Erythropoietin N-glycosylation of Therapeutic Formulations Quantified and Characterized: An Interlab Comparability Study of High-Throughput Methods

Abstract

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.