title: In vitro characterization of recombinant factor VIII concentrates reveals significant differences in protein content, activity and thrombin activation profile.
abstract: Recombinant factor VIII (rFVIII) concentrates differ due to cell lines, culture conditions, presence of the B domain and authorized potency assays. This study characterizes three commercially available rFVIII concentrates: a second-generation full length (A), a third-generation full length (B) and a third-generation B domain-deleted (BDD) product (C). rFVIII concentrates were characterized for FVIII activity (FVIII:C) by one-stage clotting and chromogenic assays, FVIII antigen (FVIII:Ag), thrombin activation profile and FXa-generation assay. The rFVIII concentrates exhibited significant differences with regard to FVIII:C, FVIII:Ag and thrombin activation profile. Product A had significantly greater FVIII:C and FVIII:Ag relative to the measured values of products B and C. In addition, product A demonstrated faster and more complete activation by thrombin than the two others. BDD product C had the slowest measured thrombin activation rate. Product A exhibited a greater in vitro FXa generation than products B and C. We found no differences in FXa generation among all three products when FXa generation was normalized for FVIII:Ag. The greater FVIII:C and FVIII:Ag values for product A compared with that for products B and C are due to application of different authorized potency assays (one-stage assay for A vs. chromogenic assay for B and C). The variation in thrombin activation profiles may arise from differences in cell line-dependent posttranslational modifications of the various recombinant proteins.
authors.affiliation: Institute of Experimental Haematology and Transfusion Medicine, University Clinic Bonn, Bonn, Germany.
authors.forename: S
authors.initials: S
authors.lastname: Pahl
authors.forename: A
authors.initials: A
authors.lastname: Pavlova
authors.forename: J
authors.initials: J
authors.lastname: Driesen
authors.forename: J
authors.initials: J
authors.lastname: Müller
authors.forename: B
authors.initials: B
authors.lastname: Pötzsch
authors.forename: J
authors.initials: J
authors.lastname: Oldenburg
mesh_terms: D015153:Blotting, Western; D004591:Electrophoresis, Polyacrylamide Gel; D057075:Enzyme Assays; D004797:Enzyme-Linked Immunosorbent Assay; D005169:Factor VIII / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D015951:Factor Xa / Q000378:metabolism; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics; D013917:Thrombin / Q000378:metabolism*
chemical_list: D011994:Recombinant Proteins; D005169:Factor VIII; D013917:Thrombin; D015951:Factor Xa