Standardization of allergen products: 2. Detailed characterization of GMP-produced recombinant Phl p 5.0109 as European Pharmacopoeia reference standard.
BACKGROUND
The Biological Standardization Programme of the European Directorate for Quality of Medicines and Healthcare (EDQM) aims at the establishment of well-characterized reference standards based on recombinant allergens and validated assays for the quantification of major allergen content. The objective of this study was to examine the detailed physicochemical and immunological characterization of recombinant Phl p 5.0109, the second available allergen reference standard.


METHODS
Recombinant Phl p 5.0109 PP5ar06007 was produced under GMP conditions and analyzed by an array of physicochemical and immunological methods for identity, quantity, homogeneity, and folding stability in bulk solution, as well as thermal denaturation, aggregation state, and biological activity when formulated for long-time storage.


RESULTS
PP5ar06007 revealed as a highly homogeneous, monomeric, well-folded preparation of rPhl p 5.0109, as documented by mass spectrometry, SDS-PAGE, isoelectric focusing, size-exclusion chromatography with light scattering, circular dichroism, and infrared spectroscopy. Upon storage at +4°C, PP5ar06007 retained the monomeric state for at least 2 months. A protein quantity of 1.56 ± 0.03 mg/ml was determined by amino acid analysis in PP5ar06007, and its biological activity was shown to be comparable to natural Phl p 5 in terms of basophil activation and T-cell reactivity.


CONCLUSIONS
Recombinant Phl p 5.0109 PP5ar06007 was characterized extensively at the physicochemical and immunological level. It revealed to be a highly stable, monomeric, and immunologically equivalent of its natural counterpart. PP5ar06007 is now available as European Pharmacopoeia allergen reference standard for grass pollen products.
['Himly|M|M|', 'Nandy|A|A|', 'Kahlert|H|H|', 'Thilker|M|M|', 'Steiner|M|M|', 'Briza|P|P|', 'Neubauer|A|A|', 'Klysner|S|S|', 'van Ree|R|R|', 'Buchheit|K-H|KH|', 'Vieths|S|S|', 'Ferreira|F|F|']
D000485:Allergens D000595:Amino Acid Sequence D052179:Antigens, Plant D006801:Humans D008970:Molecular Weight D011489:Protein Denaturation D017510:Protein Folding D055550:Protein Stability D011994:Recombinant Proteins D012015:Reference Standards D013816:Thermodynamics
European Directorate for Quality of Medicines and Healthcare BSP090 Ph. Eur. reference standard good manufacturing practice recombinant Phl p 5 allergen