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

This application note presents a highly sensitive and selective sample preparation strategy for the LC-MS/MS quantification of free/active biotherapeutic from human serum for the TNF-α targeting biotherapeutics: infliximab, adalimumab, and etanercept.

Using a typical set of standard curve and QC samples, and very low volumes of serum (≤10 μL), limits of quantification of 10–50 ng/mL were achieved. Using selective immunoaffinity enrichment and a standardized, kit-based approach to protein digestion combined with fast UPLC analysis and a highly sensitive mass spectrometer, excellent linearity and precision for all standards and QC samples were achieved with %RSDs <15%. With the appropriate immunoaffinity capture reagents, this technique can be applied to other targets and biotherapeutics, demonstrating its broad applicability for high sensitivity, quantitative bioanalysis of large molecules from complex matrices.