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Aim: IP-10 is a protein target for the treatment of Crohn's disease. Inhibition of IP-10 by anti-IP-10 mAbs neutralizes its various biological activities. The measurement of free IP-10 suppression as a target engagement biomarker is required for the assessment of drug effect on the target. Results: The development of highly sensitive immunoaffinity-LC–MS/MS assays for quantifying free and total IP-10 in cynomolgus monkey serum is reported for the first time. This paper details strategies for maximizing assay sensitivity by selecting digestion routes, and optimizing immunocapture to achieve full recovery and minimal matrix effect. For the free IP-10 assay, bioanalytical strategies have been established to minimize drug/ligand dissociation. Conclusion: The assays have been implemented for target engagement measurement, pharmacokinetic-pharmacodynamic correlation, and human dose projections.

Immunoaffinity LC–MS/MS for quantitative determination of a free and total protein target as a target engagement biomarker