Impact of the standardization unit’s definition on the in vitro biological potency of allergen extracts

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(AU) for mite allergen extracts, with a unique definition for that unit [4]. In Europe, every manufacturer develops its own standardization system, with its own unit definition [3]. It is possible for two or more European companies to use the same unit name with yet different definitions, as is in fact the case for the index of reactivity (IR), used by both ALK-Abelló in France and Stallergenes Greer worldwide (which, to our knowledge, is however a unique case in Europe). According to Stallergenes Greer's definition, 100 IR/mL indeed corresponds to a concentration that induces a mean wheal diameter of 7 mm when applied by skin prick testing to allergic patients, whereas according to ALK-Abelló's definition, it corresponds to a mean wheal diameter of 6 mm [5]. It is, however, tempting to consider that products from different companies but with the same labeling, e.g., 300 IR/mL, display the same biological potency. Hence it is important to determine whether such temptation is founded or not.
In such a context, this study aimed to characterize house dust mite (HDM) Staloral 300 IR/mL and HDM Osiris 300 IR/mL in terms of biological potency, but also in terms of concentrations of major HDM allergens, namely group 1, group 2, and group 23 allergens [6], as well as of protein content, protein profiles, and allergen profiles. As a reminder, the maintenance dose recommended for both products is 1 mL daily.
Total allergenic activity (TAA) was assayed based on the inhibition of IgE binding in enzyme-linked immunosorbent assay (ELISA), in comparison to an in-house reference extract, as described elsewhere [7]. Protein content was determined using the Kjeldahl method [8]. Der p 1 (EPC-DP1-25), Der f 1 (EPC-DF1-25), and Der p 23 (EPC-DP23-25) concentrations were obtained using commercially available ELISA kits (Indoor Biotechnologies, Charlottesville, VA, USA) essentially according to the manufacturer's instructions. The concentration of group 2 allergens (Der p 2 + Der f 2) was determined using mass spectrometry (MS), as previously described [9]. The protein profiles were obtained using non-reducing sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE) followed by SYPRO Ruby staining, and the allergen profiles were obtained by both western blotting and liquid chromatography coupled to tandem MS (LC-MS/MS). LC-MS/MS was also used to detect non-mite proteins in the extracts. Further details on the methods used are provided in Supplementary materials and Table S1.

Results
To check the suitability of the methods used for measuring TAA, as well as group 1 and group 2 allergen concentrations, those methods were applied to two batches of Orylmyte 300 IR (Stallergenes Greer), as compared to two batches of Acarizax 12 SQ-HDM (ALK-Abelló). As shown by Table 1, the resulting ratios between the two products were of the same order of magnitude as the ratios deduced from the literature [10], so that the methods used can be considered quite suitable for the purpose of this study.
Although the labeling of HDM Staloral and Osiris is the same, namely 300 IR/mL, the measured TAA was 2.4 times higher in HDM Staloral than in HDM Osiris (Table S2 and Figure 1A), when compared using the same analytical method. The higher biological potency of HDM Staloral 300 IR/mL was paralleled by higher allergen and protein contents, namely 1.5 time more Der p 1 and Der f 1, 3.0 times more group 2 allergens, 2.7 times more Der p 23, and 1.8 more time proteins (Table S2 and Figure 1B-F), again, when using the same analytical methods. Of note, Der p 1 and Der p 23 are especially important, since IgE specific to those allergens are associated with asthma [6,12,13]. Besides, HDM Staloral 300 IR/mL displays a slightly better, namely a 1.3-time higher specific activity, defined as (TAA/protein content) × 1,000 (Table S2).     . Culture medium-derived proteins found in one or more batches of HDM Staloral and Osiris 300 IR/mL farinae group 7, group 10, group 25, group 32 allergens, as well as Der p 14, were found in one or more batches of HDM Osiris but not in HDM Staloral (Figure 3). Almost all the allergens found in one product and not the other were identified with rather low identification scores (Figure 3). It is worth stressing that not all the allergens officially recognized by the World Health Organization (WHO)/International Union of Immunological Societies (IUIS; see www.allergen.org) were detected by LC-MS/MS, and/or detected in all tested batches, especially Der p 23, which was yet quantifiable by ELISA (Table S2). This can be explained by the detectability of peptides in complex samples being especially sensitive to their physicochemical properties, including their sequence specificities and their abilities to be ionized, which are highly peptide-dependent [14].
The LC-MS/MS analysis also allowed to identify proteins originating from the culture medium, namely from the wheat Triticum aestivum and the yeast Saccharomyces cerevisiae (Figure 4), which are commonly used for growing HDMs [15]. Over five times more culture medium-derived proteins were found in HDM Osiris 300 IR/mL than in HDM Staloral 300 IR/mL (Figure 4). This suggests a more thorough consumption of the culture medium by the mites in the case of HDM Staloral than in the case of HDM Osiris. Furthermore, this might explain why the specific activity of HDM Staloral is slightly higher than that of HDM Osiris (Table S2), although it is not very likely that the proteins found specifically in HDM Osiris be present in critical amounts in that product, given the similarity of its protein profile with that of HDM Staloral ( Figure 1A). Importantly, no allergen could be identified among the detected wheat and yeast proteins.

Conclusions
Qualitatively speaking, i.e. in terms of protein and allergen profiles, and save for the number of culture medium-derived proteins (Figure 4), HDM Staloral and Osiris 300 IR/mL look similar (Figures 2 and 3). On the other hand, the two products do not display the same biological potency in vitro (Table S2 and Figure 1). This was entirely expected since their labelings in IR/mL rely upon two different definitions of IR. On this basis, and because the biological potency correlates more or less with the concentrations of major allergens [3], the fact that HDM Staloral and Osiris 300 IR/mL contained differing amounts of these (Table S2 and Figure 1) was also expected.
This study partly involved in-house methods, which might be seen as a possible limitation. This limitation was overcome by benchmarking some of the used methods against independently published data ( Table 1).
In a nutshell, HDM Staloral 300 IR/mL and HDM Osiris 300 IR/mL are qualitatively similar products, but they differ quantitatively in terms of biological potency and levels of major allergens.

Supplementary materials
The supplementary material for this article is available at: https://www.explorationpub.com/uploads/ Article/file/100912_sup_1.pdf.

Conflicts of interest
All authors are employees of Stallergenes Greer, Antony, France.

Ethical approval
Individual sera constituting the pool, used as a reagent, come from blood donation at the French Blood Establishment (EFS) holding an Ethics and Professional conduct committee as per the decision n° 2019-05.