Benchmarking the Performance of a Commercial Electrostatic Linear Ion Trap (ELIT) Charge Detection Mass Spectrometer For Higher-Order Antibody Aggregate Analysis

The analysis of higher-order antibody aggregates presents significant challenges for conventional mass spectrometry due to their low abundance, broad mass distributions, and extensive heterogeneity. Researchers evaluated the practical performance of a commercial electrostatic linear ion trap charge detection mass spectrometer, the XevoTM CDMS, for therapeutic antibody analysis.

The platform demonstrated reproducible mass and charge measurements, with repeatability and intermediate-precision relative standard deviations (RSDs) below 1%. It maintained consistent mass determination across varying concentration and solution conditions.

Controlled mixtures of antibody-derived species exhibited a linear molar-ratio-dependent ion response, allowing for semi-quantitative estimation of relative species abundance under specific conditions. When analyzing heat-stressed trastuzumab, CDMS measurements showed qualitative consistency with SEC-MALS, mass photometry, and conventional native MS. Importantly, CDMS enabled direct mass determination of individual ions within highly heterogeneous high-molecular-weight (HMW) populations that displayed extensive charge-state overlap and were difficult to separate using conventional native MS.

Incorporating a high-duty-cycle acquisition increased the sampling of low-abundance, high-m/z ions, improving access to higher-order aggregate signals. Joint single ion mass-charge analysis successfully differentiated statistical ion families within the broad HMW fraction. By relating major mass differences to independently obtained compositional information, the study highlighted the XevoTM CDMS as a practical and multidimensional platform for comprehensive antibody aggregate characterization.