:1 to Cα-formylglycine (FGly). 
     
     
         4 . The cell of  claim 1 , wherein the cell, once cultivated under a cell culture condition, produces the I2S protein comprising at least about 90% conversion of the cysteine residue corresponding to Cys59 of SEQ ID NO:1 to Cα-formylglycine (FGly). 
     
     
         5 . The cell of  claim 1 , wherein the cell, once cultivated under a cell culture condition, produces the I2S protein comprising at least about 95% conversion of the cysteine residue corresponding to Cys59 of SEQ ID NO:1 to Cα-formylglycine (FGly). 
     
     
         6 . The cell of  claim 1 , wherein the cell, once cultivated under a cell culture condition, produces the I2S protein comprising at least about 97% conversion of the cysteine residue corresponding to Cys59 of SEQ ID NO:1 to Cα-formylglycine (FGly). 
     
     
         7 . (canceled) 
     
     
         8 . The cell of  claim 1 , wherein the first nucleic acid encodes an I2S protein having an amino acid sequence identical to SEQ ID NO:1. 
     
     
         9 . The cell of  claim 1 , wherein the second nucleic acid encodes an FGE protein having an amino acid sequence identical to SEQ ID NO:5. 
     
     
         10 . The cell of  claim 1 , wherein the first nucleic acid comprises a sequence at least 70% identical to SEQ ID NO:7 
     
     
         11 . The cell of  claim 1 , wherein the first nucleic acid comprises a sequence of SEQ ID NO:7. 
     
     
         12 . The cell of  claim 1 , wherein the second nucleic acid comprises a sequence at least 70% identical to SEQ ID NO:8 
     
     
         13 . The cell of  claim 1 , wherein the second nucleic acid comprises a sequence identical to SEQ ID NO:8. 
     
     
         14 - 19 . (canceled) 
     
     
         20 . The cell of  claim 1 , wherein the cell is adaptable to suspension culture. 
     
     
         21 . A method of producing recombinant iduronate-2-sulfatase (I2S) protein comprising cultivating a cell of  claim 1  under conditions such that the recombinant I2S and FGE proteins are co-expressed in the cell. 
     
     
         22 . The method of  claim 21 , wherein the cell is cultivated at a large scale. 
     
     
         23 . The method of  claim 22 , wherein the large scale is a bioreactor process. 
     
     
         24 . The method of  claim 23 , wherein the bioreactor process is a perfusion process. 
     
     
         25 . The method of  claim 23 , wherein the bioreactor is at a scale selected from 10 L, 200 L, 500 L, 1000 L, 1500 L, or 2000 L. 
     
     
         26 . (canceled) 
     
     
         27 . The method of  claim 21 , wherein the cell is cultivated in a serum-free medium. 
     
     
         28 - 30 . (canceled) 
     
     
         31 . The method of  claim 21 , wherein the method further comprises a step of purifying the recombinant I2S protein. 
     
     
         32 - 33 . (canceled)