Online Buffer Exchange Coupled to Ion Mobility-Mass Spectrometry for the Rapid, Automated Analysis of Antibodies from Formulation

<jats:p>Native ion mobility-mass spectrometry (nIM-MS) enables rapid protein structure and stability analysis, but adoption remains limited by time-consuming offline buffer exchange and manual static nanoelectrospray ionization (nESI) emitter workflows. This low throughput is incompatible with the demands of biotherapeutic development, where many excipient conditions must be screened. Here, we couple the DynaChip X1, a microfluidic tangential flow filtration device, to a drift tube IM-qTOF for the analysis of monoclonal antibodies (mAbs) directly from formulation buffer. We show that online desalting preserves native-like ion structure, producing compact, monomodal charge state distributions and collision cross sections in agreement with offline buffer exchange and static nESI emitter results. We then benchmark the desalting capacity of the DynaChip X1 by systematically introducing different formulation buffer components, including a complete commercial formulation, into a standard mAb solution. Acceptable desalting is achieved for all components after simple dilution of the mAb to 0.1 mg/mL, with most sufficiently desalted at 5x – 10x this concentration. Online collision-induced unfolding (CIU) experiments across the elution profile reveal a stable 6.5-minute window over which replicate CIU experiments are acquired from approximately 1 µg of protein sample, with gas-phase stability and run-to-run reproducibility comparable to static nESI. We then extend the platform through a series of vignettes, showing online electron-capture dissociation, heat stress analysis, and direct cleanup of a crude antibody-drug conjugate reaction mixture. Overall, these data establish tangential flow filtration as an online desalting interface for nIM-MS, eliminating the offline buffer exchange step and enabling the adoption of off-the-shelf nESI emitters that have limited adoption of nIM-MS in formulation screening.</jats:p>