BIOCHEMICAL AND FUNCTIONAL-CHARACTERIZATION OF RECOMBINANT VON-WILLEBRAND-FACTOR PRODUCED ON A LARGE-SCALE

Recombinant iron Willebrand factor (r-vWF) was produced in serum-free medium on a large scale in recombinant Chinese hamster ovary cells and was purified from fermentation supernatant by a combination of anion exchange chromatography and herapin affinity chromatography. Heparin a ffinity chromatography yielded r-vWF polymers of different degrees of multimerization. r-vWF was analysed by qualitative and quantitative fu nctional analysis. We could show that while binding of r-vWF to platel ets did not depend on multimerization of the molecule, ristocetin-indu ced platelet aggregation, binding to collagen and binding to heparin c orrelated directly with the extent of multimerization. Binding of reco mbinant coagulation factor VIII (r-FVIII) to r-vWF was studied by real -time biospecific interaction analysis and surface plasmon technology. The data indicated that binding of r-FVIII did not depend on r-vWF mu ltimerization. Real-time biospecific interaction analysis suggested a potential stoichiometry of 2 to 2.5 r-vWF subunits per r-FVIII molecul e. Kinetic analysis of the r-vWF-r-FVIII interaction gave a binding ra te constant of 3 x 10(6) M-1 s(-1) and an association constant of 2.5 x 10(9) M-1. Reaction of r-vWF with carbohydrate-specific lectins demo nstrated that r-vWF contained a high proportion of N-glycans composed of mannose, galactose, glucose, N-acetylglucosamine and terminal siali c acid. Carbohydrate moities were covalently bound to the protein stru cture and were quantitatively removed from r-vWF only after protein de naturation. The results demonstrated that r-vWF produced on large scal e under serum-free culture conditions exhibited qualitative and quanti tative functional properties comparable to human plasma-derived vWF.