Industrial production of clotting factors: Challenges of expression, and choice of host cells

Biotechnology Journal

Volumn 10, Issue 7, 2015, Pages 995-1004

  Kumar, Sampath R ^a  

^a Adimab LLC   (United States)

Author keywords

Cell engineering; Clotting factors; Expression challenges; Host cell lines; Von Willebrand Factor (VWF)

Indexed keywords

CARBOXYLATION; CELL CULTURE; CELLS; COAGULATION; CYTOLOGY; ENZYMES; GLYCOPROTEINS; PRODUCTIVITY; PROTEINS;

CLOTTING FACTORS; COMPLEX STRUCTURE; ENGINEERING SOLUTIONS; EXPRESSION CHALLENGES; HOST CELLS; INDUSTRIAL PRODUCTION; POST-TRANSLATIONAL MODIFICATIONS; VON WILLEBRAND FACTOR;

CELL ENGINEERING;

ACTIVATED PROTEIN C; BLOOD CLOTTING FACTOR; BLOOD CLOTTING FACTOR 10; BLOOD CLOTTING FACTOR 10A; BLOOD CLOTTING FACTOR 7; BLOOD CLOTTING FACTOR 7A; BLOOD CLOTTING FACTOR 8; BLOOD CLOTTING FACTOR 9; PROTEIN C; PROTHROMBIN; THROMBIN; VITAMIN K DEPENDENT CARBOXYLASE; VITAMIN K EPOXIDE REDUCTASE; VON WILLEBRAND FACTOR; F8 PROTEIN, HUMAN; RECOMBINANT PROTEIN;

BHK CELL LINE; CARBOXYLATION; CELL ENGINEERING; GLYCOSYLATION; HEK293 CELL LINE; HETEROLOGOUS EXPRESSION; HOST CELL; HUMAN; INDUSTRIAL PRODUCTION; PRIORITY JOURNAL; PROTEIN DOMAIN; PROTEIN SECRETION; REVIEW; SIGNAL TRANSDUCTION; SULFATION; VON WILLEBRAND DISEASE; ANIMAL; BIOSYNTHESIS; BLOOD CLOTTING; CHO CELL LINE; CRICETULUS; GENE EXPRESSION REGULATION; GENETICS; PROTEIN PROCESSING;

ANIMALS; BLOOD COAGULATION; CELL ENGINEERING; CHO CELLS; CRICETULUS; FACTOR VIII; GENE EXPRESSION REGULATION; HEK293 CELLS; HUMANS; PROTEIN PROCESSING, POST-TRANSLATIONAL; RECOMBINANT PROTEINS; VON WILLEBRAND FACTOR;

EID: 84935724880     PISSN: 18606768     EISSN: 18607314     Source Type: Journal    
DOI: 10.1002/biot.201400666     Document Type: Review

References (104)

