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 recombinant von Willebrand factor (r-vWF) produced by recombinant CHO cells demonstrated r-vWF to be more intact and less proteolytically degraded than plasma-derived vWF (pd-vWF) [B, Fischer et al, (1994) FEBS Lett. 351, 345-348], In contrast to pd-vWF, r-vWF preparations consisted 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 clone was transfected with the cDNA of the human propeptide processing enzyme Furin, A r-vWF/r-Furin co-expressing cell clone was cultivated at industrial scale in high cell density perfusion fermenters, r-vWF obtained from these cells was fully processed, Analysis of r-vWF by multimer analysis revealed a multimer pattern equal in number of high molecular weight multimer to pd-vWF, but absence of satellite bands, Two-dimensional electrophoretic analysis of both the primary diner and the complete multimer pattern of r-vWF shelved that the recombinant coagulation factor was composed exclusively of intact and mature subunits. Since the triplet structure typical to pd-vWF is known to reflect proteolytic degradation, r-vWF thus exhibits an integrity far superior compared to pd-vWF.