The use of high-performance anion-exchange chromatography and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry to monitor and identify oligosaccharide degradation.
High-performance anion-exchange chromatography (HPAEC) with pulsed-amperometric detection was used to monitor the consistency of the oligosaccharides released from several partially purified preparations of a tissue-type plasminogen activator mutant (TNK-tPA). Differences in the oligosaccharide map were observed, primarily in the neutral carbohydrate region of the separation. Subsequent investigations using matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI/TOF/MS) identified the neutral oligosaccharides to be primarily asialo-diantennary complex-type glycans with 2, 1, or 0 galactose residues. Additional asialo-triantennary and asialo-tetrantennary structures were also observed with varying amounts of galactose. Hydrolysis of the chromogenic substrate 4-methyl umbelliferyl-beta-galactoside confirmed that host-cell lysosomal beta-galactosidase is present at the first step in the purification process and is the cause of the observed glycan degradation. Subsequent steps in the purification process quantitatively remove this enzyme. Comparison of HPAEC and MALDI/TOF/MS analysis of time-course samples revealed quite similar rates of degradation and demonstrates the quantitative utility of these methods. HPAEC did not reveal significant changes in the sialylated structures as evidenced by nearly identical profiles for the sialic acid-containing structures.
Field
M
M
Department of Analytical Chemistry, Genentech, Inc., 460 Point San Bruno Boulevard, South San Francisco, California, 94080, USA.
Papac
D
D
Jones
A
A
D000818:Animals; D000837:Anion Exchange Resins; D016466:CHO Cells; D002851:Chromatography, High Pressure Liquid; D002852:Chromatography, Ion Exchange / Q000379:methods*; D006224:Cricetinae; D003412:Cricetulus; D005260:Female; D006863:Hydrogen-Ion Concentration; D009844:Oligosaccharides / Q000032:analysis*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*; D013997:Time Factors; D010959:Tissue Plasminogen Activator / Q000737:chemistry*; D001616:beta-Galactosidase / Q000378:metabolism
D000837:Anion Exchange Resins; D009844:Oligosaccharides; D001616:beta-Galactosidase; D010959:Tissue Plasminogen Activator