Characterization of the Candidate Reference Material of Timothy Pollen Allergen Extract in Terms of Protein Profile and Specific Allergenic Components

Abstract

Medicinal products based on pollen allergen extracts are widely used for diagnosis and therapy of pollinosis. According to the requirements of the State Pharmacopoeia of the Russian Federation, 13th ed., the allergenic potency of such products is assessed against certified reference materials whose biological activity was confirmed in vivo in subjects sensitized to particular tested allergens. The finished dosage form of a candidate In-House Reference Material (IHRM) of timothy pollen allergen extract and the corresponding allergen pollen extract (intermediate) were characterized in terms of protein profile and allergenic composition using SDS-PAGE, gel- filtration HPLC, and western blotting. The test samples were compared to finished pharmaceutical products (FPP) and pollen allergen extracts produced at different dates and made from pollen harvested at different dates, as well as to the WHO International Standard (IS) of Timothy Pollen Extract (NIBSC code: 82/520). SDS- PAGE and gelfiltration HPLC showed that the candidate IHRM protein profile was comparable in terms of major protein fractions to those of all the FPP batches and pollen allergen extracts produced at different dates. HPLC confirmed the comparability of the protein profiles of the candidate IHRM and the IS (NIBSC code: 82/520), but showed minor variations in the ratio of the main protein fractions. Western blotting confirmed the presence of the main allergenic components with relative molecular masses ranging from 50 to 60 kDa and from 27 to 35 kDa. The composition of specific allergenic components of timothy pollen allergen products manufactured by JSC «SIC «Microgen» was identical with that of the WHO IS of Timothy Pollen Extract in terms of the main protein and allergenic components.