Immunogenicity Assay Development 

Anti-drug antibodies (ADAs) may occur as an organism's response to a biotherapeutic (e.g. small oligonucleotides or antibodies) during treatment, a process also known as unwanted immunogenicity. ADAs harbor the risk of mediating unwanted biological or physiological effects, either by inhibiting the therapeutic effect of the treatment itself or by provoking adverse events.

A Multi-Step Approach for Immunogenicity Assessment

The immunogenicity assessment of a therapeutic drug is based on a three-tiered approach and involves the use of anti-drug antibodies or anti-idiotypic antibodies as positive assay control. Since the thorough examination of the occurrence of ADAs in patient samples is particularly challenging, a multi-step approach is currently recommended by regulatory authorities. This multi-step approach is offered by BioGenes together with FyoniBio. Assay development and validation are conducted according to the FDA & EMA guidelines.

BioGenes offers the development of an immunogenicity ELISA for screening of anti-drug antibodies in serum samples by using a soluble bridging ELISA. In this set-up the ADA establishes a molecular bridge between the labeled drug antibodies. For assay development and as a comparative positive control, polyclonal anti-IDs specific for the drug antibody are used. 

Screening-positive samples are subsequently analyzed in a competitive confirmation assay using the therapeutic drug antibody as competitor. 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.

Development Strategy

Each project is conducted in close cooperation with the customer and consists of different work phases.

Phase 1 - Expert consultation

  • Definition of the project´s aim
  • Thorough discussion of antigen characteristics and project requirements

Phase 2 - Immunization and development of polyclonal antibodies

  • Quality control of the antigen via SDS-PAGE
  • Pre-testing of pre-immune sera for selection of suitable animals 
  • Immunization of 5/10 rabbits according to an immunization protocol over 105 days and ELISA titer analysis

Phase 3 - Affinity purification

  • Antibody purification via affinity column chromatography for isolation of specific anti-IDs including depletion of anti-human IgGs
  • Quality control of the purified antibodies using ELISA

Phase 4 - Development and optimization of the bridging ELISA in solution

  • Production of drug substance FITC and Biotin conjugates
  • Spiking experiments to assess the suitability of the ELISA method

Phase 5 - Antibody & method transfer

  • Transfer of the antibodies or the assay method to FyoniBio or the customer’s preferred CRO 
  • Opt. consultation with hands-on training 

Approximate project duration: 25-30 weeks

Pooled Scientific Expertise

To provide you with an all-inclusive service, BioGenes works closely together with FyoniBio. By combining our scientific expertise, we can offer you an all-in-one package ranging from custom antibody and assay development to ELISA validation and the analysis of pre-clinical and clinical samples according to GCLP guidelines. However, customers can also choose to independently obtain the individual services from either company alone.



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