The European Bioanalysis Forum recommendation on establishing appropriate drug tolerance levels in antidrug antibody assays

ABSTRACT

The European Bioanalysis Forum, in collaboration with several key industry stakeholders, has recently led discussions that address international immunogenicity guidance documents, specifically the three tier approach for immunogenicity testing strategies, after more than 20 years of experience with biotherapeutics. As part of this, the strategy and methods used to assess drug tolerance across all immunogenicity assays are challenged, emphasizing that bioanalytical scientists need to consider the context-of-use of each assay. Here, recommendations for drug tolerance assessments, driven by strong scientific rationale and subject to reevaluation as needed, are provided. This includes carefully considering the drug and positive control concentrations considered to be appropriate and which tiers are most relevant for performing drug tolerance assessments.

1. Background

Immunogenicity testing strategies have been in place for over 20 years to detect unwanted immune responses to administered recombinant proteins in patient clinical studies [1]. Unwanted antidrug antibody (ADA) responses can result in negative clinical consequences, for example a deleterious impact on pharmacokinetics (PK), pharmacodynamics (PD), efficacy, or safety. Over the years the pharmaceutical industry has utilized a cross-industry accepted approach for immunogenicity assessment evaluations, which help to refine the testing strategy for ADAs in the preclinical and clinical phases of a biotherapeutic. Health authority guidelines have been in place since 2001, starting with the US FDA guidance on immunogenicity, which has been updated in recent years [2]. Guidance from the EMA [3], announcements from China’s National Medical Products Administration [4] and a draft guideline from Japan’s Pharmaceuticals and Medical Devices Agency [5] have followed. Thus, immunogenicity testing for biologics is a regulatory requirement. These health authority guidance documents have recommended the same three-tiered approach that includes screening, confirmation of the screened positive samples and titer of the confirmed positive samples.

The three-tiered immunogenicity testing strategy recommended in the guidelines is currently being challenged by the industry [6]. The European Bioanalysis Forum (EBF), in collaboration with subject matter experts across the industry, recently held a Focus Workshop in September 2023 [7], questioning whether the multi-tiered approach is still fit-for-purpose. The health authority guidelines were originally built to address adverse clinical sequelae observed in patients exposed to high-risk molecules, but many new biotherapeutics are deemed low risk [8]. During the Focus Workshop, attendees reflected on the pharmaceutical industry's experience and accumulated immunogenicity results since 2001, the discussions around context-of-use (CoU) [9–17] and the industry’s willingness to adapt immunogenicity testing strategies depending on the potential immunogenicity risk level of a molecule program. Several topics were covered, including the question: are we aiming for higher drug tolerance than needed? Subsequently, the EBF presented several case studies on drug tolerance at the 2023 Open Symposium which also challenged the need to generate highly drug tolerant assays [14].

Drug tolerance is the ability of the ADA assay to determine the presence or absence of ADAs in samples which also contain the drug molecule. Evaluation of drug tolerance for immunogenicity assays is specifically requested in the available guidances for immunogenicity and is a fundamental parameter to be included in assay validation. The EMA guidance addresses drug tolerance as such: “…the Applicant has to demonstrate that the tolerance of the assay to the therapeutic exceeds the levels of the therapeutic protein in the samples for ADA testing”. The guidance also qualifies this statement, stating there could be challenges “due to technical limitations” and therefore: “If this occurs, the best possible assay should be employed and the approach taken should be properly justified” [3]. The FDA guidance states this as: “In general, FDA recommends that sponsors develop assays that are optimized for sensitivity, specificity, selectivity, drug tolerance, precision, reproducibility and robustness...” and that: “The assessment of assay sensitivity in the presence of the expected levels of interfering therapeutic protein product, also known as the assay's drug tolerance, is critical to understanding the sensitivity and suitability of the method for detecting ADA in dosed subjects” [2].

