ELECTROPHORETIC ANALYSES OF CLOTTING FACTOR-VIII CONCENTRATES

Preparations of clotting factor VIII (FVIII) are complex protein mixtu res, regardless of whether they are made by genetically engineered cel ls or isolated from human plasma. Commercially available FVIII prepara tions contain either von Willebrand factor (vWF) or human serum albumi n (HSA), both of which function as stabilizers. Apart from these, the preparations contain other plasma proteins as impurities. Such protein mixtures were analyzed with SDS-PAGE and 2D-electrophoresis. Single b ands and spots were identified by immunoblot using monoclonal and poly clonal antibodies. The aim was to detect every preparation which has a n increased level of FVIII cleavage products. Chromatographic methods and SDS-PAGE under reducing and non-reducing conditions usually do not show any differences between single preparations. Only by a combinati on of separations under non-reducing and increasingly reducing conditi ons cleavage products of FVIII can in some cases be detected in immuno blot. In 2D electrophoresis the detection of proteins with high molecu lar masses was achieved by applying a mixture of thiourea and urea. Th e spots in 2D-electrophoresis of plasma-derived FVIII preparations hav e been identified as FVIII and vWF, but also as impurities, chiefly fr om fibrinogen. It was shown that especially 2D-electrophoresis and imm unoblot can be used for better characterization of FVIII concentrates. The 2D protein maps of FVIII concentrates will be an efficient tool f or quality control of such products. (C) 1998 Elsevier Science B.V. Al l rights reserved.