Highlightsd An ultrafast deconvolution tool for top-down mass spectrometry data is presented d A spectrum transformation that dramatically accelerates deconvolution is suggested d Our method reports more masses and substantially fewer artifacts than other tools

No abstract

Feature deconvolution, the determination of intact proteoform masses, is crucial for top-down proteomics, but currently suffers from long runtimes and quality issues. We present FLASHDeconv, an algorithm based on a simple transformation of mass spectra, which turns deconvolution into the search for constant patterns thus greatly accelerating the process. We show higher deconvolution quality and two to three orders of magnitude faster execution speed than existing approaches.

The demand for reliable comparability studies of biosimilars grows with their increased market share. These studies focus on physicochemical, structural, functional and clinical properties to ensure that a biosimilar has no significant differences to the originator product and can be released into the market without extensive clinical trials. In the current study, Enbrel® (etanercept, the originator) and Altebrel™ (the proposed biosimilar) underwent direct comparison. “Bottom-up” mass spectrometric analysis was used for primary sequence analysis, evaluation of N/O-glycosylation sites and quantification of methionine oxidation. N/O-glycans were analyzed after permethylation derivatization and the effect of N-glycans on in-vitro functionality of etanercept was assayed. Three enzyme peptide mapping resulted in complete identification of the primary structure. It was confirmed that total ion chromatograms are valuable datasets for the analysis of the primary structure of biodrugs. New N/O-glycan structures were identified and all the N-glycans were quantified. Finally, investigation of the functional properties of N-deglycosylated and non-modified etanercept samples using surface plasmon resonance analysis and in-vitro bioassay showed that N-glycosylation has no significant effect on its in-vitro functionality. Analysis of etanercept and its biosimilar, revealed a high similarity in terms of glycosylation, primary structure and in-vitro functionality.

The characterization of therapeutic glycoproteins is challenging due to the structural heterogeneity of the therapeutic protein glycosylation. This study presents an in-depth analytical strategy for glycosylation of first-generation erythropoietin (epoetin beta), including a developed mass spectrometric workflow for N-glycan analysis, bottom-up mass spectrometric methods for site-specific N-glycosylation, and a LC-MS approach for Oglycan identification. Permethylated N-glycans, peptides, and enriched glycopeptides of erythropoietin were analyzed by nanoLC-MS/MS, and de-N-glycosylated erythropoietin was measured by LC-MS, enabling the qualitative and quantitative analysis of glycosylation and different glycan modifications (e.g., phosphorylation and O-acetylation). The newly developed Python scripts enabled the identification of 140 N-glycan compositions (237 N-glycan structures) from erythropoietin, especially including 8 phosphorylated N-glycan species. The site-specificity of N-glycans was revealed at the glycopeptide level by pGlyco software using different proteases. In total, 114 N-glycan compositions were identified from glycopeptide analysis. Moreover, LC-MS analysis of de-N-glycosylated erythropoietin species identified two O-glycan compositions based on the mass shifts between non-O-glycosylated and O-glycosylated species. Finally, this integrated strategy was proved to realize the in-depth glycosylation analysis of a therapeutic glycoprotein to understand its pharmacological properties and improving the manufacturing processes.

The characterization of glycoproteins, like erythropoietin, is challenging due to the structural micro- and macro-heterogeneity of the protein glycosylation. This study presents an in-depth strategy for glycosylation analysis of a first-generation erythropoietin (epoetin beta), including a developed top-down mass spectrometric workflow for N-glycan analysis, bottom-up mass spectrometric methods for site-specific N-glycosylation and a LC-MS approach for O-glycan identification. Permethylated N-glycans, peptides and enriched glycopeptides of erythropoietin were analyzed by nanoLC-MS/MS and de-N-glycosylated erythropoietin was measured by LC-MS, enabling the qualitative and quantitative analysis of glycosylation and different glycan modifications (e.g., phosphorylation and O-acetylation). Extending the coverage of our newly developed Python script to phosphorylated N-glycans enabled the identification of 140 N-glycan compositions (237 N-glycan structures) from erythropoietin. The site-specificity of N-glycans was revealed at glycopeptide level by pGlyco software using different proteases. In total, 215 N-glycan compositions were identified from N-glycan and glycopeptide analysis. Moreover, LC-MS analysis of de-N-glycosylated erythropoietin species identified two different O-glycan compositions, based on the mass shifts between non-O-glycosylated and O-glycosylated species. This integrated strategy allows the in-depth glycosylation analysis of a therapeutic glycoprotein to understand its pharmacological properties and improving the manufacturing processes.

No abstract

A characteristic of many proteoforms, derived from a single gene, is their similarity regarding the composition of atoms, making their analysis very challenging. Many overexpressed recombinant proteins are strongly associated with this problem, especially recombinant therapeutic glycoproteins from large-scale productions. In contrast to small molecule drugs, which consist of a single defined molecule, therapeutic protein preparations are heterogenous mixtures of dozens or even hundreds of very similar species. With mass spectrometry, currently highquality spectra of intact proteoforms can be obtained only, if the complexity of the mixture of individual proteoform-ions, entering the gas phase at the same time is low. Thus, prior to mass spectrometric analysis, an effective separation is required for getting fractions with a low number of individual proteoforms. This is especially true not only for recombinant therapeutic proteins, because of their huge heterogeneity, but also relevant for top-down proteomics. Purification of proteoforms is the bottleneck in analyzing intact proteoforms with mass spectrometry. This review is focusing on the current state of the art, especially of liquid chromatography for preparing proteoforms for mass spectrometric top-down analysis. The topic of therapeutic proteins has been chosen, because this group of proteins is most challenging regarding their proteoform analysis.