High-throughput work flow for IgG Fc-glycosylation analysis of biotechnological samples
| Autor: | Niklas Engler, Maurice H. J. Selman, Dietmar Reusch, André M. Deelder, Manfred Wuhrer, Markus Haberger, Patrick Bulau |
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| Jazyk: | angličtina |
| Rok vydání: | 2013 |
| Předmět: |
Spectrometry
Mass Electrospray Ionization Glycosylation Electrospray ionization IgG glycosylation Molecular Sequence Data Biophysics Biochemistry Immunoglobulin G Chromatography Affinity Biotechnological samples chemistry.chemical_compound Automation Bioreactors Humans Trypsin Solid phase extraction Electrospray mass spectrometry Cytotoxicity Staphylococcal Protein A Molecular Biology Chromatography High Pressure Liquid Chromatography biology Solid Phase Extraction Glycopeptides Cell Biology Recombinant monoclonal antibodies Glycopeptide Recombinant Proteins High-Throughput Screening Assays Immunoglobulin Fc Fragments Immobilized Proteins chemistry Carbohydrate Sequence biology.protein Fermentation Antibody Biotechnology |
| Zdroj: | Analytical Biochemistry, 432(2), 82-89 |
| Popis: | Immunoglobulin G (IgG) fragment crystallizable (Fc) glycosylation is crucial for antibody effector functions such as antibody-dependent cellular cytotoxicity and complement-dependent cytotoxicity. To monitor IgG Fc glycosylation, high-throughput techniques for glycosylation analysis are needed in the biotechnology industry. Here we describe the development of a fully automated high-throughput method based on glycopeptide analysis. Samples are prepared in 96-well plates. The IgG’s are purified directly from fermentation broths by means of immobilized protein A followed by trypsin digestion. Glycopeptides are purified by hydrophilic interaction solid-phase extraction and analyzed by electrospray mass spectrometry in the positive-ion mode. Data are automatically processed and relative intensities of the various IgG glycopeptides are obtained. The intermediate precision of the method is below 5% for the five major glycoforms of an IgG1 antibody. The newly developed method is suitable for glycosylation profiling of IgG’s from fermentation broths. We compared the developed method to other glycoanalytical methods and successfully applied it to analyze the fermentation time course of two different clones of the same therapeutic antibody. |
| Databáze: | OpenAIRE |
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