Real-time Electron Capture Dissociation Characterization of Antibody Subunits via Microdroplet Reactions

Significance of the topic

Comprehensive structural characterization of monoclonal antibodies (mAbs) and engineered multispecific formats is essential across biopharmaceutical development for confirming primary sequence, disulfide connectivity, and product quality attributes that affect safety and efficacy. Conventional workflows rely on offline reduction and proteolysis that are time-consuming, labor intensive and can introduce artefacts. Accelerated microdroplet chemistry in an electrospray-like source combined with electron-capture dissociation (ECD) fragmentation provides a rapid middledown approach that preserves labile modifications and disulfide information while markedly reducing turnaround time.

Objectives and study overview

This poster reports implementation of microdroplet-accelerated chemical reduction and enzymatic digestion within the Agilent Jet Stream (AJS) ion source, paired with ECD fragmentation in an ExD cell on an Agilent 6545XT AdvanceBio LC/Q-TOF, to characterize subunits of a trispecific antibody. Goals were to: (1) demonstrate rapid microdroplet TCEP reduction and GlySERIAS enzymatic cleavage in-line with MS, (2) obtain ECD fragmentation maps for middledown sequence coverage, and (3) compare microdroplet products to conventional offline reactions in terms of product profile and speed.

Methodology and instrumentation

Sample preparation and online microdroplet reactions:

• Trispecific antibody desalted and prepared at 1 mg/mL in 5 mM ammonium bicarbonate.
• TCEP prepared at 10 mg/mL in 5 mM ammonium bicarbonate for rapid reduction.
• GlySERIAS enzyme (Genovis) prepared and loaded at 1 unit/µL for proteolytic cleavage of tethering Gly-Ser linkers.
• An autosampler mixing program aspirated 5 µL reagent (TCEP or enzyme) then 5 µL sample into the injector needle, mixed and injected directly into the AJS source: microdroplet reaction initiates on aspiration and during transfer to the ion source.

Mass spectrometry and fragmentation:

• Agilent Infinity II 1290 Bio UPLC coupled to Agilent 6545XT AdvanceBio LC/Q-TOF equipped with Agilent Jet Stream source and ExD cell (ECD capability).
• MS1 used to verify reaction efficiency (deconvoluted spectra); targeted MS2 isolates specific charge states for ExD/ECD fragmentation.
• ExD Viewer software used for fragment assignment and coverage mapping; ExD cell lens and filament tuning performed as described in the poster.

Main results and discussion

Microdroplet TCEP reduction and ECD fragmentation:

• Reduction of the trispecific antibody in microdroplets and isolation of the 15+ charge state at m/z 1561.49 yielded ECD fragmentation with approximately 65% sequence coverage for the analyzed subunit using ExD Viewer (restrictive scoring). Coverage included regions between inter-chain disulfide bonds, indicating effective rapid reduction of at least some disulfide linkages in the microdroplet environment.

Microdroplet GlySERIAS digestion and ECD of scFv fragment:

• GlySERIAS enzyme cleaved the [Gly4Ser]4 tethering linkers in microdroplets, producing a mixture of cleavage variants consistent with off-line digestion products. The microdroplet protocol achieved comparable product profiles without the hour-long incubation needed for offline digestion.
• ECD fragmentation of the scFv digestion product (12+ charge state at m/z 2207.34) produced only ~27% coverage. Sparse coverage mapped to regions bounded by intact intra-chain disulfide bonds that resisted reduction, consistent with measured masses indicating non-reduced intrachain disulfides. Attempts to increase reduction (additional TCEP runs) did not substantially improve coverage for those bonds. Lower precursor intensity and suboptimal charge states further limited fragmentation yield in some cases.

Interpretation:

• Microdroplet reactions markedly accelerate both chemical reduction and targeted enzymatic digestion and produce products comparable to conventional workflows, but the extent of disulfide reduction can be sequence- and context-dependent: some intrachain disulfides remain resistant on the microsecond timescale despite microdroplet acceleration.
• ECD in the ExD cell provides effective middledown fragmentation preserving labile features and enabling high coverage where disulfides are reduced; however, precursor charge state and intensity critically affect fragmentation efficiency.

