Analysis of N-glycans from recombinant immunoglobulin G by on-line reversed-phase high-performance liquid chromatography/mass spectrometry.
An on-line reversed-phase (RP) high-performance liquid chromatography/mass spectrometry (MS) method has been developed for profiling and characterizing N-glycans from recombinant immunoglobulin G antibodies. In this method, released N-glycans are derivatized at their reducing end with 2-aminobenzamide (2AB) and separated on a RP column with on-line fluorescence and MS detection. The method achieves good resolution of all major glycans and segregates glycan types (high-mannose, hybrid, and complex) to different regions of the chromatogram, thus allowing accurate quantification of N-glycans from the fluorescent signal alone. Moreover, the mobile phase used allows high quality on-line MS detection. The 2AB-labeled N-glycans demonstrate good ionization efficiency in electrospray and generate primarily doubly charged [M+2H](2+) ions. The mass and structural information can be readily obtained from the on-line MS and tandem MS data. As little as 70 fmol glycan species can be detected and identified.
Chen
Xiaoyu
X
Process and Analytical Sciences, Amgen, Inc., Thousand Oaks, CA 91320, USA.
Flynn
Gregory C
GC
D002236:Carbohydrate Conformation; D002851:Chromatography, High Pressure Liquid; D007074:Immunoglobulin G / Q000737:chemistry*; D007202:Indicators and Reagents; D013058:Mass Spectrometry; D008958:Models, Molecular; D011134:Polysaccharides / Q000737:chemistry* / Q000302:isolation & purification; D011994:Recombinant Proteins / Q000737:chemistry
D007074:Immunoglobulin G; D007202:Indicators and Reagents; D011134:Polysaccharides; D011994:Recombinant Proteins