Structural analysis of recombinant von Willebrand factor produced at industrial scale fermentation of transformed CHO cells co‐expressing recombinant furin

Fischer, Bernhard; Schlokat, Uwe; Mitterer, Artur; Reiter, Manfred; Mundt, Wolfgang; Turecek, Peter L.; Schwarz, Hans; Dorner, Friedrich

doi:10.1016/0014-5793(95)01218-4

Cited by 35 publications

(28 citation statements)

References 16 publications

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“…Sensor chip CM5 and an amine coupling kit containing N-hydroxysuccinimide (NHS), N-ethyl-N%-(3-diethylaminopropyl)-carbodiimide (EDS) and ethanolamine hydrochloride were obtained from Pharmacia Biosensor. Recombinant von Willebrand factor (r-vWF) produced by large-scale culture of recombinant CHO cells [28,29], human plasma, human plasma cryoprecipitate and purified monoclonal anti-von Willebrand factor antibody, MAb AvW8/2, were from IMMUNO (Vienna, Austria). Recombinant coagulation factor VIII (Kogenate, r-FVIII) was obtained from Bayer (Leverkusen, Germany).…”

Section: Methodsmentioning

confidence: 99%

“…Collagen-bound vWF was quantified using rabbit antihuman vWF IgG-peroxidase conjugate. Multimer analysis of vWF was performed by SDS-1% agarose gel electrophoresis, whereby individual vWF multimers were separated by electrophoresis and then blotted onto nitrocellulose membrane as described previously [28,29]. vWF multimers were detected by immunoenzymatic staining using a rabbit anti-human vWF serum as primary antibody; the second antibody was alkaline phosphatase-conjugated, affinity-purified, goat anti-rabbit IgG.…”

Section: Methodsmentioning

confidence: 99%

“…Thus, we adapted Chinese hamster ovary (CHO) cells to serum-free culture conditions and optimized the composition of the culture medium for continuous production of r-vWF on a large scale [29 -31]. Multimer structure and subunit composition of r-vWF produced under serum-free conditions have been analysed in detail [29]. Preclinical studies and characterization of rvWF in animal models have been reported [32][33][34][35].…”

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confidence: 99%

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Biochemical and functional characterization of recombinant von Willebrand factor produced on a large scale

Fischer¹,

Schlokat²,

Reiter³

et al. 1997

CMLS, Cell. mol. life sci.

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Recombinant von 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 heparin affinity chromatography. Heparin affinity chromatography yielded r-vWF polymers of different degrees of multimerization. r-vWF was analysed by qualitative and quantitative functional analysis. We could show that while binding of r-vWF to platelets did not depend on multimerization of the molecule, ristocetin-induced platelet aggregation, binding to collagen and binding to heparin correlated directly with the extent of multimerization. Binding of recombinant 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 multimerization. Real-time biospecific interaction analysis suggested a potential stoichiometry of 2 to 2.5 r-vWF subunits per r-FVIII molecule. Kinetic analysis of the r-vWF-r-FVIII interaction gave a binding rate 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 demonstrated that r-vWF contained a high proportion of N-glycans composed of mannose, galactose, glucose, N-acetylglucosamine and terminal sialic acid. Carbohydrate moities were covalently bound to the protein structure and were quantitatively removed from r-vWF only after protein denaturation. The results demonstrated that r-vWF produced on large scale under serum-free culture conditions exhibited qualitative and quantitative functional properties comparable to human plasma-derived vWF.

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

mentioning

confidence: 99%

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Biochemical and functional characterization of recombinant von Willebrand factor produced on a large scale

Fischer¹,

Schlokat²,

Reiter³

et al. 1997

CMLS, Cell. mol. life sci.

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“…Indeed, although increased furin expression improves VWF processing in CHO cells, the multimeric pattern is similar regardless of furin expression levels. 30 Moreover, cleavage of pro-VWF into the propolypeptide and the mature VWF is not essential for multimerization. 31 Multimerization, however, depends on (1) the presence of proVWF demonstrating that the information for VWF multimerization is situated in the proVWF per se, 32 (2) the presence of free sulfhydryl(s) in the propeptide, 33 and (3) the presence of an acidic cellular environment.…”

Section: Discussionmentioning

confidence: 99%

Phenotypic correction of von Willebrand disease type 3 blood-derived endothelial cells with lentiviral vectors expressing von Willebrand factor

Meyer

Vanhoorelbeke

Chuah

et al. 2006

Blood

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Von Willebrand disease (VWD) is an inherited bleeding disorder, caused by quantitative (type 1 and 3) or qualitative (type 2) defects in von Willebrand factor (VWF). Gene therapy is an appealing strategy for treatment of VWD because it is caused by a single gene defect and because VWF is secreted into the circulation, obviating the need for targeting specific organs or tissues. However, development of gene therapy for VWD has been hampered by the considerable length of the VWF cDNA

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“…Multimer analysis of vWF was performed with 1% (w/v) agarose gel electrophoresis followed by blotting onto nitrocellulose membrane as described previously [6,7]. Visualization of vWF multimers was by immunoenzymatic staining using a primary antibody (rabbit IgG to human vWF) and a secondary antibody (alkaline phosphatase-conjugated, affinity-purified, goat anti-rabbit IgG).…”

Section: Agarose Gel Electrophoresismentioning

confidence: 99%

Collagen covalently immobilized onto plastic surfaces simplifies measurement of von Willebrand factor-collagen binding activity

Dorner³

1998

Annals of Hematology

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Human collagen type III was immobilized covalently via activated carbohydrate moieties onto hydrazine-treated microtiter plates which could be used to measure von Willebrand factor (vWF) collagen binding activity (vWF:CBA) in an ELISA. Such plates were simple to prepare and remained stable at 4 degrees C and -20 degrees C for at least 2 months. Samples analyzed by this system included (a) normal human vWF fractionated according to the degree of multimerization, (b) normal citrated and EDTA plasma and corresponding serum, and (c) plasma from patients with von Willebrand disease (vWD) types 1 and 2. When related to the concentration of vWF antigen (vWF:Ag), proportionally low levels of vWF:CBA were found for samples lacking the high-molecular-weight multimers, while higher values were obtained for samples containing these multimers. The ratio of vWF:CBA/vWF:Ag sensitively reflected the functional and structural intactness of the vWF molecules for all analyzed samples. Monoclonal antibody directed to the region within the A1 domain of vWF which interacts with the glycoprotein Ib completely inhibited the vWF ristocetin cofactor (vWF:RistCof), while vWF:CBA was not affected. Thus vWF:CBA and vWF:RistCof clearly represent separate, noninterchangeable functional parameters of vWF. In conclusion, our results indicate that the newly described method for the immobilization of collagen onto microtiter plates is suitable for the determination of vWF:CBA. In conjunction with vWF:Ag and the calculated ratio of vWF:CBA/vWF:Ag, this method simplifies the detection and classification of patients with vWD and assists in quality control during the purification of normal vWF.

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Structural analysis of recombinant von Willebrand factor produced at industrial scale fermentation of transformed CHO cells co‐expressing recombinant furin

Cited by 35 publications

References 16 publications

Biochemical and functional characterization of recombinant von Willebrand factor produced on a large scale

Biochemical and functional characterization of recombinant von Willebrand factor produced on a large scale

Phenotypic correction of von Willebrand disease type 3 blood-derived endothelial cells with lentiviral vectors expressing von Willebrand factor

Collagen covalently immobilized onto plastic surfaces simplifies measurement of von Willebrand factor-collagen binding activity

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