1. Kingdon, H. S., Lundblad, R. L., An adventure in biotechnology: The development of haemophilia A therapeutics - from whole-blood transfusion to recombinant DNA to gene therapy. Biotechnol. Appl. Biochem. 2002, 35, 141-148.
2. Boedeker, B. G., Production processes of licensed recombinant factor VIII preparations. Semin. Thromb. Hemost. 2001, 27, 385-394.
3. Brasha-Mitchell, E., Collins, A., Shehu, L., Seneff, M. G. et al., Safety and dose-finding study of activated protein C (drotrecogin alfa activated) as an anticoagulant in end-stage renal disease patients treated with hemodialysis. Blood purification 2014, 37, 243-248.
4. Escobar, M. A., Advances in the treatment of inherited coagulation disorders. Haemophilia 2013, 19, 648-659.
5. Oldenburg, J., Albert, T., Novel products for haemostasis - current status. Haemophilia 2014, 20Suppl4, 23-28.
6. Berkner, K. L., Vitamin K-dependent carboxylation. Vitam. Horm. 2008, 78, 131-156.
7. Hansson, K., Stenflo, J., Post-translational modifications in proteins involved in blood coagulation. J. Thromb. Haemost. 2005, 3, 2633-2648.
8. Kaufman, R. J., Post-translational modifications required for coagulation factor secretion and function. Thromb. Haemost. 1998, 79, 1068-1079.
9. Brown, M. A., Stenberg, L. M., Biopharmaceuticals: Post-translational modification carboxylation and hydroxylation, in: Walsh, G. (Ed.), Post-translational modification of protein biopharmaceuticals, Wiley-VCH, Weinheim 2009.
10. Berkner, K. L., Expression of recombinant vitamin K-dependent proteins in mammalian cells: Factors IX and VII. Methods Enzymol. 1993, 222, 450-477.
11. de la Salle, H., Altenburger, W., Elkaim, R., Dott, K. et al., Active gamma-carboxylated human factor IX expressed using recombinant DNA techniques. Nature 1985, 316, 268-270.
12. Kaufman, R. J., Wasley, L. C., Furie, B. C., Furie, B., Shoemaker, C. B., Expression, purification, and characterization of recombinant gamma-carboxylated factor IX synthesized in Chinese hamster ovary cells. J. Biol. Chem. 1986, 261, 9622-9628.
13. Messier, T. L., Pittman, D. D., Long, G. L., Kaufman, R. J., Church, W. R., Cloning and expression in COS-1 cells of a full-length cDNA encoding human coagulation factor X. Gene 1991, 99, 291-294.
14. Busby, S., Kumar, A., Joseph, M., Halfpap, L. et al., Expression of active human factor IX in transfected cells. Nature 1985, 316, 271-273.
15. Camire, R. M., Larson, P. J., Stafford, D. W., High, K. A., Enhanced gamma-carboxylation of recombinant factor X using a chimeric construct containing the prothrombin propeptide. Biochemistry 2000, 39, 14322-14329.
16. Wu, S. M., Cheung, W. F., Frazier, D., Stafford, D. W., Cloning and expression of the cDNA for human gamma-glutamyl carboxylase. Science 1991, 254, 1634-1636.
17. Hallgren, K. W., Hommema, E. L., McNally, B. A., Berkner, K. L., Carboxylase overexpression effects full carboxylation but poor release and secretion of factor IX: Implications for the release of vitamin K-dependent proteins. Biochemistry 2002, 41, 15045-15055.
18. Rehemtulla, A., Roth, D. A., Wasley, L. C., Kuliopulos, A. et al., In vitro and in vivo functional characterization of bovine vitamin K-dependent gamma-carboxylase expressed in Chinese hamster ovary cells. Proc. Nal. Acad. Sci. U.S.A. 1993, 90, 4611-4615.
19. Van Horn, W. D., Structural and functional insights into human vitamin K epoxide reductase and vitamin K epoxide reductase-like1. Crit. Rev. Biochem. Mol. Biol. 2013, 48, 357-372.
20. Li, T., Chang, C. Y., Jin, D. Y., Lin, P. J. et al., Identification of the gene for vitamin K epoxide reductase. Nature 2004, 427, 541-544.
21. Rost, S., Fregin, A., Ivaskevicius, V., Conzelmann, E. et al., Mutations in VKORC1 cause warfarin resistance and multiple coagulation factor deficiency type 2. Nature 2004, 427, 537-541.
22. Hallgren, K. W., Qian, W., Yakubenko, A. V., Runge, K. W., Berkner, K. L., r-VKORC1 expression in factor IX BHK cells increases the extent of factor IX carboxylation but is limited by saturation of another carboxylation component or by a shift in the rate-limiting step. Biochemistry 2006, 45, 5587-5598.
