title: Standardization of allergen products: 2. Detailed characterization of GMP-produced recombinant Phl p 5.0109 as European Pharmacopoeia reference standard.
abstract: 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.
authors.affiliation: Department of Molecular Biology, University of Salzburg, Salzburg, Austria.
authors.forename: M
authors.initials: M
authors.lastname: Himly
authors.affiliation: Research and Development, Allergopharma GmbH & Co. KG, Reinbek, Germany.
authors.forename: A
authors.initials: A
authors.lastname: Nandy
authors.affiliation: Research and Development, Allergopharma GmbH & Co. KG, Reinbek, Germany.
authors.forename: H
authors.initials: H
authors.lastname: Kahlert
authors.affiliation: Research and Development, Allergopharma GmbH & Co. KG, Reinbek, Germany.
authors.forename: M
authors.initials: M
authors.lastname: Thilker
authors.affiliation: Department of Molecular Biology, University of Salzburg, Salzburg, Austria.
authors.forename: M
authors.initials: M
authors.lastname: Steiner
authors.affiliation: Department of Molecular Biology, University of Salzburg, Salzburg, Austria.
authors.forename: P
authors.initials: P
authors.lastname: Briza
authors.affiliation: Biomay AG, Vienna, Austria.
authors.forename: A
authors.initials: A
authors.lastname: Neubauer
authors.affiliation: Research and Development, Allergopharma GmbH & Co. KG, Reinbek, Germany.
authors.forename: S
authors.initials: S
authors.lastname: Klysner
authors.affiliation: Academic Medical Center, Amsterdam, The Netherlands.
authors.forename: R
authors.initials: R
authors.lastname: van Ree
authors.affiliation: European Directorate for Quality of Medicines and Healthcare, Strasbourg, France.
authors.forename: K-H
authors.initials: KH
authors.lastname: Buchheit
authors.affiliation: Department of Allergology, Paul-Ehrlich-Institut, Langen, Germany.
authors.forename: S
authors.initials: S
authors.lastname: Vieths
authors.affiliation: Department of Molecular Biology, University of Salzburg, Salzburg, Austria.
authors.forename: F
authors.initials: F
authors.lastname: Ferreira
mesh_terms: D000485:Allergens / Q000008:administration & dosage / Q000737:chemistry / Q000276:immunology*; D000595:Amino Acid Sequence; D052179:Antigens, Plant / Q000008:administration & dosage / Q000737:chemistry / Q000276:immunology; D006801:Humans; D008970:Molecular Weight; D011489:Protein Denaturation; D017510:Protein Folding; D055550:Protein Stability; D011994:Recombinant Proteins / Q000008:administration & dosage / Q000737:chemistry / Q000276:immunology* / Q000592:standards*; D012015:Reference Standards; D013816:Thermodynamics
chemical_list: D000485:Allergens; D052179:Antigens, Plant; D011994:Recombinant Proteins
keywords: BSP090; European Directorate for Quality of Medicines and Healthcare; Ph. Eur. reference standard; good manufacturing practice; recombinant Phl p 5 allergen