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Title: Recombinant clotting factor VIII concentrates: Heterogeneity and high-purity evaluation
Authors: D'Amici, Gian Maria
Timperio, Anna Maria
Gevi, Federica
Grazzini, Giuliano
Zolla, Lello
Keywords: 2-DE;Hemophilia A;Proteomics;Recombinant factor VIII
Issue Date: 2010
Publisher: Wiley-Blackwell
Source: D'Amici, G.M. et al. 2010. Recombinant clotting factor VIII concentrates: Heterogeneity and high-purity evaluation. "Electrophoresis" 31(16): 2730-2739
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 (Advates, Helixate NexGens and Refactos), 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 NexGens showed the presence of impurities, such as
Hsp70 kDa, haptoglobin and proapolipoprotein; Refactos showed glutathione S-transferase
and b-lactamase, whereas Advates 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.
L'artcoo è disponibile sul sito dell'editore
ISSN: 0173-0835
DOI: 10.1002/elps.201000216
Appears in Collections:DISA - Archivio della produzione scientifica

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