23. Sun, Y. M., Jin, D. Y., Camire, R. M., Stafford, D. W., Vitamin K epoxide reductase significantly improves carboxylation in a cell line overexpressing factor X. Blood 2005, 106, 3811-3815.
24. Wajih, N., Hutson, S. M., Owen, J., Wallin, R., Increased production of functional recombinant human clotting factor IX by baby hamster kidney cells engineered to overexpress VKORC1, the vitamin K 2, 3-epoxide-reducing enzyme of the vitamin K cycle. J. Biol. Chem. 2005, 280, 31603-31607.
25. Vermeer, C., Comparison between hepatic and nonhepatic vitamin K-dependent carboxylase. Haemostasis 1986, 16, 239-245.
26. de Castilho Fernandes, A., Fontes, A., Gonsales, N., Swiech, K. et al., Stable and high-level production of recombinant Factor IX in human hepatic cell line. Biotechnol. Appl. Biochem. 2011, 58, 243-249.
27. Enjolras, N., Dargaud, Y., Perot, E., Guillaume, F. et al., Human hepatoma cell line HuH-7 is an effective cellular system to produce recombinant factor IX with improved post-translational modifications. Thromb. Res. 2012, 130, e266-273.
28. Grinnell, B. W., Walls, J. D., Marks, C., Glasebrook, A. L. et al., Gamma-carboxylated isoforms of recombinant human protein S with different biologic properties. Blood 1990, 76, 2546-2554.
29. Hedner, U., Mechanism of action, development and clinical experience of recombinant FVIIa. J. Biotechnol. 2006, 124, 747-757.
30. Bandyopadhyay, P. K., Vitamin K-dependent gamma-glutamylcarboxylation: An ancient posttranslational modification. Vitam. Horm. 2008, 78, 157-184.
31. Bandyopadhyay, P. K., Clark, K., Stevenson, B. J., Rivier, J. E. et al., Biochemical characterization of Drosophila gamma-glutamyl carboxylase and its role in fly development. Insect Mol. Biol. 2006, 15, 147-156.
32. Robertson, H. M., Genes encoding vitamin-K epoxide reductase are present in Drosophila and trypanosomatid protists. Genetics 2004, 168, 1077-1080.
33. Vatandoost, J., Zomorodipour, A., Sadeghizadeh, M., Aliyari, R. et al., Expression of biologically active human clotting factor IX in Drosophila S2 cells: Gamma-carboxylation of a human vitamin K-dependent protein by the insect enzyme. Biotechnol. Pro. 2012, 28, 45-51.
34. Berting, A., Farcet, M. R., Kreil, T. R., Virus susceptibility of Chinese hamster ovary (CHO) cells and detection of viral contaminations by adventitious agent testing. Biotechnol. Bioeng. 2010, 106, 598-607.
35. Ghaderi, D., Zhang, M., Hurtado-Ziola, N., Varki, A., Production platforms for biotherapeutic glycoproteins. Occurrence, impact, and challenges of non-human sialylation. Biotechnol. Genet. Eng. Rev. 2012, 28, 147-175.
36. Jayapal, K. P., Wlaschin, K. F., Yap, M. G. S., Hu, W.-S., Recombinant protein therapeutics from CHO cells - 20 years and counting. Chem. Eng. Prog. 2007, 103, 40-47.
37. Blostein, M., Cuerquis, J., Landry, S., Galipeau, J., The carboxylation efficiency of the vitamin K-dependent clotting factors: Studies with factor IX. Haemophilia 2008, 14, 1063-1068.
38. Grinnell, B. W., Yan, S. B., Macias, W. L., Activated Protein C, in: McGrath, B. M., Walsh, G. (Eds.), Directory of therapeutic enzymes, CRC Press Boca Raton, FL 2006, pp. 69-96.
39. Manithody, C., Yang, L., Rezaie, A. R., Role of basic residues of the autolysis loop in the catalytic function of factor Xa. Biochemistry 2002, 41, 6780-6788.
40. Yang, L., Gopalakrishna, K., Manithody, C., Rezaie, A. R., Expression, purification and characterization of factor IX derivatives using a novel vector system. Protein Expression Purif. 2006, 50, 196-202.
41. McCue, J., Osborne, D., Dumont, J., Peters, R. et al., Validation of the manufacturing process used to produce long-acting recombinant factor IX Fc fusion protein. Haemophilia 2014, 20, e327-e335.
42. Lentz, S. R., Ehrenforth, S., Karim, F. A., Matsushita, T. et al., Recombinant factor VIIa analog in the management of hemophilia with inhibitors: Results from a multicenter, randomized, controlled trial of vatreptacog alfa. J. Thromb. Haemost. 2014, 12, 1244-1253.
43. Mena, J. A., Kamen, A. A., Insect cell technology is a versatile and robust vaccine manufacturing platform. Expert Rev. Vaccines 2011, 10, 1063-1081.
