PK and PD Ligand-Binding Assays in Large Molecule Drug Development

Ligand-binding assays play a crucial role in the bioanalysis of large molecules and remain a fundamental component of pharmacokinetic and pharmacodynamic studies both during early and late phase drug development. This chapter will review the general principles of immunoassays and ligand-binding assays, along with their respective advantages and disadvantages. We discuss different immunoassay platforms such as enzyme-linked immunosorbent assays (ELISA), electrochemiluminescence assays, Gyrolab, immuno-PCR, and Simoa Quanterix. We further discuss general considerations of regulatory compliance and the evolution of regulatory expectations throughout drug development. Finally, we provide 4 examples of immunoassays: (1) a generic pharmacokinetic sandwich ELISA for the detection of human Immunoglobulin G1 (IgG1) in mouse serum; (2) a pharmacokinetic target capture ELISA assay for the detection of therapeutic antibodies against the proprotein convertase subtilisin/kexin type 9 (PCSK9) in rhesus monkey serum; (3) a pharmacodynamic/biomarker immunoassay for measuring free vascular endothelial growth factor-A (VEGF-A) in human Aqueous Humor using the Simoa Quanterix platform; and (4) an immunocapture method for the precipitation of human IgG1 from mouse plasma for subsequent LC/MS analysis. Detailed discussions on these examples, along with their benefits, limitations, and possible modifications, are provided.