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

作者：Onami Ichio^*; Ayabe Miho; Murao Naoaki; Ishigai Masaki

来源：Journal of Chromatography A, 2014, 1334: 64-71.

DOI：10.1016/j.chroma.2014.01.082

• Receptor activator of nuclear factor-kappa B
• ligand (RANKL)
• LC-MS/MS
• Immunoaffinity
• Immune complex

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-kappa B 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.

• 出版日期2014-3-21