44. Orlova, N. A., Kovnir, S. V., Vorobiev, II., Gabibov, A. G., Vorobiev, A. I., Blood Clotting Factor VIII: From Evolution to Therapy. Acta Naturae 2013, 5, 19-39.
45. Sadler, J. E., Biochemistry and genetics of von Willebrand factor. Annu. Rev. Biochem. 1998, 67, 395-424.
46. Lenting, P. J., Christophe, O. D., Denis, C. V., von Willebrand factor biosynthesis, secretion, and clearance: Connecting the far ends. Blood 2015, 125, 2019-2028.
47. Soukharev, S., Hammond, D., Ananyeva, N. M., Anderson, J. A. et al., Expression of factor VIII in recombinant and transgenic systems. Blood Cells Mol. Dis. 2002, 28, 234-248.
48. Hoeben, R. C., Fallaux, F. J., Cramer, S. J., van den Wollenberg, D. J. et al., Expression of the blood-clotting factor-VIII cDNA is repressed by a transcriptional silencer located in its coding region. Blood 1995, 85, 2447-2454.
49. Lynch, C. M., Israel, D. I., Kaufman, R. J., Miller, A. D., Sequences in the coding region of clotting factor VIII act as dominant inhibitors of RNA accumulation and protein production. Hum. Gene Ther. 1993, 4, 259-272.
50. Dorner, A. J., Bole, D. G., Kaufman, R. J., The relationship of N-linked glycosylation and heavy chain-binding protein association with the secretion of glycoproteins. J. Cell Biol. 1987, 105, 2665-2674.
51. Pipe, S. W., Kaufman, R. J., Factor VIII C2 domain missense mutations exhibit defective trafficking of biologically functional proteins. J. Biol. Chem. 1996, 271, 25671-25676.
52. Malhotra, J. D., Miao, H., Zhang, K., Wolfson, A. et al., Antioxidants reduce endoplasmic reticulum stress and improve protein secretion. Proc. Natl. Acad. Sci. U.S.A. 2008, 105, 18525-18530.
53. Tagliavacca, L., Wang, Q., Kaufman, R. J., ATP-dependent dissociation of non-disulfide-linked aggregates of coagulation factor VIII is a rate-limiting step for secretion. Biochemistry 2000, 39, 1973-1981.
54. Zhang, B., Kaufman, R. J., Ginsburg, D., LMAN1 and MCFD2 form a cargo receptor complex and interact with coagulation factor VIII in the early secretory pathway. J.Bbiol. Chem. 2005, 280, 25881-25886.
55. Wise, R. J., Dorner, A. J., Krane, M., Pittman, D. D., Kaufman, R. J., The role of von Willebrand factor multimers and propeptide cleavage in binding and stabilization of factor VIII. J. Biol. Chem. 1991, 266, 21948-21955.
56. Pipe, S. W., The promise and challenges of bioengineered recombinant clotting factors. J.Tthromb. Haemost. 2005, 3, 1692-1701.
57. Selvaraj, S. R., Scheller, A. N., Miao, H. Z., Kaufman, R. J., Pipe, S. W., Bioengineering of coagulation factor VIII for efficient expression through elimination of a dispensable disulfide loop. Journal of thrombosis and haemostasis 2012, 10, 107-115.
58. Spencer, H. T., Denning, G., Gautney, R. E., Dropulic, B. et al., Lentiviral vector platform for production of bioengineered recombinant coagulation factor VIII. Molecular therapy 2011, 19, 302-309.
59. Ishaque, A., Thrift, J., Murphy, J. E., Konstantinov, K., Over-expression of Hsp70 in BHK-21 cells engineered to produce recombinant factor VIII promotes resistance to apoptosis and enhances secretion. Biotechnol. Bioeng. 2007, 97, 144-155.
60. Mufarrege, E. F., Antuna, S., Etcheverrigaray, M., Kratje, R., Prieto, C., Development of lentiviral vectors for transient and stable protein overexpression in mammalian cells. A new strategy for recombinant human FVIII (rhFVIII) production. Protein Expression Purif. 2014, 95, 50-56.
61. Picanco-Castro, V., Russo-Carbolante, E. M., Fontes, A. M., Fernandes, A. C., Covas, D. T., An enhancer/promoter combination strengthens the expression of blood-coagulation factor VIII in non-viral expression vectors. Genet. Mol. Res. 2008, 7, 314-325.
62. Nair, A. R., Jinger, X., Hermiston, T. W., Effect of different UCOE-promoter combinations in creation of engineered cell lines for the production of Factor VIII. BMC Res. Notes 2011, 4, 178.
63. Gomez, K., Klamroth, R., Mahlangu, J., Mancuso, M. E. et al., Key issues in inhibitor management in patients with haemophilia. Blood Transfus. 2014, 12, 1, s319-s329.
64. Kavakli, K., Yang, R., Rusen, L., Beckmann, H. et al., Prophylaxis vs. on-demand treatment with BAY 81-8973, a full-length plasma protein-free recombinant factor VIII product: results from a randomized trial (LEOPOLD II). J. Thromb. Haemost. 2014, 13, 360-369.
