Molecular Fingerprinting of Complex Allergoids
Sunday, March 6, 2016
South Exhibit Hall H (Convention Center)
Matthew Heath, Andrew Bell, Murray Skinner, Alan D. Bullimore
Rationale: Targeted reduction in IgE reactivity of native allergen extracts to produce allergoids via covalent cross-linking is beneficial in producing safe and efficacious immunotherapies. We present techniques to demonstrate the presence of the relevant allergens in allergoid preparations by tandem mass spectrometry and the molecular fingerprint of those allergoids  by high performance-size exclusion chromatography (HPLC-SEC).

Methods: The polymerization profile of sweet grass allergoids was determined by HPLC-SEC, from which separate size fractions of allergoids were collected. Proteomic analysis of each corresponding fraction was purified, subject to tryptic digest and analysed via tandem mass spectrometry. Once the peptide had been identified, it was compared to protein databases such as NCBI or SwissProt, from which the sequence identity was assessed.

Results: HPLC-SEC highlighted the spread of allergens/allergoids pre- and post-modification. The HPLC profiles of the allergoids showed a decrease in retention time (increase in molecular weight) after modification (i.e. polymerization). A greater number of allergens are identified from tandem mass spectrometry (proteomic) analysis as the predicted molecular weight range of each fraction decreases.

Conclusions: Native and modified extracts are not two discrete preparations but are instead a formula of native and modified allergens, within which IgG reactive epitopes are present. Proteomic analysis confirmed the presence of allergens from multiple grass species. This work demonstrates that IgG epitopes remain in an allergoid formulation while IgE epitopes are attenuated, allowing safe administration of a higher strength product in fewer doses.