Fit-for-purpose immunogenicity assays: What to consider during assay development and validation?

Nermina Vejzagic

by Nermina Vejzagic

Therapeutic protein products can induce an undesirable immune response resulting in the formation of anti-drug antibodies (ADAs) [1, 2]. ADAs can be differentiated into binding antibodies and neutralizing antibodies [3]. Pre-existing ADAs can be present in patients before drug treatment and have the ability to bind to a drug component [4]. The severity of clinical response induced by ADAs (especially NAbs) can range from benign, mild (e.g., loss of drug efficacy) to severe (e.g., neutralization of a life-saving drug or fatal hypersensitivity) [5]. Therefore, detection and characterization of ADAs are essential for the evaluation of their possible impact on pharmacokinetics, pharmacodynamic, patient safety, and drug efficacy [1, 2].

To evaluate immunogenicity data, a multi-tiered testing approach using sensitive and validated methods should be appied. The multi-tiered immunogenicity assessment is based on a screening assay to identify ADAs in samples (tier 1). In the confirmatory assay (tier 2), the specificity of ADAs for the product is determined through competition with a therapeutic protein product. The ADA confirmed samples are then characterized by titration and neutralization assays (tier 3). Whereas titration assays provide information on the magnitude of the immune response, neutralizing antibody assays evaluate ADAs for neutralizing activity [1, 2]. The outline of the multi-tiered approach is provided in Figure 1.

Figure 1: Multi-tiered approach for immunogenicity assessment. Adapted from [1, 2].

In general, the ADA assay should be developed and validated using the matrix representative of samples from the target population [1, 2]. However, if not accessible at the time of the assay development and validation, critical parameters such as sensitivity, cut-points, and selectivity need to be confirmed after samples from treatment-naïve subjects are available from a suitable study population [2]. Following the development, the ADA assay must be validated to ensure that critical parameters are met and that the assay is fit-for-purpose. These important parameters for the development and validation of ADA assay are shown in Table 1.

Neutralizing antibodies (NAbs) represent a subpopulation of ADAs with a potential impact on patient safety as they can block the biological activity of a drug, thus leading to a decline in product efficacy [5]. For this reason, the detection of NAbs is an important component of immunogenicity assessment. The assay to determine NAbs needs to be reliable, specific, and sensitive, taking into consideration mechanism of action (MoA) of the therapeutic protein product [5]. Two types of NAb formats can be used: Cell-based and non-cell-based competitive ligand binding assays [1, 2]. The scientific approach for the choice of appropriate assay format will be dependent on the therapeutic MoA, target and effector pathways of the drug, risk of immunogenicity, sensitivity, precision, selectivity, and robustness of the assay [1, 2, 5]. Key considerations in the development of cell-based NAb assays include choosing a suitable cell line, selecting an appropriate cellular response representing the endpoint method, choice of suitable assay controls, and assay optimization [6]. T the general requirements required for the establishment of fit-for-purpose NAb assays are outlined in Table 1.

ADA: Anti-drug antibody, NAb: Neutralizing antibody. Source [1, 2].

ADA: Anti-drug antibody, NAb: Neutralizing antibody. Source [1, 2].

Table 1: General requirements for the development and validation of immunogenicity assays

One of the challenges in the development and validation of ADA and NAb assays, and subsequent samples testing, is the interference of soluble drug target. Consequently, there is a need to identify and overcome this interference introduced by the presence of high circulating concentrations of soluble drug target [7].  

In our experience, we see that agencies in different regions thoroughly review immunogenicity packages submitted with marketing authorisation applications (MAAs) and biologics license applications (BLAs). This detailed review by agencies focusses on the confirmation that the methods are validated according to applicable guidelines and that immunogenicity results are scientifically discussed for their impact on pharmacokinetics, efficacy, and safety. Good clinical practice (GCP) inspections of laboratories performing these investigations are commonly initiated to verify that the assays are performed according to their governing procedures and to check if samples are adequately transported and stored. Therefore, the impact of immunogenicity packages on the overall success of regulatory submissions is critical.

Our team of regulatory and bioanalytical experts can help you overcome current challenges in developing and validating assays for immunogenicity assessment. If you would like us to guide you, feel free to contact us.


  1. EMA, Guideline on immunogenicity assessment of therapeutic proteins, European Medicines Agency. Retrieved from 2017, Committee for Medicinal Products for Human Use (CHMP) London, UK.

  2. FDA, Immunogenicity Testing of Therapeutic Protein Products–Developing and Validating Assays for Anti-Drug Antibody Detection (2019), FDA, US. Retrieved from 2019: Center for Drug Evaluation and Research (CDER) and Center for Biologics Evaluation and Research (CBER), Food and Drug Administration.

  3. Sethu, S., et al., Immunogenicity to biologics: mechanisms, prediction and reduction. Archivum immunologiae et therapiae experimentalis, 2012. 60(5): p. 331-344.

  4. Xue, L., et al., Recommendations for the assessment and management of pre-existing drug-reactive antibodies during biotherapeutic development. The AAPS journal, 2017. 19(6): p. 1576-1586.

  5. Wu, B., et al., Strategies to determine assay format for the assessment of neutralizing antibody responses to biotherapeutics. The AAPS journal, 2016. 18(6): p. 1335-1350.

  6. Jolicoeur, P. and R.L. Tacey, Development and validation of cell-based assays for the detection of neutralizing antibodies to drug products: a practical approach. Bioanalysis, 2012. 4(24): p. 2959-2970.

  7. Zhong, Z.D., et al., Drug target interference in immunogenicity assays: recommendations and mitigation strategies. The AAPS journal, 2017. 19(6): p. 1564-1575.

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