title: Structural analysis of the recombinant therapeutic product rFVIII and its PEGylated variants using 2-D DIGE.
abstract: 2-D DIGE is a method that circumvents the gel-to-gel variability inherent in conventional 2-DE and is particularly useful for studying proteome changes in diverse applications such as developmental biology and tissue proteomics. We developed a 2-D DIGE protocol for recombinant factor VIII (rFVIII), a therapeutic protein used for the treatment of hemophilia A. The factor VIII heterodimer is composed of heterogeneous, heavily glycosylated heavy and light chains that are held together by a divalent cation. 2-DE of rFVIII led to a separation of the various fragments whose identity could be determined by Western blot. A comparison of two rFVIII batches by 2-D DIGE revealed their identical composition, whereas an rFVIII variant lacking its central B domain was congruent with the smallest heavy and light chain fragments of rFVIII only. A simpler pattern was obtained upon removal of the terminal sialic acids of rFVIII's glycans, due to a better focusing in the first dimension. 2-D DIGE was also well suited to structurally evaluate various PEGylated rFVIII conjugates. 2-D DIGE thus proved an excellent and straightforward method for structural analysis of rFVIII. Our data suggest that the method could serve as a tool for quality control of very complex pharmaceutically active ingredients.
authors.affiliation: Baxter Innovations GmbH, Vienna, Austria.
authors.forename: Christina
authors.initials: C
authors.lastname: Monetti
authors.forename: Hanspeter
authors.initials: H
authors.lastname: Rottensteiner
authors.forename: Herbert
authors.initials: H
authors.lastname: Gritsch
authors.forename: Alfred
authors.initials: A
authors.lastname: Weber
authors.forename: Friedrich
authors.initials: F
authors.lastname: Scheiflinger
authors.forename: Peter L
authors.initials: PL
authors.lastname: Turecek
mesh_terms: D000818:Animals; D015153:Blotting, Western; D015180:Electrophoresis, Gel, Two-Dimensional / Q000379:methods*; D004591:Electrophoresis, Polyacrylamide Gel; D005169:Factor VIII / Q000737:chemistry* / Q000235:genetics; D006801:Humans; D007525:Isoelectric Focusing; D051379:Mice; D010446:Peptide Fragments / Q000737:chemistry* / Q000235:genetics; D011092:Polyethylene Glycols / Q000737:chemistry*; D021122:Protein Subunits; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics; D012756:Sheep; D012794:Sialic Acids
chemical_list: C451034:B-domain-deleted factor VIII; D010446:Peptide Fragments; D021122:Protein Subunits; D011994:Recombinant Proteins; D012794:Sialic Acids; D011092:Polyethylene Glycols; D005169:Factor VIII