Monitoring the intact light chain of therapeutic monoclonal antibodies in human serum using an Orbitrap Exploris 240 mass spectrometer for clinical research

Significance of the Topic

Monoclonal antibodies (mAbs) are increasingly used in clinical therapy, with a projected market value exceeding $200 billion by 2026. Precise quantitation of therapeutic mAbs in patient serum is critical for optimizing dosing, ensuring efficacy, and minimizing adverse effects. Traditional peptide-based assays can be time-consuming and may struggle to distinguish therapeutic mAbs from endogenous immunoglobulins.

Aims and Study Overview

This study presents a rapid and selective workflow for quantifying intact light chains of therapeutic mAbs in human serum. By employing Protein L magnetic bead capture, on‐bead IdeS digestion and disulfide reduction, and high‐resolution accurate mass (HRAM) Orbitrap analysis, the authors aimed to establish limits of detection (LOD) and quantitation (LOQ), linear range, and reproducibility for six clinically relevant mAbs.

Methodology

Sample preparation involved spiking six target mAbs (adalimumab, bevacizumab, camrelizumab, daratumumab, golimumab, rituximab) at concentrations from 1 to 100 µg/mL into human serum. Two non‐overlapping mAbs (nivolumab, pembrolizumab) served as internal standards. Protein L magnetic beads selectively bound kappa light chain–containing antibodies, excluding lambda chains. Captured mAbs underwent on‐bead IdeS digestion at 37 °C and on‐bead reduction with TCEP at 57 °C, eliminating elution steps and minimizing sample loss. Following desalting and concentration, samples were analyzed by reversed-phase UHPLC coupled to an Orbitrap Exploris 240 MS under the BioPharma option.

Instrumentation

• Thermo Scientific Orbitrap Exploris 240 mass spectrometer with BioPharma option
• Vanquish Flex Binary UHPLC system
• MAbPac RP column (2.1 × 50 mm, 4 µm)
• Protein L magnetic beads (Thermo Scientific Pierce)
• TraceFinder software for targeted data extraction

Main Results and Discussion

High-resolution MS at >120 k resolving power enabled clear separation of isotopic clusters for light, heavy (Fd′), and Fc/2 subunits. Calibration curves for all six mAbs exhibited excellent linearity (R2>0.99). LOQs ranged from 1 to 5 µg/mL; LODs were between 1 and 5 µg/mL. Inter- and intra-day reproducibility yielded retention time variation <0.05 min within lots (<0.2 min between lots) and peak area differences <20% across columns.

Benefits and Practical Applications of the Method

This streamlined workflow requires only 10 µL of serum per analysis and under 1.5 hours of sample preparation. On-bead processing reduces handling and sample loss. Quantitation at the intact light chain level avoids dependence on peptide signatures, facilitating monitoring of mAbs with limited tryptic peptides and distinguishing them from endogenous IgG.

Future Trends and Opportunities

Advances in HRAM mass spectrometry will enable multiplexed quantitation of diverse antibody formats and bispecific constructs. Automation of on-bead digestion and integration with clinical platforms may support high-throughput therapeutic drug monitoring. Further miniaturization and direct sampling techniques could expand applications to point-of-care settings.

Conclusion

The described Protein L-based purification and Orbitrap Exploris 240 MS workflow achieves sensitive, selective, and reproducible quantitation of intact mAb light chains in serum. It offers a robust alternative to peptide-based assays, with potential for broader clinical implementation in therapeutic drug monitoring.

References

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