title: Validation of a ligand binding assay using dried blood spot sampling.
abstract: Dried blood spots (DBS) technology has been introduced as a microsampling alternative to traditional plasma or serum sampling for pharmacokinetics or toxicokinetics evaluation. The application of DBS has been established for many small molecule drugs at discovery, nonclinical, and clinical stages. However, the application of DBS for large molecule therapeutics development is not yet well-established. This article describes the method validation of a ligand binding assay (LBA) for DBS sampling of a therapeutic monoclonal antibody-AMG 162 (Denosumab). The original serum LBA was modified for the DBS method. A fit-for-purpose method validation was performed to evaluate accuracy and precision, selectivity, dilutional linearity, and stability. In addition, the parameters relevant to DBS, such as spot volume, extraction recovery, whole blood stability, and hematocrit effects, were evaluated. The validation results demonstrated assay robustness with inter-assay precision of ≤ 19%, inter-assay accuracy of ≤ 9%, and total error of ≤ 24%. Selectivity, extraction recovery, dilutional linearity, and stability were demonstrated. The validation results revealed some limitations of the possible effect of blood hematocrit on therapeutic concentration measurements and the caution required using whole blood for standards and quality controls preparation. This is the first article to describe a thorough method validation of an LBA using DBS for a therapeutic monoclonal antibody. The lessons learned can serve as a model process for future method validation of other LBAs for large molecule therapeutics or biomarkers using the DBS sampling method.
authors.affiliation: Pharmacokinetics and Drug Metabolism Department, Amgen Inc., Mail Stop 30E-3-C, One Amgen Center Drive, Thousand Oaks, CA 91320, USA.
authors.forename: Daniel
authors.initials: D
authors.lastname: Burns
authors.forename: Laura
authors.initials: L
authors.lastname: Brunner
authors.forename: Surendran
authors.initials: S
authors.lastname: Rajendran
authors.forename: Beth
authors.initials: B
authors.lastname: Johnson
authors.forename: Mark
authors.initials: M
authors.lastname: Ma
authors.forename: Jin
authors.initials: J
authors.lastname: Wang
mesh_terms: D061067:Antibodies, Monoclonal, Humanized / Q000378:metabolism*; D001793:Blood Preservation; D000069448:Denosumab; D059788:Dried Blood Spot Testing / Q000379:methods*; D006400:Hematocrit; D006801:Humans; D008024:Ligands
chemical_list: D061067:Antibodies, Monoclonal, Humanized; D008024:Ligands; D000069448:Denosumab