title: Analysis of recombinant DNA-derived glycoproteins via high-performance capillary electrophoresis coupled with off-line matrix-assisted laser desorption ionization time-of-flight mass spectrometry.
abstract: This paper describes the analysis of glycoform populations of the glycoproteins ovalbumin and Desmodus salivary plasminogen activator (DSPA alpha 1) by a combination of capillary electrophoresis (CE) and off-line matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS). Ovalbumin has a single N-linked glycosylation site and DSPA alpha 1 has six sites for potential glycosylation, 2 N-linked and four O-linked. The conditions used for the electrophoretic separation of ovalbumin include a borate buffer system, together with a diamine additive such as 1,4-diaminobutane (DAB). An electropherogram of DSPA glycoforms could be obtained at pH 3.0 (phosphate buffer) using a bovine serum albumin (BSA) coated capillary. Fraction collection was performed by controlled application of pressure [5000 Pa (50 mbar)] for zone elution and MALDI-TOF-MS was performed on samples prepared by a 1:1 dilution with the UV absorbing matrix sinapinic acid. Both electrophoretic separations were successfully characterized by good quality mass spectra and distinct mass trends were observed for the collected fractions. It is likely that each of the collected fractions are still mixtures of glycoforms and explanation of relative mobilities or masses of different fractions is not possible at this stage. The ability to perform rapid off-line MALDI-TOF-MS of fractions from complex electropherograms will be a powerful tool to demonstrate product consistency in the manufacture of glycoprotein pharmaceuticals.
authors.affiliation: Analytical/Medical Laboratory, Hewlett-Packard Laboratories, Palo Alto, CA 94304, USA.
authors.forename: J A
authors.initials: JA
authors.lastname: Chakel
authors.forename: E
authors.initials: E
authors.lastname: Pungor
authors.forename: W S
authors.initials: WS
authors.lastname: Hancock
authors.forename: S A
authors.initials: SA
authors.lastname: Swedberg
mesh_terms: D000818:Animals; D002685:Chiroptera; D019075:Electrophoresis, Capillary / Q000379:methods*; D008401:Gas Chromatography-Mass Spectrometry / Q000379:methods*; D006023:Glycoproteins / Q000032:analysis*; D007834:Lasers; D010047:Ovalbumin / Q000032:analysis*; D010960:Plasminogen Activators / Q000032:analysis*; D011994:Recombinant Proteins / Q000032:analysis
chemical_list: D006023:Glycoproteins; D011994:Recombinant Proteins; C069588:salivary plasminogen activator alpha 1, Desmodus rotundus; D010047:Ovalbumin; D010960:Plasminogen Activators