D3.216 - Determination of Allergenic Activity of Native Mugwort Allergen Preparations by a Competitive IgE-Binding Test

As a manufacturer of diagnostic and therapeutic allergen products, determining the allergenic activity of these preparations is an essential element for Allergopharma to ensure their efficacy, safety, and quality. For this purpose, the IgE potency assay, a state-of-the-art analytical procedure, was developed and validated according to ICH-Q2 for the allergen source mugwort. Challenges in developing the IgE potency assay for the allergen source mugwort included providing a qualified mugwort-specific serum pool and ensuring high sensitivity of the assay while maintaining good reproducibility.

In the competitive IgE binding assay, the reference is coupled to the MTP in a defined concentration. Dilution series of reference and sample are added as inhibitors to the MTP and incubated with a qualified allergen specific serum pool. Allergen in the liquid phase competes with solid phase-bound reference material for binding of the specific IgE in the serum pool. Solid-phase bound IgE is detected with a detection cascade of biotinylated anti-human IgE-mAb and a streptavidin-polyHRP-conjugate and quantified in a colorimetric manner using TMB as substrate. The colour signal is proportional to the amount of bound IgE and reversely proportional to the extent of inhibition by the sample or reference. The relative potency is calculated as the relation of the dilution factors at 50% inhibition of sample to reference (Prel_DF).

Assay-specific parameters such as dilution of serum, reference and sample were determined to include at least four data points within the pseudo-linear range of the inhibition curve. The assay was validated according to ICH-Q2 for different allergen preparations, i.e. allergen extract, Prick-Test solution and Provocation-Test solution. Within the defined working range, the assay showed a high degree of linearity (r= 0.999), good accuracy with recovery rates between 91-111%, as well as satisfying precision (CV < 16%), respectively. Suitable SSTs were established to monitor the assay performance. Comparability to the previously used RAST inhibition test was shown.

The developed IgE Potency assay mugwort is a GMP compliant, robust, precise and reproducible method to determine the specific IgE reactivity of native mugwort allergen preparations in quality control.