iggzero endoglycosidase  (Genovis Inc)

Journal: Glycobiology

Article Title: EndoS and EndoS2 hydrolyze Fc-glycans on therapeutic antibodies with different glycoform selectivity and can be used for rapid quantification of high-mannose glycans

doi: 10.1093/glycob/cwv047

Figure Lengend Snippet: The activity of endoglycosidases EndoS and EndoS2 on therapeutic mAbs. Four approved therapeutic mAbs, cetuximab, adalimumab, panitumumab and denosumab, were deglycosylated with either EndoS or EndoS2 for 30 min at 37°C and fragmented using IdeS to generate Fc and F(ab′)2 fragments that were separated on a 4–12% SDS-PAGE Bis-Tris gel under nonreducing conditions. The first four lanes are antibodies without IdeS fragmentation. This figure is available in black and white in print and in color at Glycobiology online.

Article Snippet: The mAbs and the endoglycosidases EndoS and EndoS2 (Genovis AB, IgGZEROTM, GlycINATORTM) were incubated for 30 min at 37°C in 10 mM PBS and 150 mM NaCl, pH 7.4.

Techniques: Activity Assay, SDS Page

Journal: Glycobiology

Article Title: EndoS and EndoS2 hydrolyze Fc-glycans on therapeutic antibodies with different glycoform selectivity and can be used for rapid quantification of high-mannose glycans

doi: 10.1093/glycob/cwv047

Figure Lengend Snippet: MALDI-TOF analysis of EndoS and EndoS2 hydrolyzed antibody glycans. EndoS or EndoS2 was incubated for 30 min at 37°C and hydrolyzed N -glycans from cetuximab ( A ), panitumumab ( B ), denosumab ( C ) and adalimumab ( D ). Glycans were separated from the enzymatic reaction and analyzed using MALDI-TOF (*identified in low intensities). Glycans were detected as sodium adducts. The detected glycoforms are presented in MALDI mass spectrums as well as tables with corresponding m/z and relative intensity (%). Glycans are drawn according to the Consortium for Functional Glycomics nomenclature ( www.functionalglycomics.org ).

Article Snippet: The mAbs and the endoglycosidases EndoS and EndoS2 (Genovis AB, IgGZEROTM, GlycINATORTM) were incubated for 30 min at 37°C in 10 mM PBS and 150 mM NaCl, pH 7.4.

Techniques: Incubation, Functional Assay

Journal: Glycobiology

Article Title: EndoS and EndoS2 hydrolyze Fc-glycans on therapeutic antibodies with different glycoform selectivity and can be used for rapid quantification of high-mannose glycans

doi: 10.1093/glycob/cwv047

Figure Lengend Snippet: Hydrophilic interaction liquid chromatography (HILIC)-FLD-MS analysis of the EndoS and EndoS2 glycan profile of cetuximab. EndoS2 and EndoS hydrolyzed glycans from cetuximab in a 30 min reaction at 37°C. The glycans were 2-AB labeled and analyzed using a HILIC-FLD-MS setup. Seven major differences in the peaks were identified (marked 1–7), and the masses, retention times and relative abundances are presented in Table .

Article Snippet: The mAbs and the endoglycosidases EndoS and EndoS2 (Genovis AB, IgGZEROTM, GlycINATORTM) were incubated for 30 min at 37°C in 10 mM PBS and 150 mM NaCl, pH 7.4.

Techniques: Hydrophilic Interaction Liquid Chromatography, Glycoproteomics, Labeling

Journal: Glycobiology

Article Title: EndoS and EndoS2 hydrolyze Fc-glycans on therapeutic antibodies with different glycoform selectivity and can be used for rapid quantification of high-mannose glycans

doi: 10.1093/glycob/cwv047

Figure Lengend Snippet: Differences of EndoS and EndoS2 hydrolyzed glycans from cetuximab

Article Snippet: The mAbs and the endoglycosidases EndoS and EndoS2 (Genovis AB, IgGZEROTM, GlycINATORTM) were incubated for 30 min at 37°C in 10 mM PBS and 150 mM NaCl, pH 7.4.

Techniques: Recombinase Polymerase Amplification

Journal: Glycobiology

Article Title: EndoS and EndoS2 hydrolyze Fc-glycans on therapeutic antibodies with different glycoform selectivity and can be used for rapid quantification of high-mannose glycans

doi: 10.1093/glycob/cwv047

Figure Lengend Snippet: EndoS and EndoS2 activity on Fc and F(ab′)2 fragments. ( A ) Cetuximab was deglycosylated with EndoS and EndoS2, cleaved with IdeS and the Fc and F(ab′)2 fragments were separated using affinity purification. The F(ab′)2 and Fc fragments were separated on a 4–12% Bis-Tris gel and analyzed using a LCA lectin blot to detect remaining glycans on the antibody fragments. ( B ) Dilutions of EndoS and EndoS2 were incubated with cetuximab for 30 min, cleaved with IdeS and separated on SDS-PAGE. The amount of deglycosylated Fc fragments (Fc-glyc) were calculated based on densitometry where the Fc fragment generated with IdeS alone was set as reference.

Article Snippet: The mAbs and the endoglycosidases EndoS and EndoS2 (Genovis AB, IgGZEROTM, GlycINATORTM) were incubated for 30 min at 37°C in 10 mM PBS and 150 mM NaCl, pH 7.4.

Techniques: Activity Assay, Affinity Purification, Incubation, SDS Page, Generated

Journal: Glycobiology

Article Title: EndoS and EndoS2 hydrolyze Fc-glycans on therapeutic antibodies with different glycoform selectivity and can be used for rapid quantification of high-mannose glycans

doi: 10.1093/glycob/cwv047

Figure Lengend Snippet: Rapid quantification of mAb high mannose content. The four therapeutic mAbs, cetuximab ( A ), panitumumab ( B ), denosumab ( C ) and adalimumab ( D ), were deglycosylated with EndoS or EndoS2 for 30 min and digested to antibody subunit fragments with IdeS to generate Fc and F(ab′)2 that were separated on RP-UHPLC. The separations of the Fc fragments from all four antibodies are presented. The appearance of two peaks is due to lysine clipping. The integrated peak areas are indicated in blue (IdeS), pink (EndoS) and green (EndoS2), and peak area values are presented in Table .

Article Snippet: The mAbs and the endoglycosidases EndoS and EndoS2 (Genovis AB, IgGZEROTM, GlycINATORTM) were incubated for 30 min at 37°C in 10 mM PBS and 150 mM NaCl, pH 7.4.

Techniques:

Journal: Glycobiology

Article Title: EndoS and EndoS2 hydrolyze Fc-glycans on therapeutic antibodies with different glycoform selectivity and can be used for rapid quantification of high-mannose glycans

doi: 10.1093/glycob/cwv047

Figure Lengend Snippet: Quantification of high-mannose and hybrid peaks using EndoS and EndoS2

Article Snippet: The mAbs and the endoglycosidases EndoS and EndoS2 (Genovis AB, IgGZEROTM, GlycINATORTM) were incubated for 30 min at 37°C in 10 mM PBS and 150 mM NaCl, pH 7.4.

Techniques: