title: High-throughput glycosylation analysis of therapeutic immunoglobulin G by capillary gel electrophoresis using a DNA analyzer.
abstract: The Fc glycosylation of therapeutic antibodies is crucial for their effector functions and their behavior in pharmacokinetics and pharmacodynamics. To monitor the Fc glycosylation in bioprocess development and characterization,high-throughput techniques for glycosylation analysis are needed. Here, we describe the development of a largely automated high-throughput glycosylation profiling method with multiplexing capillary-gel-electrophoresis (CGE) with laser induced fluorescence (LIF) detection using a DNA analyzer. After PNGaseF digestion, the released glycans were labeled with 9-aminopyrene-1,3,6-trisulfonic acid (APTS) in 96-well plates, which was followed by the simultaneous analysis of up to 48 samples. The peak assignment was conducted by HILIC-UPLC-MS/MS of the APTS-labeled glycans combined with peak fractionation and subsequent CGE-LIF analysis of the MS-characterized fractions. Quantitative data evaluation of the various IgG glycans was performed automatically using an in-house developed software solution. The excellent method accuracy and repeatability of the test system was verified by comparison with two UPLC-based methods for glycan analysis. Finally, the practical value of the developed method was demonstrated by analyzing the antibody glycosylation profiles from fermentation broths after small scale protein A purification.
authors.forename: Dietmar
authors.initials: D
authors.lastname: Reusch
authors.forename: Markus
authors.initials: M
authors.lastname: Haberger
authors.forename: Tobias
authors.initials: T
authors.lastname: Kailich
authors.forename: Anna-Katharina
authors.initials: AK
authors.lastname: Heidenreich
authors.forename: Michael
authors.initials: M
authors.lastname: Kampe
authors.forename: Patrick
authors.initials: P
authors.lastname: Bulau
authors.forename: Manfred
authors.initials: M
authors.lastname: Wuhrer
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000737:chemistry*; D016466:CHO Cells; D006224:Cricetinae; D003412:Cricetulus; D019075:Electrophoresis, Capillary; D005456:Fluorescent Dyes / Q000737:chemistry*; D006031:Glycosylation; D007074:Immunoglobulin G / Q000737:chemistry*; D011134:Polysaccharides / Q000737:chemistry*; D011721:Pyrenes / Q000737:chemistry; D017422:Sequence Analysis, DNA / Q000295:instrumentation*; D012984:Software
chemical_list: C098210:1-aminopyrene-3,6,8-trisulfonic acid; D000911:Antibodies, Monoclonal; D005456:Fluorescent Dyes; D007074:Immunoglobulin G; D011134:Polysaccharides; D011721:Pyrenes