Development of a plasma- and albumin -free recombinant von Willebrand factor

Summary

Baxter has developed a recombinant therapy for treating von Willebrand‘s disease. Recombinant VWF is co-expressed with the rFVIII in CHO cells used to produce the rFVIII product Advate. This rVWF is used as a drug component for a rVWF-rFVIII complex drug product. CHO cells produce partially processed and partially un-processed rVWF still containing the pro-peptide. In order to make a consistent preparation containing mature and processed rVWF only, rVWF is exposed to recombinant furin to remove the pro-peptide. Recombinant VWF and furin are produced under serum- and protein-free conditions. It is highly purified by a series of chromatographic steps and formulated in a protein-free buffer and has a homogeneous multimer distribution. The specific activity is higher in rVWF than in commercial plasma-derived VWF-FVIII complex products. SDS agarose electrophoretic analysis shows the presence of ultra-high molecular weight multimers. The FVIII-binding capacity and affinity of rVWF to FVIII is comparable to VWF in plasma. Carbohydrate analysis shows an intact glycosylation pattern. Recombinant VWF binds to collagen and promotes platelet adhesion under shear stress. It stabilizes endogenous FVIII in VWF-deficient knock-out mice as seen by a secondary rise in murine FVIII.

Keywords

Recombinant von Willebrand factor - von Willebrand disease - hemophilia - substitution therapy

