STRUCTURAL-ANALYSIS OF RECOMBINANT VON-WILLEBRAND-FACTOR PRODUCED AT INDUSTRIAL-SCALE FERMENTATION OF TRANSFORMED CHO CELLS CO-EXPRESSING RECOMBINANT FURIN

Thorough analysis of multimer composition and molecular structure of r ecombinant von Willebrand factor (r-vWF) produced by recombinant CHO c ells demonstrated r-vWF to be more intact and less proteolytically deg raded than plasma-derived vWF (pd-vWF) [B, Fischer et al, (1994) FEBS Lett. 351, 345-348], In contrast to pd-vWF, r-vWF preparations consist ed of pro-vWF (vWF containing covalently attached propeptide) as well as mature vWF subunits forming homo- and hetero-multimers, In order to ensure complete propeptide processing, a r-vWF-producing CHO cell clo ne was transfected with the cDNA of the human propeptide processing en zyme Furin, A r-vWF/r-Furin co-expressing cell clone was cultivated at industrial scale in high cell density perfusion fermenters, r-vWF obt ained from these cells was fully processed, Analysis of r-vWF by multi mer analysis revealed a multimer pattern equal in number of high molec ular weight multimer to pd-vWF, but absence of satellite bands, Two-di mensional electrophoretic analysis of both the primary diner and the c omplete multimer pattern of r-vWF shelved that the recombinant coagula tion factor was composed exclusively of intact and mature subunits. Si nce the triplet structure typical to pd-vWF is known to reflect proteo lytic degradation, r-vWF thus exhibits an integrity far superior compa red to pd-vWF.