| Autor: |
O'Flaherty R; National Institute for Bioprocessing, Research and Training, Fosters Avenue, Blackrock, A94 X099 Dublin, Ireland.; Department of Chemistry, Maynooth University, W23 F2K8 Maynooth, Ireland., Amez Martín M; Ludger Ltd., Culham Science Centre, Abingdon OX14 3EB, UK.; Center for Proteomics and Metabolomics, Leiden University Medical Center, Albinusdreef 2, 2333 ZA Leiden, The Netherlands., Gardner RA; Ludger Ltd., Culham Science Centre, Abingdon OX14 3EB, UK., Jennings PM; National Institute for Bioprocessing, Research and Training, Fosters Avenue, Blackrock, A94 X099 Dublin, Ireland., Rudd PM; National Institute for Bioprocessing, Research and Training, Fosters Avenue, Blackrock, A94 X099 Dublin, Ireland., Spencer DIR; Ludger Ltd., Culham Science Centre, Abingdon OX14 3EB, UK., Falck D; Center for Proteomics and Metabolomics, Leiden University Medical Center, Albinusdreef 2, 2333 ZA Leiden, The Netherlands. |
| Abstrakt: |
Recombinant human erythropoietin (EPO) is a biopharmaceutical frequently used in the treatment of anemia. It is a heavily glycosylated protein with a diverse and complex glycome. EPO N -glycosylation influences important pharmacological parameters, prominently serum half-life. Therefore, EPO N -glycosylation analysis is of the utmost importance in terms of controlling critical quality attributes. In this work, we performed an interlaboratory study of glycoanalytical techniques for profiling and in-depth characterization, namely (1) hydrophilic interaction liquid chromatography with fluorescence detection after 2-aminobenzamide labeling (HILIC-FLD(2AB)) and optional weak anion exchange chromatography (WAX) fractionation and exoglycosidase digestion, (2) HILIC-FLD after procainamide labeling (PROC) optionally coupled to electrospray ionization-MS and (3) matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-MS). All techniques showed good precision and were able to differentiate the unique N -glycosylation profiles of the various EPO preparations. HILIC-FLD showed higher precision, while MALDI-TOF-MS covered the most analytes. However, HILIC-FLD differentiated isomeric N -glycans, i.e., N -acetyllactosamine repeats and O -acetylation regioisomers. For routine profiling, HILIC-FLD methods are more accessible and cover isomerism in major structures, while MALDI-MS covers more minor analytes with an attractively high throughput. For in-depth characterization, MALDI-MS and HILIC-FLD(2AB)/WAX give a similar amount of orthogonal information. HILIC-FLD(PROC)-MS is attractive for covering isomerism of major structures with a significantly less extensive workflow compared to HILIC-FLD(2AB)/WAX. |
