Comparison of methods for the analysis of therapeutic immunoglobulin G Fc-glycosylation profiles—Part 1: Separation-based methods |
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Authors: | Dietmar Reusch Markus Haberger Bernd Maier Maria Maier Ronny Kloseck Boris Zimmermann Michaela Hook Zoltan Szabo Samnang Tep Jo Wegstein Nadja Alt Patrick Bulau Manfred Wuhrer |
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Institution: | 1.Pharma Biotech Development Penzberg; Roche Diagnostics GmbH; Penzberg, Germany;2.ProZyme, Inc.; Hayward, CA USA;3.Center for Proteomics and Metabolomics; Leiden University Medical Center; Leiden, The Netherlands;4.Division of BioAnalytical Chemistry; Department of Chemistry and Pharmaceutical Sciences; VU University Amsterdam; Amsterdam, The Netherlands |
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Abstract: | Immunoglobulin G (IgG) crystallizable fragment (Fc) glycosylation is crucial for antibody effector functions, such as antibody-dependent cell-mediated cytotoxicity, and for their pharmacokinetic and pharmacodynamics behavior. To monitor the Fc-glycosylation in bioprocess development, as well as product characterization and release analytics, reliable techniques for glycosylation analysis are needed. A wide range of analytical methods has found its way into these applications. In this study, a comprehensive comparison was performed of separation-based methods for Fc-glycosylation profiling of an IgG biopharmaceutical. A therapeutic antibody reference material was analyzed 6-fold on 2 different days, and the methods were compared for precision, accuracy, throughput and other features; special emphasis was placed on the detection of sialic acid-containing glycans. Seven, non-mass spectrometric methods were compared; the methods utilized liquid chromatography-based separation of fluorescent-labeled glycans, capillary electrophoresis-based separation of fluorescent-labeled glycans, or high-performance anion exchange chromatography with pulsed amperometric detection. Hydrophilic interaction liquid chromatography-ultra high performance liquid chromatography of 2-aminobenzamide (2-AB)-labeled glycans was used as a reference method. All of the methods showed excellent precision and accuracy; some differences were observed, particularly with regard to the detection and quantitation of minor glycan species, such as sialylated glycans. |
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Keywords: | IgG glycosylation monoclonal antibody (mAb) HILIC-UPLC 2-AB labeling APTS labeling method comparison DNA analyzer HPAEC glycan analysis CE-LIF high-throughput |
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