Glycosylation impact the pharmacokinetics,pharmacodynamics and/or efficacy of glycoprotein therapeutics1.Erythropoietin(EPO)is a therapeutic glycoprotein that stimulates red blood cell production.Glycosylation heterogeneity of EPO is very complex,and its pattern has a large impact on its in vivo activity.To guarantee safety and efficacy of EPO,glycomic analysis is a regulatory requirement2.In the current paper,CE-LIF of 8-aminopyrene-1,3,6-trisulfonic labeled N-glycan present on the darbepoetin alfa is well established.We applied a combination of weak anion exchange fractionation and exoglycosidase array digestion,followed by separation analysis of the digestion products with CE-LIF.It is a rapid method to characterize monosaccharide sequence and linkage information of N-glycans that need only low amounts of starting materials.Meanwhile,the N-glycan pool of darbepoetin alfa was labeled with a 2-aminobenzamide fluorescence tag and analyzed using ultraperformance hydrophilic interaction liquid chromatography/electrospray quadruple time of fligh mass spectrometry(UPHILIC/Q-TOF-MS).Biologically relevant metrics of biotherapeutic potency such as glycan branching,sialylation and O-acetylation were reproducibly profiled from three production batches.The combined use of both of the applied technique is recommended for rapid and comprehensive characterization of N-glycosylation patterns of darbepoetin alfa and biosimilars.