Sample Preparation for N-Glycosylation Analysis of Therapeutic Monoclonal Antibodies by Electrophoresis

Szekrényes, Ákos and Partyka, Jan and Varadi, Csaba and Krenkova, Jana and Foret, Frantisek and Guttman, András (2015) Sample Preparation for N-Glycosylation Analysis of Therapeutic Monoclonal Antibodies by Electrophoresis. METHODS IN MOLECULAR BIOLOGY, 1274. pp. 183-195. ISSN 1064-3745

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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 modifi cation has become a recent target in effi cacy and safety issues. To ensure consistent quality of these products, glycosylation profi les 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 profi ling and structural elucidation of N-glycans. The presented pro- tocols include protein A affi nity partitioning of monoclonal antibodies (mAbs), enzymatic release of the N-linked glycans, labeling of the liberated carbohydrates, reaction mixture purifi cation techniques to remove the excess labeling reagent, and high-resolution and rapid capillary electrophoresis-laser-induced fl uorescence (CE-LIF)-based profi ling of the labeled and purifi ed N-glycans.

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