The European Union Biological Standardization Program: Detailed Characterization Of Gmp-Produced Recombinant Bet V 1.0101 And Phi P 5.0109 As Candidate Reference Standards

RATIONALE: Standardization of allergen extracts requires availability of well-characterized recombinant allergens used as reference standards provided by the European regulatory authorities. The objective of this study was the detailed physicochemical and immunological characterization of rBet v 1.0101 and rPhl p 5.0109, which shall be used in a ring trial within the framework of the Biological Standardization Program BSP090 of the European Directorate for Quality of Medicines and Healthcare (EDQM). METHODS: Recombinant Bet v 1.0101 and Phl p 5.0109 were produced under GMP conditions and analyzed by an array of physicochemical and immunological methods for identity, quantity, homogeneity, folding and denaturation, aggregation state and stability in solution, alum-adsorbed suspension, and lyophilized state, as well as for biological activity. RESULTS: Using mass spectrometry both preparations were shown to contain rBet v 1.0101 and rPhl p 5.0109, respectively. SDS-PAGE, isoelectric focussing, deamidation analysis, and size-exclusion chromatography with light scattering revealed sample homogeneity of >99.9%. Upon storage at +4°C they retained monomeric state up to three months. Proteins were quantified by amino acid analysis. Folding studies based on Fourier Transform-infrared spectroscopy revealed similar secondary structure and aggregation behavior in solution, alum-adsorbed suspension, and lyophilized state. Biological activity of both proteins was comparable to their natural counterparts. CONCLUSIONS: Recombinant Bet v 1.0101 and Phl p 5.0109 were characterized extensively by physicochemical and immunological methods. They were shown highly stable, monomeric, and immunologically equivalent to their natural counterparts. Thus, they represent appropriate candidate reference standards for allergen products of birch and timothy grass.