Immunogenicity Testing and Immunogenicity Assays
Immunogenicity (IM) testing is a necessary step in the development of any biotherapeutic drug. Innovative strategies have been used to reduce the potential immunogenicity of these large molecules. PEGylation, humanized structures, and chimeric constructs have proven to varying degrees to be effective measures to ensure that a biologic drug’s efficacy and safety are not compromised by the patient’s own immune response. However, none of these inventive designs ensure that biotherapeutics will be immune from the problem of patient rejection and toxicity.
With the arrival of biosimilars or the equivalent of biotherapeutic generic drugs, immunogenicity testing will likely still be required even when the original name brand drug has acceptable immunogenicity.
This is because unlike small molecule generic drugs which have exactly the same chemical structures as their predecessors, biosimilars, produced by different processes, or at different scale, even when using the same process, may have minor variations in chemical structures and impurity levels, thus eliciting different immunogenic reactions when administered in humans.
The latest draft guidance on Biosimilars released by the FDA in February 2012, “Scientific Considerations in Demonstrating Biosimilarity to a Reference Product” states that, at the very least, two separate immunogenicity studies should be conducted to compare any biosimilar to its reference product:
“(1) a premarket study powered to detect major differences in immune responses between the two products and (2) a postmarket study designed to detect more subtle differences in immunogenicity.”
(see lines 548-559)
In addition, the draft guidance goes on to state the following:
“The follow-up period should be determined based on (1) the time course for the generation of immune responses (such as the development of neutralizing antibodies, cell-mediated immune responses), and expected clinical sequelae (informed by experience with the reference product), (2) the time course of disappearance of the immune responses and clinical sequelae following cessation of therapy, and (3) the length of administration of the product. For example, the minimal follow-up period for chronically administered agents should be one year, unless a shorter duration can be justified by the sponsor.” (See lines 592-599)
Choice of Methods for Immunogenicity Testing
The biopharmaceutical scientist can choose from several technologies to perform immunogenicity testing. A double antigen bridging assay has been preferred since such a method, once optimized, can be applied to immunogenicity testing in any host species. Thus, the same IM assay can be used for early animal studies and clinical studies in humans. Each technology platform has its advantages and disadvantages:
ELISA is a well proven, low cost, open technology platform for detecting high affinity anti-drug antibodies (ADAs). It has superior drug tolerance, but may miss low affinity ADAs due to the requirement of high sample dilution and multiple wash steps that may disrupt weakly bound ADA-drug complexes. ELISA can detect ADAs after acid dissociation of drug complexed antibodies.
ANP’s NIDS® IM ELISA assay can be used in neat serum without the need for sample dilution. In addition, it only requires one wash step, thus significantly improving the IM ELISA assay performance (related publication). PEG IM ELISA has also been successfully developed using this method (related publication).
Surface Plasmon Resonance (SPR, Biacore) IM assay, has been shown to be efficient in the detection of low affinity ADAs, but overall is not as sensitive as ELISA due to the label free assay configuration and the requirement for sample dilution. It shows higher drug tolerance for low affinity ADAs, but cannot be used with acid dissociation of circulating complexes. The method requires investment in costly dedicated instrumentation. Similar problems also exist in the Bio-Layer Interferometry (BLI) Dip and Read based IM assays.
Electrochemiluminescence (ECL) IM assay is very similar to ELISA in performance with the claims of improved sensitivity from the use of an electrochemiluminescent label. However, similar shortcomings with the detection of low affinity ADAs due to the need for sample dilution and a final wash step still exist in addition to a significantly higher cost in equipment and reagents, when compared to regular ELISA. The method requires investment in costly dedicated instrumentation.
Rapid Immunogenicity assay using immunochromatographic test strips is a newly developed IM assay method that requires no sample dilution and wash steps, thus capable of detecting both high and low affinity ADAs. It is very tolerant of acid dissociated samples. ANP’s NIDS® rapid IM assay can be utilized for not only patient sample testing during clinical trials, but more importantly the near-patient monitoring of immunogenic reactions, particularly after the biologic drug/biosimilar is approved (related publication). ANP offers various rapid IM assay products and services using both a handheld reader and a high throughput screening (HTS) reader.
Challenges in the Detection of ADAs: Immune Complexes and Endogenous Interfering Proteins
ADAs in an immune patient may already be bound to the biotherapeutic drug in circulating immune complexes, especially in the presence of excess drug. Unless dissociated from these complexes, the ADA will not be detectable in any IM assay of any format. The typical approach to this challenge is to perform an acid dissociation pre-treatment of the sample to liberate ADA from the immune complexes, then after neutralization, immediately run the IM assay. The IM assay has to be run immediately after neutralization to prevent the immune complexes from reforming.
Endogenous Protein and Target Interference
Endogenous protein interferences can cause erroneous results in immunogenicity tests in whatever format. For monoclonal antibodies and similar biotherapeutics that function by binding and blocking disease-associated active proteins, the drug’s target molecule can by itself create a bridging or sandwich complex with the drug conjugate reagents in the IM assay. This leads to a false positive result in IM assays in the absence of ADA.
