Antibody-Drug Conjugates (ADCs) are a complex class of targeted therapeutics that combine the specificity of a monoclonal antibody with the cell-killing potency of a small molecule drug, or payload. The two components are joined by a chemical linker designed to remain stable in the bloodstream and release the payload only within the target cell. Because ADCs merge the properties of both large biological molecules and small chemical drugs, their behavior in the body is highly complex, creating unique challenges for measurement. Accurate quantification is necessary to establish the pharmacokinetics, which informs decisions about dosing, safety, and therapeutic effectiveness during drug development.
Species Requiring Measurement
The inherent complexity of ADCs means that a single measurement is insufficient to understand their full activity and fate within a patient’s body. Three distinct entities must be quantified in circulation to accurately characterize the drug’s profile.
Total Antibody
The Total Antibody measures all antibody molecules present in the sample, irrespective of whether they still carry the cytotoxic payload. This measurement provides information on the overall absorption, distribution, and clearance characteristics, which often mirror the behavior of the parent monoclonal antibody.
Conjugated Antibody
The Conjugated Antibody, sometimes called the active ADC, specifically measures only the antibody molecules that still retain the attached drug payload. Monitoring the concentration of the conjugated antibody is essential for assessing the concentration of the active, targeted therapeutic over time.
Free Payload
The third, and most safety-relevant, entity is the Free Payload, which is the small molecule drug that has been cleaved from the antibody and is circulating on its own. Since these payloads are highly potent, quantifying their concentration is paramount for assessing potential off-target toxicity to healthy tissues.
Ligand Binding Assay Techniques
Ligand Binding Assays (LBAs) are the industry standard for quantifying the large-molecule components of the ADC, namely the Total Antibody and the Conjugated Antibody. These immunological methods, such as Enzyme-Linked Immunosorbent Assay (ELISA) and Electro-Chemiluminescence Immunoassay (ECLIA), leverage the high-affinity binding between antigens and antibodies. For measuring the Total Antibody, the assay typically uses non-specific reagents, such as anti-human IgG antibodies, to capture all therapeutic antibody molecules.
To quantify the Conjugated Antibody, the assay format must be highly specific, often employing a capture reagent that binds to the antibody framework and a detection reagent that recognizes the drug payload. This “sandwich” approach ensures that only those antibody molecules that still possess the attached payload are counted.
LBAs are favored for their high sensitivity and capacity for high-throughput analysis. However, a major limitation is their inability to distinguish minor structural changes in the large ADC molecule or to accurately quantify the small-molecule payload itself.
Mass Spectrometry Approaches
Mass Spectrometry (MS) approaches provide a necessary complement to LBAs, particularly for quantifying the small-molecule components and offering structural insights. Liquid Chromatography coupled with tandem Mass Spectrometry (LC-MS/MS) is the preferred technique for quantifying the highly potent Free Payload in biological fluids. The method involves separating the payload from the complex sample matrix using chromatography before it is ionized and precisely measured by the mass spectrometer, offering exceptional specificity and sensitivity for safety monitoring.
For analyzing the large ADC molecule, MS can be used in a “middle-up” or “top-down” fashion to characterize the protein structure. This involves either partially or fully breaking down the ADC before mass analysis to confirm the identity of the conjugated payload and its attachment site.
A hybrid approach, combining the specificity of immunoaffinity capture with the sensitivity of LC-MS/MS, is often employed to quantify the conjugated payload. In this scenario, the ADC is first isolated from the plasma matrix, enzymatically digested to release the payload, and the resulting small molecule is then quantified using the MS instrument.
Determining the Drug-to-Antibody Ratio
The Drug-to-Antibody Ratio (DAR) is a structural attribute defined as the average number of cytotoxic drug molecules linked to each antibody. This ratio is a critical quality attribute because it directly influences the ADC’s potency, pharmacokinetics, and homogeneity. The starting ADC material is typically a mixture of species with varying drug loads (e.g., DAR 0, DAR 2, DAR 4), which is referred to as the drug load distribution.
Hydrophobic Interaction Chromatography (HIC-HPLC) is the primary method used to determine the DAR and resolve this distribution of drug-loaded species. HIC separates the ADC species based on the number of attached hydrophobic drug molecules, causing species with a higher DAR to elute later than those with a lower DAR.
High-Resolution Mass Spectrometry (HRMS) is often coupled with separation techniques to confirm the precise mass of each separated species. Monitoring the DAR is also important in vivo because drug deconjugation after administration can lead to a decrease in the ratio, impacting therapeutic efficacy and increasing the concentration of the circulating free payload.