Development of a Hybrid Immunoaffinity-LC-MS/MS Method for the Quantification of Active Biotherapeutics Targeting TNF-α in Serum

Significance of the Topic

The accurate measurement of active, free biotherapeutic agents targeting tumor necrosis factor alpha (TNF-α) in serum is critical for pharmacokinetic and pharmacodynamic assessment during drug development and clinical monitoring. Traditional immunoassays can suffer from cross-reactivity and limited specificity, while LC-MS/MS offers higher selectivity. Combining immunoaffinity enrichment using TNF-α with sensitive LC-MS/MS quantification addresses the dual need for specificity and low detection limits from minimal sample volumes.

Objectives and Study Overview

This study aimed to develop and validate a hybrid immunoaffinity-LC-MS/MS method to quantify free/active infliximab, adalimumab, and etanercept in human serum. Key goals included achieving low ng/mL limits of quantification (LLOQs) using microsampling (2.5–10 µL), ensuring reproducibility, and demonstrating a kit-based, generic workflow compatible with routine bioanalysis.

Methodology and Instrumentation

Samples spiked with known concentrations of each biotherapeutic (10–50,000 ng/mL) were mixed with stable isotope labeled internal standards and subjected to immunopurification. Biotinylated TNF-α was bound to streptavidin magnetic beads to capture free drug from serum. Bound proteins were eluted with formic acid, neutralized, and digested using a standardized ProteinWorks eXpress Digest Kit. Peptides were separated on an ACQUITY UPLC Peptide BEH C18 column and detected on a Xevo TQ-XS triple quadrupole mass spectrometer operating in ESI+ MRM mode. Skyline software guided peptide selection and transition optimization.

Main Results and Discussion

The assay exhibited excellent linearity over 3–3.7 orders of magnitude (R² > 0.99) and achieved LLOQs of 10 ng/mL for infliximab, 25 ng/mL for adalimumab, and 50 ng/mL for etanercept using only 2.5 µL of serum. Increasing sample volume to 10 µL improved signal intensity and peak shapes. Precision and accuracy across quality control levels met regulatory guidelines, with %RSD < 15% and accuracy between 85–110%. Chromatographic gradients were optimized individually to resolve target peptides from endogenous interferences.

Benefits and Practical Applications

• High specificity through target-ligand immunocapture minimizes matrix effects.
• Low sample volume requirement supports studies in preclinical species or limited clinical samples.
• Generic, kit-based digestion provides reproducibility and simplifies workflow.
• Applicable to multiple TNF-α inhibitors and adaptable to other biotherapeutics with corresponding ligands.

Instrumentation Used

• Waters ACQUITY UPLC I-Class System
• ACQUITY UPLC Peptide BEH C18 Column (1.7 µm, 2.1 × 150 mm)
• Waters Xevo TQ-XS Mass Spectrometer
• ProteinWorks eXpress Digest Kit
• MassLynx v4.2 and TargetLynx Software

Future Trends and Potential Applications

The hybrid immunoaffinity-LC-MS/MS strategy can be extended to emerging biotherapeutics by selecting appropriate capture ligands. Advances in microfluidics and high-throughput bead handling could further reduce assay time and sample volume. Integration with multiplexed MRM panels may enable simultaneous quantification of multiple drugs and biomarkers in personalized medicine and therapeutic drug monitoring.

Conclusion

This method delivers a robust, sensitive, and reproducible approach for quantifying free TNF-α inhibitors in serum from minimal sample volumes. Its generic, kit-based workflow and high specificity support broad bioanalytical applications in drug development and clinical monitoring.

References

1. El Amrani M, van den Broek MP, Göbel C, et al. J Chromatogr A. 2016;1454:42–48.
2. Lee JW, Kelley M, King LE, et al. AAPS J. 2011;13(1):99–110.
3. FDA Guidance for Industry: Bioanalytical Method Validation.
4. EMA Guideline on Bioanalytical Method Validation; 2011.