Structural Characterization of Glycated Monoclonal Antibodies Using Collision Induced Unfolding on the SELECT SERIES™ Cyclic™ IMS Mass Spectrometer

Importance of the Topic

Monoclonal antibodies are cornerstone biotherapeutics whose safety and efficacy depend on precise control of their higher order structures and stability. Traditional calorimetric methods provide thermodynamic insights in solution but often require lengthy analysis and substantial material. Native ion mobility–mass spectrometry (IM-MS) coupled with collision-induced unfolding (CIU) offers a rapid, solvent-free alternative to probe protein conformations and stability, supporting developability assessments and quality control workflows.

Study Objectives and Overview

This study applies native IM-MS and CIU to characterize the unfolding pathway of the NISTmAb reference monoclonal antibody under increasing levels of glycation. By incubating the antibody with glucose for defined intervals, researchers aimed to evaluate how covalent glycation influences gas-phase unfolding transitions and to demonstrate CIU as a high-throughput approach for monitoring subtle structural changes.

Methodology and Instrumentation

Methodology:

• Glycation of NISTmAb at 5 mg/mL with 1 M glucose for 30 minutes, 7 days, and 16 days at 30 °C.
• Buffer exchange into ammonium acetate and dilution to 0.1 mg/mL for native analysis.
• Acquisition of native mass spectra to confirm hexose additions (+162 Da) and proteoform distribution.
• CIU workflow: stepwise collision voltage ramp in the trap cell to induce gas-phase unfolding with mobility separation at each energy step.
• Data analysis and fingerprint generation using CIUSuite3 to map unfolding intermediates and calculate CIU50 values.

Instrumentation Used

• SELECT SERIES Cyclic IMS™ Mass Spectrometer with quadrupole selection and Cyclic IMS separation.
• Nanolockspray ionization source with borosilicate glass nanocapillaries.
• High-resolution TOF analyzer operating in positive mode over 50–8 000 m/z.
• Software: MassLynx™ v4.2, Driftscope™ v3.0, and CIUSuite3 for fingerprint extraction and quantitative comparisons.

Main Results and Discussion

Native MS confirmed progressive glycation, with up to 11 hexose modifications after extended incubation. CIU fingerprints for the 25+ charge state consistently revealed five conformational states. Increasing glycation levels markedly stabilized the fourth unfolding intermediate, shifting its transition to higher collision voltages. Pairwise fingerprint comparisons yielded a significant RMSD of 15.4 between non-glycated and 16-day-glycated samples (intra-condition RMSD 2.8). CIU50 analysis showed minimal change in the first three transitions but a pronounced elevation for transition 4 (from 102 V to 148 V), underscoring glycation-induced stabilization.

Benefits and Practical Applications

• Rapid assessment of protein folding pathways in minutes without chromatographic separation.
• High-throughput screening of biotherapeutic stability under different stress conditions.
• Quantitative metrics (RMSD, CIU50) enable objective comparison of formulation or modification effects.
• Reduced sample consumption and interference due to native, solvent-free analysis.

Future Trends and Opportunities

Integration of CIU with other native MS techniques, such as hydrogen/deuterium exchange and crosslinking, could yield multidimensional stability profiles. Advances in automation and AI-driven data interpretation promise to accelerate developability screening and real-time quality monitoring. Expansion of CIU libraries and calibration standards will facilitate cross-laboratory reproducibility and regulatory acceptance.

Conclusion

This application note demonstrates that CIU on the SELECT SERIES Cyclic IMS instrument, combined with CIUSuite3 software, provides a fast, sensitive, and quantitative approach to detect structural stabilization of glycated monoclonal antibodies. The methodology supports early developability assessments and offers a robust platform for high-throughput biotherapeutic characterization.

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