Recombinant glycoproteins and uses thereof
Provided herein are recombinant glycoproteins (e.g., recombinant human α-galactosidase-A proteins) with an altered (e.g., improved) glycosylation profile, and pharmaceutical compositions and kits including one or more of these proteins. Also provided are methods of generating a mammalian cell useful for recombinant expression of a glycoprotein (e.g., recombinant human α-galactosidase-A), methods of producing recombinant glycoproteins, and methods of treatment that include administering to a subject at least one of the recombinant glycoproteins (e.g., recombinant human α-galactosidase-A protein).
1. A recombinant human α-galactosidase-A (rhAGA) protein produced by a method comprising:
(a) providing a Chinese hamster ovary (CHO) cell produced by a method comprising:
(i) providing a CHO DXB11 cell line;
(ii) sequentially culturing the CHO DXB11 cell line in:
a cell culture medium comprising 5% fetal bovine serum for 3 days to 5 days;
a cell culture medium comprising 2.5% fetal bovine serum for 3 days to 5 days; and
a cell culture medium comprising no animal serum for about 5 days to 10 days; and
(iii) generating a single-cell clone from the CHO cell resulting from step (ii), wherein the single-cell clone, when transfected with red fluorescent protein (RFP), would have a transfection efficiency of >30% RFP positive, a cell growth doubling time in a serum-free medium of <35 hours, and >75% culture viability, and further wherein the single-cell clone grows in a protein-free, animal derived component (ADC) free medium;
(b) stably transfecting the single-cell clone from step (a) with an expression vector comprising a sequence encoding the rhAGA protein;
(c) selecting a transfected clone that produces rhAGA;
(d) culturing the clone in a protein-free, ADC-free medium; and
(e) harvesting the rhAGA protein from the cell culture,
wherein the rhAGA protein comprising a percentage of total N-linked oligosaccharides that are bis-mannose-6-phosphorylated oligosaccharides between 9.0% and 11.0%.
2. The rhAGA protein of claim 1 , wherein the expression vector comprises a sequence encoding the rhAGA protein that is at least 90% identical to SEQ ID NO: 1.
3. The rhAGA protein of claim 1 , wherein the amino acid sequence of the rhAGA protein comprises SEQ ID NO:3.
4. The rhAGA protein of claim 1 , wherein the amino acid sequence of the rhAGA protein consists of SEQ ID NO:3.
5. The rhAGA protein of claim 1 , wherein the amino acid sequence of the rhAGA protein consists of SEQ ID NO:2.
6. A recombinant human α-galactosidase-A (rhAGA) protein comprising:
a percentage of total N-linked oligosaccharides that are bis-mannose-6-phosphorlyated oligosaccharides between 9.0% and 11.0%.
7. The rhAGA protein of claim 6 , further comprising a percentage of total N-linked oligosaccharides that are monosialylated, nonfucose-containing oligosaccharides between 0.3% and 1.2%.
8. The rhAGA protein of claim 6 , wherein the amino acid sequence of the rhAGA protein comprises SEQ ID NO:3.
9. The rhAGA protein of claim 6 , wherein the amino acid sequence of the rhAGA protein consists of SEQ ID NO:3.
10. The rhAGA protein of claim 6 , wherein the amino acid sequence of the rhAGA protein comprises SEQ ID NO:2.
11. The rhAGA protein of claim 6 , further comprising:
a percentage of total N-linked oligosaccharides that are triantennary, trisialylated oligosaccharides of form 2 that is between 6.0% and 8.0%; and
a molar ratio of mannose-6-phosphate to protein that is between 1.8-3.0.
12. The rhAGA protein of claim 6 , further comprising at least one of:
a percentage of total N-linked oligosaccharides that are neutrally-charged oligosaccharides between 0.1% and 2.0%;
a percentage of total N-linked oligosaccharides that are monosialylated fucose-containing oligosaccharides between 0.1% and 2.0%;
a percentage of total N-linked oligosaccharides that are triantennary, trisialylated oligosaccharides of form 1 between 0.1% and 5.0%;
a percentage of total N-linked oligosaccharides that are tetrasialylated oligosaccharides between 6.0% and 8.0%; and
a percentage of total N-linked oligosaccharides that are monosialylated and monophosphorylated between 8.5% and 10.5%.
13. A pharmaceutical composition comprising the rhAGA protein of claim 1 , and a pharmaceutically acceptable carrier.
14. A pharmaceutical composition comprising the rhAGA protein of claim 2 , and a pharmaceutically acceptable carrier.
15. A pharmaceutical composition comprising the rhAGA protein of claim 3 , and a pharmaceutically acceptable carrier.
16. A pharmaceutical composition comprising the rhAGA protein of claim 4 , and a pharmaceutically acceptable carrier.
17. A pharmaceutical composition comprising the rhAGA protein of claim 5 , and a pharmaceutically acceptable carrier.
18. A pharmaceutical composition comprising the rhAGA protein of claim 6 , and a pharmaceutically acceptable carrier.
19. A pharmaceutical composition comprising the rhAGA protein of claim 7 , and a pharmaceutically acceptable carrier.
20. A pharmaceutical composition comprising the rhAGA protein of claim 8 , and a pharmaceutically acceptable carrier.
21. A pharmaceutical composition comprising the rhAGA protein of claim 9 , and a pharmaceutically acceptable carrier.
22. A pharmaceutical composition comprising the rhAGA protein of claim 10 , and a pharmaceutically acceptable carrier.
23. A pharmaceutical composition comprising the rhAGA protein of claim 11 , and a pharmaceutically acceptable carrier.
24. A pharmaceutical composition comprising the rhAGA protein of claim 12 , and a pharmaceutically acceptable carrier.