To monitor the Fc glycosylation of therapeutic immunoglobulin G in bioprocess development, product characterization and release analytics, reliable techniques for glycosylation analysis are needed. Several analytical methods are suitable for this application. We recently presented results comparing detection methods for glycan analysis that are separation-based, but did not include mass spectrometry (MS). In the study reported here, we comprehensively compared MS-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 investigated were compared with respect to precision, accuracy, throughput and analysis time. Emphasis was put on the detection and quantitation of sialic acid-containing glycans. Eleven MS methods were compared to hydrophilic interaction liquid chromatography of 2-aminobenzamide labeled glycans with fluorescence detection, which served as a reference method and was also used in the first part of the study. The methods compared include electrospray MS of the heavy chain and Fc part after limited digestion, liquid chromatography MS of a tryptic digest, porous graphitized carbon chromatography MS of released glycans, electrospray MS of glycopeptides, as well as matrix assisted laser desorption ionization MS of glycans and glycopeptides. Most methods showed excellent precision and accuracy. Some differences were observed with regard to the detection and quantitation of low abundant glycan species like the sialylated glycans and the amount of artefacts due to in-source decay.
Keywords: 2-AB, 2-aminobenzamide; CE, capillary electrophoresis; ESI-MS; ESI-MS, electrospray ionization-mass spectrometry; Fab, fragment antigen-binding; Fc, fragment crystallizable; HILIC-UHPLC, hydrophilic interaction liquid chromatography-ultra high performance liquid chromatography; HILIC-UPLC; HPAEC-PAD, high-performance anion exchange chromatography with pulsed amperometric detection; IdeS protease, proteolytic enzyme like protease from Streptococcus pyrogenes; IgG glycosylation; IgG, immunoglobulin G; LC-MS; LCMS, liquid chromatography-mass spectrometry; MALDI, matrix assisted laser desorption ionization; MALDI-MS; PGC-MS, porous graphitized carbon chromatography- mass spectrometry; PNGase F, Peptide-N-Glycosidase F; RP-HPLC, reversed phase high performance liquid chromatography; TIC, total ion chromatogram; glycan analysis; mAb, monoclonal antibody; mass spectrometry; method comparison; monoclonal antibody (mAb).