Immunogenicity Testing

Assessment of the ADA Response for a Wide Range of Biotherapeutics

FyoniBio has many years of experience in immunogenicity testing services including assay development, validation and bioanalysis in the course of clinical trials. All bioanalytical studies are performed according to the current ICH, FDA and EMA guidelines in our ISO 9001 certified and GCLP compliant laboratories. Get in touch with us, we will be happy to support you throughout your clinical development program.


What is immunogenicity and why it is important?

Biotherapeutics from small oligonucleotides to large proteins can elicit immune responses when applied in animals or humans. The immune response may result in the formation of anti-drug antibodies (ADA). The probability to develop ADAs depends on the application route and design of the biotherapeutic. It ranges from a few patients to high incidences in the mid double-digit range.

Binding of ADA to the drug results in clinical responses that range from no symptoms to severe side effects, and may even be health- or life-threatening. The presence of ADA may alter the pharmacokinetics, may neutralize the drug’s activity, and affect the mode of action, may support hypersensitivity or autoimmunity reactions, or may cross-react with endogenous counterparts of the drug.

Potential impact of ADA generation on biotherapeutic safety and efficacy

In order to evaluate the safety, efficacy and pharmacokinetics of a biotherapeutic drug, its immunogenic potential has to be assessed during preclinical and clinical development. Immunogenicity testing means, that it is examined whether or not anti-drug antibodies are detectable in the blood after drug application. In preclinical animal studies the occurrence of ADA is mainly analyzed to evaluate any effects on the bioavailability of the drug. In humans, immunogenicity testing as a key safety aspect is included in clinical trials from phase I onwards.


Immunogenicity Analysis

Samples are usually analyzed in a three-tier approach using

i) a screening assay to identify negative and potentially positive samples

ii) a confirmation assay to verify the potentially positive samples as negative or positive

iii) a titration assay to determine a serological titer.

Analytical approach for immunogenicity testing

Major challenges which have to be solved during ADA assay development are the availability of a suitable positive control, sensitivity, drug tolerance, matrix effects including pre-existing antibodies, and the determination of the assay cut point.

Clinical samples are analyzed by using all three assays of the approach whereas preclinical samples are typically only analyzed using the screening and confirmation assay.

Immunogenicity Assay Design

At FyoniBio, anti-drug antibodies are detected using an electrochemiluminescence (ECL) assay in a homogeneous bridging format. Samples are diluted at the minimally required dilution to overcome potential matrix effects. Diluted samples are subjected to an acid dissociation step to dissolve complexes of anti-drug antibodies and the drug, thereby improving greatly the drug tolerance. Next, samples are neutralized and simultaneously incubated with biotinylated and sulfoTAG-labelled drug to start immune complexes formation. Subsequently, the solution is transferred to a blocked streptavidin coated 96-well plate. Unbound proteins are removed by washing and ECL is measured on a MESO QuickPlex SQ 120 (MSD Inc.). The measured light signal is proportional to the concentration of anti-drug antibodies in the sample.

Scheme of ECLIA bridging format

Immunogenicity Assay Evaluation

Evaluation of the measured signal is done qualitatively, using assay specific cut-points established during validation. As a result of the immunogenicity assay, a sample’s signal may be above or below the cut point, classifying the sample as positive or negative. Screening-positive samples are subsequently analyzed in a confirmation assay in absence and presence of the drug. Sufficient ADA signal inhibition by the drug verifies a sample as truly positive. Finally, confirmed positive samples are analyzed in the semi-quantitative titration assay where the Log2 titer of a sample is determined from a 2-fold serial dilution.

For positive samples, further characterization of the isotype and the neutralizing activity of the ADA might be performed.

Need more information about our Immunogenicity Testing Services?


Immunogenicity Testing – Downloads

Application note: How to perform immunogenicity testing

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Dr. Doreen Weigelt