Journal of Chromatography B, 877 (2009) 1827–1832 Contents lists available at ScienceDirect Journal of Chromatography B journal homepage: www.elsevier.com/locate/chromb Development of a high performance anion exchange chromatography analysis for mapping of oligosaccharides Carl Grey a,b,∗ , Per Edebrink a,c , Maria Krook a,c , Sven P. Jacobsson d a BioPR&D, AstraZeneca R&D, SE-151 85 Södertälje, Sweden b Department of Biotechnology, Lund University, P.O. Box 124, SE-221 00 Lund, Sweden c Recipharm Biologics, Gärtunavägen 10, SE-152 57 Södertälje, Sweden d Department of Analytical Chemistry, Stockholm University, SE-106 91 Stockholm, Sweden article info Article history: Received 3 February 2009 Accepted 3 May 2009 Available online 13 May 2009 Keywords: HPAEC-PAD MALDI-TOF MS Oligosaccharides N-glycosylation Glycoprotein Monoclonal antibodies abstract In the present study a HPAEC-PAD method is described that was developed for monitoring the consistency of N-glycosylation during the production and purification of recombinant proteins and monoclonal anti- bodies. The method successfully separated 18 neutral and sialylated oligosaccharides. Results obtained were compared with MALDI-TOF MS and it was shown that both methods gave similar results. In addi- tion, a method validation was performed showing that the HPAEC-PAD analysis was well suited for the mapping and characterization of oligosaccharides. The method was found to be robust and additionally the precision was significantly better compared to the MALDI-TOF MS method. © 2009 Elsevier B.V. All rights reserved. 1. Introduction The biopharmaceutical market of therapeutic proteins is under- going a rapid expansion, not least in the field of monoclonal antibodies (mAbs) where there are currently 20 therapeutic mAbs on the market and about 500 antibody based development pro- grams in pipeline [1]. Characterization of protein therapeutics is naturally a vital part in drug development, where both the primary sequences and post-translational modifications such as glycosylation need to be studied closely. Protein glycosylation affects many different physicochemical properties, such as sol- ubility, viscosity and through stabilizing protein conformation and protein folding, as well as biological properties such as modulating activity, participating in cell–cell interactions and determining circulation half-life [2–4]. Since proper glycosyla- tion is often critical for the protein function, the production of therapeutic glycoproteins needs to be closely monitored, as both reactor conditions, improvements in cell productivity and downstream processing can affect the glycan heterogeneity [5–7]. Oligosaccharide analysis is thus important in the produc- ∗ Corresponding author at: Department of Biotechnology, Lund University, P.O. Box 124, SE-221 00 Lund, Sweden. Tel.: +46 46 2220858; fax: +46 46 2224713. E-mail address: carl.grey@biotek.lu.se (C. Grey). tion of glycoprotein pharmaceuticals as well as in fundamental research. Since introduced in the 1980s, high-performance anion exchange chromatography (HPAEC) coupled with pulsed ampero- metric detection (PAD) has become a valuable tool in glycoprotein characterization [8,9]. The advantage is that it can separate and detect oligosaccharides without any derivatization and yet offers excellent resolution and a high sensitivity, with a detection limit in the 300 fmol region [10]. Mass spectrometry based methods are also commonly used for both quantitative analysis using MALDI- TOF MS [11,12], LC–MS [13] and structural analysis using MS/MS based techniques [14]. Also, an interesting combination of MS and HPAEC-PAD has been suggested [15]. Other alternatives are the use of HPLC or capillary electrophoresis in combination with UV or fluorescence detection, where chromophores often are intro- duced to the sugars [10,13,16,17]. Previously, several studies have been published using HPAEC-PAD for the characterization of pro- tein glycosylation [2,8,18–24], but none of these has been shown to separate all glycans found in recombinantly produced mAbs and simultaneously separate both sialylated and neutral oligosac- charides. The aim of the present study was thus to develop an HPAEC-PAD method that could separate neutral as well as sialy- lated oligosaccharides more efficiently than previously described methods, by optimizing both gradient and eluent composition, including pusher concentration and pH. The developed method 1570-0232/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.jchromb.2009.05.003