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 lysosomal enzyme to degrade natural substrates, comprising:
(a) culturing an isolated human cell deficient in the lysosomal enzyme under conditions in which natural substrates for the lysosomal enzyme accumulate;
(b) contacting the cell with the lysosomal enzyme;
(c) lysing the cell;
(d) adding to the cell lysate an enzyme that (i) is specific for natural substrates and (ii) cleaves small oligosaccharides from the natural 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 lysosomal enzyme to degrade natural substrates by comparing (i) the amount of labeled small oligosaccharides from cells contacted with the lysosomal enzyme with (ii) the amount of labeled small oligosaccharides from cells not contacted with the lysosomal enzyme, wherein a reduction in (h)(i) as compared to (h)(ii) indicates the activity of the lysosomal enzyme to degrade natural substrates.
2. The method of claim 1 , wherein the lysosomal enzyme is a lysosomal sulfatase enzyme selected from the group consisting of, iduronate-2-sulfatase (IDS), sulfamidase/heparin-N-sulfatase (SGSH), N-acetylglucosamine-sulfatase (G6S) and N-acetylgalactosamine-6-sulfatase (GALNS).
3. The method of claim 2 , wherein the lysosomal sulfatase enzyme is GALNS.
4. The method of claim 1 , wherein the lysosomal enzyme is α-L-iduronidase (IDU).
5. The method of claim 1 , wherein the lysosomal enzyme is acid α-glucosidase (GAA).
6. The method of claim 1 , wherein the lysosomal enzyme is β-glucuronidase (GUSB).
7. The method of claim 1 , wherein the lysosomal enzyme is β-galactosidase (GLB1).
8. The method of claim 2 , wherein the lysosomal sulfatase enzyme is IDS.
9. The method of claim 2 , wherein the lysosomal sulfatase enzyme is SGSH.
10. The method of claim 2 , wherein the lysosomal sulfatase enzyme is G6S.
11. The method of claim 3 , wherein the natural substrates are keratan sulfate (KS) substrates.
12. The method of claim 11 , wherein the enzyme in step (d) is Keratanase II and the small oligosaccharides in step (d) are KS disaccharides.