Cells for producing recombinant iduronate-2-sulfatase
View Patent ↗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 90% 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 90% 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), and wherein the level of iduronate-2-sulfatase protein expressed by the cell is between 0.3-fold and 10-fold higher than the level of formylglycine generating enzyme protein expressed by the cell.
2. A cell comprising
a first nucleic acid encoding an iduronate-2-sulfatase (I2S) protein comprising an amino acid sequence at least 90% 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 90% 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) and at a specific productivity rate of greater 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. The cell of claim 1 , wherein the first and/or the second nucleic acid is operably linked to a hCMV promoter.
8. The cell of claim 1 , wherein the first nucleic acid comprises a sequence at least 70% identical to SEQ ID NO:7.
9. The cell of claim 1 , wherein the second nucleic acid comprises a sequence at least 70% identical to SEQ ID NO:8.
10. The cell of claim 1 , wherein both of the first and second nucleic acids are exogenous.
11. The cell of claim 1 , wherein the cell is a mammalian cell.
12. The cell of claim 11 , wherein the mammalian cell is a CHO cell.
13. 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.
14. The method of claim 13 , wherein the cell is cultivated in a large scale bioreactor process at a scale selected from 10 L, 200 L, 500 L, 1000 L, 1500 L, or 2000 L.
15. The method of claim 14 , wherein the bioreactor process is a perfusion process.
16. The method of claim 13 , wherein the cell is cultivated in a serum-free medium.
17. The method of claim 13 , wherein the cell is cultivated in suspension.
18. The method of claim 13 , wherein the method further comprises a step of purifying the recombinant I2S protein.
19. The cell according to claim 1 , wherein the first nucleic acid encodes an iduronate-2-sulfatase (I2S) protein comprising an amino acid sequence at least 95% identical to SEQ ID NO:1.
20. The cell according to claim 1 , wherein the second nucleic acid encodes a formylglycine generating enzyme (FGE) protein comprising an amino acid sequence at least 95% identical to SEQ ID NO:5.
21. The cell according to claim 1 , wherein the first nucleic acid encodes an iduronate-2-sulfatase (I2S) protein comprising an amino acid sequence identical to SEQ ID NO:1.
22. The cell according to claim 1 , wherein the second nucleic acid encodes a formylglycine generating enzyme (FGE) protein comprising an amino acid sequence identical to SEQ ID NO:5.