Characterization of Depigmented-Polymerized Pollen Extracts for Allergen Immunotherapy: Presence of Relevant Allergens and Molecular Size Consistency

Chemically modified allergens (allergoids) are extensively used for allergen immunotherapy since they have a reduced allergenicity respect to native extracts while maintaining their immunogenicity. However, modification makes characterization of these molecules complicated, requiring specific techniques. The objective was to characterize different depigmented-polymerized pollen extracts. These allergoids have been previously purified (depigmentation process) in order to eliminate allergologically irrelevant low-molecular weight components. Depigmented-polymerized extracts of birch (Betula alba), olive tree (Olea euopaea), grass (Phleum pratense) and pellitory wall (Parietaria judaica) pollens were manufactured from native extracts. The presence of the relevant allergens in the modified molecule was determined by mass spectrometry and the profile of polymerization of the allergoids was determined by high performance-size exclusion chromatography (HPSEC) using a Bio SEC-3 Column (Agilent) in a HPLC system. Peptide sequencing confirmed the presence of the relevant allergens and their isoforms in the allergoids. Thus, allergens of groups 1, 2, 6 and 7 were detected in B. alba, groups 1, 2, 4, 5, 6, 7, 11, 12 and 13 in P. pratense; groups 1, 3, 6, 8, 9 and 10 in O. europaea; and groups 1, 2 and 4 in P. judaica. The HPLC profiles of the allergoids showed high consistency between batches with a decrease in retention time (increase in molecular weight) after polymerization. The depigmented-polymerized allergen extracts of different pollens have been characterized, demonstrating the presence of the relevant allergens and the consistency batch-to-batch in the molecular size of the modified molecule.