title: Characterization of factor VIII pharmaceutical preparations by means of MudPIT proteomic approach.
abstract: For a good clinical outcome of Haemophilia A substitutive therapy a detailed characterization of factor VIII (FVIII) concentrates is required, in order to disclose the eventual relations between differently composed concentrates and their biological effects. This preliminary work could be a first step towards a deep structural characterization of FVIII concentrates, using the fast and simply manageable MudPIT technology, which enables the identification and characterization of protein mixtures taking advantage of both the high separation capacity of two-dimensional chromatography and the powerful peptide characterization ability of tandem mass spectrometry. The aim of this study was to evaluate the suitability of for the characterization of FVIII molecule in complex mixtures such its commercial concentrates, both plasma-derived and recombinant, and for the determination of the protein composition of different FVIII preparations. By means of Multidimensional Protein Identification Technology (MudPIT) it was possible to assess the presence of factor VIII in its preparations and to identify most of the contaminant proteins without gel separation. In particular, 125 and 42 proteins were identified in plasma-derived and recombinant concentrates, respectively. Concerning investigation of FVIII, 24 different peptides were identified in plasma-derived corresponding to 7, 29, 27, 19 and 67 of percentage coverage for A1, A2, A3, C1 and C2 domains, respectively. About its multimeric carrier von Willebrand factor (VWF), we have sequenced 42% of domain interacting with A3 and C2 domains of FVIII. Finally, it has been observed that normalized parameters, such as total peptide hits obtained by SEQUEST may be used for evaluation of the relative abundance of FVIII in different preparations.
authors.affiliation: ITB/CNR, via Fratelli Cervi 93, 20090 Segrate (MI), Italy.
authors.forename: Fabrizio
authors.initials: F
authors.lastname: Basilico
authors.forename: Ilaria
authors.initials: I
authors.lastname: Nardini
authors.forename: Filippo
authors.initials: F
authors.lastname: Mori
authors.forename: Elena
authors.initials: E
authors.lastname: Brambilla
authors.forename: Louise
authors.initials: L
authors.lastname: Benazzi
authors.forename: Antonella
authors.initials: A
authors.lastname: De Palma
authors.forename: Enrico
authors.initials: E
authors.lastname: Rosti
authors.forename: Claudio
authors.initials: C
authors.lastname: Farina
authors.forename: PierLuigi
authors.initials: P
authors.lastname: Mauri
mesh_terms: D053801:Capillary Electrochromatography / Q000379:methods*; D004340:Drug Contamination / Q000517:prevention & control; D005169:Factor VIII / Q000032:analysis*; D010455:Peptides / Q000032:analysis; D004364:Pharmaceutical Preparations / Q000032:analysis*; D011506:Proteins / Q000032:analysis; D040901:Proteomics / Q000379:methods*; D011994:Recombinant Proteins / Q000032:analysis; D053719:Tandem Mass Spectrometry / Q000379:methods
chemical_list: D010455:Peptides; D004364:Pharmaceutical Preparations; D011506:Proteins; D011994:Recombinant Proteins; D005169:Factor VIII