High-throughput Glycosylation Analysis of Therapeutic Immunoglobulin G by Capillary Gel Electrophoresis Using a DNA Analyzer

Overview

Journal MAbs

Specialty Allergy & Immunology

Date 2013 Oct 19

PMID 24135630

Citations 21

Authors

Dietmar Reusch

Markus Haberger

Tobias Kailich

Anna-Katharina Heidenreich

Michael Kampe

Patrick Bulau

Manfred Wuhrer

Affiliations

Soon will be listed here.

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.

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