Acid dissociation by itself will not resolve this problem. Other approaches that may work involve using a separate blocking antibody that will bind interfering target proteins prior to running an immunogenicity assay. Once blocked, the target molecules can no longer form bridging complexes with the drug conjugate reagents. However, these blocking antibodies can be dissociated from the target molecules upon acid dissociation, thus removing their corrective effect. If added immediately after the neutralization step in the acid dissociation process, the blocker will not have enough time to bind target proteins since the IM assay has to be run immediately.
The simplest approach is to irreversibly extract or scavenge the interfering endogenous proteins prior to running the IM assay. The NIDS® NPX4000 Interfering Protein Extraction Kit enables the irreversible removal of these interfering endogenous proteins by attaching your specific blocking antibody or binder to activated particles, pretreating the sample with these particles, then cleanly separating them with the bound interferents . More than 99% of the interfering drug target can be removed by these easy to use particles without affecting ADA levels. We have found that these particles use significantly less blocking antibody or binder than conventional methods.
ANP Reagents: an Open Platform for Immunogenicity Testing
ANP has developed many IM ELISAs and rapid IM assays for its clients. It now offers the biopharmaceutical scientist all its tools so that IM assays in either format are now within easy reach. The IM assay developer can now acquire all the reagents to develop IM assays from ANP. ANP’s quality reagents are easy to use and are based on ANP’s nanomanipulation technology, the Nano-Intelligent Detection System (NIDS®). ANP offers the following products to assist you in developing immunogenicity assays for your specific biotherapeutic drug:
NIDS® HyperBind Streptavidin/Neutravidin coated plates: The NIDS® technology, specifically designed for large molecule testing, significantly improves the binding efficiency of biotinylated proteins and increases the sensitivity of IM ELISAs.
NIDS® DIY Biotin Labeling Kits: Complete Do-It-Yourself kits for the biotinylation of proteins using linkers optimized for use with our HyperBind plates. Included are conjugation and purification reagents, as well as rapid QC strips to confirm that your protein has been successfully biotinylated.
NIDS® DIY Digoxigenin Labeling Kits: Complete Do-It-Yourself kits for the digoxigenin-labeling and purification of proteins using linkers optimized for use with our HyperBind plates. Included are conjugation and purification reagents, as well as rapid QC strips to confirm that your protein has been successfully labeled with digoxigenin.
NIDS® DIY Immunogenicity ELISA kit: A complete Do-It-Yourself kit containing 10 HyperBind plates, biotinylation and digylation kits and all the reagents to optimize an IM ELISA for your drug.
NIDS® DIY Rapid Immunogenicity Assay Development Kit: Under development is a complete Do-It-Yourself kit to develop a rapid IM assay using ANP’s NIDS® reagents. Included is a dedicated handheld reader for use with assay strips.
PEG IM ELISA kit: This is a pioneering universal method for the detection of PEG-specific ADAs to any PEGylated drug. It detects ADAs to PEG molecules of various structures, lengths, and linking groups.
PEG Rapid IM Assay: Under development is a rapid assay for the detection of PEG-specific ADAs to any PEGylated drug. It detects ADAs to PEG molecules of various structures, lengths, and linking groups.
NIDS® NPX4000 Interfering Protein Extraction kit: A simple and easy-to-use kit capable of irreversible removal of interfering endogenous proteins such as drug target compounds, which can cause erroneous results in immunogenicity and PK assays, using ANP’s proprietary nanoparticles.
ANP Assay Development Services
ANP offers its bioconjugation and assay development expertise free of charge to any user of our DIY kits. ANP can also develop immunogenicity methods in ELISA or rapid assay format for clients who prefer to do so.
- European Medicines Agency: “Guideline on Immunogenicity Assessment of Biotechnology-Derived Therapeutic Proteins” EMEA/CHMP/BMWP/14327/2006
- European Medicines Agency “Guideline on immunogenicity assessment of monoclonal antibodies intended for in vivo clinical use” EMA/CHMP/BMWP/86289/2010
- US Food and Drug Administration , “Scientific Considerations in Demonstrating Biosimilarity to a Reference Product” February 2012
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- Pan J, Small T., Qin D, Pauley C, Kaplanski C, Verch T, Chen D, Bakhtiar R, Naling Y, Vallejo Y, Yin R. (2010) NIDS Rapid Assays for the Detection of Anti-Drug Antibodies to Various Polyethylene Glycol (PEG) Polymers. American Association of Pharmaceutical Scientists National Biotechnology Conference, San Francisco, CA May 2010
- Pan J, Small T, Qin D, Vallejo YR, Yin R. (2010) NIDS® Rapid Assays for the Detection of Anti-Drug Antibodies to Peptide Drugs. American Association of Pharmaceutical Scientists National Biotechnology Conference, San Francisco, CA May (Abstract, received the “Excellence in Ligand Binding Assays” award)
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