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 method for measuring the activity of a recombinant human arylsulfatase B (ARSB) to degrade dermatan sulfate (DS) substrates, comprising:
(a) culturing an isolated human cell deficient in the ARSB under conditions in which DS substrates for the ARSB accumulate;
(b) contacting the cell with the ARSB;
(c) lysing the cell;
(d) adding to the cell lysate an enzyme that (i) is specific for DS substrates and (ii) cleaves small oligosaccharides from the DS substrates;
(e) labeling the small oligosaccharides with a detectable moiety;
(f) optionally separating the labeled small oligosaccharides;
(g) detecting the labeled small oligosaccharides; and
(h) determining the activity of the ARSB to degrade DS substrates by comparing (i) the amount of labeled small oligosaccharides from cells contacted with the ARSB with (ii) the amount of labeled small oligosaccharides from cells not contacted with the ARSB, wherein a reduction in (h)(i) as compared to (h)(ii) indicates the activity of the ARSB to degrade DS substrates.
2. The method of claim 1 , wherein the enzyme in step (d) is chondroitin ABC lyase, and the small oligosaccharides in step (d) are DS disaccharides.