Native Top-Down Mass Spectrometry combined with High-Resolution Charge Variant Analysis of Trastuzumab Originator and Biosimilars

Comprehensive characterisation of monoclonal antibody (mAb) charge heterogeneity is essential for ensuring product quality, maintaining batch consistency, and supporting biosimilar development. Charge variant analysis (CVA) is widely used to separate acidic and basic proteoforms from the main species. However, cation-exchange chromatography coupled to mass spectrometry provides limited information and cannot localise the post-translational modifications (PTMs) responsible for mAb heterogeneity. Here, we present the coupling of pH-gradient CVA with native top-down mass spectrometry (TD-MS) for proteoform-specific analysis of trastuzumab. Individual charge variants were chromatographically separated under native conditions and directly fragmented on the chromatographic timescale using higher-energy collision dissociation (HCD), electron-transfer dissociation (ETD), and ultraviolet photodissociation (UVPD). The addition of proton-transfer charge reduction (PTCR) helped to reduce spectral congestion and enhanced the detection of high-mass fragment ions, resulting in improved sequence coverage. This workflow enabled the complete sequencing of complementarity-determining region (CDR) 3 and the direct identification and insights on the location of key PTMs at the intact-protein level, including deamidation, succinimide, and N-terminal pyroGlu, on individual proteoforms. Comparison of five trastuzumab samples (originator and biosimilars) demonstrated high reproducibility in fragmentation patterns, sequence coverage, and variant assignment, highlighting the robustness of the method. Although limitations remain due to the challenges of fragmentating intact mAbs under native conditions, this work establishes a proof of concept for CVA native TD-MS characterisation of mAbs to complement bottom-up and middle-down analyses and has potential for broad applicability on antibody-based biopharmaceuticals, from characterisation to biosimilarity evaluation.