Improved and universal inline electrochemical reduction and subunits LC-MS analysis of multiple classes of monoclonal antibodies

Significance of Inline Electrochemical Reduction in mAb Analysis

Monoclonal antibodies (mAbs) are critical biotherapeutics whose structural complexity demands robust analytical workflows. Traditional reduction methods rely on chemical reagents and extensive sample cleanup, which can compromise labile modifications and extend analysis times. Integrating inline electrochemical (EC) reduction with liquid chromatography–mass spectrometry (LC-MS) streamlines sample preparation, minimizes reagent use, and preserves sensitive glycoforms, supporting rapid and detailed characterization of mAbs in research and quality control settings.

Goals and Study Overview

This study aimed to develop and validate a universal inline EC-LC-MS workflow for mAb and subunit analysis across multiple IgG subclasses. Key objectives included:

• Demonstrating complete disulfide bond reduction inline without chemical reducing agents.
• Verifying method compatibility with intact mAbs and IdeS-digested subunits (Lc, Fd, scFc).
• Ensuring accurate profiling of acid-labile modifications such as sialylated N-glycans.
• Applying the workflow to diverse antibody classes (IgG1, IgG2, IgG4) and a heterogeneous drug product (cetuximab).

Methodology and Instrumentation

The optimized platform combined an Antec Scientific ROXY Exceed potentiostat with a μ-PrepCell SS reactor, a Thermo Scientific Vanquish Tandem LC system, and a Thermo Scientific Q Exactive Plus Hybrid Quadrupole-Orbitrap mass spectrometer. Key parameters:

• Electrochemical reduction: 1 V square-wave pulse (1 s reduction, 0.1 s clean) at 60 °C in 20% acetonitrile, 1% formic acid.
• Trap and analytical columns: MAbPac RP (2.1 × 50 mm trap; 2.1 × 100 mm analytical) with gradient elution over 23.5 min.
• MS settings: positive ion mode, 3.8 kV spray voltage, m/z 600–5000, resolution up to 140 000 for subunits.
• Sample prep: buffer exchange into 1% formic acid, 20% ACN; IdeS digestion for subunit generation; vacuum centrifugation and resuspension.

Main Results and Discussion

The inline EC-LC-MS workflow achieved complete reduction of intact mAbs and IdeS-digested subunits in 23.5 min without chemical reagents. Performance was consistent across IgG1 (rituximab), IgG2 (denosumab), and IgG4 (nivolumab), confirming universal applicability despite subclass-dependent disulfide arrangements. Analysis of cetuximab subunits revealed well-resolved isotopic profiles for scFc, Fd, and Lc with deconvoluted glycoform distributions. Relative abundances of sialylated species matched literature values, demonstrating that EC conditions do not degrade acid-labile modifications.

Benefits and Practical Applications

The presented workflow offers multiple advantages for biopharmaceutical analysis:

• Single-platform reduction and LC-MS analysis without chemical reducing agents.
• Elimination of extra cleanup steps, reducing hands-on time and potential sample loss.
• Maintenance of labile post-translational modifications, enabling reliable quantitation of sialylated glycans.
• Scalability to various mAb subclasses and complex drug products.

Future Trends and Applications

Inline EC-LC-MS is poised to evolve further, with potential developments including:

• Integration with automated sample preparation and real-time data analysis for high-throughput screening.
• Extension to other biotherapeutic formats, such as bispecific antibodies and fusion proteins.
• Miniaturized EC cells for low-volume and single-cell applications.
• Coupling with alternative detectors (e.g., ion mobility) to enhance proteoform separation and characterization.

Conclusion

This optimized inline EC-LC-MS workflow delivers rapid, universal, and reagent-free reduction of mAbs and their subunits, preserving sensitive modifications and supporting detailed structural profiling across IgG subclasses. Its simplicity and robustness make it an attractive solution for routine analytical and quality control laboratories in the biopharmaceutical industry.

Reference

• Morgan T.E. et al. Analyst 2021, 146(21), 6547–6555.
• Füssl F. et al. Anal. Chem. 2020, 92(7), 5431–5438.
• Ayoub D. et al. mAbs 2013, 5(5), 699–710.