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

Comprehensive characterization of monoclonal antibody (mAb) charge heterogeneity is essential to ensure product quality, maintain batch consistency and support 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 localize the post-translational modifications (PTMs) responsible for mAb heterogeneity. Here, the coupling of pH-gradient CVA with native top-down mass spectrometry (TD-MS) for proteoform-specific analysis of trastuzumab is presented. Individual charge variants were chromatographically separated under native conditions and directly fragmented on the chromatographic time scale using higher-energy collision dissociation (HCD), electron-transfer dissociation (ETD) and ultraviolet photodissociation (UVPD). The addition of proton-transfer charge reduction (PTCR) helped reduce spectral congestion and enhanced the detection of high-mass fragment ions, resulting in improved sequence coverage. This workflow enabled the complete sequencing of the complementarity-determining region (CDR) 3 and the direct identification and insights into the location of key PTMs at the intact-protein level, including deamidation, succinimide and N-terminal pyroGlu for 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 fragmenting intact mAbs under native conditions, this work establishes a proof of concept for CVA native TD-MS characterization of mAbs to complement bottom-up and middle-down analyses, and has potential for broad applicability for antibody-based biopharmaceuticals.