Abstract
EPO has a complex glycosylation pattern with differently branched and charged glycans. A combination of hydrophilic interaction chromatography (HILIC) with weak anion exchange chromatography (WAX) enables highly orthogonal separation. Comprehensive 2D-LC analysis with HILIC in the first and WAX in the second dimension provides high resolution 2D chromatography together with simultaneous charge profiling. Meanwhile, multiple heart-cutting 2D-LC analysis combining WAX and HILIC separation provides a flexible alternative whereby the user can select multiple peaks to be analyzed in the second dimension and, moreover, run longer gradients in the second dimension.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
Similar content being viewed by others
References
Mocini D et al (2007) Structure, production and function of erythropoietin: implications for therapeutical use in cardiovascular disease. Curr Med Chem 14(21):2278–2287
Egrie JC et al (1986) Characterization and biological effects of recombinant human erythropoietin. Immunobiology 172:213–224
Rush RS et al (1995) Microheterogeneity of erythropoietin carbohydrate structure. Anal Chem 67:1442–1452
Egrie JC, Browne JK (2001) Development and characterization of novel erythropoiesis stimulating protein (NESP). Br J Cancer 84(Supplement 1):3–10
Melmer M et al (2010) HILIC analysis of fluorescence-labeled N-glycans from recombinant biopharmaceuticals. Anal Bioanal Chem 398:905–914
Bones C et al (2011) 2D-LC analysis of BRP 3 erythropoietin N-glycosylation using anion exchange fractionation and hydrophilic interaction UPLC reveals long poly-N-acetyl lactosamine extensions. Anal Chem 83(11):4154–4162
Jelkmann W (1992) Erythropoietin: structure, control of production, and function. Physiol Rev 72(2):449–489
Nwosu CC et al (2011) Simultaneous and extensive site-specific N- and O-glycosylation analysis in protein mixtures. J Proteome Res 10(5):2612–2624
Buckenmaier S (2015) Agilent 1290 Infinity 2D-LC Solution for Multiple Heart-Cutting. Agilent Technologies Application Note, Publication Number 5991-5615EN. Agilent Technologies, Inc., Waldbronn, Germany
Schneider S (2016) N-Glycan Analysis of mAbs and Other Glycoproteins with UHPLC and Fluorescence Detection. Agilent Technologies Application Note, publication number 5990-9774EN. Agilent Technologies, Inc., Waldbronn, Germany
Schneider S, Naegele E, Krieger S (2017) Online 2D-LC Analysis of Complex N-Glycans in Biopharmaceuticals Using the Agilent 1290 Infinity 2D-LC Solution. Agilent Technologies Application Note, publication number 5991-5349EN. Agilent Technologies, Inc., Waldbronn, Germany
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2021 Springer Science+Business Media, LLC, part of Springer Nature
About this protocol
Cite this protocol
Schneider, S., Naegele, E., Krieger, S. (2021). Online 2D-LC for Complex N-Glycan Analysis from Biopharmaceuticals. In: Delobel, A. (eds) Mass Spectrometry of Glycoproteins. Methods in Molecular Biology, vol 2271. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-1241-5_16
Download citation
DOI: https://doi.org/10.1007/978-1-0716-1241-5_16
Published:
Publisher Name: Humana, New York, NY
Print ISBN: 978-1-0716-1240-8
Online ISBN: 978-1-0716-1241-5
eBook Packages: Springer Protocols