Nature Communications / Native mass spectrometry combined with enzymatic dissection unravels glycoform heterogeneity of biopharmaceuticals

Robust manufacturing processes resulting in consistent glycosylation are critical for the efficacy and safety of biopharmaceuticals. Information on glycosylation can be obtained by conventional bottomup methods but is often limited to the glycan or glycopeptide level. Here, we apply high-resolution native mass spectrometry (MS) for the characterization of the therapeutic fusion protein Etanercept to unravel glycoform heterogeneity in conditions of hitherto unmatched mass spectral complexity. Higher spatial resolution at lower charge states, an inherent characteristic of native MS, represents a key component for the successful revelation of glycan heterogeneity. Combined with enzymatic dissection using a set of proteases and glycosidases, assignment of specific glycoforms is achieved by transferring information from subunit to whole protein level. The application of native mass spectrometric analysis of intact Etanercept as a fingerprinting tool for the assessment of batch-to-batch variability is exemplified and may be extended to demonstrate comparability after changes in the biologic manufacturing process.(VLID)261711

Site-Specific Characterization and Absolute Quantification of Pegfilgrastim Oxidation by Top-Down High-Performance Liquid Chromatography–Mass Spectrometry

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 <i>pegfilgrastim</i>, a poly­(ethylene glycol) modified recombinant human granulocyte-colony stimulating factor. <i>Pegfilgrastim</i> 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 <i>pegfilgrastim</i> batch was analyzed as a a real biopharmaceutical sample to confirm the feasibility of our approach for monitoring low levels of oxidation variants