Journal of the American Society for Mass Spectrometry· 2026Q2
Non-Ensemble, Conformation-Resolved Stability Analysis of Trispecific Antibodies by Differential Melting Voltage
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- Q2SCImago
- 2026year
Short summary
A new method combining Tandem-TIMS with Differential Melting Voltage (DMV) quantifies gas-phase stability differences between distinct conformational subpopulations of trispecific antibodies (TriAbs), revealing heterogeneity not seen in ensemble measurements.
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Key points
- Tandem-TIMS coupled with DMV analysis provides non-ensemble, conformation-resolved gas-phase stability measurements for trispecific antibodies (TriAbs).
- Distinct conformational subpopulations within TriAbs exhibit different activation-induced unfolding trajectories and relative stabilities.
- Wild-type TriAbs showed stepwise increases in melting voltage, indicating comparable stability across subpopulations, while an Fc-mutated construct displayed a more gradual stability increase.
- These conformational stability differences were not apparent from ensemble-level cross sections or weakly reflected in solution-phase nanoDSF measurements.
AI-generated from the title and abstract; the full text is not read.
Abstract
Abstract 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.
The authors' abstract, as published at the source. Journal of the American Society for Mass Spectrometry, 2026 · DOI ↗
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Field: Spectroscopy
SpectroscopyChemistry