For all types and tiers of immunogenicity assays (screening, titer, confirmatory ADA assays or domain specificity, cell- or ligand-binding-based neutralizing ADA assays), optimizing the assay for increased drug tolerance is possible. However, in the health authority guidance documents there is no specific recommendation on whether drug tolerance should be evaluated across all these types of assays. Developing a drug tolerant method for all assay tiers can be challenging in some circumstances, as it usually requires the addition of several complicated assay steps to a method. Several publications over the years have documented many approaches to improve drug tolerance for immunogenicity assays [18–27]. Additional method steps like acid dissociation, additional capture, precipitation or elution steps are included in many drug tolerant assay formats. These pretreatment steps may destroy drug or ADA and these effects are challenging to measure. In addition, drug tolerant formats can be time-consuming and can result in poor assay performance. Presentations at the 2023 EBF Open Symposium have done the same, while questioning the need to conduct so much resource-intensive work [28,29]. Recent publications have also questioned the need to do so much to address drug tolerance in nonclinical and clinical immunogenicity assays [30–33]. Clinically meaningful data requires understanding the capabilities of the assay and the interactions taking place. Recent developments have proposed an alternative scientific approach that adds the additional step of characterizing the binding of the positive control (PC) and with this calculate drug tolerance of an assay [34].

In this recommendation paper, the EBF would like to continue to challenge the three-tiered paradigm and stress that bioanalytical scientists need to consider the CoU and therefore the purpose, of the assay itself. Overall, the EBF supports a strategy based on scientific knowledge, driven by the unique needs of each situation. Assessing drug tolerance should not been seen as a ‘tick box’ exercise. Drug tolerance assessments should be part of an iterative process, with assay strategy decisions supported on strong scientific rationale and subject to reevaluation as needed.

2. CoU for immunogenicity assays

The value and implementation of CoU principles is broadening now, in response to several years of discussion and published recommendation papers [9–17], including open industry debate such as the EBF Focus Workshop in 2022 [14] and the EBF Open Symposium in 2023 [15] on biomarker assay CoU. The EBF and others [16] recommend that the bioanalytical scientist understands the CoU of all bioanalytical requests [17] and that this CoU statement is appropriately documented. This includes immunogenicity assays.

The purpose of the assay and the decisions being made with the data can impact the parameters investigated with appropriate acceptance criteria. The documented CoU statement can be impacted by the stage of development (nonclinical, clinical, Phase I versus Phase III or postmarketing commitment), the scientific value of the results and the immunogenicity risk (low to medium versus high risk). These factors will affect which tier(s) of immunogenicity assessment will be utilized, which assay characteristics are appropriate and what limitations the assays may have. Because the CoU includes the study population the assay will be used for, a change in CoA may lead toa change of population specific cut points, which also impacts drug tolerance.

In considering the purpose of the assay itself, we also need to consider the modality of the biotherapeutic when it comes to the extent to which immunogenicity may affect safety and/or efficacy and therefore in which contexts ADAs need to be detectable by the assay. This is particularly applicable to nonantibody-based therapeutics such as hormones, cytokines, adeno-associated virus, chimeric antigen receptor T-cell therapies and any other modalities. Another factor in CoU is that the first indicator of an immunogenicity response, is often an abnormal PK profile and immunogenicity is often determined later to confirm the reason for these observations. The CoU of the assay and therefore the rationale to determine drug tolerance and the level of drug tolerance should always be considered. The real question should be: what could be the clinical impact of immunogenicity and what level of drug tolerance is appropriate? The CoU questions should include:

• 1.What type of study is the assay for, e.g., clinical or nonclinical, early or registrational?
• 2.Will false negative results, in the presence of a certain level of drug, prevent interpretation of impact?
• 3.What is the immunogenicity risk category?
• 4.Would the presence of unexpected ADAs be a safety concern?
• 5.Is there a likely impact on efficacy or PK/PD?

