Ligand-Binding Assay Development: What Do You Want to Measure Versus What You Are Measuring?

The analysis of biotherapeutics by ligand-binding assay (LBA) is associated with some unique challenges that are unlike those commonly encountered in chromatographic methods for chemically based small molecule drugs. While small molecule drugs are typically highly protein bound, the use of an extraction procedure disrupts these interactions and thus allows for measurement of total drug concentrations; modeling is subsequently required to estimate the amount of free drug based upon protein-binding data. Protein-binding interactions for small molecule drugs are typically low affinity and high capacity in nature (i.e., albumin binding). In contrast, LBAs are most often used for the quantitation of large molecule proteins or peptides, where the use of an extraction procedure is replaced by sample dilution. Interferences in ligand-binding assays are typically of high affinity and low capacity and include target, receptors, anti-drug antibodies, and binding proteins (1,2), however some are typically low affinity such as anti-drug antibodies. While ligand-binding assays are traditionally the methodology of choice for the quantification of biotherapeutics in biological matrices, there is a trend towards increased use of chromatographic methods. Immunoenrichment and enzymatic digestion followed by the LC-MS/MS monitoring of specific biotherapeutic peptide fragments for use as a surrogate to quantify the whole molecule are becoming emergent techniques in the bioanalyst toolbox (3,4). This diversity in analytical technology often times leads to good-natured debate among bioanalytical scientists who tend to gravitate towards one of the two technological approaches. Yet, during assay development, there are two critical questions that often go unanswered; what do you want to measure and what are you measuring