title: Sample preparation for N-glycosylation analysis of therapeutic monoclonal antibodies by electrophoresis.
abstract: There are a considerable number of biopharmaceuticals that have been approved for clinical use in the past decade. Over half of these new generation drugs are glycoproteins, such as monoclonal antibodies or other recombinant glycoproteins, which are mostly produced in mammalian cell lines. The linked carbohydrate moieties affect not only their physicochemical properties and thermal stability but also crucial features like receptor-binding activity, circulating half-life, as well as immunogenicity. The structural diversity of these attached glycans can be manifested in altered monosaccharide composition and linkages/positions among the monosaccharide building blocks. In addition, as more and more biosimilar products hit the market, understanding the effects of their glycosylation modification has become a recent target in efficacy and safety issues. To ensure consistent quality of these products, glycosylation profiles have to be monitored and controlled in all steps of the manufacturing process, i.e., from clone selection to lot release. In this paper, we describe some of the recently introduced and commonly used sample preparation techniques for capillary electrophoresis (CE)-based profiling and structural elucidation of N-glycans. The presented protocols include protein A affinity partitioning of monoclonal antibodies (mAbs), enzymatic release of the N-linked glycans, labeling of the liberated carbohydrates, reaction mixture purification techniques to remove the excess labeling reagent, and high-resolution and rapid capillary electrophoresis-laser-induced fluorescence (CE-LIF)-based profiling of the labeled and purified N-glycans.
authors: Horváth Laboratory of Bioseparation Sciences, University of Debrecen, Nagyerdei krt 98, Elméleti tömb, 1/111, 4032, Debrecen, Hungary.
authors: Ákos
authors: Á
authors: Szekrényes
authors: Jan
authors: J
authors: Partyka
authors: Csaba
authors: C
authors: Varadi
authors: Jana
authors: J
authors: Krenkova
authors: Frantisek
authors: F
authors: Foret
authors: András
authors: A
authors: Guttman
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000737:chemistry*; D019075:Electrophoresis, Capillary / Q000379:methods*; D006031:Glycosylation; D006801:Humans; D011134:Polysaccharides / Q000737:chemistry; D013194:Staining and Labeling / Q000379:methods
chemical_list: D000911:Antibodies, Monoclonal; D011134:Polysaccharides