3. Questions on drug tolerance assessments for immunogenicity assays

As a result of several EBF immunogenicity team discussions and at an interim Focus Workshop which addressed the three-tiered paradigm as mentioned and touched on these topics on drug tolerance, the EBF asked the following questions:

• 1.What drug and PC concentrations do you consider to be appropriate for drug tolerance assessment (e.g., around relative sensitivity of assay and a few concentrations above, Ctrough of drug, etc.)?
• 2.Which tiers are most relevant for performing drug tolerance assessments?
• 3.How do we move back to scientific approaches on a case-by-case basis, rather than a ‘tick-box’ exercise for drug tolerance requirements?
• 4.What recommendations can we draw from these discussions?

The cross-industry points that were raised during these discussions included the following:

3.1. What concentrations of PC & drug do you test?

The ADA PC tested in method development and validation can only be surrogate relative to the actual ADA response in patients and therefore is only utilized to characterize the assay. Nonetheless, several publications have recommended that different levels of ADA and drug concentrations be tested in drug tolerance experiments in immunogenicity assays [35,36].

In this paper, we recommend that the surrogate concentrations of ADA used should match the intended CoU for the assay and be scientifically justified. In simpler terms, they should align with the risk associated with the molecule and the potential clinical impact of immunogenicity. This includes considering whether a false negative for low- to medium-risk molecules could be clinically meaningful, particularly if the method and therefore the assay cut point threshold are being tested at the limit of the assay technology. It is critical that high-risk molecules with nonredundant endogenous counterparts require drug tolerant assay that detect even low responses; however low- to medium-risk molecules are unlikely to have clinically relevant, low-signal positives. As a consequence of this recommendation, the EBF also recommends that there is no need to assess drug tolerance at the low PC (LPC) concentration in a hyper-sensitive assay with very low LPC concentration. Generally, it should not be a requirement to test drug tolerance at less than 100 ng/ml of the PC, the sensitivity recommended by the FDA guidance [2]. While testing only 100 ng/ml is suggested, the levels tested should be study and context related. And if the assay is hyper-sensitive, it is not necessary to determine drug tolerance at the LPC concentration. Testing at the high PC concentration should also be carefully considered given the CoU of the assay.

The EBF supports testing drug tolerance early in method development as recommended by the FDA guidance, including at 250 ng/ml and 500 ng/ml PC and other levels as appropriate. For example, if an assay can measure positive low-titer ADA responses in clinical study samples at the trough level of the drug, the assay may be acceptable even if the drug tolerance at 100 ng/ml of the PC is lower than the trough level. During these assessments, it is important to bear in mind that the drug tolerance measured with the PC does not necessarily translate to the drug tolerance with actual study sample ADAs; the PC is a surrogate, often chosen as the ‘best’ surrogate to support optimal assay characteristics and is not representative of ADA present in samples. Furthermore, it has been shown that assay sensitivity and drug tolerance are likely driven by multiple factors such as cut-point confidence level, stoichiometry of PC–drug complexes and presentation of epitopes and this highlights the importance of PC selection to reliable detection and clinical interpretation of the presence and magnitude of ADA responses [37,38]. Testing multiple PCs is suggested to better characterize the ADA assay.

The EMA guidance [3] also states:

“Several product-related factors will influence the development of an immune response against a therapeutic protein... Therefore, the sampling schedule for detection of an immune response should be adapted and applied individually for each product, also taking into account its pharmacokinetics (e.g. elimination half-life) and the drug tolerance of the ADA-assay(s). Baseline samples should always be collected.”

The aim is to have the appropriate degree of drug tolerance at the interested drug levels, which is often at Ctrough levels. When asked during the 2023 Focus Workshop discussion, a common response from delegates was that drug tolerance should be achieved up to C_max. Many cited Myler et al. [36], where it states: “It is very important that the maximum drug concentrations expected to be present in study samples be described in the text of the validation report and discussed in relation to the validated drug tolerance result.” and “Drug concentrations are selected to cover the range of concentrations expected to be present in study samples.”). However, this should be understood to refer to the maximum concentration of drug in ADA samples, not the C_max of the PK samples.

