Allergen characteristics, quality, major allergen content and galenics for mite allergen-specific immunotherapy preparations

House dust mite extracts for allergen-specific immunotherapy (AIT) require in Germany as other common allergens (pollen from sweet grasses [except maize], birch, alder, hazel; bee and wasp venom) marketing authorisation according to the German Therapy Allergen Ordinance (“Therapieallergene-Verordnung”, [TAV]). Mite allergen extracts that have been approved and also those which are in the approval process are subject to government batch testing. Batch test passing is a prerequisite for marketability. Appropriate quality, efficacy, and safety are prerequisites for the approval of house dust mite extracts. Five HDM allergen extracts from four manufacturers are currently approved in Germany for subcutaneous or sublingual therapy; further extracts are in the approval process. The allergen strength of different products is not comparable; manufacturers use company-specific units to describe the strength. Of the three known major allergens (Group 1 allergens: Der p 1, Der f 1, Group 2 allergens: Der p 2, Der f 2 and Group 23: Der p 23, Der f 23) only Group 1 and Group 2 allergens are usually used to standardize the extracts. Group 23 allergens are localized in the outer membrane of mite faeces, and elution requires special extraction methods. To be efficacious in a single patient an allergen extracts used for AIT must contain all allergen components against which the patient is sensitised. Based on post hoc analyses of large clinical studies, it has been proven for house dust mite tablets that Der p 23 is also contained. In Germany, the Paul Ehrlich Institute is responsible for the approval of therapeutic allergens. For the marketing authorisation information on production procedures, and quality are necessary efficacy and safety (positive benefit risk ratio) of the product must be demonstrated in clinical trials according to the current state of the art.

dust mite tablets that Der p 23 is also contained. In Germany, the Paul Ehrlich Institute is responsible for the approval of therapeutic allergens. For the marketing authorisation information on production procedures, and quality are necessary efficacy and safety (positive benefit risk ratio) of the product must be demonstrated in clinical trials according to the current state of the art.

AIT
Allergen-specific immunotherapy EMA European Medicines Agency IgE Immunoglobulin E PEI Paul Ehrlich Institute TAV Therapy allergen ordinance

Raw material
Different raw materials can be used for the production of house dust mite extracts. On the one hand, purified mite bodies are used and, on the other hand, mite culture material in which, in addition to house dust mites in various stages of development (from egg to adult mite), faeces particles and components of the culture medium are also found. By using sieves with different mesh sizes, particles can be separated depending on their diameter. Protein and allergen profile depend on the raw material; group 2 allergens dominate in extracts from purified mite bodies, while group 1 allergens dominate in mite faeces. The allergen composition of the raw material is influenced by the culture medium on which the house dust mites were grown [1]. Also, the concentration of allergens varies in mites at different stages of development [2].
Furthermore, bacteria and fungi are also found in and on mites, even though little is known to date about the microbiome of mites. Thus, proteins from bacteria and fungi are also to be expected in mite extracts.

Protein concentration
The protein concentration of house dust mite extracts can be measured by different methods. For batch consistency, the protein concentration of an extract must be in the range of 50-150% of the manufacturer's specification [3].

Allergen profile
From a regulatory point of view, extract quality can first be defined by the fact that extracts must be characterized and standardized and, in particular, that different batches must have comparable allergenic potency (allergenic strength) and a homogeneous allergenic profile. For batch consistency, the concentration of investigated major allergens of an extract must be in the range of 50-200% of the specification given by the manufacturer [3].
From a clinical point of view, allergen quality includes clinical efficacy and safety in the treatment of allergic patients. For efficacy, it is essential that a therapeutic extract contains all individually relevant allergens in a sufficient dose.

Regulatory view
In Europe, allergen products have been regulated by European law since 1989. From a regulatory point of view, the EMA has published in its Guideline on Allergen Products Production and Quality of 20 November 2008, essential comments on the quality of allergen extracts [4]. In addition to the description of the raw material and the culture medium, production processes including methods of extraction, filtration, dialysis, concentration and freeze drying have to be described and validated in detail.
As house dust mites are cultured by different manufacturers, it is necessary to have the exact specification of the raw material, with a description of the mite species (morphology, e.g. biochemical or genetic properties), cultivation conditions and especially also information about the culture medium used to cultivate the mites [5]. The allergenicity of the components of the culture medium should be as low as possible to prevent nondust-mite-specific reactions to the aller-gen extract. In particular, components from animal materials may not be used.
The qualitative and quantitative composition of a house dust mite extract, which is a complex mixture of allergens and nonallergenic substances, depends on the extraction method but also on the raw material used, which in turn is influenced by the culture conditions of the mites.
The allergenic potency of the allergen extract can be investigated using competitive IgE binding assays, with the potency of extracts expressed in manufacturer-specific units. In addition to total allergen activity, relevant allergens must be quantified by immunological methods. If standardization programs for the quantification of individual allergens are available, these should be applied. The allergen strength of chemically modified allergens (allergoids) cannot be determined by IgE binding tests in the end product.
The EMA provides guidance on the establishment and characterization of reference materials in terms of in-house materials with which the respective end product is compared. Pool serum, which is necessary for the batch control studies, should consist of 10-15 individual sera from well-characterized patients. These assays are particularly useful for consistency of the extracts. The extract strength can be tested by reactivity in the prick test on house dust mite allergic patients. The finished product must be described in detail; the complete list of all active substances must be submitted. The stability of extracts must also be checked; here, in particular, the storage capability is analyzed.
The total allergenic potency of the final product, as well as the protein concentration, must be in the range of 50 to 150% of the given specification. At the same time, the range to be observed for individual allergens is somewhat larger at 50-200% [3]. This means that it is difficult for the manufacturer to adjust the total allergic potency of the final product while maintaining the specifications for individual allergens.
A list of the house dust mite therapy allergens currently listed in Germany on the website of the Paul Ehrlich Institute (PEI) can be found in Table 1. Here, the currently approved house dust mite extracts are listed alongside the extracts that are currently in the approval process. Also listed are the manufacturerspecific units mentioned here to describe the strength of the allergen extracts.
No comparable data on the exact composition of the therapeutic extracts on the market can be found in the literature. Five sublingual house dust mite extracts available in Spain were studied in 2015 with respect to group 1 and group 2 allergen concentrations [6]. Der p 1 concentrations in the preparations ranged from 0.6-14.5 µg/ml, Der f 1 ranged from 0.2-12.4 µg/ml, and group 2 allergens ranged from 0.2-1.5 µg/ml which is likely to result in a wide variation in the clinical effectiveness of therapy with these extracts. The administered amount of other important allergens such as Der p 23 cannot be determined based on the published studies. The authors criticize that the clinical effectiveness of the various preparations has been insufficiently studied. When four house dust mite extracts available in Korea were examined [7], the protein concentration of the extracts ranged from 404-1057 µg/ml, and the Der f 1 concentration ranged from 3.59-40,688 µg/ml. The allergenic potency was also investigated, which ranged from 37,038-60,491 PAU/ml.

