Novel strategy using tryptic peptide immunoaffinity-based LC-MS/MS to quantify denosumab in monkey serum

Area of Science:

• Pharmacokinetics and Drug Metabolism
• Immunology and Immunoassays
• Analytical Chemistry

Background:

• Denosumab, a fully human IgG2 antibody, targets human RANKL with high affinity and specificity.
• Existing methods using Fc-fused RANKL are unsuitable for indirect ELISA development.
• Urgent need for a robust and accurate method to characterize denosumab pharmacokinetics.

Purpose of the Study:

• To develop and validate a sensitive bioanalytical method for denosumab quantification.
• To establish an alternative method for pharmacokinetic studies in the absence of specific reagents.

Main Methods:

• Immunoaffinity enrichment coupled with liquid chromatography-tandem mass spectrometry (LC-MS/MS).
• Method validation including linearity, precision, and accuracy assessment.

Main Results:

• The LC-MS/MS method demonstrated a linear range from 0.1 to 30 μg/ml.
• High precision was achieved with intra- and inter-run coefficients of variation (CV%) within 11.5% and 10.5%, respectively.
• Pharmacokinetic data obtained via LC-MS/MS were consistent with traditional ELISA results.

Conclusions:

• The developed LC-MS/MS method accelerates denosumab quantification and reduces associated costs.
• This approach provides a valuable alternative for pharmacokinetic analysis when specific antigens or biotinylated reagents are unavailable.
• The method ensures reliable characterization of denosumab pharmacokinetics.