Benefits and practical applications

• Significant time savings: microdroplet reactions eliminate lengthy offline incubation steps (e.g., hour-long digests), enabling near-real-time middledown characterization directly at the ion source.
• Reduced sample handling lowers risk of artefacts and improves throughput suitable for rapid analytical support during antibody engineering and development.
• Combining microdroplet chemistries with ECD preserves post-translational modifications (PTMs) and provides informative fragmentation across disulfide-cleaved regions, supporting disulfide mapping and sequence confirmation.
• Appropriate for automated flow-injection or LC-coupled screening in biopharma QC, method development, and research contexts where rapid subunit information is valuable.

Limitations and considerations

• Incomplete reduction of certain intrachain disulfides limits ECD coverage in some subunits; reaction conditions, reagent concentration, or droplet residence time may need optimization per construct.
• Fragmentation efficiency depends on precursor charge state and intensity; selection of optimal charge states and enrichment of target ions may be required for maximal coverage.
• Microdroplet reaction outcomes can produce heterogeneous cleavage products (especially for flexible tether sequences), necessitating careful spectral deconvolution and interpretation.

Future trends and potential applications

• Expansion of the enzyme/reagent toolbox for microdroplet-accelerated chemistries to include additional proteases, glycan-modifying enzymes, and selective chemistries to broaden middledown capabilities.
• Integration of complementary fragmentation modes (ECD combined with HCD/ETD) and improved precursor charge-state control to increase coverage and PTM localization.
• Automation and inline QC workflows using flow-injection microdroplet reactions for lot release screening, comparability studies and accelerated lead optimization.
• Further optimization of microdroplet residence time, reagent stoichiometry, and ion source geometry to overcome resistant disulfides and enhance reproducibility across diverse antibody formats.
• Adoption of native-pH microdroplet approaches and high-resolution MS platforms to preserve noncovalent features while enabling rapid structural interrogation.

Conclusions

The combination of microdroplet-accelerated reduction/digestion in the Agilent Jet Stream source with ECD fragmentation in an ExD cell on the 6545XT AdvanceBio Q-TOF enables rapid middledown characterization of complex antibody formats. The approach delivers comparable product profiles to conventional offline workflows with major reductions in processing time and sample handling. ECD provides strong sequence information where disulfides are reduced; however, some intrachain disulfides remain resistant and limit coverage without further optimization. The workflow shows promise for high-throughput biopharma applications and motivates additional development of microdroplet chemistries and MS fragmentation strategies.

Used instrumentation

• Agilent Infinity II 1290 Bio UPLC
• Agilent 6545XT AdvanceBio LC/Q-TOF with Agilent Jet Stream (AJS) ion source
• Agilent ExD cell (ECD capability) installed on 6545XT
• Autosampler with programmable injector mixing (5 µL reagent + 5 µL sample)
• Amicon 10 kDa spin filters for desalting
• TCEP (tris(2-carboxyethyl)phosphine) for reduction
• GlySERIAS® enzyme (Genovis) for linker/tether cleavage
• ExD Viewer software for fragment assignment and coverage mapping

References

1. Agilent Application Note: 5994-6752EN.
2. Gunawardena, H. P.; Ai, Y.; Gao, J.; Zare, R. N.; Chen, H. Rapid Characterization of Antibodies via Automated Flow Injection Coupled with Online Microdroplet Reactions and Native-pH Mass Spectrometry. Analytical Chemistry 2023, 95 (6), 3340–3348. DOI: 10.1021/acs.analchem.2c04535.
3. Yang, Y.; Xiao, M.; Lau, J.; Knierman, M.; Zhao, H.; Qiu, X.; Luo, K.; Sausen, J.; Gunawardena, H. P.; Chen, H. Ultrafast Microdroplet Digestion of Antibodies with Fc-Silencing Mutations. Analytical Chemistry 2025, 97 (24), 12813–12823. DOI: 10.1021/acs.analchem.5c01856.