Method of analyzing the charge profile of heparan N-sulfatase

Patent metadata

Field	Value
Publication number	US-9290793-B2
Application number	US-201414210058-A
Country	US
Kind code	B2
Filing date	Mar 13, 2014
Priority date	Mar 13, 2013
Publication date	Mar 22, 2016
Grant date	Mar 22, 2016

Abstract

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.

First claim

I claim:

1. A method of analyzing a charge profile of a heparan N-sulfatase (HNS) protein comprising characterizing the charge profile of the HNS protein by capillary zone electrophoresis (CZE),
wherein the CZE separates peak groups indicative of absence or presence of charge variants and,
wherein the charge variants are associated with absence or presence of varying amounts of sialic acid and/or mannose-6-phosphate.

2. The method of claim 1 , wherein the charge profile comprises at least 14 peak groups.

3. The method of claim 1 , wherein the characterizing comprises quantitatively determining relative migration time and/or relative peak area of each peak group.

4. The method of claim 1 , wherein the HNS protein is produced by mammalian cells.

5. The method of claim 1 , wherein the HNS protein is produced at a large scale.

6. 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.

7. 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.

8. The method of claim 1 , wherein the capillary zone electrophoresis is conducted using a buffer system comprising Tris.

9. The method of claim 8 , wherein the buffer system has a pH ranging from approximately 7.8-8.2.

10. The method of claim 1 , wherein the capillary zone electrophoresis is conducted using a capillary with a length ranging between 56-112.5 cm.

11. The method of claim 1 , wherein the charge profile comprises at least 5 peak groups.

Assignees

• Shire Human Genetic Therapies

Inventors

• Roseman Daniel

Classifications

• G01N27/44791

  Microapparatus (sample containers with integrated microfluidic structures B01L3/5027) · CPC title

• C12N9/16

  acting on ester bonds (3.1) · CPC title

• C12Q1/44 Primary

  involving esterase · CPC title

• A61K38/00

  Medicinal preparations containing peptides (peptides containing beta-lactam rings A61K31/00; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, A61K31/00; ergot alkaloids of the cyclic peptide type A61K31/48; containing macromolecular compounds having statistically distributed amino acid units A61K31/74; medicinal preparations containing antigens or antibodies A61K39/00; medicinal preparations characterised by the non-active ingredients, e.g. peptides as drug carriers, A61K47/00) · CPC title

• G01N27/44739

  Collecting the separated zones, e.g. blotting to a membrane or punching of gel spots · CPC title