# Immunological In Vitro Assay for Quantification of Adjuvanted Allergoids

## TL;DR

A new in vitro assay was developed to measure allergoid content in adjuvanted allergy treatments, ensuring product consistency and quality.

## Contribution

The study introduces a novel fluorescence-based assay for quantifying allergoids in adjuvanted AIT products, enabling animal-free quality control.

## Key findings

- The assay demonstrated high specificity and low cross-reactivity with other allergen types.
- It successfully quantified allergoid content across four different grass pollen AIT drug products.
- The method detected product alterations caused by heat stress and confirmed batch consistency.

## Abstract

Many IgE‐mediated allergic disorders can be treated with allergen immunotherapy (AIT). In order to improve safety and efficacy, some AIT products contain allergen extracts which are chemically cross‐linked to generate allergoids and are adsorbed to aluminum hydroxide adjuvant. The modification and adsorption impair accessibility of the protein and quantification of the allergoid content.

An ELISA‐like assay to quantify the allergoid content in adjuvanted grass pollen allergoid AIT products (from here on called: AIT drug products; AIT‐DPs) was developed using a fluorescence detection system. The high density of the aluminum hydroxide particles enabled pelleting the antigen complexes by centrifugation. Rabbit anti‐grass pollen allergoid sera or a mouse anti‐Phl p 5 monoclonal antibody (mAb) was used as the primary antibody. Protein content of the samples was quantified by nitrogen analysis.

High specificity of the primary antibodies was confirmed by isoelectric focusing, gel‐electrophoresis, and immunoblotting. Performance of the allergoid content assay was demonstrated in grass pollen AIT‐DPs with high specificity and low/absent cross‐reactivity with tree pollen or mite AIT‐DPs. It was used to confirm batch‐to‐batch consistency and allergoid content of distinct grass pollen AIT‐DPs. Overall, in relation to their total protein content, the allergoid content ranged between 0.8 and 2.1 relative to an in‐house reference for all grass pollen AIT‐DPs, whereas use of mAb revealed product‐specific differences in the Phl p 5 amount. Additionally, the assay detects product alteration by heat stress.

The described assay is suitable to quantify the allergoid content and quality of allergoids in complex with aluminum hydroxide. It is suitable for animal‐free final product testing in vitro, for example, for batch release to ensure the quality of AIT‐DPs.

An assay was developed to measure the allergoid content in adjuvanted grass allergoid AIT drug product (AIT‐DPs) at the finished product level. Both polyclonal rabbit sera or a mouse anti‐Phl p 5 mAb can be used, allowing differential product analysis. All four tested grass pollen AIT‐DPs of different manufacturers could be quantified using a defined reference, demonstrating broad applicability of the assay. ACA, allergoid content assay; AIT, allergen immunotherapy; AIT‐DP, adjuvanted allergoid AIT drug product; Al(OH)3, aluminum hydroxide; FITC, fluorescein isothiocyanate; mAb, monoclonal antibody; Phl p 5, Group 5 allergen
Phleum pratense
; RAC, relative allergoid content.