Characterization of a quantitative method to measure free proprotein convertase subtilisin/kexin type 9 in human serum

Abstract

Proprotein convertase subtilisin/kexin type 9 (PCSK9) is a serine protease that plays an important role in the regulation of serum low-density lipoprotein (LDL) cholesterol by downregulation of LDL receptor, and as such is considered a novel target in cholesterol lowering therapy. In support of the drug development program for Evolocumab, a fully human IgG2 antibody that targets PCSK9, a quantitative ELISA to measure free PCSK9 in human serum was developed. PCSK9 serves as a biomarker of pharmacological response during treatment, and measuring levels of the free ligand post-dosing was of interest as an aid to establishing the pharmacokinetic and pharmacodynamic properties of the therapeutic. Given the complexities associated with the measurement of free ligand in the presence of high concentrations of circulating drug, it was important to challenge the method with experiments designed to assess ex vivo conditions that have the potential to affect the binding equilibrium of drug and ligand within test samples during routine sampling handling and assay conditions. Herein, we report results of experiments that were conducted to characterize the assay in alignment with regulatory guidance and industry standards, and to establish evidence that the method is measuring the free ligand in circulation at the time serum was collected. A robust supporting data package was generated that demonstrates the method specifically and reproducibly measures the free ligand, and is suitable for its intended use.

Tutorial on Monoclonal Antibody Pharmacokinetics and Its Considerations in Early Development

“…When the target antigen is soluble, free and total antigen concentrations may also be required for appropriate PK/PD assessment. For example, an anti‐ID antibody was used to measure unbound anti‐PCSK9 antibody (evolocumab), while unbound PCSK9 was measured using evolocumab for capture and a second biotin‐conjugated anti‐PCSK9 antibody for detection . PK/PD analysis using unbound evolocumab, unbound PCSK9, and LDL‐C data has enabled dose and regimen selection in clinical trials (see “Target‐mediated or specific clearance” for further details).…”

Clinical Pharmacokinetics and Pharmacodynamics of Evolocumab, a PCSK9 Inhibitor

“…A quantitative enzyme-linked immunosorbent assay (ELISA) was developed to evaluate the effects of evolocumab on unbound PCSK9 in human serum [ 41 ]. The precision and accuracy of the assay to detect endogenous PCSK9 was demonstrated, with coefficients of variation of ≤ 15% and bias of ≤ 20%.…”

Influence of Renal Function on Evolocumab Exposure, Pharmacodynamics, and Safety

“…4,10 In the assay, evolocumab was used for capture, and a second bioconjugated anti-PCSK9 antibody was used for detection to ensure PCSK9 bound to evolocumab was not measured. 4,10 In the assay, evolocumab was used for capture, and a second bioconjugated anti-PCSK9 antibody was used for detection to ensure PCSK9 bound to evolocumab was not measured.…”

“…Unbound PCSK9 was measured in human serum using an ELISA method. 4,10 In the assay, evolocumab was used for capture, and a second bioconjugated anti-PCSK9 antibody was used for detection to ensure PCSK9 bound to evolocumab was not measured. The accuracy and precision of the analytical method for the quantification of PCSK9 in human serum were ±15% and ࣘ20%, respectively, for standards and ±25% and ࣘ20%, respectively, for quality control samples.…”

“…Unbound PCSK9 was measured in human serum using an ELISA method . In the assay, evolocumab was used for capture, and a second bioconjugated anti‐PCSK9 antibody was used for detection to ensure PCSK9 bound to evolocumab was not measured.…”