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Evolving immunogenicity assay performance expectations and a lack of harmonized anti-drug antibody validation testing and reporting tools have resulted in significant time spent by health authorities and sponsors on resolving filing queries. Following debate at the American Association of Pharmaceutical Sciences National Biotechnology Conference, a group was formed to address these gaps. Over the last 3 years, 44 members from 29 organizations (including 5 members from Europe and 10 members from FDA) discussed gaps in understanding immunogenicity assay requirements and have developed harmonization tools for use by industry scientists to facilitate filings to health authorities. Herein, this team provides testing and reporting strategies and tools for the following assessments: (1) pre-study validation cut point; (2) in-study cut points, including procedures for applying cut points to mixed populations; (3) system suitability control criteria for in-study plate acceptance; (4) assay sensitivity, including the selection of an appropriate low positive control; (5) specificity, including drug and target tolerance; (6) sample stability that reflects sample storage and handling conditions; (7) assay selectivity to matrix components, including hemolytic, lipemic, and disease state matrices; (8) domain specificity for multi-domain therapeutics; (9) and minimum required dilution and extraction-based sample processing for titer reporting., (© 2021. The Author(s).)

In September 2018, the American Association of Pharmaceutical Scientists (AAPS) conducted an Annual Guidance Forum on the considerations related to immunogenicity testing for therapeutic protein products. In addition to a broad representation by the pharmaceutical industry, the event included strong representation by leading scientists from the US Food and Drug Administration (FDA). The agency and industry perspectives and updates to the guidance were presented. Specific topics that were discussed included the strategies of anti-drug antibody (ADA) assay cut-point assessments, the selection of ADA-positive controls (PCs), and the evaluation of PC performance. Assessment strategies and relevance of ADA assay attributes were also discussed, including assay drug tolerance and ADA assay sensitivity. The following is a summary of the discussion.

Sensitive and specific methodology is required for the detection and characterization of anti-drug antibodies (ADAs). High-quality ADA data enables the evaluation of potential impact of ADAs on the drug pharmacokinetic profile, patient safety, and efficacious response to the drug. Immunogenicity assessments are typically initiated at early stages in preclinical studies and continue throughout the drug development program. One of the potential bioanalytical challenges encountered with ADA testing is the need to identify and mitigate the interference mediated by the presence of soluble drug target. A drug target, when present at sufficiently high circulating concentrations, can potentially interfere with the performance of ADA and neutralizing antibody (NAb) assays, leading to either false-positive or, in some cases, false-negative ADA and NAb assay results. This publication describes various mechanisms of assay interference by soluble drug target, as well as strategies to recognize and mitigate such target interference. Pertinent examples are presented to illustrate the impact of target interference on ADA and NAb assays as well as several mitigation strategies, including the use of anti-target antibodies, soluble versions of the receptors, target-binding proteins, lectins, and solid-phase removal of targets. Furthermore, recommendations for detection and mitigation of such interference in different formats of ADA and NAb assays are provided.

Anti-drug antibodies (ADA) pose a potential risk to patient safety and efficacy and are routinely monitored during clinical trials. Pre-existing drug-reactive antibodies are present in patients without prior drug exposure and are defined by their ability to bind to a component of the drug. These pre-existing drug-reactive antibodies are frequently observed and could represent an adaptive immune response of an individual who has been previously exposed to antigens with structural similarities to the biotherapeutic. Clinical consequences of these antibodies can vary from no impact to adverse effects on patient safety, exposure, and efficacy, and are highly dependent on biotherapeutic modality, disease indications, and patient demographics. This paper describes how the immunogenicity risk assessment of a biotherapeutic integrates the existence of pre-existing drug-reactive antibodies, and provides recommendations for risk-based strategies to evaluate treatment-emergent ADA responses.

Bioanalytical methods must enable the delivery of data that meet sound, scientifically justified, fit-for-purpose criteria. At early phases of biotherapeutic drug development, suitable criteria of a ligand-binding assay could be met for pharmacokinetic (PK) in-study sample testing without a full validation defined by regulatory guidelines. To ensure fit-for-purpose methods support PK testing through all phases of biotherapeutic development, three tiers of method validation - regulatory, scientific and research validations - are proposed. The three-tiered framework for method validation outlines the differences in the parameters that should be assessed, the acceptance criteria that may be applied, and the documentation necessary at each level. The criteria for selecting the appropriate application of each of these PK method validation workflows are discussed.

