The CREATE project: Development of certified reference materials for allergenic products and validation of methods for their quantification

Abstract

Standardization of allergen extracts for diagnosis and immunotherapy of allergies is based on biological standardization, i.e., IgE-binding potencies. Skin tests and competitive IgE-binding assays are important elements of allergen standardization, especially from a safety perspective. However, biological standardization does not provide specific information about the major allergen content of allergen vaccines, i.e., the content of the active ingredients needed for attaining efficacy of immunotherapy. Another disadvantage of the current system is that allergen manufacturers express potencies of their products in company??specific units that do not allow product comparison and this situation is not tenable as an international system of standardization. In the 1980s, the WHO/IUIS Allergen Standardization Subcommittee developed International Reference Preparations (IRP) of several extracts to facilitate product comparison. Unfortunately, these IRP standards were not adopted by the industrial or regulatory authorities. During the 1990s, most major respiratory allergens became available as recombinant molecules, and the dependence of effective immunotherapy on administration of defined quantities of major allergen had become well-accepted. That was the moment for the IUIS-WHO committee to revive the endeavor for setting up a system of allergen standardization that would allow comparison of products and at the same time give accurate information on the content of active ingredients of the major allergens. The initiative was supported by a grant from the European Union, and the CREATE project was born. From November 2001 to April 2005, a consortium of basic and clinical researchers, regulators, allergen manufacturers and biotech companies carried out the project that aimed at evaluating the potential of recombinant allergens to serve as future major allergen reference materials and the potential of available sandwich ELISAs for their accurate measurement. The approach was to produce purified recombinant allergens and compare these to their natural counterparts serving as a gold standard. Nine recombinant molecules representing 8 major allergens were produced: Rbet v 1, rPhl p 1, rPhl p 5a and rPhl p 5b, rOle e 1, rDer p 1, rDer p 2, rDer f 1 and rDer f 2. They were compared with purified natural allergens for physicochemical (identity, purity, folding, aggregation state, solubility and stability) and immunological (IgE-binding potency, biological activity and dose-response behavior in ELISA) characteristics. As part of these studies, panels of sera from allergic patients with seasonal or perennial rhinitis and/or asthma were collected from clinical centers in 8 countries for use in comparing IgE antibody responses and in 9.1 WHO/IUIS Allergen Standardization Initiatives assessments of biological potency. Overall, 150??350 sera with IgE antibodies to each of the allergens were obtained, comprising a bank of 961 sera. Three recombinant allergens displayed sufficient structural and immunological similarity, and biological potency, to their natural counterparts to be selected for a follow-up project that should ultimately lead to their establishment as international reference materials: Rbet v 1, rPhl p 5a and rDer p 2. In parallel with these studies, several ELISAs were evaluated for their measurement of major allergens. For most allergens, one or two ELISAs were identified, which showed comparable dose response curves for the recombinant and natural allergens (a requirement for measurement of major allergens in allergenic products, using a recombinant reference). This fulfilled an important aim of the CREATE project which was to develop purified allergen references together with complementary ELISA systems. A follow-up project has recently been initiated by the CREATE partnership and the European Directorate for the Quality of Medicines (EDQM). Together, three allergen manufacturers from CREATE provided EDQM with sufficient GMP-grade rBet v 1 and rPhl p 5a and with two suitable ELISA kits per allergen. After physico-chemical characterization of both allergens, these reagents will be used in a ring trial together with the selected ELISAs to validate their applicability in allergen standardization based on mass units of major allergen. One of the conclusions from CREATE is that certified recombinant references will have to be linked to certified assays so that products can be directly compared on the basis of mass units of major allergens. The rBet v 1 and rPhl p 5a allergens developed through CREATE will soon be established as international references and will become part of the European Pharmacopeia. The task for the future is to apply the approach used in CREATE to produce a repertoire of purified allergens that can be used as international references for standardization purposes and to harmonize allergen measurements worldwide.