Streamline Immunogenicity Assay Development: Expedite Research Workflows by Understanding the Issues with ADA Assays

Streamline Immunogenicity Assay Development_ Expedite Research Workflows by Understanding the Issues with ADA Assays

The biotech industry is rapidly modifying and developing biotherapeutics into complex large molecules with specificity moieties and multiple functional domains to produce a specific biological method of action. (ADC, ACC, Bi-specific antibodies, Fusion proteins, recombinant-modified proteins, etc.)

Therefore, developing LBA assays to detect the immunogenicity of these biotherapeutics is becoming very complex due to this complexity of the biotherapeutics and lack of available reagents.

Immunogenicity is the ability of an antigen or epitope to elicit an immune response in the body of an animal or Human. In short, it is the ability to induce a humoral and/or cell-mediated immune responses, which eliminates it from the body. In the case of biotherapeutics, the therapeutic is the “antigen” and Anti-Drug-Antibodies (ADA) produced are binding to the therapeutic.

This can result in the biotherapeutic no longer being effective, or worse, adverse reactions, such as life-threatening anaphylaxis. Because of these dangers, immunogenicity testing is essential in the drug development process.

In the drug development process, immunogenicity testing is an essential step to ensure drug safety and efficacy. This post discusses what anti-drug antibodies (ADAs) are, how to test for them, their connection with drug tolerance, and FDA recommendations for safe testing practices.

Immunogenicity Testing

As described above, the immunogenicity effect takes the form of the immune system producing antibodies against the biotherapeutic. So how do you test a novel drug’s immunogenicity, or its tendency to trigger ADA production?

Since the ADAs are generated in the body, specifically in the serum, we can approximate the relative amount using the same biotherapeutic which is “labeled” in two ways. Then a “Bridging ADA assay” is generated as shown below:

Bridging ADA Assay Generation

The FDA recommends a multi-tiered ADA testing approach, wherein there are three tiers: a screening assay, a confirmatory assay, and titration and neutralization assays. The FDA Multi-Tier approach is shown below:

Expanded FDA ADA Multi-Tiered Approach

ADA Screening Assay

Screening assays, also known as binding antibody assays, are used to detect antibodies that bind to the therapeutic protein product. As mentioned above, since we cannot measure the ADAs directly, we “approximate” with statistical equations to determine a set false-positive rate in patient serum samples. To determine if the sample is positive or negative for immunogenicity, a “cut point,” or level of ADA response, is decided on.

A Cut-Point is a statistically determined threshold value for the distinction of positive and negative results in an immunogenicity assay (ADA and Nab). The cut point is determined statistically and depends on a group of naive subjects who have not been dosed with similar therapeutics. Factors such as participants’ pre-existing antibodies and statistical outliers influence the cut point

ADA Confirmatory Assay

Confirmatory assays determine the specificity of ADA/Nab for the therapeutic protein product by competition with the therapeutic protein. Due to the set false-positive detection rate in the screening tier, the FDA recommends creating confirmatory assays to verify that the antibodies are bonding to the therapeutic treatment. This assay is essential for accurately measuring the immunogenicity of the tested drug. Generally, the confirmatory assay format can be like the screening assay format. An example of a confirmatory assay is a competition assay, which verifies that the antibody is specifically bonding to the therapeutic protein, and not interfering material such as other proteins in the serum.

ADA Titration and Neutralization Assay

This ​​tier takes the confirmed ADA samples from the previous two stages a step further. The titration assay defines the magnitude of the ADA response, and the neutralizing assay analyzes the antibody for neutralizing activity. In the titration assay, the positive sample is titrated to a reactivity like the assay cut point. Then, the titer estimates the ADA concentration. This is important to measure because, with some drugs, the harm is due to the persistence of ADAs, or their concentration levels. 

It is also essential to determine if the ADAs neutralize the therapeutic protein. If the drug is neutralized, it cannot treat the patient’s condition. A neutralization assay answers this question and is often done as a cell-based assay. Ligand binding and enzymatic activity neutralizing antibody (NAb) assays are also used. The FDA recommends at least 30 samples for neutralizing antibody analysis.

ADA In Clinical Trials

There are some testing differences between preclinical and clinical studies. In preclinical assays, the study uses a high drug concentration and high dose levels, whereas, in clinical assays, a lower dose level is utilized. NAb assays are usually not necessary in preclinical studies but are required in the clinical setting. Another aspect that the FDA looks at is “Drug Tolerance”. In essence, with high concentrations of the therapeutic in the serum, what levels of ADA will the immunogenicity assay detect. Our Director of Scientific Solutions, John Pirro, was instrumental in dealing with a complicated ADA issue. In one of his past roles, John had a client that was having trouble with drug tolerance for a clinical trial and the solution below demonstrates what can be done to streamline the assay:

High Drug Tolerant Immunogenicity Testing Issue and Solution Case Study

FDA Recommendations

The Food and Drug Administration has set out recommendations for developing and validating ADA assays. The immunological assays need to address the different immunoglobulins in human serum. They are IgG, IgM, IgA, and IgE. These can also form complexes that could initiate cytokine reactions. Size exclusion chromatography could help in these possible situations. In addition to a tiered approach, as discussed earlier, and advised sample number for NAb assays, the FDA has sensitivity for screening and confirmation assays, the minimum number of samples needed for cut-point determinations, what statistical equations to be used, etc.

Understanding all the criteria and knowing which assay modifications will meet these standards can streamline the assay development process. Knowledge and Scientists with experience in multiple ADA assay tiers can deliver these acceptable immunological data to regulatory agencies like the FDA. 


Immunogenicity testing is a mandatory regulatory step in developing a new drug. At the least, the testing ensures the given drug can serve its function. It can even save lives. Contact us today to learn how Sword Bio can optimize your immunogenicity analysis and accelerate your drug development process.