http://http://dspace.unitus.it:80 The DSpace digital repository system captures, stores, indexes, preserves, and distributes digital research material. Thu, 23 May 2013 16:26:48 GMT 2013-05-23T16:26:48Z http://hdl.handle.net/2067/1843 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 Abstract: 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. Description: L'artcoo è disponibile sul sito dell'editore http://onlinelibrary.wiley.com/ Thu, 31 Dec 2009 23:00:00 GMT http://hdl.handle.net/2067/1843 2009-12-31T23:00:00Z http://hdl.handle.net/2067/1844 Title: Comparison among plasma-derived clotting Factor VIII by using Authors: Timperio, Anna Maria; Gevi, Federica; Grazzini, Giuliano; Vaglio, Stefania; Zolla, Lello Abstract: Background. Deficiency or dysfunction of coagulation factor VIII (FVIII) is the underlying cause of haemophilia A. Haemophilic patients are at present treated with plasma-derived FVIII (pdFVIII) or recombinant FVIII (rFVIII) in order to correct their clotting deficiency. pdFVIII concentrates are exclusively produced from human plasma upon pooling from multiple donors. It is not know whether the presence of excess of other plasma proteins, in addition to von Willebrand factor, could stimulate untoward immune responses in the recipient. Thus, information regarding the presence of contaminants in commercial products is of concern. Materials and Methods. Two commercially available pdFVIII concentrates were characterized through SDS-PAGE and mass spectrometry Emoclot® and Beriate® . Results. The components of two pdFVIII products considered in this study were well identified by mass spectrometry analysis, in both cases we found abundant components coming from blood plasma, and some other contaminants. Only in Beriate® we also found truncated form of pdFVIII. Conclusion. The two pdFVIII examined showed the presence of vWF, Fibrinogen in excess, and other substances that could be considered as contaminants or impurities. Description: L'articolo è disponibile sul sito dell'editore http://www.bloodtransfusion.it Thu, 31 Dec 2009 23:00:00 GMT http://hdl.handle.net/2067/1844 2009-12-31T23:00:00Z