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Teaser: Everything you need to know about the FDA’s current thinking on immunogenicity testing for therapeutic protein products. Major points of two relevant documents presented together.

FDA Guidance On Immunogenicity Testing Assays For Therapeutic Protein Products

In 2018, the FDA published its recommendations on assays necessary for detecting anti-drug antibodies (ADA) generated by therapeutic protein products (TPPs). These recommendations apply to ADA assays at the clinical trial stage.

This latest guidance document adds to the FDA’s guidelines on immunogenicity testing of TPPs issued in  2014.

Core Recommendations in the 2014 Document

The FDA recommends adopting a risk-based approach for the immunogenicity assays of TPPs. The purpose of the document is to indicate the most significant clinical risks associated with the immunogenetic impact of TPPs and suggest mitigation strategies.

The guidelines also focus on product-specific and patient-specific risk factors, along with mitigation strategies for the same.

Recommendations Related to Product Efficacy

●       The FDA recommends early immunogenicity testing of TPPs to ensure patient safety and product efficacy.

●       The FDA encourages detailing any specific underlying immunologic mechanism of adverse events related to immunology witnessed during the process of developing a therapeutic protein product.

●       Antibody development has the potential of compromising the efficacy of TPPs. That can happen through both neutralizing and non-neutralizing antibodies.

●       Pharmacodynamic biomarkers may help in evaluating interferences on product activity due to antibody mediation.

●       To assess clinical relevance, it is necessary to test the correlation with the clinical response of antibody generated interference with product activities.

Recommendations Related to Patient Safety

●       The FDA emphasizes that adverse events caused by TPPs are often unpredictable. This requires applying a risk-oriented approach even if the initial immunogenetic assays reflect low immunogenicity risk.

●       Applicants for the clinical trial of TPPs need to furnish a justification of the paradigm selected for immunogenetic assays with a focus on patient- and product-specific concerns.

●       The FDA lists the following as some of the most critical adverse immunogenetic responses of TPPs:

○       Anaphylaxis: The FDA recommends meeting all cases of clinical diagnosis of anaphylaxis, with no reference to the presumed pathophysiology.

However, anaphylaxis with confirmed IgE involvement has implications both for prognosis and mitigation.

○       Cytokine Release Syndrome: The FDA recommends a risk-based assessment with a focus on the mechanism of action, along with animal and in vitro results, through the collection of cytokine levels before and after administering the therapeutic protein product.

○       Infusion Reactions: The FDA recommends the use of descriptive terminology for the range of acute reactions that a therapeutic protein product may generate.

○       Non-acute Reactions: Appropriate immunogenicity assays need to be employed to test possible delayed adverse reactions. 

○       Cross-Reactivity to Endogenous Proteins: The recommendation is to use appropriate immunogenicity assays from a risk-oriented perspective.

Recommended Mitigation Strategies During Clinical Trial

●       Developing ADA assays

●       Considering product-specific antibody sampling

●       Staggered dosing for individual patients and the use of dosing cohorts

●       Taking a risk-based approach to dose escalation

●       Intense monitoring of possible adverse events and focusing on the underlying mechanism in case of an adverse event.

●       Preparing a strong rationale for comparative immunogenicity studies and using prespecified criteria wherever possible.

●       Robust safety monitoring in the post-marketing phase.

Recommended Mitigation Strategies for Product-Specific Immunogenicity Issues

●       Immunogenicity assays for products related to novel antibodies should incorporate measures to determine if the novel epitopes are the target of ADA responses.

●       In the case of therapeutic proteins that have been engineered, as also for fusion molecules, use assays to study ADA response to the whole molecule.

●       The latest methods for detecting protein aggregates need to be employed before closing any assays.

●       For evaluating the level and range of subdivisible particles both at the initial stage and over shelf-life, there needs to be an effort to characterize particles in size range of  0.1- 2 microns.

●       For glycosylated protein products, the FDA recommends the use of a cell-substrate production system. The manufacturing process should be able to glycosylate the therapeutic protein product in a non-immunogenic manner. 

●       ADA assays for pegylated TPPs should be able to detect both antibodies against PEG moiety and anti-protein antibodies. The same principle is applicable to non-pegylated TPPs modified with some other entity of high molecular weight, such as hydroxyethyl starch.

●       The FDA stresses the need to minimize all IIRMIs (innate immune response modulating impurities). The use of biomarkers such as cytokine release and defined cell populations activating the transcription factor receive special emphasis.

●       Sponsors need to monitor the immunomodulatory properties of TPPs from the earliest stage. Special attention to be given to:

○       Formulation

○       Container Closure

○       Product Custody

Significant Additions in the 2018 Document

The latest guidance document contains recommendations about screening assays, confirmatory assays, titration assays, and neutralization assays with reference to the immunogenicity testing of TPPs.

The focus is on assay development and validation for ADA detection.

The FDA Recommends a Multi-Tiered Approach to ADA Testing

●       A sensitive screening assay to be used at the initial stage of clinical samples evaluation.

●       A confirmatory assay will be necessary for samples that test positive in the screening assay. The aim is to demonstrate that the ADAs are specific to the TPP under consideration.

●       The FDA recommends titration and neutralization assays for samples that test positive in the confirmatory test.

●       It may be necessary in some cases to use assays to detect cross-activity to other proteins.

●       The FDA cautions that comparing ADA incidence across products can be misleading, even for products that share structural homology or incidence.

●       The FDA recommends getting in touch with the FDA for product-specific guidance, especially for products with endogenous counterparts having functions that are not redundant.

●       The document provides detailed recommendations on assay development, validation, the implementation process of assay testing, and documentation.

●       The document also provides detailed recommendations on the following:

○       Isotypes or Subtypes of Immunoglobulins

○       Specificity of Domains

○       Assay Cut-Point

○       Assay Sensitivity, with special focus on Drug Tolerance

○       Assay Specificity

○       Assay Selectivity, with special focus on Matrix Interference and Minimal Required Dilution

○       Assay Precision

○       Reproducibility of Assay,

○       Robustness and Sample Stability, with special focus on Format Selection

○       Reagents Selection, with special focus on the Development of Positive Control Antibodies, Negative Controls Development, and Non-Specific Binding Controls

○       Reporting Parameters for Quasi-Quantitative and  Qualitative Assays

●       Among other relevant points to consider, the FDA draws attention to:

○       Pre-Existing Antibodies

○       Rheumatoid Factor

○       Monoclonal Antibodies

○       Conjugated Proteins

The document also provides detailed guidelines on developing and validating assays at each recommended tier.