PRODUCTION OF HIGHLY HOMOGENEOUS AND STRUCTURALLY INTACT RECOMBINANT VON-WILLEBRAND-FACTOR MULTIMERS BY FURIN-MEDIATED PROPEPTIDE REMOVAL IN-VITRO

Recombinant human von Willebrand Factor (rvWF), a multimeric glycoprot ein essential to haemostasis, has been developed as a potential therap eutic agent for treatment of von Willebrand disease (vWD), Permanent C hinese-hamster ovary (CHO)-rvWF cell clones co-expressing recombinant furin (rfurin) were established in order to ensure complete wWF propep tide removal [Fischer, Schlokat, Mitterer, Reiter, Mundt, Turecek, Sch warz and Dorner(1995) FEES Lett, 375, 259-262], Large quantities of ma terial are required for in vivo tests and clinical studies. This deman d is commonly met by achieving high-yield expression of the desired pr otein via amplification, Go-amplification of rfurin, necessary to comp letely process increasing amounts of wWF precursor, could not be accom plished, presumably due to lethal effects of overexpressed rfurin for the host cells [Creemers (1994) Ph,D. Thesis, University of Leuven], R ecent reports have inferred that rfurin can only mediate wWF processin g intracellularly [Rehemtulla and Kaufman (1992) Blood 79, 2349-2355; Rehemtulla, Dorner and Kaufman (1992) Proc, Natl, Acad, Sci, U.S.A. 89 , 8235-8239], We report here that rvWF-precursor processing, however, occurs predominantly extracellularly upon rfurin co-expression, Mixing experiments employing rfurin- as well as rvWF-precursor-containing co nditioned media demonstrate that wWF precursors are accessible and cle avable by rfurin in vitro, Exposure to rfurin in vitro converts the he terogeneous multimer pattern typical of incompletely processed wWF mul timers into highly homogeneous and structurally intact multimers super ior to the ones exhibited by plasma-derived vWF, These findings thus d emonstrate the feasibility of large-scale production of a completely p rocessed, intact and homogeneous rvWF preparation, based on individual rvWF-precursor high-yield expression and subsequent propeptide remova l by rfurin in vitro.