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Recombinant clotting factor VIII concentrates: Heterogeneity and high-purity evaluation.
MedLine Citation:
PMID:  20737444     Owner:  NLM     Status:  In-Process    
Factor VIII is an important glycoprotein involved in hemostasis. Insertion of expression vectors containing either the full-length cDNA sequence of human factor VIII (FLrFVIII) or B-domain deleted (BDDrFVIII) into mammalian cell lines results in the production of recombinant factor VIII (rFVIII) for therapeutic usage. Three commercially available rFVIII concentrates (Advate, Helixate NexGen and Refacto), either FLrFVIII or BDDrFVIII, were investigated by 1- and 2-DE and MS. The objective of this study was to compare the heterogeneity and the high purity of both rFVIII preparations before and after thrombin digestion. In particular, the 2-D gel was optimized to better highlight the presence of contaminants and many unexpected proteins. Recombinant strategies consisting of insertion of expression vectors containing BDDrFVIII and FLrFVIII resulted in homogeneous and heterogeneous protein products, respectively, the latter consisting in a heterogeneous mixture of various B-domain-truncated forms of the molecule. Thrombin digestion of all the three rFVIII gave similar final products, plus one unexpected fragment of A2 domain missing 11 amino acids. Regarding the contaminants, Helixate NexGen showed the presence of impurities, such as Hsp70 kDa, haptoglobin and proapolipoprotein; Refacto showed glutathione S-transferase and beta-lactamase, whereas Advate apparently did not contain any contaminants. The proteomic approach will contribute to improving the quality assurance and manufacturing processes of rFVIII concentrates. In this view, the 2-DE is mandatory for revealing the presence of contaminants.
Gian Maria D'Amici; Anna Maria Timperio; Federica Gevi; Giuliano Grazzini; Lello Zolla
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Publication Detail:
Type:  Journal Article; Research Support, Non-U.S. Gov't    
Journal Detail:
Title:  Electrophoresis     Volume:  31     ISSN:  1522-2683     ISO Abbreviation:  Electrophoresis     Publication Date:  2010 Aug 
Date Detail:
Created Date:  2010-08-25     Completed Date:  -     Revised Date:  -    
Medline Journal Info:
Nlm Unique ID:  8204476     Medline TA:  Electrophoresis     Country:  Germany    
Other Details:
Languages:  eng     Pagination:  2730-9     Citation Subset:  IM    
Department of Environmental Sciences, Tuscia University, Viterbo, Italy.
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