Immunogenicity Testing Assay

The body produces Anti-drug antibodies (ADA) as an immunogenic response against therapeutic proteins, enzymes, antibodies, peptides, and other biologic combination products. ADAs, somewhat identical to the proteins found in the human body, are complex macromolecules composed of hundreds of amino acids. The generated ADA antibodies could bind to various epitopes of the biologics, making them ineffective. Additionally, these antibodies can neutralize or break down the biologics or drug products into their components in many ways. The human body makes five different antibodies called immunoglobulins (Ig) to combat different antigens, namely IgA, IgG, IgM, IgE, and IGD. We test all relevant Ig isotypes in the initial ADA screening assay, depending on the route of administration and clinical evidence or risk of anaphylaxis. Generally, the FDA doesn’t mandate the determination of Ig isotype in screening assays. That said, assessment of antigen-specific Ig assays and evaluation of the generated Ig ADA subtype is sometimes deemed informative based on the drug therapy.

Immunogenicity is defined as any drug compound’s ability to evoke an immune response. Immunogenicity testing is necessary because sponsors seeking regulatory approval must ensure that their drug doesn’t elicit an adverse immune response upon administration. Typically, immunogenicity results from the detection of drug biomaterial as a foreign object by the body’s immune response. Depending upon the biomaterial, the T-helper cells usually form a cascade and migrate towards the drug. It is challenging to predict how these T- helper cells would respond to a specific therapy. This immunogenic response could be the reason for the limited binding between the biomaterial and the site of action or the rejection of biomaterial altogether. The immunogenicity prediction for clinical trials could be based upon drug performance in preclinical testing. NorthEast BioLab performs immunogenicity assays such as ADA and NAb Testing to evaluate antibodies produced against your biologics.

Immunogenicity assays can be involved, given the complexities and interferences associated with the various sample matrices, drug therapy, and targeted site of action. That said, the Anti-drug Antibody (ADA) assay (screening, confirmation, and titration) and Neutralizing Antibody (NAb) assay are the most common assays during the drug development process. The ADA and NAb assays are completed to identify and evaluate the antibody that binds with any therapeutic product. Our scientists obtain rigorous and actionable data for your drug development through highly sensitive and precise Anti-drug Antibody and Neutralizing Antibody assays.

Anti-drug antibody (ADA) assay development and validation is an essential step in the clinical and preclinical testing of biologics for managing immune responses that affect drug therapy safety and efficacy. Our immunogenicity experts are well versed in the development and validation of qualitative and semi-qualitative immunoassays using the FDA recommended multi-step approach to measure ADAs and NAbs. As the first step, our scientists screen patient samples to detect ADA immunogenicity. Next, the samples are evaluated for cut point analysis, which is a test to eliminate false-positives and perform the confirmation assay for potential positives. As a confirmatory assay, we determine the titer assessment and specificity of the binding. Afterward, all confirmed ADA positive samples are assayed for the presence of neutralizing antibodies by NAb assay.

ADA assay development is a multi-step bioanalytical approach that includes several assay formats and method validation for suitable immunoassays. In the first tier, our scientists utilize a sensitive screening assay, aka binding antibody assay, to analyze biological samples for low and high-affinity ADA antibodies. In the second tier of our ADA testing, we perform a cut point analysis to determine positive or negative samples. Any samples that demonstrate potential binding get re-tested for specificity by competition with the therapeutic protein in a confirmatory assay. At this point, the FDA recommends that sponsors conduct risk-based characterization testing for any cross-reactivity, isotypes assessment, and epitope specificity. After ADA confirmatory assay, positive samples need to be tested further for neutralizing and titration assays.

Neutralizing Antibody (NAb) assays are a necessary component for clinical trials when anti-drug antibodies are detected in patient samples. NAbs can neutralize the effects of biomaterials and are evoked in the body as an adverse immune response to therapeutic proteins. In these NAb assay, it is crucial to observe whether i) patients are producing anti-drug antibodies and ii) these antibodies are neutralizing the therapeutic drug effects. NorthEast BioLab immunogenicity professionals design and develop your customized and specific Neutralizing Antibody assay. We utilize MSD Quickplex 120 to increase NAb assay sensitivity and dynamic range, as well as limit matrix effects as compared to traditional ELISA.

Immunogenicity test is crucial in clinical trials from Phase I through Phase III. Enzyme-linked immunosorbent assays (ELISA) offer the traditional approach to quantitate ADA antibodies from administered drug therapy. First, we coat the polystyrene plate with a solution of capture antigen or antibody and wash the plate. Next, we add a blocking solution, incubate, and rewash. Afterward, we add biological samples from the drug therapy administration and rewash. At this time, the ADA antibodies can bind with the capture antigen or antibody. Finally, we add the detection substrate that emits a color if the ADA target is identified. Notably, the MSD immunogenicity assay has several advantages over conventional ELISA, including higher sensitivity, matrix tolerance, free drug tolerance, and lower sample volume requirement. It is essential to perform both ADA and NAb assays if patients are showing immunogenicity against your drug therapy. Our MSD Quickplex 120 platform delivers high-performance ADA immunogenicity assay for broad range multiplex detection using electrode plate binding and electrochemiluminescence quantitation.

Immunogenicity testing is crucial because the protein-based therapeutics, such as monoclonal antibodies and vaccines, are sometimes recognized as foreign particles by the body and induce an unwanted immunogenic response. Thus, immunogenicity assessment of biologics or therapeutic proteins is mandatory for regulatory approval. Often immune response involves B or T cell epitopes. For immunogenicity prediction, histocompatibility complex class II/ MHC is identified as an immunogenic molecule that shows T-epitopes as the receptors for cascade responses. These types of cells are responsible for producing antibodies and inactivating the administered protein-based drugs. Other risk factors for immunogenicity testing include dosage, administration route, delivery vehicle, and immune system activation to predict the immunogenicity of protein. Thus, measuring the capacity of producing antibodies is an essential aspect for the safety of clinical trials and for the success of therapeutic drugs. Immune tolerance can also significantly affect the therapeutics product design in many cases. Rheumatoid Arthritis and Hemophilia A exemplify health conditions that have complicated treatments due to immunogenicity.

Immunogenicity assays during clinical and preclinical studies evaluate the tendency of biologics to produce an immune response in the form of ADAs and NAbs. Immunogenic response against biologics and therapeutic proteins is a common concern for scientists and clinicians focused on drug development. These responses could result in adverse clinical events due to the inactivation of drug therapy for patients. Several factors determine whether any biologic does or does not evoke an immune response, including drug structure, variants, immunogenic status, dosage, route of administration, and genetic profile of treated patients. The overall purpose of immunogenicity study, ADA assay specifically, is to determine whether ADA antibodies are generated and can cause adverse events or loss of efficacy. NorthEast BioLab experts develop and validate your immunogenicity assays, including ADA and NAb assays, to analyze your novel compounds in clinical and preclinical studies.