Non-Ensemble, Conformation-Resolved Stability Analysis of Trispecific Antibodies by Differential Melting Voltage

Trispecific antibodies (TriAbs) integrate multiple antigen-binding modules within a single construct, creating greater architectural complexity and conformational heterogeneity than conventional monoclonal antibodies. Conventional stability measurements report ensemble-averaged behavior and may therefore conceal stability differences among distinct conformational subpopulations within a TriAbs construct. Here, tandem-trapped ion mobility spectrometry (Tandem-TIMS) was combined with differential melting voltage (DMV) analysis to investigate the conformation-resolved gas-phase stability of wild-type and Fc-mutated TriAbs. Under native conditions, both antibody constructs retained compact, native-like structures with nearly identical ensemble-level cross sections. Nonensemble Tandem-TIMS measurements resolved distinct conformational subpopulations that followed different activation-induced unfolding trajectories, confirming that they represented structurally distinct conformational states. DMV analysis quantified these differences using a single, quantifiable metric: the melting voltage, Vm. The wild-type construct showed a stepwise increase in the melting voltage, with several conformational subpopulations displaying comparable relative stability toward collisional activation. In contrast, the AAS-mutated construct exhibited a more gradual increase in stability with increasing cross section. These differences were not apparent from the ensemble-level cross section distributions and were only weakly reflected in solution-phase nanoDSF measurements. The results demonstrate that similar intact antibody size and bulk thermal properties do not necessarily imply similar conformational landscapes. Most importantly, the integration of Tandem-TIMS with DMV analysis provides a nonensemble strategy for directly connecting structural heterogeneity with the relative gas-phase stability of conformational subpopulations in complex antibody therapeutics.