Characterization of lysosomal enzymes by capillary zone electrophoresis
The present invention provides, among other things, methods for the characterization of recombinant Heparan N-Sulfatase (HNS) during manufacture. The present invention uses capillary zone electrophoresis to determine the charge profile, isoform distribution, and/or glycan profile of recombinant HNS; and represents a quality feature for the batch consistency, storage stability, biological half-life, pharmacokinetic, pharmacodynamic and biological activity of the enzyme. In particular, such characterization methods may be beneficial to optimize conditions and ensure consistency for the manufacture of HNS for the treatment of a patient diagnosed with Sanfilippo syndrome using enzyme replacement therapy.
1. A method of manufacturing a heparan N-sulfatase (HNS) protein comprising a step of analyzing a HNS protein manufactured at a large scale by capillary zone electrophoresis, wherein the analyzing step comprises characterizing the charge profile of the HNS protein.
2. The method of claim 1 , further comprising a step of adjusting a manufacturing condition based on the analysis of the charge profile.
3. The method of claim 1 , wherein the analyzing step is conducted before releasing a manufacturing lot.
4. The method of claim 1 , wherein characterizing the charge profile comprises a step of separating peak groups indicative of absence or presence of charge variants by capillary zone electrophoresis.
5. The method of claim 1 , wherein the charge profile comprises at least 14 peak groups.
6. The method of claim 4 , wherein the charge variants are associated with the absence, presence of varying amounts of sialic acid and/or M6P groups.
7. The method of claim 4 , wherein the charge variants are associated with the absence, presence of varying amount of mono-, di-, tri-sialylated glycans, mono-, di-M6P groups, and combination thereof.
8. The method of claim 1 , wherein the characterizing step comprises quantitatively determining relative migration time and/or relative peak area of each peak group.
9. The method of claim 8 , wherein the relative migration time of each peak group is determined relative to an electroosmotic flow (EOF) marker.
10. The method of claim 8 , wherein the relative peak area of each peak group is calculated by peak area percentage as compared to the total peak areas.
11. The method of claim 1 , wherein the method further comprises determining the quality of the HNS protein.
12. The method of claim 1 , wherein the HNS protein is produced by mammalian cells.
13. The method of claim 12 , wherein the mammalian cells are human cells.
14. The method of claim 1 , wherein the method comprises determining if there is variation in the charge profile of the HNS protein as compared to a reference.
15. The method of claim 14 , wherein the reference is the charge profile of a HNS protein produced from a different manufacturing batch.
16. The method of claim 15 , wherein the method further comprises a step of assessing batch-to-batch charge variability comprising comparing graphs trending the relative peak areas versus peak groups for each HNS protein produced by different batches.
17. The method of claim 1 , wherein the capillary zone electrophoresis is conducted under conditions such that longer migration times correspond to species of increasing negative charges.
18. The method of claim 1 , wherein the capillary zone electrophoresis is conducted using a buffer system comprising Tris at a concentration ranging from about 20-30 mM.
19. The method of claim 1 , wherein the buffer system has a pH ranging from approximately 7.8-8.2.
20. The method of claim 1 , wherein capillary zone electrophoresis is conducted using a capillary with a length ranging between 56-112.5 cm.