Exploring the Chemical Space of Protein Glycosylation in Noncovalent Protein Complexes: An Expedition along Different Structural Levels of Human Chorionic Gonadotropin by Employing Mass Spectrometry

Modern analytical approaches employing high-resolution mass spectrometry (MS) facilitate the generation of a vast amount structural data highly complex glycoproteins. Nevertheless, systematic interpretation this at different levels remains an challenge. The glycoprotein utilized as model system in study, human chorionic gonadotropin (hCG), exists heterodimer composed two heavily glycosylated subunits. In order to unravel multitude glycoforms recombinant hCG (drug product Ovitrelle), we combine established techniques, such released glycan and glycopeptide analysis, with novel high-performance liquid chromatography-mass (HPLC-MS) characterize protein subunits native MS analyze noncovalent complex. Starting from deconvoluted spectrum dimeric comprising about 50 signals, it was possible explore chemical space elucidate complexity that hides behind just signals. Systematic, stepwise integration obtained glycans, glycopeptides, using computational annotation tool allowed us reveal 1031 underlying glycoforms. Additionally, critical quality attributes sialylation core fucosylation were compared for batches Ovitrelle assess potential variability.