Fig. 1

Generation and structural assessment of wild type and glyco-engineered EPO. a Depiction of the workflow used in this study. EPO is overexpressed in CHO cells whereby specific glyco-genes are manipulated to achieve the desired glycosylation, followed by EPO purification and native MS analysis. High-resolution native MS spectra of different clones are correlated, leading to a similarity matrix. b Depiction of EPO glycosylation sites. c Deconvoluted high-resolution native mass spectra of WT EPO (top) and two KO-based glyco-engineered clones: C23 (middle) and C21 (bottom). These two KO clones would supposedly delete β-4 and β-6branching of N-glycans as indicated by the pictograms. In these deconvoluted spectra the main glycoproteoforms are color coded, wherein each color corresponds to a unique Hex_x+3HexNAc_xF₃ composition, where F denotes fucose, and the numbers placed above the annotated peaks indicate the number of sialic acid residues. d Comparison of native mass spectra of EPO from two biological replicate clones C18 (black) and C19 (orange).The correlation coefficient between these two spectra over all ion signals is 0.93, as depicted in the top-right. All other high-resolution native mass spectra of EPO, purified from each of the prepared 25 clones, are provided in Supplementary Fig 4