DiscussionThe development and production of recombinant biosimilars is a challenging field due to their structural complexity. When implementing a manufacturing process for biosimilars, differences in the protein structure or post-translational modifications can occur. Therefore a characterization program is required following the biosimilar guidelines. Of great interest are protein glycosylations which can have a great influence on efficacy and safety of the biopharmaceutical. Glycosylations are usually analyzed after isolation from the protein by HPAEC-PAD or HILIC-FLD. Alternatively, LC-ESI-MS analysis of the intact protein can be employed to yield the glycosylation pattern or monosaccharide profile. Here, we analyzed Ustekinumab, Erythropoietin, and Etanercept with an UHR-TOF mass spectrometer. The method was qualified by comparison to data achieved by HILIC-FLD and HPAEC-PAD.