Glycan profiling of monoclonal antibodies using zwitterionic-type hydrophilic interaction chromatography coupled with electrospray ionization mass spectrometry detection.
We present a new method for the analysis of glycans enzymatically released from monoclonal antibodies (MAbs) employing a zwitterionic-type hydrophilic interaction chromatography (ZIC-HILIC) column coupled with electrospray ionization mass spectrometry (ESI-MS). Both native and reduced glycans were analyzed, and the developed procedure was compared with a standard HILIC procedure used in the pharmaceutical industry whereby fluorescent-labeled glycans are analyzed using a TSK Amide-80 column coupled with fluorescence detection. The separation of isobaric alditol oligosaccharides present in monoclonal antibodies and ribonuclease B is demonstrated, and ZIC-HILIC is shown to have good capability for structural recognition. Glycan profiles obtained with the ZIC-HILIC column and ESI-MS provided detailed information on MAb glycosylation, including identification of some less abundant glycan species, and are consistent with the profiles generated with the standard procedure. This new ZIC-HILIC method offers a simpler and faster approach for glycosylation analysis of therapeutic antibodies.
Mauko
Lea
L
Pfizer Analytical Research Centre (PARC), Australian Centre for Research on Separation Science (ACROSS), School of Chemistry, University of Tasmania, Hobart, TAS 7001, Australia.
Nordborg
Anna
A
Hutchinson
Joseph P
JP
Lacher
Nathan A
NA
Hilder
Emily F
EF
Haddad
Paul R
PR
D000911:Antibodies, Monoclonal / Q000737:chemistry*; D002240:Carbohydrate Sequence; D002850:Chromatography, Gel / Q000379:methods*; D005456:Fluorescent Dyes / Q000737:chemistry; D006031:Glycosylation; D057927:Hydrophobic and Hydrophilic Interactions; D008969:Molecular Sequence Data; D009844:Oligosaccharides / Q000032:analysis*; D012260:Ribonucleases / Q000737:chemistry; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*
D000911:Antibodies, Monoclonal; D005456:Fluorescent Dyes; D009844:Oligosaccharides; D012260:Ribonucleases; C029600:ribonuclease B