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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. Graphical Abstract

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.

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.

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.

Critical reagents are essential components of ligand binding assays (LBAs) and are utilized throughout the process of drug discovery, development, and post-marketing monitoring. Successful lifecycle management of LBA critical reagents minimizes assay performance problems caused by declining reagent activity and can mitigate the risk of delays during preclinical and clinical studies. Proactive reagent management assures adequate supply. It also assures that the quality of critical reagents is appropriate and consistent for the intended LBA use throughout all stages of the drug development process. This manuscript summarizes the key considerations for the generation, production, characterization, qualification, documentation, and management of critical reagents in LBAs, with recommendations for antibodies (monoclonal and polyclonal), engineered proteins, peptides, and their conjugates. Recommendations are given for each reagent type on basic and optional characterization profiles, expiration dates and storage temperatures, and investment in a knowledge database system. These recommendations represent a consensus among the authors and should be used to assist bioanalytical laboratories in the implementation of a best practices program for critical reagent life cycle management.