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Electrophoretic analyses of clotting factor VIII concentrates

Authors
  • Pock, Katharina
  • Jungbauer, Alois
  • Kannicht, Christoph
  • Löster, Klemens
  • Buchacher, Andrea
  • Josic, Djuro
Type
Published Article
Journal
Analytica Chimica Acta
Publisher
Elsevier
Publication Date
Jan 01, 1998
Volume
372
Issue
1
Pages
219–229
Identifiers
DOI: 10.1016/S0003-2670(98)00348-1
Source
Elsevier
Keywords
License
Unknown

Abstract

Preparations of clotting factor VIII (FVIII) are complex protein mixtures, regardless of whether they are made by genetically engineered cells or isolated from human plasma. Commercially available FVIII preparations contain either von Willebrand factor (vWF) or human serum albumin (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 bands and spots were identified by immunoblot using monoclonal and polyclonal antibodies. The aim was to detect every preparation which has an 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 combination of separations under non-reducing and increasingly reducing conditions cleavage products of FVIII can in some cases be detected in immunoblot. In 2D electrophoresis the detection of proteins with high molecular masses was achieved by applying a mixture of thiourea and urea. The spots in 2D-electrophoresis of plasma-derived FVIII preparations have been identified as FVIII and vWF, but also as impurities, chiefly from fibrinogen. It was shown that especially 2D-electrophoresis and immunoblot can be used for better characterization of FVIII concentrates. The 2D protein maps of FVIII concentrates will be an efficient tool for quality control of such products.

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