Sample preparation for N-glycosylation analysis of therapeutic monoclonal antibodies by electrophoresis

Ákos Szekrényes, Jan Partyka, Csaba Varadi, Jana Krenkova, Frantisek Foret, A. Guttman

Research output: Contribution to journalArticle

8 Citations (Scopus)

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.

Original languageEnglish
Pages (from-to)183-195
Number of pages13
JournalMethods in molecular biology (Clifton, N.J.)
Volume1274
DOIs
Publication statusPublished - 2015

Fingerprint

Glycosylation
Polysaccharides
Electrophoresis
Monoclonal Antibodies
Monosaccharides
Capillary Electrophoresis
Biosimilar Pharmaceuticals
Glycoproteins
Carbohydrates
Staphylococcal Protein A
Therapeutics
Half-Life
Lasers
Clone Cells
Hot Temperature
Fluorescence
Safety
Cell Line
Pharmaceutical Preparations

Keywords

  • Biopharmaceuticals
  • Capillary electrophoresis
  • Fluorophore labeling
  • N-glycan analysis
  • Therapeutic monoclonal antibody

ASJC Scopus subject areas

  • Molecular Biology
  • Genetics
  • Medicine(all)

Cite this

Sample preparation for N-glycosylation analysis of therapeutic monoclonal antibodies by electrophoresis. / Szekrényes, Ákos; Partyka, Jan; Varadi, Csaba; Krenkova, Jana; Foret, Frantisek; Guttman, A.

In: Methods in molecular biology (Clifton, N.J.), Vol. 1274, 2015, p. 183-195.

Research output: Contribution to journalArticle

Szekrényes, Ákos ; Partyka, Jan ; Varadi, Csaba ; Krenkova, Jana ; Foret, Frantisek ; Guttman, A. / Sample preparation for N-glycosylation analysis of therapeutic monoclonal antibodies by electrophoresis. In: Methods in molecular biology (Clifton, N.J.). 2015 ; Vol. 1274. pp. 183-195.
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