A global comparability approach for biosimilar monoclonal antibodies using LC–tandem MS based proteomics

•A 3-step comparison strategy is demonstrated for biosimilars.•Comparisons include sequencing, targeted modifications and non-targeted tryptic map.•Anti-Her2 trastuzumab is compared with the correct and mutated biosimilars.•Non-targeted intensity comparisons are shown to correctly identify the variants.•The method provides extra-dimensional view to assess the complexity for biosimilars.

Liquid chromatography–tandem mass spectrometry-based proteomics for peptide mapping and sequencing was used to characterize the marketed monoclonal antibody trastuzumab and compare it with two biosimilar products, mAb A containing D359E and L361M variations at the Fc site and mAb B without variants. Complete sequence coverage (100%) including disulfide linkages, glycosylations and other commonly occurring modifications (i.e., deamidation, oxidation, dehydration and K-clipping) were identified using maps generated from multi-enzyme digestions. In addition to the targeted comparison for the relative populations of targeted modification forms, a non-targeted approach was used to globally compare ion intensities in tryptic maps. The non-targeted comparison provided an extra-dimensional view to examine any possible differences related to variants or modifications. A peptide containing the two variants in mAb A, D359E and L361M, was revealed using the non-targeted comparison of the tryptic maps. In contrast, no significant differences were observed when trastuzumab was self-compared or compared with mAb B. These results were consistent with the data derived from peptide sequencing via collision induced dissociation/electron transfer dissociation. Thus, combined targeted and non-targeted approaches using powerful mass spectrometry-based proteomic tools hold great promise for the structural characterization of biosimilar products.

Graphical abstractA global non-specific comparison method, which compares the base peak chromatogram of the enzymatic map, is demonstrated as a useful tool to supplement the current methods for the structural characterization of biosimilar products.Figure optionsDownload full-size imageDownload as PowerPoint slide