• References

• 1 Sadler JE. Biochemistry and genetics of von Willebrand factor. Annu Rev Biochem 1998; 67: 395-424
• 2 Ofosu FA, Freedman J, Semple JW. Plasma-derived biological medicines used to promote haemostasis. Thromb Haemost 2008; 99: 851-862
• 3 Franchini M, Targher G, Lippi G. Prophylaxis in von Willebrand disease. Ann Hematol 2007; 86: 699-704
• 4 Gill JC. Treatment of urgent bleeding in von Willebrand disease. Thromb Res 2007; 120 (Suppl. 01) S21-S25
• 5 Nichols WL, Hultin MB, James AH. et al. Von Willebrand disease (VWD): Evidence-based diagnosis and management guidelines, the National Heart, Lung, and Blood Institute (NHLBI) Expert Panel report (USA). Haemophilia 2008; 14: 171-232
• 6 Ruggeri ZM, Zimmerman TS. The complex multi-meric composition of factor VIII/von Willebrand factor. Blood 1981; 57: 1140-1143
• 7 Aihara M, Sawada Y, Ueno K. et al. Visualization of von Willebrand factor multimers by immunoenzymatic stain using avidin-biotin peroxidase complex. Thromb Haemost 1986; 55: 263-267
• 8 Denis C, Methia N, Frenette PS. et al. A mouse model of severe von Willebrand disease: Defects in hemostasis and thrombosis. Proc Natl Acad Sci USA 1998; 95: 9524-9529
• 9 Blanchette VS, Shapiro AD, Liesner RJ. et al. rAHFPFM Clinical Study Group. Plasma and albumin-free recombinant factor VIII: pharmacokinetics, efficacy and safety in previously treated pediatric patients. J Thromb Haemost 2008; 6: 1319-1326
• 10 Turecek PL, Váradi K, Schlokat U. et al. In vivo and in vitro processing of recombinant pro-von Willebrand factor. Histochem Cell Biol 2002; 117: 123-129
• 11 Turecek PL, Pichler L, Auer W. et al. Evidence for extracellular processing of pro-von Willebrand factor after infusion in animals with and without severe von Willebrand disease. Blood 1999; 94: 1637-1647
• 12 Schlokat U, Fischer BE, Herlitschka S. et al. Production of highly homogeneous and structurally intact recombinant von Willebrand factor multimers by furin-mediated propeptide removal in vitro. Biotechnol Appl Biochem 1996; 24: 257-267
• 13 Wise RJ, Dorner AJ, Krane M. et al. The role of von Willebrand factor multimers and propeptide cleavage in binding and stabilization of factor VIII. J Biol Chem 1991; 266: 21948-21955
• 14 Varadi K, Rottensteiner H, Vejda S. et al. Species-dependent variability of ADAMTS13-mediated proteolysis of human recombinant von Willebrand factor. J Thromb Haemost 2009; 7: 1134-1142
• 15 Hubbard AR, Heath AB. Standardization of factor VIII and von Willebrand factor in plasma: calibration of the WHO 5^th International Standard (02/150). J Thromb Haemost 2004; 2: 1380-1384
• 16 Hubbard AR. Von Willebrand factor standards for plasma and concentrate testing. Semin Thromb He-most 2006; 32: 522-528
• 17 Lethagen S, Carlson M, Hillarp A. A comparative in vitro evaluation of six von Willebrand factor concentrates. Haemophilia 2004; 10: 243-249
• 18 Budde U, Metzner HJ, Müller HG. Comparative analysis and classification of von Willebrand factor/ factor VIII concentrates: impact on treatment of patients with von Willebrand disease. Semin Thromb Hemost 2006; 32: 626-635
• 19 Federici AB. Management of von Willebrand disease with factor VIII/von Willebrand factor concentrates: results from current studies and surveys. Blood Coagul Fibrinolysis 2005; 16 (Suppl. 01) S17-S21
• 20 Federici AB, Castaman G, Thompson A, Berntorp E. Von Willebrand‘s disease: clinical management. Haemophilia 2006; 12 (Suppl. 03) 152-158
• 21 Parti R, Schoppmann A, Lee H, Yang L. Stability of lyophilized and reconstituted plasma/albumin-free recombinant human factor VIII (ADVATE rAHFPFM). Haemophilia 2005; 11: 492-496
• 22 Shapiro AD. Anti-hemophilic factor (recombinant), plasma/albumin-free method (octocog-alpha; ADVATE) in the management of hemophilia A. Vasc Health Risk Manag 2007; 3: 555-565
• 23 Fischer BE, Schlokat U, Mitterer A. et al. Structural analysis of recombinant von Willebrand factor produced at industrial scale fermentation of transformed CHO cells co-expressing recombinant furin. FEBS Lett 1995; 375: 259-262
• 24 Preininger A, Schlokat U, Mohr G. et al. Strategies for recombinant Furin employment in a biotechnological process: complete target protein precursor cleavage. Cytotechnology 1999; 30: 1-16
• 25 Turecek PL, Furlan M, Lämmle B. et al. Cleavage of recombinant von Willebrand factor by a VWF-depolymerizing protease. Poster presented at the 38th Annual Meeting of the American Society of Hematology (ASH), Orlando, FL/USA, Dec. 6–10, 1996. Blood 1996; 88 (Suppl. 01) 326a
• 26 Roussi J, Turecek PL, André P. et al. Effects of human recombinant, plasma-derived and porcine von Willebrand factor in pigs with severe von Willebrand disease. Blood Coagul Fibrinolysis 1998; 9: 361-372
• 27 Plaimauer B, Schlokat U, Turecek PL. et al. Recombinant von Willebrand factor: preclinical development. Semin Thromb Hemost 2001; 27: 395-403
• 28 Schwarz HP, Dorner F, Mitterer A. et al. Evaluation of recombinant von Willebrand factor in a canine model of von Willebrand disease. Haemophilia 1998; 4 (Suppl. 03) 53-62
• 29 Schwarz HP, Dorner F, Mitterer A. et al. Preclinical evaluation of recombinant von Willebrand factor in a canine model of von Willebrand disease. Wien Klin Wochenschr 1999; 111: 181-191
• 30 Turecek PL, Gritsch H, Pichler L. et al. In vivo characterization of recombinant von Willebrand factor in dogs with von Willebrand disease. Blood 1997; 90: 3555-3567
• 31 Schwarz HP, Schlokat U, Mitterer A. et al. Recombinant von Willebrand factor-insight into structure and function through infusion studies in animals with severe von Willebrand disease. Semin Thromb Hemost 2002; 28: 215-226