Allergic diseases affect about one third of the world’s population and are increasing in prevalence, particularly among children. Many patients receive allergen immunotherapy (also known as desensitization or hypo-sensitization, typically administered by subcutaneous injection.
In more recent sublingual immunotherapy (SLIT) approaches, allergens are provided as tablets that are placed underneath the tongue. The allergen is then absorbed through the oral mucosa, a natural site of immune tolerance. In 2013, the World Allergy Organisation (WAO) emphasized the benefits of sublingual immunotherapy (read more). Especially in children, SLIT allergy treatment has been shown to stop progression from rhinitis to asthma, with long-lasting effects beyond treatment time (read more).
Currently marketed SLIT tablets contain mixtures of natural extracts, with varying allergen concentrations and compositions. To better standardize dosage, recombinant allergens are being developed. For example, recombinant birch allergens have been shown to possess equivalent properties to natural allergen extracts (read more).
However, to meet pharmaceutical production standards, recombinant allergens need to comply with stringent quality control, which includes delivering information on isoforms, secondary structure, thermal stability, and functional activity. Mass spectrometry emerged as a powerful method for precise characterization of recombinant allergens (read more).
More than 50% of allergic disease cases are attributed to house dust mite (HDMs), and more than 80% of children with asthma are allergic to mites. Bordas-Le Floch and colleagues conducted a study to evaluate whether the recombinant form of one of the major allergens of HDM, the 14 kDa Der p2 protein, could replace natural extracts. Recombinant Der p2 protein was produced in two hosts: the yeast Pichia pastoris and E.coli. Yields, purity, and endotoxin content were determined to be similar for both recombinant allergens and the natural extract. Activity measurements including antigen binding activity, basophil and T-cell activation, and efficacy in mouse sublingual immunotherapy did not reveal any significant differences between the natural and the two recombinant proteins.
However, when circular dichroism spectroscopy was applied to determine secondary structure and thermal stability, Der p2 produced in E.coli showed major deviations in secondary structure and impaired thermal stability compared to the natural and the yeast recombinant allergens.
To analyze these variations in more detail, all three preparations were analyzed using liquid chromatography electrospray ionization quadrupole time-of-flight detection mass spectrometry (LC-ESI-QTOF MS). In a top down MS approach, the precise masses of four isoforms in the natural extract and one isoform in the recombinant preparations were confirmed.
In a second step, cysteine pairing was analyzed in LC-tandem MS bottom up experiments after fragmentation by alkylation and trypsin digests. While the natural and yeast recombinant proteins showed very similar peak patterns, incorrect pairing of several cysteines was detected in the allergen obtained from E.coli expression. These deviations were likely responsible for the altered secondary structure and thermal instability. Results were confirmed in mass spectrometry-based quality control experiments of three independent production batches.
Based on MS data, it was possible to give a clear recommendation to use the yeast Pichia pastoris as a host for future preparations of recombinant Der p2. Documentation of correct cysteine pairing by MS was determined to be an indispensable parameter in quality control procedures of recombinant allergens.
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