RAPID MONITORING OF SITE-SPECIFIC GLYCOSYLATION MICROHETEROGENEITY OFRECOMBINANT HUMAN INTERFERON-GAMMA

An analytical system is presented for rapid assessment of site-specifi c microheterogeneity of the two potential N-linked glycosylation sites of recombinant human interferon-gamma (IFN-gamma) derived from Chines e hamster ovary cell culture. The target protein is first purified fro m culture supernatant by immunoaffinity chromatography, and the acidic eluent is neutralized via an in-line mixing tee. Online proteolysis i s rapidly performed by an immobilized trypsin cartridge, and reversed- phase chromatography isolates the two pools of glycopeptides represent ing the potential glycosylation sites, Following off-line analysis by matrix-assisted laser-desorption ionization/time-of-flight (MALDI/TOF) mass spectrometry, observed mass shifts of glycopeptides relative to the known masses of their amino acid portions are correlated to site-s pecific oligosaccharide structures. Desialylation of glycopeptides by sialidase treatment on the MALDI sample plate allows for quantitative estimations of asialoglycan structures by MALDI/TOF. This methodology permits glycoprotein microheterogeneity to be evaluated in a time fram e of similar to 2 h, utilizing as little as 0.5 mu g (25 pmol) of prod uct. Results of monitoring a batch culture are presented as well as an alysis of a culture containing deoxymannojirimycin, an inhibitor of gl ycoprotein processing.