title: Critical role of bioanalytical strategies in investigation of clinical PK observations, a Phase I case study.
abstract: RG7652 is a human immunoglobulin 1 (IgG1) monoclonal antibody (mAb) targeting proprotein convertase subtilisin/kexin type 9 (PCSK9) and is designed for the treatment of hypercholesterolemia. A target-binding enzyme-linked immunosorbent assay (ELISA) was developed to measure RG7652 levels in human serum in a Phase I study. Although target-binding assay formats are generally used to quantify free therapeutic, the actual therapeutic species being measured are affected by assay conditions, such as sample dilution and incubation time, and levels of soluble target in the samples. Therefore, in the presence of high concentrations of circulating target, the choice of reagents and assay conditions can have a significant effect on the observed pharmacokinetic (PK) profiles. Phase I RG7652 PK analysis using the ELISA data resulted in a nonlinear dose normalized exposure. An investigation was conducted to characterize the ELISA to determine whether the assay format and reagents may have contributed to the PK observation. In addition, to confirm the ELISA results, a second orthogonal method, liquid chromatography tandem mass spectrometry (LC-MS/MS) using a signature peptide as surrogate, was developed and implemented. A subset of PK samples, randomly selected from half of the subjects in the 6 single ascending dose (SAD) cohorts in the Phase I clinical study, was analyzed with the LC-MS/MS assay, and the data were found to be comparable to the ELISA data. This paper illustrates the importance of reagent characterization, as well as the benefits of using an orthogonal approach to eliminate bioanalytical contributions when encountering unexpected observations.
authors.affiliation: a Department of BioAnalytical Sciences; Genentech Inc ; South San Francisco , CA USA.
authors.forename: Kun
authors.initials: K
authors.lastname: Peng
authors.forename: Keyang
authors.initials: K
authors.lastname: Xu
authors.forename: Luna
authors.initials: L
authors.lastname: Liu
authors.forename: Robert
authors.initials: R
authors.lastname: Hendricks
authors.forename: Reginald
authors.initials: R
authors.lastname: Delarosa
authors.forename: Rich
authors.initials: R
authors.lastname: Erickson
authors.forename: Nageshwar
authors.initials: N
authors.lastname: Budha
authors.forename: Maya
authors.initials: M
authors.lastname: Leabman
authors.forename: An
authors.initials: A
authors.lastname: Song
authors.forename: Surinder
authors.initials: S
authors.lastname: Kaur
authors.forename: Saloumeh K
authors.initials: SK
authors.lastname: Fischer
mesh_terms: D000911:Antibodies, Monoclonal / Q000097:blood / Q000276:immunology* / Q000493:pharmacokinetics*; D061067:Antibodies, Monoclonal, Humanized; D002853:Chromatography, Liquid; D015331:Cohort Studies; D004305:Dose-Response Relationship, Drug; D004311:Double-Blind Method; D004797:Enzyme-Linked Immunosorbent Assay; D006801:Humans; D007074:Immunoglobulin G / Q000097:blood / Q000276:immunology*; D000071449:Proprotein Convertase 9; D043484:Proprotein Convertases / Q000276:immunology*; D012697:Serine Endopeptidases / Q000276:immunology*; D053719:Tandem Mass Spectrometry
chemical_list: D000911:Antibodies, Monoclonal; D061067:Antibodies, Monoclonal, Humanized; D007074:Immunoglobulin G; C000600650:RG7652; C472125:PCSK9 protein, human; D000071449:Proprotein Convertase 9; D043484:Proprotein Convertases; D012697:Serine Endopeptidases
keywords: 5, 5′-tetramethylbenzidine;; BSA, bovine serum albumin; CDR, complementarity-determining region; ELISA, enzyme-linked immunosorbent assay; HRP, horseradish peroxidase; IS, internal standard; IgG1, immunoglobulin G1; LC-MS/MS; LC-MS/MS, liquid chromatography tandem mass spectrometry; LDL-c, low density lipoprotein cholesterol; LDLR, low density lipoprotein receptor; LLOQ, lower limit of quantification; MAD, multiple-ascending dose; MQC, minimum quantifiable concentration; MRM, multiple reaction monitoring; NHS, normal human sera; PBS, phosphate buffered saline; PCSK9, proprotein convertase subtilisin/kexin type 9;; PD, pharmacodynamics; PK, pharmacokinetics; RG7652; RT, room temperature; S/N, signal-to-noise; SA, streptavidin; SAD, single-ascending dose; SIL, stable isotope-labeled; TMB, 3, 3′; clinical pharmacokinetic assay; enzyme-linked immunosorbent assay; mAbs, monoclonal antibodies; proprotein convertase subtilisin/kexin type 9; rhuPCSK9, recombinant human PCSK9; signature peptide