title: High-throughput analysis of therapeutic and diagnostic monoclonal antibodies by multicapillary SDS gel electrophoresis in conjunction with covalent fluorescent labeling.
abstract: Capillary gel electrophoresis (CGE) in the presence of sodium dodecyl sulfate (SDS) is a well-established and widely used protein analysis technique in the biotechnology industry, and increasingly becoming the method of choice that meets the requirements of the standards of International Conference of Harmonization (ICH). Automated single channel capillary electrophoresis systems are usually equipped with UV absorbance and/or laser-induced fluorescent (LIF) detection options offering general applicability and high detection sensitivity, respectively; however, with limited throughput. This shortcoming is addressed by the use of multicapillary gel electrophoresis (mCGE) systems with LED-induced fluorescent detection (LED-IF), also featuring automation and excellent detection sensitivity, thus widely applicable to rapid and large-scale analysis of biotherapeutics, especially monoclonal antibodies (mAb). The methodology we report in this paper is readily applicable for rapid purity assessment and subunit characterization of IgG molecules including detection of non-glycosylated heavy chains (NGHC) and separation of possible subunit variations such as truncated light chains (Pre-LC) or alternative splice variants. Covalent fluorophore derivatization and the mCGE analysis of the labeled IgG samples with multi-capillary gel electrophoresis are thoroughly described. Reducing and non-reducing conditions were both applied with and without peptide N-glycosidase F mediated deglycosylation.
authors: Horváth Laboratory of Bioseparation Sciences, University of Debrecen, Debrecen, Hungary.
authors: Ákos
authors: Á
authors: Szekrényes
authors: Udo
authors: U
authors: Roth
authors: Márta
authors: M
authors: Kerékgyártó
authors: Andrea
authors: A
authors: Székely
authors: István
authors: I
authors: Kurucz
authors: Karen
authors: K
authors: Kowalewski
authors: András
authors: A
authors: Guttman
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000032:analysis* / Q000302:isolation & purification; D002417:Cattle; D019075:Electrophoresis, Capillary / Q000191:economics / Q000379:methods*; D005456:Fluorescent Dyes / Q000032:analysis*; D057166:High-Throughput Screening Assays / Q000191:economics / Q000379:methods*; D007074:Immunoglobulin G / Q000032:analysis* / Q000302:isolation & purification; D057230:Limit of Detection; D051379:Mice; D012967:Sodium Dodecyl Sulfate / Q000737:chemistry*
chemical_list: D000911:Antibodies, Monoclonal; D005456:Fluorescent Dyes; D007074:Immunoglobulin G; D012967:Sodium Dodecyl Sulfate