ANALYSIS OF RECOMBINANT DNA-DERIVED GLYCOPROTEINS VIA HIGHPERFORMANCECAPILLARY ELECTROPHORESIS COUPLED WITH OFF-LINE MATRIX-ASSISTED LASER-DESORPTION IONIZATION TIME-OF-FLIGHT MASS-SPECTROMETRY

This paper describes the analysis of glycoform populations of the glyc oproteins ovalbumin and Desmodus salivary plasminogen activator (DSPA alpha 1) by a combination of capillary electrophoresis (CE) and off-li ne matrix-assisted laser desorption ionization time-of-flight mass spe ctrometry (MALDI-TOF-MS). Ovalbumin has a single N-linked glycosylatio n 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 berate buffer system, together with a diamine additive such as 1,4-diaminobutane (DAB), An electropherogr am of DSPA glycoforms could be obtained at pH 3.0 (phosphate buffer) u sing a bovine serum albumin (BSA) coated capillary. Fraction collectio n was performed by controlled application of pressure [5000 Pa (50 mba r)] for zone elution and MALDI-TOF-MS was performed on samples prepare d by a 1:1 dilution with the UV absorbing matrix sinapinic acid. Both electrophoretic separations were successfully characterized by good qu ality mass spectra and distinct mass trends were observed for the coll ected fractions. It is likely that each of the collected fractions are still mixtures of glycoforms and explanation of relative mobilities o r masses of different fractions is not possible at this stage. The abi lity to perform rapid off-line MALDI-TOF-MS of fractions from complex electropherograms will be a powerful tool to demonstrate product consi stency in the manufacture of glycoprotein pharmaceuticals.