New opportunities in immunogenicity

Immunogenicity is critical for measuring the potential for immune responses to ensure safe and effective therapies. As the field evolves and new approaches emerge, sponsors have a range of immunogenic considerations for therapeutic proteins and associated analytical methodologies for detecting and characterising anti-drug antibodies (ADAs). Here we share some industry best practices for achieving high-quality ADA assays and explore the emerging trends including the advantages and limitations of singlicate ADA analysis compared with alternative techniques.

How immunogenicity impacts the development and efficacy of therapeutic proteins

Understanding the propensity of a therapeutic protein to generate an immune response is important for several reasons. Dosing with a biotherapeutic can stimulate an immune response to the product itself or to proteins with a related structure. The immune response can potentially impact the efficacy of the therapeutic and when endogenous proteins with a related structure are targeted by ADAs, that presents a significant safety risk for the patient.

Therapeutic efficacy can be impacted via reductions in the amount of the drug in circulation or reduction of the ability of a therapeutic to bind to its target, which is referred to as a neutralising antibody or a NAb. A variety of factors �C such as formulation, protein modifications and structure, target, route of administration and dose �C all have the potential to impact the immunogenicity of a therapeutic and this should be carefully considered during development.

Over the years, regulations have been implemented to monitor immunogenicity as its importance is better understood. The field is still rapidly evolving and discussions on new approaches continue with the goal of monitoring effectively while also being pragmatic and efficient.

Selecting the right approach to immunogenicity

The field of immunogenicity is very broad in terms of drug modalities and analytical techniques, presenting unique challenges and opportunities in selecting the best approach. Different therapeutics each present their own challenges when choosing an assay format, determining assessments to include in validation or determining assay tiers to include. There are also a variety of analytical approaches to choose from to address a sponsor��s needs. For example, there are different platforms, assay formats, types of automation and different techniques at the bench that may better suit each study. The qualitative nature of immunogenicity assays also means that there are different opinions across the industry regarding the analysis and interpretation of data and the ideal approach for meeting regulatory requirements.

At 91��, we frequently see slight variations in the preferred statistical analysis approach for the determination of assay cut points among our sponsors. Experiments and criteria are not as clear as for pharmacokinetic (PK) assays regulated by ICH M10 harmonised guidance. These different aspects can often be challenging, but they also offer us the opportunity for continued development of our own scientific knowledge and expertise, and for collaborative engagement with our sponsors.

Anti-drug antibodies and current analytical methodologies

There are multiple approaches to detecting and characterising anti-drug antibodies (ADAs). Most often we use electrochemiluminescence assays (ECLA), typically with the Meso Scale Discovery (MSD) platform. We also use enzyme-linked immunosorbent assays (ELISAs), and occasionally enzyme activity assays. We also have experience using flow cytometry for readout of ADAs and mass spectrometry for IgG subclass typing of immune responses.

There are a variety of different method formats, including simple bridging assay as well as more drug-tolerant formats. These include:

affinity capture elution (ACE)
solid phase extraction with acid dissociation (SPEAD)
bead extraction and acid dissociation (BEAD)
precipitation and acid dissociation (PandA)
high ionic strength dissociation assays (HISDA)
The most appropriate format is selected based on considerations such as the therapeutic, its target and the level of drug tolerance that��s needed.

Best practices for ensuring high-quality ADA detection and characterisation

The current industry standard for the analysis of clinical samples is a three-tiered approach: screening, confirmatory and titration. Additional tiers should be considered where domain characterisation is necessary for multi-domain therapeutics such as bispecific antibodies or antibody-drug conjugates (ADCs). The addition of antibody isotyping assays may also be appropriate where there are relevant clinical concerns. It is also important to consider the required assay parameters during the early stages of method development, ensuring the assay can reliably tolerate ADAs in the presence of the therapeutic, its target, other co-dosed medications, and other matrix components. And finally, the therapeutic��s mechanism of action and assay parameters that can be achieved should be considered when determining if a cell-based or a plate-based assay is most appropriate to monitor for the neutralising effects of ADAs.

Careful selection of controls and cut-points

A good surrogate positive control is important for assessing and monitoring assay parameters, as is having either sufficient volume or reproducibility for long-term support of an assay. Monoclonal and polyclonal controls both have their own advantages and limitations. It can be challenging to generate a positive control, especially against some therapeutics such as peptides. The negative control pool should be representative of the samples used to establish cut-points.

When determining cut-points, it is important to apply the appropriate statistical technique to establish meaningful cut-points and obtain meaningful assay results.

Typically, data are assessed for normality followed by selection of a parametric or nonparametric cut-point calculation. Outliers are identified and removed, but excessive removal of outliers can be problematic. There is new interest in the industry around alternative approaches such as the application of random effects modeling. Consideration of the need for disease state population cut-points is also of importance, as is monitoring in-study false positive rates as an indication that in-study cut-points are needed.

Evolving industry standards

91�� scientists and managers stay connected to current opinions in industry via attendance at conferences and meetings, participation in publications and conversations with our sponsors. Our three bioanalytical labs in Lenexa, Kansas; Assen, Netherlands; and Whitesboro, New York afford us insight in local environments and industry landscapes globally. Additionally, with approximately 500 bioanalytical staff members, 91�� has the added benefit of a large team of experts and the exchange of information among our sites.

Advantages and limitations of singlicate ADA analysis

Singlicate ADA analysis is a topic of interest in industry. There are a few obvious advantages to single-well analysis:

the ability to analyse twice as many samples per plate
higher sample throughput
reduction in utilisation of potentially costly reagents, materials and labor

From a regulatory perspective, there��s not an explicit requirement for duplicate-well analysis, provided an assay has very good duplicate precision, as many modern ligand binding assays do, particularly on platforms such as MSD. Therefore, moving to single-well analysis should theoretically have minimal impact.

Furthermore, ADA samples typically undergo multiple analyses within the three-tier approach, so a switch to a single-well analysis for each tier would likely have minimal impact. However, due in part to the qualitative nature of immunogenicity assays, there��s still ongoing discussion in the industry around how best to employ single-well analysis in assay validation and its impact on the statistical determination of parameters such as assay cut-point and 1% failure rate LPC (low positive control).

Questions driving industry discussion on the topic of singlicate ADA analysis include: Should the validation be performed in duplicate wells with the results from duplicate- and single-wells compared to support the use of single-well analysis for samples? Or can the validation be performed in single wells without such a comparison? What are the impacts to variability in cut-point assessments? There��s certainly evidence in support of the use of single-well analysis for ADA. But at this point, it��s still a relatively new approach that I��m sure will become better characterised as it��s more widely used. Currently, we see more uptake with single-well analysis for PK.

Emerging trends in bioanalysis of immunogenicity

As biotherapeutics evolve, so must the methods we use to assess immunogenicity. 91�� has invested in examining the best analytical approach for therapeutics such as gene therapies, which are increasingly common. In addition to classic ligand binding assays, techniques such as cell-based assays with flow cytometry readout for immunogenicity assessments for cell-based therapeutics, or hybrid LBA/LC-MS assays for IgG subclass-typing to further characterise immune responses can be advantageous.

As our understanding of immunogenicity continues to evolve, we can expect an industry-wide movement toward a more risk-based approach informed by upfront assessment of therapeutics and an improved understanding of the safety implications of an immunogenic response. This will allow for a more pragmatic and focused approach to assay design and validation.

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