RESEARCH PAPER LC-MS analysis combined with principal component analysis and soft independent modelling by class analogy for a better detection of changes in N-glycosylation profiles of therapeutic glycoproteins Ana Planinc 1 & Bieke Dejaegher 2,3 & Yvan Vander Heyden 3 & Johan Viaene 3 & Serge Van Praet 4 & Florence Rappez 4 & Pierre Van Antwerpen 1 & Cédric Delporte 1 Received: 15 April 2016 /Revised: 25 May 2016 /Accepted: 1 June 2016 # Springer-Verlag Berlin Heidelberg 2016 Abstract Therapeutic proteins are among the top selling drugs in the pharmaceutical industry. More than 60 % of the approved therapeutic proteins are glycosylated. Nowadays, it is well accepted that changes in glycosyl- ation may affect the safety and the efficacy of the ther- apeutic proteins. For this reason, it is important to char- acterize both the protein and the glycan structures. In this study, analytical and data processing methods were developed ensuring an easier characterization of glycoprofiles. N-glycans were (i) enzymatically released using peptide-N-glycosidase F (PNGase F), (ii) reduced, and (iii) analyzed by hydrophilic interaction liquid chro- matography coupled to a high-resolution mass spectrom- eter (HILIC-HRMS). Glycosylation changes were ana- lyzed in human plasma immunoglobulin G samples which had previously been artificially modified by adding other glycoproteins (such as ribonuclease B and fetuin) or by digesting with enzyme (neuraminidase). Principal component analysis (PCA) and classification through soft independent modelling by class analogy (SIMCA) were used to detect minor glycosylation changes. Using HILIC-MS-PCA/SIMCA approach, it was possible to detect small changes in N-glycosylation, which had not been detected directly from the extracted- ion chromatograms, which is current technique to detect N-glycosylation changes in batch-to-batch analysis. The HILIC-MS-PCA/SIMCA approach is highly sensitive approach due to the sensitivity of MS and appropriate data processing approaches. It could help in assessing the changes in glycosylation, controlling batch-to-batch consistency, and establishing acceptance limits according to the glycosylation changes, ensuring safety and efficacy. Keywords Therapeutic glycoproteins . N-Glycans . Principal component analysis (PCA) . Soft independent modelling by class analogy (SIMCA) . High-resolution mass spectrometry (HRMS) . Hydrophilic interaction liquid chromatography (HILIC) Published in the topical collection Glycomics, Glycoproteomics and Allied Topics with guest editors Yehia Mechref and David Muddiman. Ana Planinc, Bieke Dejaegher, Yvan Vander Heyden, Johan Viaene, Pierre Van Antwerpen, and Cédric Delporte—Joint Research Group ULB-VUB Electronic supplementary material The online version of this article (doi:10.1007/s00216-016-9683-9) contains supplementary material, which is available to authorized users. * Ana Planinc aplaninc@ulb.ac.be 1 Analytical Platform of the Faculty of Pharmacy and Laboratory of Pharmaceutical Chemistry, Faculty of Pharmacy, Université Libre de Bruxelles (ULB), Boulevard du Triomphe, Campus Plaine, CP 205/ 05B, 1050 Brussels, Belgium 2 Laboratory of Instrumental Analysis and Bioelectrochemistry, Faculty of Pharmacy, Université Libre de Bruxelles (ULB), Boulevard du Triomphe, 1050 Brussels, Belgium 3 Department of Analytical Chemistry and Pharmaceutical Technology (FABI), Center for Pharmaceutical Research (CePhaR), Faculty of Medicine and Pharmacy, Vrije Universiteit Brussel (VUB), Laarbeeklaan 103, 1090 Brussels, Belgium 4 Pharmacy, Centre Hospitalier Universitaire Saint-Pierre, Brussels, Belgium Anal Bioanal Chem DOI 10.1007/s00216-016-9683-9