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.
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).