The EBF also recommends having the appropriate sampling time points and if possible, for a study, more than one washout sample time point. It is important to choose the appropriate drug levels for testing, depending on your study and to have a solid idea of drug concentrations in sampled time points in advance, using modelling data, if necessary, to know what drug tolerance is relevant for the study. For example, the clearance of some drug candidates can be quite fast and typically ADA sampling should happen close to five-times the half-life of the biotherapeutic, as per the FDA guidance [2]. Adhering to this principle, the appropriate drug levels for testing is often at Ctrough levels. This will depend on the study design and it is important to use the best possible estimate of drug concentrations in sampled time points, using modelling data if necessary. The aim is to have appropriate drug tolerance at the relevant drug levels only. Drug tolerance can be assessed during method development by titrating drug versus PC in a checkboard experiment. In method validation, a single run, in singlicate, of drug tolerance tests at the relevant PC and drug concentrations is sufficient.

3.2. Which tiers are you performing drug tolerance in?

For clinical assays testing drug tolerance in the screening assay during development and validation experiments is important and for nonclinical assays, this may be evaluated as needed [39]. Most responses from the EBF Focus Workshop [7] indicated that the industry is tending toward testing drug tolerance only in the screening assay for low- to medium-risk molecules and that there often was not a strong rationale to test drug tolerance in confirmatory assays, unless the molecule was deemed high risk for immunogenicity. Additionally for confirmatory assays, it can be difficult, because of the presence of excess drug and potentially unnecessary, given the lack of orthogonality between confirmatory and screening assays that has been shown over the years in case studies [40].

For titer assays, the EBF challenges the usefulness of evaluating drug tolerance in this tier. The EBF continues to challenge the value of performing a titer tier at all, preferring the reporting of signal-to-noise, with increasing publications to support this [7,41–43].

3.3. How do we move back to scientific approaches rather than a ‘tick-box’ exercise for drug tolerance requirements?

The EBF recommends that the bioanalytical community consider the CoU of the assays and then decide on what assay characteristics are needed. The immunogenicity risk assessment should be a major consideration in determining whether drug tolerance is sought, whether it is evaluated and to what levels in which tiers. The key is to employ strong scientific rationale to justify the use of resources on an immunogenicity testing strategy, rather than simply following guidelines written for high-risk molecules, as some specific recommendations of regulatory guidance may not be applicable for all biotherapeutics. Early consultation with the health authorities is recommended, including when drug tolerance of the assay is a concern. Sponsors should remain proactive in approaching health authorities on their immunogenicity testing strategies, including why a certain level of drug tolerance is scientifically and strategically appropriate given an assay’s CoU and fully acknowledging, that clinical impact is more important than ADA incidence.

3.4. What recommendations can we draw from these discussions?

For these recommendations, it is important to reiterate that the drug tolerance of the PC does not necessarily reflect the drug tolerance of actual study samples; the PC is chosen as the ‘best’ surrogate to optimize assay characteristics and may not accurately represent ADA present in samples.

As a result, the EBF has the following recommendations for drug tolerance:

