Manufacture of Active Highly Phosphorylated Human N-Acetylgalactosamine-6-Sulfatase and Uses Thereof

摘要

This invention provides compositions of active highly phosphorylated human N-acetylgalactosamine-6-sulfatase (GALNS), and pharmaceutical compositions and formulations thereof, methods of producing and purifying GALNS, and its 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 GALNS enzyme, e.g., Mucopolysaccharidosis IVa (MPS IVa or Morquio A syndrome).

主权项

1. A method of purifying a 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, wherein said GALNS enzyme:
(i) has a purity of at least about 95% as determined by Coomassie Blue staining when subjected to SDS-PAGE under non-reducing conditions, (ii) has at least about 50% conversion of the cysteine residue at position 53 to Cα-formylglycine (FGly), and (iii) optionally, has between 0.5 to 0.8 bis-phosphorylated oligomannose chains per monomeric protein chain, and wherein at least 97% of said GALNS enzyme is in the precursor form as determined by SDS-capillary gel electrophoresis (SDS-CGE), comprising: a) filtering a culture medium containing the GALNS enzyme secreted from a mammalian cell line that expresses human sulfatase modifying factor 1 (SUMF1) and the recombinant human GALNS enzyme, ultrafiltering/diafiltering the filtered culture medium, and charcoal filtering the ultrafiltered/diafiltered culture medium; b) loading the charcoal filtered ultrafiltered/dialfiltered culture medium from step a) onto a capture column, washing the capture column under conditions such that the GALNS enzyme is retained on the capture column, and eluting the GALNS enzyme from the capture column; c) optionally, filtering the eluate from the capture column in step b) through a filter to remove viruses; d) adjusting the pH of the eluate from the capture column from step b) or the filtrate from step c) to an acid pH, and filtering the acid pH-adjusted eluate from the capture column or acid pH-adjusted filtrate; e) loading the acid pH-adjusted eluate from the capture column or acid pH-adjusted Q filtrate from step d) onto an intermediate column, washing the intermediate column under conditions such that the GALNS enzyme is retained on the intermediate column, and eluting the GALNS enzyme from the intermediate column; f) adjusting the eluate from the intermediate column in step e) to low pH for viral inactivation; and g) loading the low pH viral inactivated eluate from step f) onto a polishing column, washing the polishing column under conditions such that the GALNS enzyme is retained on the polishing column, and eluting the GALNS enzyme from the polishing column.