title: Evaluation of an immunoassay for human-specific quantitation of therapeutic antibodies in serum samples from non-human primates.
abstract: Pharmacokinetic characterization of therapeutic antibodies plays an important role during preclinical and clinical development. However, accurate pharmacokinetic evaluation of therapeutic antibodies in serum samples from non-human primates is often complicated by insufficient specificity of the assays to measure drug levels. The present paper describes the use of a murine monoclonal antibody in an immunoassay format to specifically and quantitatively measure human therapeutic antibodies in serum from non-human primates. This murine antibody is directed against a unique epitope on the constant region CH2 domain of all isotypes of human immunoglobulin G (IgG). The antibody, designated anti-human Fcgamma-pan: R10Z8E9, does not cross-react with serum from mouse, rat, and the non-human primates marmoset, rhesus macaque, cynomolgus monkey and baboon when using an enzyme-linked immunosorbent assay (ELISA) or surface plasmon resonance technology (Biacore) format for measurement of the therapeutic antibody. Use of the antibody anti-human Fcgamma-pan: R10Z8E9 as capturing and detection reagent allowed human-specific quantitation of total therapeutic antibody anti-IGF-1R in spiked cynomolgus monkey serum via a Sandwich ELISA format. In contrast, a commercially available polyclonal antibody (PAB) directed to the Fcgamma fragment of human IgG only specifically measured the therapeutic antibody in buffer samples, but not in serum from cynomolgus monkeys. This generic human IgG assay was already applied in several pharmacokinetic studies in cynomolgus monkeys to determine serum levels of different therapeutic antibodies, including the anti-IGF-1R. Validation of the assay for a humanized IgG1 therapeutic antibody against a membrane protein revealed a lower limit of quantitation of 8 ng/mL in undiluted serum. Intra-assay and inter-assay precision was characterized by a coefficient of variation of less than 10% and accuracy was within 15%. Dilutional linearity was evidenced by a recovery of 98.7-114% of expected concentrations. In conclusion, the monoclonal antibody anti-human Fcgamma-pan: R10Z8E9 provides a standard means for human-specific quantitation of therapeutic antibodies with high sensitivity in serum samples from non-human primates in a generic human IgG assay.
authors.affiliation: Pharma Research Penzberg, Roche Diagnostics GmbH, Penzberg, Germany. kay-gunnar.stubenrauch@roche.com
authors.forename: Kay
authors.initials: K
authors.lastname: Stubenrauch
authors.forename: Uwe
authors.initials: U
authors.lastname: Wessels
authors.forename: Helmut
authors.initials: H
authors.lastname: Lenz
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000097:blood* / Q000493:pharmacokinetics / Q000627:therapeutic use; D000918:Antibody Specificity; D002138:Calibration; D003429:Cross Reactions; D004797:Enzyme-Linked Immunosorbent Assay; D006801:Humans; D007118:Immunoassay / Q000379:methods*; D007141:Immunoglobulin Fc Fragments / Q000276:immunology; D007074:Immunoglobulin G / Q000276:immunology; D008252:Macaca fascicularis; D012015:Reference Standards; D015203:Reproducibility of Results
chemical_list: D000911:Antibodies, Monoclonal; D007141:Immunoglobulin Fc Fragments; D007074:Immunoglobulin G