Clinical data
In Germany, only the two house dust mite tablets for sublingual immunotherapy on the market have undergone a complete study program that meets the current requirements for the approval of therapeutic allergens.
The first tablet to be approved is a rapidly dissolving lyophilized tablet made from extracts of both house dust mites (Dermatophagoides pteronyssinus and Dermatophagoides farinae). Production ensures that the four major allergens (Der p 1 and 2; Der f 1 and 2) are contained in equal proportions [8]. Dose-finding studies were conducted in children and adults, and the investigational product was tested for safety at doses ranging from 0.5-32 DU [9]. Serious adverse effects were not observed; the preparation in the dosage of 12 DU was favored for further development. Several efficacy studies have demonstrated the clinical effectiveness of the tablet in patients with allergic rhinoconjunctivitis [10][11][12] and also allergic asthma [13]. In Germany, this tablet is regularly approved for patients aged 12 to 65 years (https:// s3.eu-central-1.amazonaws.com/prod-cerebro-ifap/ media_all/101695.pdf).
The second tablet is a pressed formulation, also contains allergens from Dermatophagoides pteronyssinus and Dermatophagoides farinae. In the course of dose finding, patients were treated with different allergen doses (100, 300 and 500 index of reactivity [IR]), efficacy was tested by exposure in an exposure chamber [14]. The clinical efficacy of the favored dosage of 300 IR was confirmed in a large phase 3 study [15]. In further efficacy studies, it was shown that no further increase in efficacy could be achieved by increasing the allergen dose [16]. The tablet is approved in Germany for adolescents (12-17 years) and adults (https://www.stallergenesgreer.de/sites/default/files/ documents/1714_Fachinfo_Orylmyte_11.2021.pdf).
Both tablets contain group 1 and group 2 allergens of Dermatophagoides pteronyssinus and farinae. By means of a post hoc analysis it could be proven for the first approved lyophilisate tablet that also patients with a sensitization against Der p 23 benefit from the treatment and that IgG-4 antibodies against Der p 23 are induced by the therapy [17]. Also for the pressed tablet it was shown that the therapy induces specific IgG-4 antibodies against Der p 1 and Der p 2 as well as specific IgG-4 antibodies against Der p 23, which also prove a therapeutic amount of the allergen in this tablet [18].
For house dust mite extracts for subcutaneous immunotherapy, the study situation is not as good; dose finding with clinical endpoint has been published for a native allergen extract, but the target parameter was the effect on lung function [19]. For another one, dose finding was based on skin tests [20].

mini-review Conclusion
House dust mite extracts contain allergens as well as nonallergenic material, and the composition of extracts from different manufacturers is heterogeneous. The allergen composition depends on the raw material and extraction methods.
For allergen-specific immunotherapy, extracts are currently available for subcutaneous and sublingual immunotherapy. In Germany, house dust mite extracts are subject to the German Therapy Allergen Ordinance ("Therapieallergene-Verordnung") and must be approved by the Paul Ehrlich Institute. Currently, three preparations for subcutaneous and two preparations for sublingual therapy are approved in Germany. For further preparations, studies required for approval are in progress; they are on the market as marketable preparations and are subject to batch control.
To prove the quality of allergen extracts here of house dust mite extracts, extensive data on the quality are demanded from the manufacturers, the range of variation for the allergen strength is specified and, if allergen components are determined, the ranges in which the concentration of these components must lie. Clinical studies are necessary for dose finding (phase 2 trials) and in the sense of large phase 3 studies for proof of efficacy and safety. For older preparations, even if they are approved, no such extensive studies are available.
The user can hardly detect quality differences between different mite allergen extracts, and a comparison of the results of published phase 3 studies is not possible due to different patient collectives and target parameters.
Funding Open Access funding enabled and organized by Projekt DEAL.
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