Advantages of Ion Mobility QTOF for Characterization of BioPharma Molecules

Significance of the Topic

Ion mobility combined with quadrupole time-of-flight mass spectrometry (IM-QTOF) introduces a crucial separation dimension based on ion size, shape and charge. This enhancement is vital for characterizing complex biopharmaceutical molecules—such as proteins, antibodies, carbohydrates and their conjugates—by improving spectral purity, sensitivity and structural insight.

Objectives and Study Overview

This work evaluates Agilent’s new drift tube IM-QTOF system to demonstrate:

• Enhanced ion mobility resolution while retaining QTOF mass accuracy
• Increased sensitivity via electrodynamic ion funnels
• Direct collision cross section (CCS) measurements
• Application across a range of bio-molecules from small saccharides to large protein complexes and antibody-drug conjugates

Methodology and Instrumentation

The IM-QTOF platform integrates a uniform-field drift tube with a high-resolution QTOF mass analyzer. Key components include:

• Multiple ion sources: ESI, nano-ESI, Agilent Jet Stream, APCI
• Front and rear electrodynamic funnels for efficient ion focusing and desolvation
• Trap funnel for ion accumulation and precise pulse introduction into the drift cell
• Uniform low-field drift cell for direct CCS determination
• High-speed QTOF data acquisition matched to mobility timescales

Instrumentation Used

Dual Agilent Jet Stream ESI source coupled to a 6560 IM-QTOF MS with the following settings:

• Gas temperature: 250 °C; nebulizer: 20 psig; sheath gas: 275 °C at 12 L/min
• VCap: 4000 V; nozzle: 2000 V; fragmentor: 400 V
• Mass range: 300–10000 m/z; scan rate: 0.9 frames/s
• IM trap fill: 50 ms; release: 0.3 ms

Key Results and Discussion

• Carbohydrate isomer separation: Raffinose and melezitose baseline resolved by drift time differences (~26.7 vs 25.8 ms).
• Oligosaccharide mixtures: Lacto-N-difucohexaose I and II distinguished in a single run, highlighting isomeric discrimination in complex glycan analysis.
• Disulfide bond variants: Siamycin II isoforms detected via distinct mobility drift times, revealing mis-formed linkages.
• Protein conformers: Cytochrome C charge states under low RF preserved native structures (S1 vs denatured S2–S5 states).
• Monoclonal antibodies: Native versus denatured IgG-2 showed significant drift time shifts; IgG-1 and IgG-2 isoform distributions compared, with IgG-2 exhibiting a higher proportion of B-type conformer.
• Biosimilar comparison: Rituximab innovator and biosimilar showed subtle CCS differences in the 27+ charge state, reflecting glycan size variations.
• Antibody–drug conjugates: Herceptin and its ADC form resolved; deconvoluted spectrum revealed eight major drug attachments and an average DAR of ~3.4.
• Large protein complexes: Bovine glutamate dehydrogenase hexamer (≈337 kDa) characterized under native conditions, with measured CCS ~11869 Ų for the 40+ charge state.

Benefits and Practical Applications

• Increased peak capacity and spectral purity for complex mixtures
• Lower detection limits by filtering background and interferences
• Direct CCS measurements support confident compound identification and structural assignments
• Preservation of native biomolecular conformations for stability and folding studies
• Comparability assessment of therapeutic antibodies and biosimilars
• Quantitation of drug-to-antibody ratios in ADC development

Future Trends and Potential Applications

Integration of IM-QTOF with advanced data analytics and 4D feature finding (mass, drift time, retention time, intensity) will enable high-throughput glycomics, proteoform profiling and native complex analysis. Anticipated developments include higher mobility resolution, faster acquisition rates, improved CCS libraries and AI-driven structural predictions for next-generation biotherapeutic characterization.

Conclusion

Agilent’s drift tube IM-QTOF system delivers a powerful combination of high sensitivity, mobility resolution and accurate mass measurement. Its ability to resolve isomers, detect structural variants and measure CCS directly makes it an indispensable tool for bio-pharmaceutical research, quality control and biosimilarity evaluation.