HYBRID LC-MS/MS FOR QUANTIFICATION OF INFLIXIMAB IN CROHN’S DISEASE PATIENT SAMPLES: DOES IT ADD VALUE?

Importance of the Topic

Therapeutic drug monitoring (TDM) of infliximab is essential for optimizing treatment in Crohn’s disease and reducing the incidence of primary and secondary non-response. Variability among commercial ELISA kits has driven the search for more specific, reproducible methods. LC-MS/MS techniques offer improved specificity, broader dynamic range, and the ability to distinguish total versus free drug.

Objectives and Study Overview

This study compared three quantification approaches—ELISA (LISA TRACKER), direct digestion LC-MS/MS, and hybrid immunoaffinity LC-MS/MS—using trough serum samples from 89 Crohn’s disease patients on maintenance infliximab therapy. The goal was to assess assay performance, agreement in drug‐level classification, and practical value for standardized TDM.

Methodology

Trough samples were analyzed by:

• LISA TRACKER ELISA for free infliximab (0.3–16.0 µg/mL)
• Direct digestion LC-MS/MS measuring total infliximab (1.0–100.0 µg/mL)
• Hybrid immunoaffinity LC-MS/MS using biotinylated TNF-α capture (0.1–50.0 µg/mL)

All LC-MS/MS samples underwent tryptic digestion with ProteinWorks eXpress kit before analysis.

Instrumentation Used

• Waters ACQUITY I-Class UPLC PLUS
• Waters Xevo TQ-XS tandem quadrupole MS (ESI+)
• BEH C18, 300 Å, 1.7 µm, 2.1 × 150 mm column
• ProteinWorks eXpress Digest Kit

Results and Discussion

Both LC-MS/MS methods demonstrated excellent linearity (r² > 0.99) across their dynamic ranges. Classification into subtherapeutic (<1.0 µg/mL), intermediate (1.0–2.0 µg/mL), and therapeutic (>2.0 µg/mL) levels showed:

• Therapeutic‐level agreement ≈ 90% across assays
• Overall concordance ≈ 60% for intermediate and subtherapeutic categories
• Bias: hybrid vs ELISA +47%, direct vs ELISA +23%, hybrid vs direct –15%
• One peptide (SINSATHYAESVK) excluded due to in vivo deamidation

Immunoaffinity capture enhanced assay specificity and alignment with ELISA results, supporting its value for standardized TDM.

Benefits and Practical Applications

• Improved specificity and confidence compared to ELISA
• Broader dynamic range for measuring total and free drug
• Ability to tailor clinical cutoffs and improve treatment decisions

Future Trends and Applications

Anticipated developments include application to additional biotherapeutics, integration of anti‐drug antibody monitoring, refinement of surrogate peptide selection, and automation for high-throughput clinical workflows.

Conclusion

Two LC-MS/MS approaches for infliximab quantification in Crohn’s disease sera were validated. The hybrid immunoaffinity method offered superior specificity and closer agreement with ELISA, providing a pathway toward standardized, reliable TDM.

References

• Dunning CM, Lame ME. Development of a Hybrid Immunoaffinity-LC-MS/MS Method for the Quantification of Active Biotherapeutic Targeting TNF-α in Serum. Waters Corporation; 2018:720006317EN.
• Samaan MA, Arkir Z, Ahmad T, Irving PM. Wide variation in the use and understanding of therapeutic drug monitoring for anti-TNF agents in inflammatory bowel disease: an inexact science? Expert Opin Biol Ther. 2018;18(12):1271–1279.
• National Institute for Health and Care Excellence. Therapeutic monitoring of TNF-α inhibitors in Crohn’s disease (DG22). 2016.