By Indiwari Gopallawa, Integrated Bioanalysis, Clinical Pharmacology and Safety Sciences, Biopharmaceuticals R&D
What is immunogenicity?
Immunogenicity describes the immune response when a substance, such as a biotherapeutic drug, is administered to a patient. Despite their reputation, all biological therapeutics can elicit immune-mediated responses. These responses can lead to diminished clinical efficacy of the biotherapeutic drug and lead to detrimental effects such as hypersensitivity, allergic reactions, and cytokine release syndrome. Immunogenicity testing is a vital part of drug development.
What are anti-drug-antibodies?
In recent years, large-molecule biotherapeutic drugs have been pursued for the treatment of various human diseases. After administration of the therapeutic drug, the immune system induces a humoral response that generates anti-drug antibodies (ADA). The effects of these ADA can weaken the clinical efficacy of biotherapeutics. Furthermore, ADA often affects drug pharmacokinetics (PK), pharmacodynamic (PD) responses, and patient safety. Therefore, assessing the immunogenicity risk of potential biotherapeutics for producing ADA in the pre-clinical and clinical phases of drug development is important.
What are the methods and platforms for detecting ADA?
There are several platforms that are used to detect ADA levels such as MSD, ELISA, and SPR.
The most common ADA assay formats are the bridging assay and the enzyme-linked immunosorbent assay (ELISA). However, novel immunoassay platforms such as Gyrolab, and AlphaLISA have been developed with improved sensitivity.
What is drug tolerance?
One of the major challenges in developing an ADA assay is drug interference. The drug tolerance of an assay is defined as the maximum concentration of free drug that can be present in a sample without preventing the ability to detect ADA. When the circulating concentration of the free drug surpasses the drug tolerance limit in an assay, binding of the free drug to ADA can prevent ADA from being detected by the assay. Therefore, methods for improving drug tolerance during ADA assay development are required.
Strategies to minimize drug interference?
ADA assays are an integral part of the drug development process. Circulating biotherapeutics can interfere with the ADA assay resulting in false negative results. There are several strategies that bioanalytical scientists utilize to improve the drug tolerance of ADA assays.
An early step to improve drug tolerance is to increase the incubation time. It has been shown that overnight incubation may improve the drug tolerance. However, sample pretreatment can be used if overnight incubation does not attain the required drug tolerance. Sample pretreatment often uses a weak acid such as acetic acid to interrupt ADA/drug complexes, releasing the ADA so that it can be detected. Several other methods use acid dissociation to improve drug tolerance such as affinity capture elution (ACE), precipitation and acid dissociation (PandA), biotin-drug extraction and acid dissociation (BEAD), and solid-phase extraction with acid dissociation (SPEAD) methods.
In the ACE method, the sample is treated with a weak acid to dissociate ADA/drug complexes and then neutralized in a plate that is coated with the drug (solid-phase) allowing ADA to be affinity captured. After washing the excess drug, ADA is diluted off with the second acid treatment and subsequently bound to a fresh surface.
Another bioanalytical method known as PandA is used to improve drug tolerance. In this method, excess drug is used to saturate ADA followed by precipitation of drug: ADA complexes with polyethylene glycol (PEG). Next, the complexes are dissociated with acid and transferred to high bind plates and are detected with a sulfo-tagged drug.
In the BEAD method, acid is used to dissociate ADA: drug complexes similar to other methods. Next, excess biotinylated drug in the neutralizing buffer is used to capture the ADA followed by extraction with streptavidin-coated magnetic beads. A second acid treatment is used to elute the ADA from the biotinylated drug and streptavidin-coated magnetic beads.
In the SPEAD, acid dissociated drug-ADA complex is neutralized and then transferred to the plate that has the biotinylated drug captured on a streptavidin-coated plate. Next, a second acid treatment is performed to elute the ADA.
All these methods must be optimized to reduce and eliminate the interference problems caused by drug interference in ADA detection.
Case Study #1:
It was shown by Chen et al., that without the affinity capture elution (ACE) method,
less than 2 μg/mL of the therapeutic protein was able to significantly minimize the ability to detect 500 ng/mL of ADA in the bridging assay. This low drug tolerance possibly hindered the ability to detect low ADA-positive samples. However, after performing the ACE method, the authors were able to demonstrate considerably improved tolerance (> 400 μg/mL of therapeutic protein) in a sample containing 500ng/mL of ADA(1).
Case Study #2:
This study describes how the PandA method eliminated drug interferences in ADA immunoassays. This method showed complete removal of drug interference at high drug concentrations in contrast to the ECL bridging assay without or with acid dissociation. The authors demonstrated that without any pretreatment, ADA was inhibited at 1µg/mL of the drug. However, after acid dissociation 15ng/mL of ADA was detected in the presence of 10 μg/mL of drug. After the PandA method, 14 ng/mL of ADA was detected despite the drug being present at 100 μg/mL(2).
What are the FDA guidelines on ADA testing?
Regulatory agencies are looking to understand the effects of immunogenicity against biotherapeutics and are directing to integrate programs for immunogenicity risk assessment starting in pre-clinical and continuing through clinical and post-clinical phases. The ICH (International Conference on Harmonization) and FDA have published guidelines for the development and optimization of immunoassays to detect anti-drug-antibodies against therapeutic products.
References
1. Chen YQ, Pottanat TG, Carter QL, Troutt JS, Konrad RJ, Sloan JH. Affinity capture elution bridging assay: A novel immunoassay format for detection of anti-therapeutic protein antibodies. J Immunol Methods. 2016;431:45-51. Epub 2016/02/14. doi: 10.1016/j.jim.2016.02.008. PubMed PMID: 26874304.
2. Zoghbi J, Xu Y, Grabert R, Theobald V, Richards S. A breakthrough novel method to resolve the drug and target interference problem in immunogenicity assays. J Immunol Methods. 2015;426:62-9. Epub 2015/08/11. doi: 10.1016/j.jim.2015.08.002. PubMed PMID: 26255760.
Recent White papers:
2022 White Paper on Recent Issues in Bioanalysis: FDA Draft Guidance on Immunogenicity Information in Prescription Drug Labeling, LNP & Viral Vectors Therapeutics/Vaccines Immunogenicity, Prolongation Effect, ADA Affinity, Risk-based Approaches, NGS, qPCR, ddPCR Assays (Part 3 - Recommendations on Gene Therapy, Cell Therapy, Vaccines Immunogenicity & Technologies; Immunogenicity & Risk Assessment of Biotherapeutics and Novel Modalities; NAb Assays Integrated Approach)
2021 White Paper on Recent Issues in Bioanalysis: TAb/NAb, Viral Vector CDx, Shedding Assays; CRISPR/Cas9 & CAR-T Immunogenicity; PCR & Vaccine Assay Performance; ADA Assay Comparability & Cut Point Appropriateness (Part 3 - Recommendations on Gene Therapy, Cell Therapy, Vaccine Assays; Immunogenicity of Biotherapeutics and Novel Modalities; Integrated Summary of Immunogenicity Harmonization)