| Article ID | Journal | Published Year | Pages | File Type |
|---|---|---|---|---|
| 1200028 | Journal of Chromatography A | 2014 | 8 Pages |
•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.
