Demonstrating the potency of a biopharmaceutical is critical to the successful development and market release of a drug. For therapeutic antibodies, binding to the target antigen is the first step in its mechanism of action and is therefore a critical quality attribute (CQA) for the product. Since the potency assays need to reflect the mechanism of action of a drug and are the only analytical technique which can correlate to the ultimate clinical efficacy of a drug there is a requirement to characterize binding to the antigen. This forms part of the characterization package along with components of the innate immune response such as Fc receptors and C1q.
Measuring binding to the target antigen is required throughout the drug development life cycle, from initial clone selection and continuing into market release. Regulatory agencies require the determination of target binding (potency) for the release of drug products under GMP (ICH Topic Q6B; Specifications: Test Procedures and Acceptance Criteria for Biotechnological/Biological Products). Throughout a product life cycle, different antigen binding assays may be utilized, depending on the phase of the drug development process. Indeed, during the early development phase, orthogonal potency methods should be explored to fully characterize the interaction. This will also identify lead candidate platforms/assays for further development. Data from orthogonal methods will also provide supportive evidence for the final assay(s) chosen for the release testing. Potency assays may be ligand binding assays, such as ELISA (Enzyme Linked Immunosorbent Assay) and surface plasmon resonance (SPR), or functional assays such as cell-based assays and flow cytometry.
During early product development, it is important to fully characterize the interactions of the therapeutic antibody with the target antigen. Label free systems such as surface plasmon resonance (SPR) can provide insights into the binding kinetics of the interaction. This can support selection/optimization of lead drug candidates to ensure that the binding properties, such as the dissociation rate/half-life, match the intended mechanism of action. Furthermore, label free binding assays can provide a rapid yes/no answer, which can be used to support species selection decisions for toxicology studies by confirming the interaction of the drug with the target antigen from different species.
While label free binding assays can provide a wealth of data on protein interactions, the instruments required are generally expensive and have a lower throughput when compared to traditional ELISA. Therefore, in later stages of the drug development life-cycle, ELISA is often used to measure the binding of a drug lot relative to a well characterized reference standard. Depending on the mechanism of action, ELISA can be developed as an antigen binding assay or competition/inhibition assay.
While ligand binding assays are capable of measuring the binding of a therapeutic antibody to its target antigen, the biological activity resulting from the binding event will also need to be characterized and monitored. Depending on the mechanism of action, binding of the antibody to its target antigen can trigger or inhibit a variety of effects such as cell proliferation, apoptosis or differentiation. A number of vendors are offering reporter gene assays, which often have benefits over bespoke cell-based assays, such as reducing development time, assay variability and run time. However, regulators still require that the data from reporter gene assays are correlated with the results from cell-based assays measuring the biological activity of the drug. Therefore, it is likely that a bespoke cell-based assay will need to be developed to demonstrate the potency. These assays can be developed to simulate the mechanism of action, with appropriate end points to measure the biological response.
As well as measuring the activity/strength of a therapeutic antibody lot, potency assays should also be stability indicating by being able to differentiate between target and degraded products. As such, potency assays are required during forced degradation and stability studies. In conjunction with structural characterization, ligand binding and cell-based assays can identify which degradation pathways, and which amino acid residues, lead to a reduction in potency. Furthermore, depending on the mechanism of action, ligand binding assays can be used to determine whether a loss of activity is due to degradation of the antigen binding site or through loss of binding to Fc binding proteins involved in the innate immune response.
Potency assays are vital in order to fully characterize the binding interaction of a monoclonal antibody to the target antigen. A matrix of potency assays should be developed and investigated to generate a comprehensive data set defining the mechanism of action of the drug. Development should be performed early in the product life cycle to ensure that the methods can accurately identify changes in active concentration between manufacturing lots and during stability studies. This also ensures that a comprehensive data set is available to fully characterize the assays selected for release testing, to ensure that the methods are robust and appropriate method acceptance criteria can be applied.
Ligand binding assays are generally more accurate and reproducible than cell-based bioassays, due to the lower complexity of cell free assay formats, and therefore are more quality control testing friendly. Due to this, validated ligand binding assays can be utilized as surrogates for cell-based bioassays for release testing. However, this requires the data from the surrogate assay to be substantiated by correlation to a cell-based bioassay. Therefore, cell-based assays are still critical to demonstrate the mechanism of action and the biological activity of the drug.