D2.226 - Evaluation of the Immunogenicity and Stability of an Aluminum-Adsorbed Allergen Immunotherapy Using Specific IgG Titration in a Murine Model

Allergen-specific immunotherapies (AITs) formulated with aluminum-based adjuvants present significant limitations for direct allergen quantification and potency assessment due to adsorption of the allergen to the aluminum hydroxide matrix. This adsorption prevents the use of conventional immunoassays on the final product, hindering both routine evaluation and stability studies.

Therefore, alternative strategies to assess the immunogenic potential over time are highly relevant. The aim of this study was to evaluate the immunogenicity and stability of a molecular Cup a 1 immunotherapy adsorbed to aluminum by analyzing the humoral response induced in a murine model.

The molecular Cup a 1 AIT with aluminum hydroxide underwent a pre-wash procedure to remove unadsorbed proteins. NMRI mice were immunized by repeated subcutaneous administrations with the product at initial time and samples stored for three months. Controls included diluent-treated and untreated animals.

After the immunization period, sera were collected to evaluate the humoral response. Immunogenicity was assessed by specific IgG titration using ELISA. Titration curves were compared using a reference batch and acceptance criteria based on reproducibility, discrimination versus controls, and correlation between samples.

Immunization with the molecular Cup a 1 AIT induced a specific humoral response in immunized animals, clearly distinguishable from controls. Titration curves obtained from the product at initial time and from stored samples showed comparable profiles, supporting the use of this approach to monitor and evaluate the immunogenic stability of the product over time.

Comparison of initial-time values with those from samples stored for three months under different conditions revealed storage-dependent variations while maintaining a high correlation between titration curves, confirming the sensitivity of the technique to detect changes in the product’s immunogenic capacity.

The use of in vivo immunogenicity tests is an appropriate tool for monitoring and evaluating the stability of aluminun-adsorbed treatments over time, due to their ability to induce an immune response in mouse models. This approach overcomes the analytical limitations associated with direct quantification and potency determination, which are not feasible for adjuvanted products. This contributes to confirm the stability of the Cup a 1 molecular treatment in depot suspension.