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.