title: Remodeling the oligosaccharides on β-glucocerebrosidase using hydrophobic interaction chromatography and applications of hydroxyl ethyl starch for improving remodeling and enhancing protein stability.
abstract: In this article, we describe a hydrophobic interaction chromatography (HIC) method to remodel the carbohydrates on recombinant human β-glucocerebrosidase (GCR) and the use of hydroxyl ethyl starch (HES) an ethylated starch polymer, to improve this process. GCR is a therapeutic protein used in the treatment of Gaucher disease, a life threatening condition in which patients lack sufficient functional levels of this enzyme. Gaucher disease is the most common inherited lysosomal storage disorder resulting in hepatomegaly, splenomegaly, and bone and lung pathology due to the accumulation of glucosylceramide in the lysosomes of macrophages (Beutler and Grabowski, 2001). The oligosaccharide remodeling of GCR, performed on HIC using three enzymes that remove sugars, increases macrophage uptake through the mannose receptor and thereby lowers its therapeutic dose versus unmodified GCR (Furbish et al., 1981; Van Patten et al., 2007). In this article we describe findings that the addition of HES lowered the amounts of three deglycosylating enzymes needed for remodeling GCR. HES also stabilized the activity of α-glucosidase, α-galactosidase, and GCR under conditions in which these three enzymes rapidly lose activity in the absence of this polymer. Circular dichroism (CD) and second derivative UV spectroscopy revealed that the secondary and tertiary structure of α-glucosidase was unchanged while for GCR there was a slight compaction of the secondary structure but no apparent affect on the tertiary structure. The thermal stability of both GCR and α-glucosidase were enhanced by HES as both molecules showed an increased transition midpoint (T(m)).
authors.affiliation: Purification Development, Genzyme Corporation, Framingham, Massachusetts 01701, USA. frank.riske@genzyme.com
authors.forename: Frank
authors.initials: F
authors.lastname: Riske
authors.forename: Andrea
authors.initials: A
authors.lastname: Hamilton
authors.forename: Cheng
authors.initials: C
authors.lastname: Zhang
authors.forename: Mike
authors.initials: M
authors.lastname: Hayes
mesh_terms: D002845:Chromatography / Q000379:methods; D002942:Circular Dichroism; D004795:Enzyme Stability; D005962:Glucosylceramidase / Q000737:chemistry*; D006801:Humans; D057927:Hydrophobic and Hydrophilic Interactions; D009844:Oligosaccharides / Q000032:analysis*; D011487:Protein Conformation; D055550:Protein Stability; D013056:Spectrophotometry, Ultraviolet; D013213:Starch / Q000378:metabolism; D013696:Temperature; D000519:alpha-Galactosidase / Q000737:chemistry; D000520:alpha-Glucosidases / Q000737:chemistry
chemical_list: D009844:Oligosaccharides; D013213:Starch; D000520:alpha-Glucosidases; D000519:alpha-Galactosidase; D005962:Glucosylceramidase