title: Characterization of oligosaccharides in recombinant tissue plasminogen activator produced in Chinese hamster ovary cells: two decades of analytical technology development.
abstract: Recombinant tissue plasminogen activator (rt-PA) is a well-characterized glycoprotein with a great deal of published information on its structure, post-translational modifications, and O- and N-glycosylation. Most of the characterization was accomplished in the late 1980s. During the past 2 decades, however, mass spectrometry has made a quantum leap forward offering new capabilities in soft electrospray ionization, speed, resolution, and accuracy of mass measurements. From this point of view, it is worthwhile to revisit the characterization of familiar proteins, such as rt-PA, using the new capabilities of modern analytical technology. In this work, we applied LC-MS with state-of-the-art instrumentation to the characterization of glycoforms of rt-PA. This method takes advantage of accurate mass measurements along with a fast "in-source" voltage switching for the detection of characteristic oxonium ions of saccharides. This method confirmed previously identified glycan structures based on existing knowledge of rt-PA glycans. In addition, we identified two novel glycan structures in rt-PA. A low level of Asn142 N-glycosylation was detected at an atypical Asn-Xaa-Cys consensus motif. It was found to be modified predominantly by biantennary hybrid structures. This N-glycosylation site was confirmed using a recently developed electron-transfer dissociation (ETD) technique. Also using this method, we detected low levels of elongation of fucose-O-Thr61 to di-, tri-, and tetrasaccharides, not previously observed in rt-PA. The results demonstrate that use of state-of-the-art analytical methods can reveal low-level, previously undetected modifications of well-characterized biopharmaceuticals.
authors: Protein Analytical Chemistry Department, Genentech, Incorporated, 1 DNA Way, South San Francisco, California 94080, USA. oborisov@gene.com
authors: Oleg V
authors: OV
authors: Borisov
authors: Matthew
authors: M
authors: Field
authors: Victor T
authors: VT
authors: Ling
authors: Reed J
authors: RJ
authors: Harris
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D016466:CHO Cells; D002240:Carbohydrate Sequence; D002623:Chemistry Techniques, Analytical; D002853:Chromatography, Liquid; D006224:Cricetinae; D003412:Cricetulus; D005643:Fucose / Q000378:metabolism; D006020:Glycopeptides / Q000737:chemistry / Q000378:metabolism; D006031:Glycosylation; D006801:Humans; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D009584:Nitrogen / Q000378:metabolism; D009844:Oligosaccharides / Q000032:analysis* / Q000737:chemistry / Q000378:metabolism; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000378:metabolism; D010959:Tissue Plasminogen Activator / Q000096:biosynthesis* / Q000737:chemistry*
chemical_list: D006020:Glycopeptides; D009844:Oligosaccharides; D011994:Recombinant Proteins; D005643:Fucose; D010959:Tissue Plasminogen Activator; D009584:Nitrogen