Fig 1.
Schematic representation of the workflow used for mAbs analysis, highlighting the four characterization levels explored.
Fig 2.
Protein level analysis of intact recombinant monoclonal antibody (characterization level 1).
(A) TOF MS spectra of monoclonal recombinant antibody, from 25 ng to 200 ng on column injection. The inset shows a zoom view of the m/z 2728.31. (B) TOF MS spectra of untreated (blue line) and PNGase-treated (pink line) antibody. A mass shift and reduction of heterogeneity are observed upon PNGase treatment. (C) Deconvolution results of intact (blue line) and deglycosylated antibody (pink line). Glycosylation heterogeneity is clearly detected in the intact antibody. The two major glycosylation species detected are highlighted in the zoom view.
Fig 3.
Protein level analysis of reduced monoclonal antibody (characterization level 2).
The reduced antibody is shown in the blue and PNGase-treated reduced antibody in the pink. (A) TOF MS spectra (left panel) and deconvolution result (right panel) for the light chain. No mass differences were observed after PNGase treatment. (B) TOF MS spectra (left panel) and deconvolution result (right panel) for the heavy chain. A clear mass shift and reduction of heterogeneity are observed upon PNGase treatment. The mass difference between the two major peaks allowed the determination of the major glycosylation specie, in agreement with the results obtained for the intact antibody.
Fig 4.
Peptide level analysis of trypsin digested monoclonal antibody (characterization level 3).
(A) Total ion chromatogram of 2 μg trypsin digested mAb analysed in microLC-MSMS. (B) IDA dependents profile showing the distribution of precursor m/z values over time. (C) Extracted ion chromatograms (XICs) of heavy chain N-terminal Q modifications. The inset shows the respective mass spectrum. (D) MSMS data of the modified N-terminal peptide PGQQVQLQESGPGLVK. The green boxes highlighted in the table corresponds to the assigned fragment ions.
Table 1.
mAb post-translational modifications detected by peptide mapping analysis with the number of modified peptides identified and the relative abundance of the modification.
Results from a representative experiment are shown.
Fig 5.
Glycopeptide analysis of tryptic digested monoclonal antibody.
(A) XICs of detected glycopeptides. The doubly and triply charged ions were detected for the glycopeptide with the G0F, G1F and G2F glycans structures. (B) Mass spectrum of the doubly charged glycopeptides. (C) MSMS data of the glycopeptide with the G0F structure. Relevant fragments were manually annotated.
Fig 6.
PNGase-released N-glycans analysis (characterization level 4).
(A) Total ion current mass chromatogram of N-glycans analysed with PGC nanoLC-MSMS. (B) MSMS data of G0F glycan structure with the higher abundant fragments highlighted. A full analysis of the MSMS data for G0F is shown in supplementary data. (C) XICs of the two most abundant glycan structures, G0F and G1F, and the mass spectra of the corresponding doubly charged m/z ions of these structures. (D) XICs of all other detected glycans. A zoom view of G0F-GlcNAc and G1F-GlcNAc is also shown.
Fig 7.
Glycans structures detected in the free N-glycan analysis with the determination of the relative abundance.
Results from a representative experiment are shown.
Fig 8.
Relative glycosylation quantification using the MS data of the reduced mAb analysis, glycopeptide analysis and free glycan analysis.
For the higher abundant glycans structures (G0F and G1F) similar results were obtained. Diverse low abundant glycan structures were only detected in the free glycans analysis.