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Immunogenicity Testing - Clinical Trial Support

Pacific BioLabs’s Bioanalytical team has developed and validated in vitro assays for many of our biopharmaceutical clients to screen and quantify immunogenicity caused by anti-drug antibodies (ADAs) and impurities. The team uses a tiered approach for these assays – a screening assay followed by titer and confirmation assays. Based on the risk profile of the biotherapeutic protein or antibody and its intended use, our team works closely with clients to develop a custom panel of immunogenicity assays. Pacific Biolabs specializes in ELISA method development as well as cell based assay development.

Aggregation Assessment & Reagent Characterization Services

PBL can assess the aggregation potential of a biotherapeutic using size-exclusion chromatography and HPLC combined with laser-light scattering. This can help to provide a more detailed picture of the product’s overall immunogenic profile. We can also characterize critical reagents used in the biotherapeutic’s development for stability, post-translational modifications and degradants.

Available Immunogenicity Services

  • Ligand Binding Assay
  • Cell Based Assays
  • Total Antibody Assay
  • Neutralizing Antibody Assay
  • Particle Size Characterization
  • Critical Reagent Characterization
  • Protein Aggregation Analysis

Instrumentation and Platforms Available

  • Enzyme Linked Immuno Sorbent Assay (ELISA)
  • High Performance Liquid Chromatography (HPLC)
  • Size Exclusion Chromatography (SEC)
  • Laser Light Scattering - Static (LLS)
  • Instrumentation Details

Importance of Immunogenicity Testing

Immunogenicity is the ability of a substance to elicit an immune response. Any substance administered to the human body can potentially elicit an immune response. However, biotherapeutics can be especially immunogenic because the large size of these molecules can trick the body into thinking that they are foreign invaders, triggering action by the immune system. Products with an increase potential for causing immunogenicity include therapeutic antibodies, enzyme therapies, peptides and combination products. These large molecules can also denature, which changes their profile, or aggregate, creating even larger particles. Ultimately, denatured products and aggregates can alter or even increase the biotherapeutic’s immunogenicity profile.

Because an immune response may impact a drug's safety and efficacy, immunogenicity testing is a key aspect of any biotherapeutic development program. FDA and EMA guidelines require sponsors to monitor immunogenicity of protein therapeutics. The FDA requires that assays should be designed in such a way that they provide sufficient sensitivity, are free from confounding interference, can detect physiological consequences, and account for potential risks based on the profile of the therapeutic and the target patient population. By designing assays with these factors in mind, it is possible to gather predictive data about the strength and type of immune response that a drug may produce in humans. Therefore, immunogenicity risk assessment is a crucial aspect of biotherapeutic drug development.

Immunogenicity testing is also needed for a biosimilar development program. The structure and composition of biosimilars can differ slightly from their parent biologics. This is because they almost always undergo different manufacturing processes than the parent biologics. Small changes in the manufacturing processes of biotherapeutics can affect small changes in their structure and hence large changes in their immunogenicity profiles.

Immunogenicity Case Study

The case study below describes our team’s experience in developing and validating an immunogenicity testing program for a client’s protein biotherapeutic. Our team routinely tests over 10,000 samples each year. This particular testing program supported the therapeutic’s clinical development, proceeding from Phase I through Phase III clinical trials.


The biotherapeutic’s intended target population was predominantly composed of women, so our team had to source human female plasma for assay development and validation.

Our Methodology

Using ELISA, our team developed the three focal assays of the immunogenicity testing program:

  • A screening assay was developed using a cut point factor that was calculated based on the study population of mostly women. Cutpoint factor is the assay threshold at or above which samples can be categorized as positive for anti-drug antibodies (ADA).
  • A confirmatory assay was performed in the presence of an excess of the drug product. By comparing the results from the screening assay (unspiked samples) with the results from the confirmatory assay (drug-spiked samples), the team could minimize the number of false positives.
  • A final titer assay was conducted to measure the amount of ADAs produced in response to the drug product.


During the course of the immunogenicity program, the client made a few minor changes to the biotherapeutic and its manufacturing processes, which could have had a large impact on its immunogenicity profile. Our team had to determine whether the immunogenicity profile of the biotherapeutic had changed significantly.

Our Solution

Our team developed custom bridging assays for new materials (such as critical reagents) that were introduced into the manufacturing processes, to determine their individual immunogenicity profiles. We also re-validated the prior assays with a new positive control that was comparable to the altered biotherapeutic. We then compared the results from these newly validated assays to those from the previous assays to measure any changes in the immunogenicity risk profile of the therapeutic.

Immunogenicity Testing Standards

Immunogenicity Related Testing Services

Read More About Immunogenicity


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