Immunogenicity refers to the nature of drugs that stimulate the body to form specific antibodies or sensitized lymphocytes. In the development of biotechnological drugs, immunogenicity testing is an important task. Immunogenicity can affect the safety and efficacy of drugs, and even life-threatening risks due to the intersection of drug-resistant antibodies and endogenous proteins. Properly designing and validating a method for detecting anti-drug antibodies (ADA) and accurately interpreting the results of sample analysis is an indispensable part of the development of biosimilar drugs. Creative Biolabs can offer experienced guidance about ADA assay method development & validation and provide theoretical basis and technical support for clinical drug delivery and reduction of a drug-induced immune response.

Development Status of ADA Assay Method

Protein drugs often trigger an inevitable immune response in the body of the patient, producing ADA. Current methods for immunogenicity detection and screening are based on a variety of immunoassays to confirm the type of ADA. In other words, the reaction mechanism of the drug in vivo is observed by detecting the neutralizing antibody on the positive sample of ADA. The commonly used assay to detect immunogenicity is a bridging immunoassay. In this assay, the drugs can be labeled separately, with different labels, and any drug-resistant antibodies present in the sample will form a bridge between the two labeled molecules. The biggest advantage of this method is the ability to simultaneously detect multiple types of markers, such as IgG, IgM, and IgA. This method is also applicable to multiple species because all immunoglobulins are capable of forming immune complexes with two labeled drugs.

Fig.1 Acid dissociation to detect ADA and drug. (Kelley, 2013)

Clinical Application of ADA Assay Method

In recent studies, ADA assays have been widely used in developing recombinant human thrombopoietin mimetic peptide-Fc fusion protein (TMP-Fc). It is a candidate drug for the treatment of chronic idiopathic thrombocytopenic purpura. In general, TMP-Fc is injected into the body as a recombinant protein, which usually triggers the body to produce the drug itself and even an antibody to an endogenous protein. Scientists have established and validated immunogenicity assays based on surface plasmon resonance (SPR) technology for the immunogenicity of two TMP-Fc proteins in cynomolgus monkeys in a comparative study. The results showed that the two methods (anti-L, anti-R) were highly consistent with the positive samples of R samples and their detection time points. Therefore, the instant messaging (IM) analysis method based on the SPR system has met the requirements of biological product research and has successfully applied to the ADA detection of two TMP-Fc biological samples.

Highlight

• Comprehensive knowledge reserve
• System planning
• Accurate data analysis
• Extensive testing

Creative Biolabs owns quality technical and experimental conditions. Please contact us in time for more information and we will be glad to help you.

Other optional SIAT® Anti-drug Antibodies (ADA) Assays:

• SIAT® Positive Control & Reagent Formation for ADA Assay
• SIAT® ADA Assay Sample Analysis
• SIAT® ADA PK/TK analysis

Published Data

• Development and Validation of an ELISA for the Detection of Binding ADAs against Interferon Beta

Fig. 1 Drug interference was tested by spiking both the high-positive control (HPC), and the low-positive control (LPC). (Kathleen Ingenhoven, 2017)

The research presents the development and validation of a highly sensitive ELISA for detecting binding anti-drug antibodies (ADAs) against interferon beta (IFN-β) in human serum. This ADA assay is part of a larger effort to assess and mitigate the immunogenicity of biopharmaceuticals. The study successfully established an assay with a confirmed sensitivity of detecting ADAs at 26 ng/mL, a significant enhancement given the typical sensitivity ranges of 250-500 ng/mL for clinical antibody assays. This improved sensitivity is crucial for early detection of ADAs, which can impact the clinical efficacy and safety of IFN-β therapy in multiple sclerosis. Through rigorous method development and validation, including optimization of assay conditions and comprehensive testing for specificity, sensitivity, and robustness, the study sets a benchmark for ADA detection that could support personalized treatment approaches and improve therapeutic outcomes.

FAQ

1. What is the purpose of developing and validating an ADA assay?

   The development and validation of an anti-drug antibody (ADA) assay ensure that the test is reliable, reproducible, and accurate for detecting antibodies against biopharmaceuticals. Validation confirms that the assay can consistently identify ADAs under different conditions and across different samples, which is crucial for assessing drug safety and efficacy.

2. What are the critical parameters to consider during ADA assay development?

   Key parameters include the assay's sensitivity, specificity, drug tolerance, and ability to detect all relevant ADA isotypes. Additionally, the assay must maintain its performance despite potential drug interference and matrix effects from patient samples. Developing an assay that balances these factors is vital for accurate immunogenicity assessment.

3. What is meant by assay specificity in the context of ADA testing?

   Assay specificity refers to the ability of the ADA assay to correctly identify only the ADAs specific to the drug and not react with other substances in the sample. High specificity is essential to avoid false positives, ensuring that only true ADAs are detected, which impacts clinical decision-making about drug safety.

4. How do you validate an ADA assay?

   ADA assay validation involves demonstrating that the assay performs reliably under specified conditions. This includes proving its accuracy, precision, specificity, sensitivity, and reproducibility through a series of experiments that assess the assay's ability to detect ADAs consistently across a range of conditions and sample types.

5. What is the role of positive and negative controls in ADA assay validation?

   Positive controls (typically ADA-positive serum) and negative controls (ADA-negative serum) are critical for ensuring that the ADA assay functions correctly. Positive controls verify that the assay can detect ADAs when present, while negative controls confirm that the assay does not produce false positives. Regular use of these controls helps maintain assay reliability.

6. Why is drug tolerance important in ADA assays?

   Drug tolerance refers to the assay's capacity to detect ADAs in the presence of the drug. High drug tolerance is essential because patient samples often contain residual amounts of the drug, which can interfere with ADA detection. An assay with good drug tolerance can accurately measure ADA levels without being affected by the drug.

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