Characterization of the N-linked oligosaccharides from human chorionic gonadotropin expressed in the methylotrophic yeast Pichia pastoris

Abstract

Human chorionic gonadotropin (hCG) is a heterodimeric, placental glycoprotein hormone involved in the maintenance of the corpus luteum during the first trimester of pregnancy. Biologically active hCG has been successfully expressed in the yeast Pichia pastoris (phCG). In the context of structural studies and therapeutic applications of phCG, detailed information about its glycosylation pattern is a prerequisite. To this end N-glycans were released with peptide-N⁴-(N-acetyl-β-glucosaminyl)asparagine amidase F and fractionated via anion-exchange chromatography (Resource Q) yielding both neutral (80%) and charged, phosphate-containing (20%) high-mannosetype structures. Subfractionations were carried out via normal phase (Lichrosorb-NH2) and high-pH anion-exchange (CarboPac PA-1) chromatography. Structural analyses of the released N-glycans were carried out by using HPLC profiling of fluorescent 2-aminobenzamide derivatives, MALDI-TOF mass spectrometry, and 500-MHz ¹H-NMR spectroscopy. Detailed neutral oligosaccharide structures, in the range of Man 8GlcNAc₂ to Man₁₁GlcNAc₂ including molecular isomers, could be established, and structures up to Man₁5GlcNAc₂ were indicated. Phosphatecontaining oligosaccharides ranged from Man9PGlcNAc2 to Man ₁₃PGlcNAc₂. Mannosyl O-glycans were not detected. Profiling studies carried out on different production batches showed that the oligosaccharide structures are similar, but their relative amounts varied with the culturing media.