A critical part of the biopharmaceutical drug development process is to identify and characterize Anti-drug Antibodies (ADA). Immune responses have varying clinical effects on subjects. They can range from something not measurable to something severe.
Detecting ADA formation and its analysis assists in understanding possible immune responses. Immunogenicity testing must be conducted in a manner to detect ADA that can manage unwarranted physiological consequences – like hypersensitivity or neutralizing responses.
Developing and validating ADA assayis a key step in preclinical and clinical biologics testing. These help to manage immune responses that have an effect on the efficacy of drug therapy and its safety.
The FDA, in 2019, has updated the guidelines regarding the development and validation of immunogenicity assays. Based on them, we adopt a risk-based approach to manage and evaluate immune responses that therapeutic proteins elicit. Updates in three main areas have a significant effect on the development and validation, as below:
Assessing Neutralizing Antibodies
Clinical scientists might detect anti-drug antibodies while they are conducting immunogenicity testing. They need to assess if any such antibodies are Neutralizing Antibodies (NAbs). NAbs can affect the therapeutic protein’s clinical activity.
Early immunogenicity testing might cause ADA responses that are above the cut point. In such cases, our scientists need to determine the presence of NAbs. This they do by carrying out assays. It is important to ensure the requisite testing before in-human trials. Otherwise, there’s the risk of the clinical hold.
Any NAb assays can detect NAbs with the requisite selectivity, specificity, precision, and sensitivity. That poses a challenge to the task of developing neutralization assays. The cell-based and non-cell-based differing assay formats carry specific challenges.
Earlier, scientists used mostly cell-based assays for the detection of Nab, but certain actions were unresponsive to cell-based assays. For example, enzymatic replacement not requiring a cellular update. But now, we have higher flexibility to design the most suitable assay for detecting NAbs.
Determining the Cut Point
The assay cut point is the point at which the sample response will test positive or negative. Thus, it is critical to establish a suitable cut point to cut down on false-negative results. Assay designing should happen in a manner where they can ensure a 5% false-positive rate. This helps the assay to identify the subjects with the potential to develop antibodies to a therapeutic protein.
To estimate the cut point for the confirmatory assay, we apply an 80-90% one-sided lower confidence interval for the 99th percentile of the negative control population. The aim of the confirmatory assay is to remove false-positive samples that arise because of non-specific binding.
Documenting the Immunogenicity Data
The immunogenicity summary report should clearly mention the data to support a therapeutic protein’s regulatory filings. The report needs to show the following:
● Immunogenicity risk assessment
● Tiered bioanalytical strategy
● Assay validation summary
● Immunogenicity data analysis
● Risk evaluation strategies.
This process demonstrates the performance features of the employed ADA assay are appropriate for the intended purpose. The validation process happens through particular laboratory investigations and experiments.
In the primary stages of clinical development, we assess assay precision, cut point, and tolerance to drugs. This is a partial validation. However, for a high-risk product, we conduct a full validation before we proceed for clinical studies..