title: Quantitative capillary zone electrophoresis method for the precise determination of charge differences arising from the manufacture of heparan-N-sulfatase.
abstract: A rapid and reproducible high-resolution capillary zone electrophoresis (CZE) method capable of resolving the charge isoforms of intact heparan-N-sulfatase (HNS) has been developed to monitor the charge consistency across different batches of HNS. Separation was carried out using a bare fused silica capillary with a buffer system composed of 25 mM Tris, pH 8.0. This CZE method allowed the separation and integration of 14 peaks, each arising from differences in the amount of sialic-acid and mannose-6-phosphate bearing glycoforms, which were confirmed using enzymatically modified samples. Standard conditioning and rinsing conditions of the capillary were used to achieve optimal repeatability. Excellent day-to-day precision was obtained for migration times of each peak relative to the electroosmotic flow marker with relative standard deviation (RSD)≤ 0.5%. The precision of the relative peak areas (peak area percentages) ranged from 0.6% to 2.8% RSD for the major isoforms (peaks 3-12), from 4.0% to 5.0% RSD for peaks 1 and 2, and from 7.4% to 23.2% RSD for peaks 13 and 14. The method was able to discriminate charge variation across different batches of HNS, including those with both significant and minor process changes.
authors.affiliation: Shire, Lexington, MA, USA. droseman@shire.com
authors.forename: Daniel S
authors.initials: DS
authors.lastname: Roseman
authors.forename: Robert
authors.initials: R
authors.lastname: Weinberger
mesh_terms: D019075:Electrophoresis, Capillary / Q000379:methods*; D006863:Hydrogen-Ion Concentration; D013429:Sulfatases / Q000737:chemistry*
chemical_list: D013429:Sulfatases; C021346:heparan sulfate sulfatase
keywords: 4-(2-hydroxyethyl)piperazine-1-ethanesulfonic acid; BGE; CIEF; CITP; CZE; Capillary zone electrophoresis; Charge variant; EDTA; EOF; Glycosylation; Gly–Gly; HEPES; HNS; HPAE-PAD; Heparan-N-sulfatase; Heterogeneity; Isoform; M6P; NP40; RSD; SDS; Tergitol-type NP40 nonyl phenoxypolyethoxylethanol; Tris(hydroxymethyl)aminomethane; Tris(hydroxymethyl)aminomethane hydrochloride; Tris–HCl; Tris–base; background electrolyte buffer; capillary isoelectric focusing; capillary isotachophoresis; capillary zone electrophoresis; diglycine; electroosmotic flow; ethylenediaminetetraacetic acid; heparan-N-sulfatase; high-performance anion-exchange pulsed amperometric detection; mannose-6-phosphate; relative standard deviation; sodium dodecyl sulphate; tergitol-type NP-40 nonyl phenoxypolyethoxylethanol