title: Separation by hydrophobic interaction chromatography and structural determination by mass spectrometry of mannosylated glycoforms of a recombinant transferrin-exendin-4 fusion protein from yeast.
abstract: Obtaining sufficient amounts of pure glycoprotein variants to characterize their structures is an important goal in both functional biology and the biotechnology industry. We have developed preparative HIC conditions that resolve glycoform variants on the basis of overall carbohydrate content for a recombinant transferrin-exendin-4 fusion protein. The fusion protein was expressed from the yeast Saccharomyces cerevisiae from high density fermentation and is post-translationally modified with mannose sugars through O-glycosidic linkages. Overall hydrophobic behavior appeared to be dominated by the N-terminal 39 amino acids from the exendin-4 and linker peptide sequences as compared to the less hydrophobic behavior of human transferrin alone. In addition, using LC techniques that measure total glycans released from the pure protein combined with new high resolution technologies using mass spectrometry, we have determined the locations and chain lengths of mannose residues on specific peptides derived from tryptic maps of the transferrin-exendin-4 protein. Though the protein is large (80,488kDa) and contains 78 possible serine and threonine residues as potential sites for sugar addition, mannosylation was observed on only two tryptic peptides located within the first 55 amino acids of the N-terminus. These glycopeptides were highly heterogeneous and contained between 1 and 10 mannose residues scattered among the various serine and threonine sites which were identified by electron transfer dissociation mass spectrometry. Glycan sequences from 1 to 6 linear mannose residues were detected, but mannose chain lengths of 3 or 4 were more common and formed 80% of the total oligosaccharides. This work introduces new technological capabilities for the purification and characterization of glycosylated variants of therapeutic recombinant proteins.
authors.affiliation: Analytical Research and Development, Pfizer Global Biologics, Chesterfield, MO 63017, USA.
authors.forename: Melissa D
authors.initials: MD
authors.lastname: Zolodz
authors.forename: John T
authors.initials: JT
authors.lastname: Herberg
authors.forename: Halyna E
authors.initials: HE
authors.lastname: Narepekha
authors.forename: Emily
authors.initials: E
authors.lastname: Raleigh
authors.forename: Matthew R
authors.initials: MR
authors.lastname: Farber
authors.forename: Robert L
authors.initials: RL
authors.lastname: Dufield
authors.forename: Denis M
authors.initials: DM
authors.lastname: Boyle
mesh_terms: D000595:Amino Acid Sequence; D002853:Chromatography, Liquid / Q000379:methods*; D000077270:Exenatide; D006020:Glycopeptides / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D006801:Humans; D057927:Hydrophobic and Hydrophilic Interactions; D008358:Mannose / Q000737:chemistry* / Q000378:metabolism; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D010455:Peptides / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics; D053719:Tandem Mass Spectrometry; D014168:Transferrin / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D014508:Urea; D014688:Venoms / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
chemical_list: D006020:Glycopeptides; D010446:Peptide Fragments; D010455:Peptides; D011993:Recombinant Fusion Proteins; D014168:Transferrin; D014688:Venoms; D014508:Urea; D000077270:Exenatide; D008358:Mannose