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.
journal_name
MAbsjournal_title
mAbsauthors
Reusch D,Haberger M,Kailich T,Heidenreich AK,Kampe M,Bulau P,Wuhrer Mdoi
10.4161/mabs.26712subject
Has Abstractpub_date
2014-01-01 00:00:00pages
185-96issue
1eissn
1942-0862issn
1942-0870pii
26712journal_volume
6pub_type
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