Characterization of Intact Neo-Glycoproteins by Hydrophilic Interaction Liquid Chromatography

Alice Pedrali; Sara Tengattini; Giorgio Marrubini; Teodora Bavaro; Petrus Hemström; Gabriella Massolini; Marco Terreni; Caterina Temporini

doi:10.3390/molecules19079070

Molecules (Jun 2014)

Characterization of Intact Neo-Glycoproteins by Hydrophilic Interaction Liquid Chromatography

Alice Pedrali,
Sara Tengattini,
Giorgio Marrubini,
Teodora Bavaro,
Petrus Hemström,
Gabriella Massolini,
Marco Terreni,
Caterina Temporini

Affiliations

Alice Pedrali: Department of Drug Sciences and Italian Biocatalysis Center, University of Pavia, Via Taramelli, 12, 27100 Pavia, Italy
Sara Tengattini: Department of Drug Sciences and Italian Biocatalysis Center, University of Pavia, Via Taramelli, 12, 27100 Pavia, Italy
Giorgio Marrubini: Department of Drug Sciences and Italian Biocatalysis Center, University of Pavia, Via Taramelli, 12, 27100 Pavia, Italy
Teodora Bavaro: Department of Drug Sciences and Italian Biocatalysis Center, University of Pavia, Via Taramelli, 12, 27100 Pavia, Italy
Petrus Hemström: Merck SeQuant AB, S-90719 Umeå, Sweden
Gabriella Massolini: Department of Drug Sciences and Italian Biocatalysis Center, University of Pavia, Via Taramelli, 12, 27100 Pavia, Italy
Marco Terreni: Department of Drug Sciences and Italian Biocatalysis Center, University of Pavia, Via Taramelli, 12, 27100 Pavia, Italy
Caterina Temporini: Department of Drug Sciences and Italian Biocatalysis Center, University of Pavia, Via Taramelli, 12, 27100 Pavia, Italy

DOI: https://doi.org/10.3390/molecules19079070
Journal volume & issue: Vol. 19, no. 7
pp. 9070 – 9088

Abstract

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In this study, an HPLC HILIC-UV method was developed for the analysis of intact neo-glycoproteins. During method development the experimental conditions evaluated involved different HILIC columns (TSKgel Amide-80 and ZIC-pHILIC), and water-acetonitrile mixtures containing various types of acids and salts. The final selected method was based on a TSKgel Amide-80 column and a mobile phase composed of acetonitrile and water both containing 10 mM HClO4. The influence of temperature and sample preparation on the chromatographic performances of the HILIC method was also investigated. The method was applied to the separation of neo-glycoproteins prepared starting from the model protein RNase A by chemical conjugation of different glycans. Using the method here reported it was possible to monitor by UV detection the glycosylation reaction and assess the distribution of neo-glycoprotein isoforms without laborious sample workup prior to analysis.

Published in Molecules

ISSN: 1420-3049 (Online)
Publisher: MDPI AG
Country of publisher: Switzerland
LCC subjects: Science: Chemistry: Organic chemistry
Website: http://www.mdpi.com/journal/molecules

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