• 1.The risk assessment and the potential clinical consequences should drive the need and the extent of the drug tolerance assessments performed in validation.
• 2.Whenever possible, thoughtfully select sampling time points, including washout samples, depending on the (known or predicted) PK of the drug candidate and choose appropriate drug levels for testing in development and validation, depending on your study.
• 3.Choose an appropriate target for drug tolerance depending on your study, considering Ctrough rather than C_max.
• 4.When selecting drug concentrations for testing in validation, the relevance to the study(ies) should be considered and drug tolerance only needs to be achieved at levels of drug expected in samples that will need to be tested to understand the impact of the presence of ADA.
• 5.Testing drug tolerance at 100 ng/ml of PC is recommended for validation. If suitable drug tolerance is not achieved at 100 ng/ml, then 250 ng/ml and 500 ng/ml PC levels may be included; other levels should be risk- and study-related. Testing at the LPC level is not recommended. High PC level is not required.
• 6.For most molecules with low immunogenicity risk, assessment of drug tolerance in the screening assay is deemed sufficient. Depending on the risk and consequences of immunogenicity, assessment of drug tolerance in the confirmatory tier might be necessary, but drug tolerance is not needed to be assessed in the titer assay.
• 7.One validation run is sufficient for drug tolerance parameters.
• 8.It is recommended to engage with the health authorities early in a drug development program, including when the desired level of drug tolerance is not achieved.

Although these recommendations provide valuable guidance, decisions regarding drug tolerance assessments should ultimately be made based on scientific judgment, prioritizing what is most appropriate and relevant in the specific CoU.

4. Conclusion

Safety and efficacy are key to any clinical study and therefore essential to consider in any immunogenicity testing strategy. Clinical relevance is investigated by integrating all clinical end points, including safety, PK/PD and efficacy. Immunogenicity needs to be considered in the context of not only the performance of the assay, including drug tolerance, but also alongside all available clinical data. Most important is the clinical impact of the presence of ADA. Since PK often provides the initial insight into immunogenicity responses, with immunogenicity data typically validating these observations later on, it is vital to consider the potential for time saving and appropriate and effective resource allocation by prioritizing effective scientific strategies in drug development. This approach, centered on patient well-being and time efficiency, underscores the importance of conducting thorough risk assessments for every drug candidate from concept stage onwards. It emphasizes the necessity of crafting appropriate bioanalytical strategies and determining the right CoU for each type and tier of immunogenicity testing. Accordingly, the right assay parameters can then be appropriately evaluated, including drug tolerance. The risk of a false negative is unlikely to be impactful for low to medium risk molecules but could be more consequential for high-risk molecules with nonredundant endogenous counterparts. Drug tolerance might be reassessed as needed based on study specificities. Additionally, target or other drug-specific interference should be equally considered and if the soluble target has the potential to affect the immunogenicity readout, its interference in the ADA assay should be assessed based on its characteristics, such as multivalent drug binding or endogenous levels. With all the considerations that impact the CoU of an immunogenicity testing strategy, we should remain mindful of how to approach drug tolerance assessments in the interest of patients and clinicians alike.

5. Future perspective

In both FDA and EMA immunogenicity guidance documents, there has been a request for the integrated summary for immunogenicity submissions at BLA. With the increasing experience of integrated summary for immunogenicities and over 20 years of accumulated immunogenicity data across a huge and ever-expanding variety of modalities, our community of scientists are in a position to cite examples, precedence and scientific rationale to push back on conservative requests for unnecessary data, including highly drug tolerant assays, when the CoU would dictate much less is required to assist clinicians in data interpretation and thus use resources appropriately to speed drug discovery and development in the right direction. With so many end-users of immunogenicity data and knowing that oftentimes the clinically relevant cut points are many times higher than highly sensitive assay cut points, it is critical that open discussions with health authorities and stakeholders continue so that more meaningful immunogenicity testing strategies are employed. In this effort to generate scientifically meaningful data, rather than simply following guidance as written, the EBF will continue to collaborate with scientists globally to ensure that the industry is consistently striving to provide scientifically meaningful results for patients.

Disclaimer

The views and conclusions presented in this paper are those of the European Bioanalysis Forum (EBF) and do not necessarily reflect the representative affiliation or individual company's position of the authors on the subject.

Financial disclosure

The authors have no relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the recommendation. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties.

Competing interests disclosure

The authors have no competing interests or relevant affiliations with any organization or entity with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, stock ownership or options and expert testimony.

Writing disclosure

No writing assistance was utilized in the production of this manuscript.