The Importance of Immunogenicity Testing in Drug Development

Somewhere back in the 1970s, the world witnessed the emergence of a new concept in the immunogenicity, i.e., biotherapeutics. This is a process wherein small drug molecules developed using recombinant DNA technology forms the basis of the immunogenicity testing. This technology allows for cost-efficient and large-scale production of proteins within the cultured cell system using controlled conditions.

In contrast to the chemical entities available in the market, biotherapeutics such as antibodies, proteins, and peptides are widely employed in boosting the efficiency of antibody-mediated immune responses. Determination of the drug exposure rate forms a critical part of the drug discovery process. It is, therefore, essential to evaluate how both the anti-drug antibodies and the anti-therapeutic antibodies affect the bioavailability of the drugs being exposed to the immune system of humans. Either of the antibodies produced by the body in response to the frequent drug exposure may belong to any of the three categories – sustaining, clearing, or neutralizing. All three of them have sufficient potential to affect both the pharmacodynamic and pharmacokinetic characteristics of a drug candidate.

The early immune response produced by a human body is usually of low affinity, where the body produces IgM molecules in meagre quantities. As it is low in its magnitude, the initial immune response may peak within a window of 7 to 14 days. Thus, determination of the magnitude of the early immune response includes – response duration; the occurrence of adverse events, evaluation of the neutralizing antibody production; and correlating the changes observed during pharmacokinetics and toxicokinetic studies.

Importance of ADA assays –

Drug screening assays are alternatively known as binding antibody assays. Generally, they assess the effects produced by an antibody binding to the therapeutic protein product. The specificity for the immunogenicity testing is usually established by competing it with a therapeutic protein in a confirmatory ADA assay. These anti-drug antibodies are further characterized through the use of both the titration and neutralization assays.

Results obtained from the immunogenicity testing facilitates an equal understanding of different characteristics of the therapeutic protein candidate under analysis – pharmacodynamics, pharmacokinetics, efficacy, and safety profiling.

Essential elements used in the process of designing an immunogenicity assay –

Following are the critical elements that are evaluated in immunogenicity testing –


  • Testing Strategy
  • Assay Cut-point
  • Sensitivity
  • Specificity
  • Selectivity
  • Precision
  • Reproducibility
  • Robustness and Sample Stability
  • Format Selection
  • Reagent Selection
  • Qualitative and Quasi-quantitative Assays


In addition to the above key factors, there are other factors that a clinician should consider before performing immunogenicity testing. These are inclusive of pre-existing antibodies, rheumatoid factor, monoclonal antibodies, and conjugated proteins.


Methodology Applied for Immunogenicity Testing –

Immunogenicity testing is a tiered approach where you have three key steps to be accomplished –

Initial screening –

Assessment of the study samples to determine whether they contain the specific ADAs or not.

Confirmatory Assay –

Determines whether there is any positive reaction possible between a putatively positive sample and free drug.

Titre Evaluation –

Assessment of the relative level of the presence of ADAs in the study sample.

To assess the potential of a neutralizing antibody, the testing involves a fourth tier where the study sample is analyzed for the efficacy of the ADA to prevent the binding of the drug to its targeted site.