Pollen allergen products: current standardisation issues

Abstract

Scientific relevance . Pollen allergen medicines are in high demand, and their therapeutic benefits directly correlate with their standardisation. Better diagnosis and treatment of allergic diseases require state-of-the-art procedures for assessing the allergenic activity of pollen allergen products using reference standards and physicochemical testing methods. Aim . The study aimed at developing methodological approaches to the standardisation of pollen allergen products in order to shift to measuring their potency in allergenic activity units (AAU) and bring their quality in line with the requirements of the State Pharmacopoeia of the Russian Federation. Materials and methods . The study used pollen allergen reference standards by Microgen, the WHO International Standard for timothy grass ( Phleum pratense ) pollen extract, a gel filtration standard kit of molecular weight markers ranging from 1.35 to 670 kDa, bovine serum albumin, serum samples with specific IgE obtained from donors sensitised to the study pollen allergens, labelled anti-human IgE antibodies, and reference standards for determining residual volatile solvents by gas chromatography. The identification of in-house reference standards for the potency of pollen allergens involved Western blotting (for allergenic components). The total protein content was determined by Bradford’s assay. In addition, the authors used high-performance liquid chromatography to study protein fractions and gas–liquid chromatography to determine the content of residual organic solvents. Results . To substitute the existing method of non-specific characterisation of allergenic activity in protein nitrogen units (PNU), the authors developed and tested a new method to control allergenic activity in allergenic activity units (AAU) based on an in vitro competitive enzyme-linked immunosorbent assay (ELISA) procedure developed and validated in this study. Furthermore, the authors developed and certified 15 primary in-house reference standards with allergenic activity established in AAU/mL using skin tests in vivo . The experimental data were analysed to standardise the allergenic activity of the pollen allergens manufactured by Microgen. The authors developed physicochemical methods for the certification of in-house reference standards and validated these methods in accordance with the State Pharmacopoeia of the Russian Federation. The study involved selecting chromatographic separation conditions for residual organic solvents (acetone and diethyl ether) and establishing system suitability criteria for the chromatographic system. The allergenic activity of secondary in-house reference standards was certified against that of primary in-house reference standards using competitive ELISA. Thus, the authors managed to shift to the standardisation of pollen allergen products in vitro . Conclusions . The authors developed their competitive ELISA-based method to standardise pollen allergen products by comparing the inhibition of immune responses to a product and a standard. The study demonstrated the feasibility of substituting allergenic activity quantification (in AAU) for protein nitrogen content determination (in PNU) and showed the first example of using AAU for the certification of in-house reference standards. Additionally, the authors developed and validated an analytical procedure for determining the content of residual organic solvents in pollen allergen products by gas–liquid chromatography.