title: 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.
abstract: 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 3.13-200ng/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.
authors.affiliation: Fuji-Gotemba Research Labs. Chugai Pharmaceutical Company, Ltd., 1-135 Komakado, Gotemba, Shizuoka 412-8513, Japan. Electronic address: ohnami.ichio39@chugai-pharm.co.jp.
authors.forename: Ichio
authors.initials: I
authors.lastname: Onami
authors.affiliation: Fuji-Gotemba Research Labs. Chugai Pharmaceutical Company, Ltd., 1-135 Komakado, Gotemba, Shizuoka 412-8513, Japan.
authors.forename: Miho
authors.initials: M
authors.lastname: Ayabe
authors.affiliation: Fuji-Gotemba Research Labs. Chugai Pharmaceutical Company, Ltd., 1-135 Komakado, Gotemba, Shizuoka 412-8513, Japan.
authors.forename: Naoaki
authors.initials: N
authors.lastname: Murao
authors.affiliation: Fuji-Gotemba Research Labs. Chugai Pharmaceutical Company, Ltd., 1-135 Komakado, Gotemba, Shizuoka 412-8513, Japan.
authors.forename: Masaki
authors.initials: M
authors.lastname: Ishigai
mesh_terms: D000818:Animals; D061067:Antibodies, Monoclonal, Humanized / Q000097:blood / Q000276:immunology / Q000493:pharmacokinetics*; D002846:Chromatography, Affinity / Q000379:methods*; D002853:Chromatography, Liquid / Q000379:methods*; D000069448:Denosumab; D004797:Enzyme-Linked Immunosorbent Assay; D006801:Humans; D018189:Immunomagnetic Separation; D051379:Mice; D008810:Mice, Inbred C57BL; D010455:Peptides / Q000737:chemistry; D053245:RANK Ligand / Q000097:blood* / Q000276:immunology*; D053719:Tandem Mass Spectrometry / Q000379:methods*
chemical_list: D061067:Antibodies, Monoclonal, Humanized; D010455:Peptides; D053245:RANK Ligand; C505688:TNFSF11 protein, human; D000069448:Denosumab
keywords: Immune complex; Immunoaffinity; LC–MS/MS; Receptor activator of nuclear factor-κB ligand (RANKL)