title: Comparison of methods for the analysis of therapeutic immunoglobulin G Fc-glycosylation profiles--part 1: separation-based methods.
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.
authors.affiliation: a Pharma Biotech Development Penzberg; Roche Diagnostics GmbH ; Penzberg , Germany.
authors.forename: Dietmar
authors.initials: D
authors.lastname: Reusch
authors.forename: Markus
authors.initials: M
authors.lastname: Haberger
authors.forename: Bernd
authors.initials: B
authors.lastname: Maier
authors.forename: Maria
authors.initials: M
authors.lastname: Maier
authors.forename: Ronny
authors.initials: R
authors.lastname: Kloseck
authors.forename: Boris
authors.initials: B
authors.lastname: Zimmermann
authors.forename: Michaela
authors.initials: M
authors.lastname: Hook
authors.forename: Zoltan
authors.initials: Z
authors.lastname: Szabo
authors.forename: Samnang
authors.initials: S
authors.lastname: Tep
authors.forename: Jo
authors.initials: J
authors.lastname: Wegstein
authors.forename: Nadja
authors.initials: N
authors.lastname: Alt
authors.forename: Patrick
authors.initials: P
authors.lastname: Bulau
authors.forename: Manfred
authors.initials: M
authors.lastname: Wuhrer
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000737:chemistry*; D016466:CHO Cells; D002851:Chromatography, High Pressure Liquid; D006224:Cricetinae; D003412:Cricetulus; D006031:Glycosylation; D007141:Immunoglobulin Fc Fragments / Q000737:chemistry*; D013058:Mass Spectrometry
chemical_list: D000911:Antibodies, Monoclonal; D007141:Immunoglobulin Fc Fragments
keywords: 2-AB labeling; 2-AB, 2-aminobenzamide; ANTS, 8-aminonaphthalene-1, 3, 6-trisulfonate; APTS labeling; APTS, 8-aminopyrene-1, 3, 6-trisulfonic acid; CCGE, cartridge-based capillary gel electrophoresis; CE-LIF; CE-LIF, capillary electrophoresis-laser induced fluorescence; CHO, Chinese hamster ovary; DNA analyzer; DSA-FACE, DNA-sequencer-aided fluorophore-assisted carbohydrate electrophoresis; ESI-MS, electrospray ionization-mass spectrometry; Fab, fragment, antigen-binding; Fc, fragment crystallizable; HILIC-UPLC; HILIC-UPLC, hydrophilic interaction liquid chromatography-ultra high performance liquid chromatography; HPAEC; HPAEC-PAD, high-performance anion exchange chromatography with pulsed amperometric detection; HPLC, high performance liquid chromatography; HR, high resolution; IAB, InstantAB labeling; IgG glycosylation; IgG, immunoglobulin G; MALDI-MS, matrix-assisted laser desorption/ionization-mass spectrometry; glycan analysis; high-throughput; mAb, monoclonal antibody; method comparison; monoclonal antibody (mAb)