Manufacture of active highly phosphorylated human lysosomal sulfatase enzymes and uses thereof
View Patent ↗This invention provides compositions of active highly phosphorylated lysosomal sulfatase enzymes, their pharmaceutical compositions, methods of producing and purifying such lysosomal sulfatase enzymes and compositions and their use in the diagnosis, prophylaxis, or treatment of diseases and conditions, including particularly lysosomal storage diseases that are caused by, or associated with, a deficiency in the lysosomal sulfatase enzyme.
1. A purified preparation of recombinant human N-acetylgalactosamine-6-sulfatase (GALNS) enzyme, said GALNS enzyme comprising an amino acid sequence at least 95% identical to amino acids 27 to 522 of SEQ ID NO: 4, and
(a) having a purity of at least about 95% as determined by Coomassie Blue staining when subjected to SDS-PAGE under non-reducing conditions,
(b) having at least about 90% conversion of the cysteine residue at position 53 to C α -formylglycine (FGly), and
(c) having between 0.5 to 0.8 bis-phosphorylated oligomannose chains per monomeric protein chain,
wherein at least 70% of said GALNS enzyme is in a precursor form as determined by Coomassie Blue staining when subjected to SDS-PAGE under reducing conditions.
2. A sterile composition comprising
(a) a recombinant human N-acetylgalactosamine-6-sulfatase (GALNS) enzyme that comprises an amino acid sequence at least 95% identical to amino acids 27 to 522 of SEQ ID NO: 4, said GALNS enzyme
(i) having a purity of at least about 95% as determined by Coomassie Blue staining when subjected to SDS-PAGE under non-reducing conditions,
(ii) having at least about 90% conversion of the cysteine residue at position 53 to C α -formylglycine (FGly), and
(iii) having between 0.5 and 0.8 bis-phosphorylated oligomannose chains per monomeric protein chain,
wherein at least 70% of said GALNS enzyme is in a precursor form as determined by Coomassie Blue staining when subjected to SDS-PAGE under reducing conditions; and
(b) a sterile pharmaceutically acceptable diluent.
3. The purified preparation of claim 1 , wherein the GALNS enzyme is at least 95% pure as determined by RP-HPLC.
4. The purified preparation of claim 1 , wherein at least 80% of the GALNS enzyme is in the precursor form as determined by Coomassie Blue staining when subjected to SDS-PAGE under reducing conditions.
5. The purified preparation of claim 1 , wherein at least 80% of the GALNS enzyme is in the precursor form as determined by SDS-capillary gel electrophoresis.
6. The purified preparation of claim 1 , wherein between 50% to 80% of the GALNS enzyme binds to a mannose-6-phosphate receptor column.
7. The purified preparation of claim 1 , wherein the GALNS enzyme exhibits a specific uptake (Kuptake) into fibroblasts that is about 1 to 5 nM.
8. The purified preparation of claim 1 , wherein the GALNS enzyme exhibits a specific uptake (Kuptake) into fibroblasts that is about 1 to 3.5 nM.
9. The sterile composition of claim 2 , wherein the GALNS enzyme is at least 95% pure as determined by RP-HPLC.
10. The sterile composition of claim 2 , wherein at least 80% of the GALNS enzyme is in the precursor form as determined by SDS-capillary gel electrophoresis.
11. The sterile composition of claim 2 , wherein at least 90% of the GALNS enzyme is in the precursor form as determined by SDS-capillary gel electrophoresis.
12. The sterile composition of claim 2 , wherein between 50% to 80% of the GALNS enzyme binds to a mannose-6-phosphate receptor column.
13. The sterile composition of claim 2 , wherein the GALNS enzyme exhibits a specific uptake (Kuptake) into fibroblasts that is about 1 to 5 nM.
14. The sterile composition of claim 2 , wherein the GALNS enzyme exhibits a specific uptake (Kuptake) into fibroblasts that is about 1 to 3.5 nM.
15. A purified preparation of recombinant human N-acetylgalactosamine-6-sulfatase (GALNS) enzyme, said GALNS enzyme comprising an amino acid sequence at least 95% identical to amino acids 27 to 522 of SEQ ID NO: 4, and
(a) having a purity of at least about 95% as determined by Coomassie Blue staining when subjected to SDS-PAGE under non-reducing conditions,
(b) having at least about 90% conversion of the cysteine residue at position 53 to C α -formylglycine (FGly), and
(c) having between 0.5 to 0.8 bis-phosphorylated oligomannose chains per monomeric protein chain,
wherein at least 70% of said GALNS enzyme is in a precursor form as determined by SDS/PAGE Coomassie blue under reducing conditions, and
wherein said GALNS enzyme exhibits a specific uptake (Kuptake) into fibroblasts that is about 1 to 5 nM.
16. The purified preparation of claim 15 , wherein the GALNS enzyme is at least 95% pure as determined by RP-HPLC.
17. The purified preparation of claim 15 , wherein at least 80% of the GALNS enzyme is in the precursor form as determined by Coomassie Blue staining when subjected to SDS-PAGE under reducing conditions.
18. The purified preparation of claim 15 , wherein at least 80% of the GALNS enzyme is in the precursor form as determined by SDS-capillary gel electrophoresis.
19. The purified preparation of claim 15 , wherein at least 90% of the GALNS enzyme is in the precursor form as determined by SDS-capillary gel electrophoresis.
20. The purified preparation of claim 15 , wherein the GALNS enzyme exhibits a specific uptake (Kuptake) into fibroblasts that is about 1 to 3.5 nM.