A versatile method for protein-based antigen bioanalysis in non-clinical pharmacokinetics studies of a human monoclonal antibody drug by an immunoaffinity liquid chromatography–tandem mass spectrometry

• LC/MS method for total antigen protein after dosing of mAb drug with LLOQ 3.13 ng/mL.
• Immunoaffinity enrichment by anti-human Fc region antibody to capture immune complex.
• No need to prepare antigen specific antibody that is different epitope from mAb drug.
• Applicable to multivalent antigen protein forming bulky immune complex with mAb drug.
• LC/MS method without interference by coexistent mAb unlike ELISA.

A versatile immunoaffinity liquid chromatography–tandem mass spectrometry (LC–MS/MS) method was developed to quantify the total concentration of a protein-based antigen in non-clinical pharmacokinetics (PK) studies of a human monoclonal antibody drug. The method combines using magnetic beads that have been coated with a commercial anti-human Fc region antibody to capture an immune complex of the antigen and antibody drug, with subsequent digestion and quantification of the antigen-derived tryptic peptide via LC–MS/MS. Although a typical immunoassay or an immunoaffinity LC–MS/MS assay requires an antigen-specific antibody that uses a different epitope from the antibody drug, this method requires only a commercial anti-human Fc region antibody. The method was applied to quantify total receptor activator of nuclear factor-κB ligand (RANKL) in the presence of denosumab, a humanized monoclonal antibody (mAb) specific to RANKL. The assay was validated as fit-for-purpose and found to be accurate (<115% interbatch accuracies) and precise (<15%, interbatch coefficient of variation) across a range of 3.13–200 ng/mL RANKL. Commercial enzyme-linked immunosorbent assay (ELISA) kit was not able to determine the total RANKL because interference by denosumab decreased recovery. In contrast, the antibody drug had less effect on the LC–MS/MS method. The method now provides a bioanalytical platform for developing other protein-based antigen assays in the early drug stage.