title: High-throughput work flow for IgG Fc-glycosylation analysis of biotechnological samples.
abstract: 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.
authors.affiliation: Pharma Biotech Development Penzberg, Roche Diagnostics GmbH, 82377 Penzberg, Germany. dietmar.reusch@roche.com
authors.forename: Dietmar
authors.initials: D
authors.lastname: Reusch
authors.forename: Markus
authors.initials: M
authors.lastname: Haberger
authors.forename: Maurice H J
authors.initials: MH
authors.lastname: Selman
authors.forename: Patrick
authors.initials: P
authors.lastname: Bulau
authors.forename: André M
authors.initials: AM
authors.lastname: Deelder
authors.forename: Manfred
authors.initials: M
authors.lastname: Wuhrer
authors.forename: Niklas
authors.initials: N
authors.lastname: Engler
mesh_terms: D001331:Automation; D019149:Bioreactors; D001709:Biotechnology; D002240:Carbohydrate Sequence; D002846:Chromatography, Affinity; D002851:Chromatography, High Pressure Liquid; D006020:Glycopeptides / Q000032:analysis / Q000302:isolation & purification; D006031:Glycosylation; D057166:High-Throughput Screening Assays; D006801:Humans; D055767:Immobilized Proteins / Q000378:metabolism; D007141:Immunoglobulin Fc Fragments / Q000032:analysis* / Q000235:genetics / Q000378:metabolism; D007074:Immunoglobulin G / Q000032:analysis* / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000032:analysis / Q000096:biosynthesis / Q000235:genetics; D052616:Solid Phase Extraction; D021241:Spectrometry, Mass, Electrospray Ionization; D013205:Staphylococcal Protein A / Q000378:metabolism; D014357:Trypsin / Q000378:metabolism; D006023:Glycoproteins
chemical_list: D006020:Glycopeptides; D055767:Immobilized Proteins; D007141:Immunoglobulin Fc Fragments; D007074:Immunoglobulin G; D011994:Recombinant Proteins; D013205:Staphylococcal Protein A; D014357:Trypsin; C046179:glycosylated IgG; D006023:Glycoproteins