title: Quantitative comparison of global carbohydrate structures of glycoproteins using LC-MS and in-source fragmentation.
abstract: A comparative method for the quantitative analysis of the ratio of oxonium fragment (reporter) ions derived from sialic acid and N-acetylhexosamine residues on a large intact glycoprotein, the B domain of recombinant human factor VIII (rhFVIII), was developed. The method utilized liquid chromatography-electrospray ionization mass spectrometry (LC-ESI MS) on a single-quadrupole instrument. During development, systematic approaches such as full-matrix and simplex strategies were used for the optimization of the signal-to-noise ratio by controlling source temperature and cone voltage. The method was found to be precise (RSD = 0.84%), sensitive (capable of differentiating 1 sialic acid residue change among at least 29 sialic acids on a 103-kDa glycoprotein that is 38% carbohydrate), applicable to a wide range of loading (11.6-372 micrograms of FVIII), and accurate according to a comparison to matrix-assisted laser desorption-ionization time-of-flight mass spectrometry. Combining the method with enzymatic removal of N-glycans, selective O-glycan analysis was also performed leading to differential fragment ion analysis ascribed to N- and O-glycans. Quantitative ESI in-source dissociation MS combined with LC can generally be used for glycoproteins, as one of the indicators, to compare the distribution of carbohydrate residues over N- and O-glycans, to investigate their isoforms, and compare batch-to-batch characteristics of biopharmaceuticals.
authors.affiliation: Genetics Institute, Inc, Andover, Massachusetts 01810, USA.
authors.forename: I
authors.initials: I
authors.lastname: Mazsaroff
authors.forename: W
authors.initials: W
authors.lastname: Yu
authors.forename: B D
authors.initials: BD
authors.lastname: Kelley
authors.forename: J E
authors.initials: JE
authors.lastname: Vath
mesh_terms: D000581:Amidohydrolases / Q000378:metabolism; D002236:Carbohydrate Conformation; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D005169:Factor VIII / Q000737:chemistry* / Q000378:metabolism; D006023:Glycoproteins / Q000737:chemistry* / Q000378:metabolism; D006801:Humans; D013058:Mass Spectrometry / Q000379:methods*; D010446:Peptide Fragments / Q000737:chemistry; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods; D013917:Thrombin / Q000378:metabolism
chemical_list: D006023:Glycoproteins; D010446:Peptide Fragments; D005169:Factor VIII; D013917:Thrombin; D000581:Amidohydrolases; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase