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In contrast with conventional drugs, biopharmaceuticals are highly complex molecules with remarkable heterogeneity. Protein glycosylation is an inherent source of this heterogeneity and also affects the safety, efficacy, and serum half-life of therapeutic glycoproteins. Therefore analysis of the glycan pattern is an important issue for characterization and quality control in the biopharmaceutical industry. In this publication we describe a complete workflow for the analysis of protein N-glycans. The sample-preparation procedure, consisting of the release of the N-glycans by PNGase-F, followed by fluorescence labeling with 2-aminobenzamide and removal of excess label, was optimized to avoid alteration of the glycan sample. Subsequently, labeled glycans were analyzed by hydrophilic-interaction liquid chromatography (HILIC) with fluorescence detection. The developed method was validated for analysis of antibody N-glycans. To demonstrate the accuracy of the method an antibody sample was additionally analyzed by an orthogonal method. The antibody was digested with lysyl endopeptidase and the (glyco-)peptides were analyzed by RP-HPLC-MS. The consistency of the results between these two methods demonstrates the reliability of the glycan analysis method introduced herein.

The characterization and absolute quantification of protein biopharmaceuticals and their product-related impurities, e.g., oxidation variants, is essential due to their potential impact on biological activity and immunogenicity. Here, we present site assignment and absolute quantification of oxidation variants of pegfilgrastim, a poly(ethylene glycol) modified recombinant human granulocyte-colony stimulating factor. Pegfilgrastim stressed with 1.0% hydrogen peroxide served as a model protein for developing a top-down high-performance liquid chromatography-mass spectrometry (HPLC-MS) platform that allowed direct site assignment of Met122, Met127, and Met138 oxidation within a total analysis time of 30 min. Three different absolute quantification methods, namely, UV absorption spectroscopy, full-scan MS, and all-ion fragmentation (AIF) MS were compared. Additionally, the monitoring of all generated fragment ions or selected sets of fragment ions were evaluated for the AIF method. Linearity of calibration curves from 5.0 to 25 ng μL(-1), 25 to 250 ng μL(-1), and 100 to 1000 ng μL(-1) was confirmed. The AIF method achieved a lower limit of detection of 0.85 ng μL(-1) and a lower limit of quantification of 2.54 ng μL(-1). On the basis of the comparison of relative standard deviations of interday measurements, AIF was concluded to be the method of choice for concentrations up to 50 ng μL(-1), and UV measurements should be carried out above this concentration. Finally, an expired pegfilgrastim batch was analyzed as a a real biopharmaceutical sample to confirm the feasibility of our approach for monitoring low levels of oxidation variants.