Rapid monitoring of site-specific glycosylation microheterogeneity of recombinant human interferon-gamma.
An analytical system is presented for rapid assessment of site-specific microheterogeneity of the two potential N-linked glycosylation sites of recombinant human interferon-gamma (IFN-gamma) derived from Chinese hamster ovary cell culture. The target protein is first purified from culture supernatant by immunoaffinity chromatography, and the acidic eluent is neutralized via an in-line mixing tee. On-line proteolysis is rapidly performed by an immobilized trypsin cartridge, and reversed-phase chromatography isolates the two pools of glycopeptides representing 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-specific 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 frame of approximately 2 h, utilizing as little as 0.5 microgram (25 pmol) of product. Results of monitoring a batch culture are presented as well as analysis of a culture containing deoxymannojirimycin, an inhibitor of glycoprotein processing.
Harmon
B J
BJ
Biotechnology Process Engineering Center, Massachusetts Institute of Technology, Cambridge 02139-4308, USA.
Gu
X
X
Wang
D I
DI
D017485:1-Deoxynojirimycin / Q000032:analysis; D000595:Amino Acid Sequence; D000818:Animals; D001212:Asialoglycoproteins / Q000032:analysis; D016466:CHO Cells; D002240:Carbohydrate Sequence; D006224:Cricetinae; D006023:Glycoproteins / Q000032:analysis; D006031:Glycosylation; D006801:Humans; D007371:Interferon-gamma / Q000032:analysis* / Q000737:chemistry; D008969:Molecular Sequence Data; D011994:Recombinant Proteins; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*
D001212:Asialoglycoproteins; D006023:Glycoproteins; D011994:Recombinant Proteins; C046697:glycosylated gamma interferon; D017485:1-Deoxynojirimycin; D007371:Interferon-gamma