Antibody subunit analysis via inline electrochemical reduction of an intact antibody using the ROXY potentiostat coupled to a Vanquish UHPLC – Q Exactive Plus MS system

Importance of the topic

Monoclonal antibodies are cornerstone biotherapeutics whose structural integrity and post-translational modifications must be rigorously characterized to ensure safety, efficacy, and regulatory compliance. Disulfide bond mapping and subunit analysis are essential for understanding antibody heterogeneity. Integrating inline electrochemical reduction with LC-MS offers a rapid, reagent-free method to generate subunits for high-resolution mass spectrometry without extensive sample preparation or enzymatic digestion.

Objectives and study overview

This application note demonstrates complete reduction of an intact mAb (NISTmAb) using a ROXY Exceed potentiostat and μ-PrepCell SS reactor inline with a Vanquish UHPLC–Q Exactive Plus MS system. The goal was to cleave inter- and intrachain disulfide bonds directly in the flow path, produce light and heavy chain subunits, and analyze them using existing LC-MS methods with only the addition of trap-and-release column switching.

Methodology and employed instrumentation

• Sample preparation: Buffer exchange of NISTmAb into 1% formic acid (with or without 20% acetonitrile) at 1 mg/mL; optional IdeS digestion followed by drying and resuspension.
• Electrochemical reduction: Inline μ-PrepCell SS flow-through cell controlled by ROXY Exceed potentiostat in pulse-mode (E1=1.0 V reduction, E2=0 V cleaning; t1=1 s, t2=0.1 s), temperature varied between 20 °C and 60 °C.
• LC-MS setup: Vanquish Flex Duo UHPLC with dual pump, trap-release valve, and MAbPac RP columns (50 × 2.1 mm for trapping, 100 × 2.1 mm for analysis); Q Exactive Plus Orbitrap operated in positive mode (MS1 range m/z 600–5000; resolution 140 k for Lc, 35 k for Hc).
• Data processing: Thermo BioPharma Finder 4.1 using Xtract for isotopically resolved spectra and ReSpect for unresolved spectra; Chromeleon CDS for electrochemical method control and current monitoring.

Main results and discussion

Inline electrochemical reduction effectively cleaved interchain disulfide bonds to yield distinct light (Lc) and heavy (Hc) chains. Increasing reaction potential raised current and promoted bond cleavage. Addition of 20% acetonitrile and heating the cell to 60 °C further enhanced reduction completeness, confirmed by mass shifts (+1 Da per interchain, +2 Da per intrachain) and charge state distribution shifts matching theoretical profiles. The trap-and-release valve switching enabled solvent compatibility without altering chromatographic gradients.

Benefits and practical applications

• Reagent-free, inline workflow eliminates chemical reductants and proteolytic enzymes, reducing sample handling time and potential artefacts.
• Compatible with existing LC-MS protocols; only electrochemical cell and valve switching are introduced.
• Supports detailed subunit analysis for glycoform profiling and disulfide mapping in biopharmaceutical QC and R&D.

Future trends and opportunities

Extending inline electrochemical reduction to top- and middle-down proteomics could enable comprehensive proteoform characterization. Integration with automated tandem MS workflows may refine non-canonical disulfide bond mapping. Further miniaturization and multiplexed electrochemical cells could increase throughput for high-capacity quality control environments.

Conclusion

Inline electrochemical reduction coupled to high-resolution LC-MS provides a streamlined, enzyme- and reagent-free method for antibody subunit analysis. Optimization of potential, temperature, and solvent conditions enables complete cleavage of NISTmAb disulfide bonds while preserving existing chromatographic workflows.

References

1. International Conference on Harmonisation Q6B: Specifications for Biotechnological Products, 1999.
2. Nicolardi S. et al., J Am Soc Mass Spectrom 2013, 24, 1980–1987.
3. Switzar L. et al., J Am Soc Mass Spectrom 2016, 27, 50–58.
4. Morgan T. et al., Analyst 2021, doi:10.1039/D1AN01184G.