title: Ultrasensitive capillary electrophoretic analysis of potentially immunogenic carbohydrate residues in biologics: galactose-α-1,3-galactose containing oligosaccharides.
abstract: With the recent growth of the global monoclonal antibody market, ultrasensitive techniques are required for rapid analysis of possible immunogenic residues, such as galactose-α-1,3-galactose (α-1,3-Gal) on therapeutic proteins expressed in murine or CHO cell lines. We report a capillary electrophoretic approach in conjunction with exoglycosidase digestion for structural elucidation of IgG N-glycans containing the above immunogenic epitope. The method uses commercially available reagents and instrumentation, thus making it readily available for implementation and validation within the biotechnology industry. The method was first evaluated using polyclonal mouse IgG N-glycans which are known to contain α-1,3-Gal containing epitopes. High reproducibility in migration time enabled determination of GU values for five α-1,3-Gal containing structures when the data from all samples analyzed was combined. The method was successfully applied to the analysis of a NCI reference standard monoclonal antibody and two development phase monoclonal antibodies. The limit of detection and limit of quantitation were 1 and 2 μg of intact protein IgG starting material, respectively, further indicating the high sensitivity of the described method.
authors.affiliation: Barnett Institute, Northeastern University, Boston, Massachusetts 02115, United States.
authors.forename: Zoltan
authors.initials: Z
authors.lastname: Szabo
authors.forename: Andras
authors.initials: A
authors.lastname: Guttman
authors.forename: Jonathan
authors.initials: J
authors.lastname: Bones
authors.forename: Randi L
authors.initials: RL
authors.lastname: Shand
authors.forename: David
authors.initials: D
authors.lastname: Meh
authors.forename: Barry L
authors.initials: BL
authors.lastname: Karger
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology; D002241:Carbohydrates / Q000737:chemistry* / Q000276:immunology; D019075:Electrophoresis, Capillary / Q000379:methods*; D005690:Galactose / Q000737:chemistry* / Q000276:immunology; D006026:Glycoside Hydrolases / Q000378:metabolism; D051379:Mice; D009844:Oligosaccharides / Q000737:chemistry* / Q000276:immunology / Q000378:metabolism
chemical_list: D000911:Antibodies, Monoclonal; D002241:Carbohydrates; D009844:Oligosaccharides; D006026:Glycoside Hydrolases; D005690:Galactose