Affinity purification of erythropoietin from cell culture supernatant combined with MALDI-TOF-MS analysis of erythropoietin N-glycosylation

Abstract Erythropoietin (EPO) is a heavily glycosylated hormone whose recombinant forms are used for treatment of anaemia. EPO glycosylation is important for its pharmacological properties. An analytical workflow, which can determine EPO glycosylation in an accurate and high-throughput fashion from...

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Autores principales:	David Falck, Markus Haberger, Rosina Plomp, Michaela Hook, Patrick Bulau, Manfred Wuhrer, Dietmar Reusch
Formato:	article
Lenguaje:	EN
Publicado:	Nature Portfolio 2017
Materias:	Medicine R Science Q
Acceso en línea:	https://doaj.org/article/6d50a3c6a01c4ababda8c549c01c7c0a
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spelling	oai:doaj.org-article:6d50a3c6a01c4ababda8c549c01c7c0a2021-12-02T15:05:45ZAffinity purification of erythropoietin from cell culture supernatant combined with MALDI-TOF-MS analysis of erythropoietin N-glycosylation10.1038/s41598-017-05641-12045-2322https://doaj.org/article/6d50a3c6a01c4ababda8c549c01c7c0a2017-07-01T00:00:00Zhttps://doi.org/10.1038/s41598-017-05641-1https://doaj.org/toc/2045-2322Abstract Erythropoietin (EPO) is a heavily glycosylated hormone whose recombinant forms are used for treatment of anaemia. EPO glycosylation is important for its pharmacological properties. An analytical workflow, which can determine EPO glycosylation in an accurate and high-throughput fashion from cell culture supernatant (CCS) in approximately 24 h, offers the possibility to follow changes during production. To address this challenge, we present a complete workflow consisting of protein purification, glycan release, sialic acid derivatization, solid phase extraction, matrix-assisted laser desorption/ionization - mass spectrometry (MALDI-MS) analysis and MassyTools data processing. EPO purification from CCS by anti-EPO antibody coupled Sepharose beads yielded excellent purity with acceptable recovery and was free of glycoform bias. Glycosylation profiles obtained by MALDI-MS were highly comparable to those obtained with an established capillary gel electrophoresis–laser induced fluorescence method. Our method delivers accurate results for the analysis of changes of important glycosylation parameters, such as sialylation and number of N-acetyllactosamine units, for the time course of a fermentation. We could resolve differences in glycosylation between several CCS samples.David FalckMarkus HabergerRosina PlompMichaela HookPatrick BulauManfred WuhrerDietmar ReuschNature PortfolioarticleMedicineRScienceQENScientific Reports, Vol 7, Iss 1, Pp 1-10 (2017)
institution	DOAJ
collection	DOAJ
language	EN
topic	Medicine R Science Q
spellingShingle	Medicine R Science Q David Falck Markus Haberger Rosina Plomp Michaela Hook Patrick Bulau Manfred Wuhrer Dietmar Reusch Affinity purification of erythropoietin from cell culture supernatant combined with MALDI-TOF-MS analysis of erythropoietin N-glycosylation
description	Abstract Erythropoietin (EPO) is a heavily glycosylated hormone whose recombinant forms are used for treatment of anaemia. EPO glycosylation is important for its pharmacological properties. An analytical workflow, which can determine EPO glycosylation in an accurate and high-throughput fashion from cell culture supernatant (CCS) in approximately 24 h, offers the possibility to follow changes during production. To address this challenge, we present a complete workflow consisting of protein purification, glycan release, sialic acid derivatization, solid phase extraction, matrix-assisted laser desorption/ionization - mass spectrometry (MALDI-MS) analysis and MassyTools data processing. EPO purification from CCS by anti-EPO antibody coupled Sepharose beads yielded excellent purity with acceptable recovery and was free of glycoform bias. Glycosylation profiles obtained by MALDI-MS were highly comparable to those obtained with an established capillary gel electrophoresis–laser induced fluorescence method. Our method delivers accurate results for the analysis of changes of important glycosylation parameters, such as sialylation and number of N-acetyllactosamine units, for the time course of a fermentation. We could resolve differences in glycosylation between several CCS samples.
format	article
author	David Falck Markus Haberger Rosina Plomp Michaela Hook Patrick Bulau Manfred Wuhrer Dietmar Reusch
author_facet	David Falck Markus Haberger Rosina Plomp Michaela Hook Patrick Bulau Manfred Wuhrer Dietmar Reusch
author_sort	David Falck
title	Affinity purification of erythropoietin from cell culture supernatant combined with MALDI-TOF-MS analysis of erythropoietin N-glycosylation
title_short	Affinity purification of erythropoietin from cell culture supernatant combined with MALDI-TOF-MS analysis of erythropoietin N-glycosylation
title_full	Affinity purification of erythropoietin from cell culture supernatant combined with MALDI-TOF-MS analysis of erythropoietin N-glycosylation
title_fullStr	Affinity purification of erythropoietin from cell culture supernatant combined with MALDI-TOF-MS analysis of erythropoietin N-glycosylation
title_full_unstemmed	Affinity purification of erythropoietin from cell culture supernatant combined with MALDI-TOF-MS analysis of erythropoietin N-glycosylation
title_sort	affinity purification of erythropoietin from cell culture supernatant combined with maldi-tof-ms analysis of erythropoietin n-glycosylation
publisher	Nature Portfolio
publishDate	2017
url	https://doaj.org/article/6d50a3c6a01c4ababda8c549c01c7c0a
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Affinity purification of erythropoietin from cell culture supernatant combined with MALDI-TOF-MS analysis of erythropoietin N-glycosylation

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