Pre-existing antibodies to biotherapeutic drugs have been detected in drug-naïve subjects for a variety of biotherapeutic modalities. Pre-existing antibodies are immunoglobulins that are either specific or cross-reacting with a protein or glycan epitopes on a biotherapeutic compound. Although the exact cause for pre-existing antibodies is often unknown, environmental exposures to non-human proteins, glycans, and structurally similar products are frequently proposed as factors. Clinical consequences of the pre-existing antibodies vary from an adverse effect on patient safety to no impact at all and remain highly dependent on the biotherapeutic drug modality and therapeutic indication. As such, pre-existing antibodies are viewed as an immunogenicity risk factor requiring a careful evaluation. Herein, the relationships between biotherapeutic modalities to the nature, prevalence, and clinical consequences of pre-existing antibodies are reviewed. Initial evidence for pre-existing antibody is often identified during anti-drug antibody (ADA) assay development. Other interfering factors known to cause false ADA positive signal, including circulating multimeric drug target, rheumatoid factors, and heterophilic antibodies, are discussed.

The Global Bioanalysis Consortium (GBC) set up an international team to explore the impact of immunogenicity on pharmacokinetic (PK) assessments. The intent of this paper is to define the field and propose best practices when developing PK assays for biotherapeutics. We focus on the impact of anti-drug antibodies (ADA) on the performance of PK assay leading to the impact on the reported drug concentration and exposure. The manuscript describes strategies to assess whether the observed change in the drug concentration is due to the ADA impact on drug clearance rates or is a consequence of ADA interference in the bioanalytical method applied to measure drug concentration. This paper provides the bioanalytical scientist guidance for developing ADA-tolerant PK methods. It is essential that the data generated in the PK, ADA, pharmacodynamic and efficacy/toxicity evaluations are viewed together. Therefore, the extent for the investigation of the PK sensitivity to the presence of ADA should be driven by the project needs and risk based.

Fulranumab, a human IgG2 monoclonal antibody that neutralizes nerve growth factor (NGF), is currently in development for the treatment of pain. Our initial immunogenicity test method was found to be prone to NGF interference, leading to a high apparent incidence of anti-drug antibody (ADA) in phase 1 studies. The ADA immunoassay comprised a homogeneous bridging electrochemiluminescence (ECL) format with biotin and ruthenium-labeled fulranumab bound together ("bridged") by ADA in test samples for detection. In this assay, NGF produced a false-positive signal due to its ability to bridge fulranumab molecules. Thus, we developed a specificity assay to eliminate the NGF false-positive results. We encountered the challenge of eliminating drug interference as well as drug target interference, and discovered that the acid-dissociation-based pretreatment of samples used for mitigating drug interference dramatically increased drug target interference. Several strategies were investigated to eliminate the NGF interference; yet only one strategy specifically removed NGF and produced true fulranumab-specific ADA results by using competitive inhibition with fulranumab and utilizing an alternative NGF binding antibody to eliminate NGF interference. Using this new method, we confirmed that the high apparent anti-fulranumab antibody incidence (>60%) in clinical study samples was in fact due to fulranumab-bound NGF released during the acid-dissociation step of the ADA testing method. We conclude that our revised method accurately identifies anti-fulranumab antibodies by incorporating steps to eliminate fulranumab and NGF interference. We advise that acid-dissociation pretreatment must not be universally applied to improve ADA assays without investigating its bioanalytical risks versus benefits.

Administration of biological therapeutic proteins can lead to unwanted immunogenicity in recipients of these products. The assessment and characterization of such immune reactions can be helpful to better understand their clinical relevance and how they relate to patient safety and therefore, have become an integral part of a product development program for biological therapeutics. Testing for anti-drug antibodies (ADA) to biological/biotechnology-derived therapeutic proteins generally follows a tiered approach. Samples are initially screened for binding antibodies; presumptive positives are then confirmed in a confirmatory assay; subsequently, confirmed-positive samples may be further characterized by titration and with a neutralizing antibody (NAb) assay. Regulatory guidances on immunogenicity state that assessing the neutralizing capacity of antibodies should preferably be done using functional bioassays, while recognizing that competitive ligand-binding (CLB) assays may be substituted when neutralizing bioassays are inadequate or not feasible. This manuscript describes case studies from four companies in which CLB assays and functional bioassays were compared for their ability to detect neutralizing ADA against a variety of biotechnology-derived therapeutic proteins. Our findings indicate that CLB assays are comparable to bioassays for the detection of NAbs, in some cases offering better detection sensitivity, lower variability, and less matrix interference., (Copyright © 2010 Elsevier B.V. All rights reserved.)