65. Campos-da-Paz, M., Costa, C. S., Quilici, L. S., de Carmo Simoes, I. et al., Production of recombinant human factor VIII in different cell lines and the effect of human XBP1 co-expression. Mol. Biotechnol. 2008, 39, 155-158.
66. Chen, C., Fang, X. D., Zhu, J., Wu, X. F. et al., The gene expression of coagulation factor VIII in mammalian cell lines. Thromb. Res. 1999, 95, 105-115.
67. Guo, X., Wang, H., Chu, H., Wang, X. et al., High level expression of human factor VIII in mammalian cells after retroviral-mediated gene transfer. Chin. Med. J. 2001, 114, 690-693.
68. He, B., Pan, Y., Chen, L., Xu, Y. et al., Stable expression of native Coagulation factor VIII using the 2A self-processing sequence and furin cleavage site. Thromb. Res. 2011, 128, e148-153.
69. Herlitschka, S. E., Schlokat, U., Falkner, F. G., Dorner, F., High expression of a B-domain deleted factor VIII gene in a human hepatic cell line. J. Biotechnol. 1998, 61, 165-173.
70. Webb, E., Tkalcevic, J., Edwards, S., Hocking, D., Nisbet, I., Expression of biologically active human factor VIII using a baculovirus vector. Biochem. Biophys. Res. Commun. 1993, 190, 536-543.
71. Kannicht, C., Ramstrom, M., Kohla, G., Tiemeyer, M. et al., Characterisation of the post-translational modifications of a novel, human cell line-derived recombinant human factor VIII. Thromb. Res. 2013, 131, 78-88.
72. Peters, R. T., Toby, G., Lu, Q., Liu, T. et al., Biochemical and functional characterization of a recombinant monomeric factor VIII-Fc fusion protein. J. Thromb. Haemost 2013, 11, 132-141.
73. De Meyer, S. F., Deckmyn, H., Vanhoorelbeke, K., von Willebrand factor to the rescue. Blood 2009, 113, 5049-5057.
74. Turecek, P. L., Schrenk, G., Rottensteiner, H., Varadi, K. et al., Structure and function of a recombinant von Willebrand factor drug candidate. Semin. Thromb. Hemost. 2010, 36, 510-521.
75. Hannah, M. J., Williams, R., Kaur, J., Hewlett, L. J., Cutler, D. F., Biogenesis of Weibel-Palade bodies. Semin. Cell Dev. Biol. 2002, 13, 313-324.
76. Turecek, P. L., Mitterer, A., Matthiessen, H. P., Gritsch, H. et al., Development of a plasma- and albumin-free recombinant von Willebrand factor. Hamostaseologie 2009, 29, 1, S32-S38.
77. Harrison, S., Adamson, S., Bonam, D., Brodeur, S. et al., The manufacturing process for recombinant factor IX. Semin. Hematol. 1998, 35, 4-10.
78. Peters, R. T., Low, S. C., Kamphaus, G. D., Dumont, J. A. et al., Prolonged activity of factor IX as a monomeric Fc fusion protein. Blood 2010, 115, 2057-2064.
79. Walsh, G., Post-translational modifications in the context of therapeutic proteins: An introductory overview, in: Walsh, G. (Ed.), Post-translational modification of protein biopharmaceuticals, Wiley-VCH, Weinheim 2009, pp. 1-14.
80. Zhang, M., Lu, Q., Mei, B., Peters, R. T., Detection of galactose-alpha-1, 3-galactose (a-4Gal) and N-glycolylneuraminic acid (NGNA) in recombinant and plasma derived FIX products. J. Thromb. Haemost. 2013, 11, 320 Abstract.
81. Kanan, Y., Al-Ubaidi, M. R., Tyrosine O sulfation: An overview. JSM Biotechnol. Biomed. Eng. 2013, 1, 1003.
82. Ezban, M., Vad, K., Kjalke, M., Turoctocog alfa (NovoEight®) - from design to clinical proof of concept. European Haematol. 2014, 93, 369-376.
83. Stone, M. J., Chuang, S., Hou, X., Shoham, M., Zhu, J. Z., Tyrosine sulfation: An increasingly recognised post-translational modification of secreted proteins. New Biotechnol. 2009, 25, 299-317.
84. Zhong, X., Wright, J. F., Biological insights into therapeutic protein modifications throughout trafficking and their biopharmaceutical applications. Int. J. Cell Biol. 2013, 2013, 1-19.
85. Desai, S. G., Continuous and semi-continuous cell culture for production of blood clotting factors. J. Biotechnol. 2015, DOI: 10.1016/j.jbiotech.2015.02.021.
86. Windyga, J., Solano Trujillo, M. H., Hafeman, A. E., BAX326 (RIXUBIS): A novel recombinant factor IX for the control and prevention of bleeding episodes in adults and children with hemophilia B. Ther. Adv. Hematol. 2014, 5, 168-180.
87. Santagostino, E., Negrier, C., Klamroth, R., Tiede, A. et al., Safety and pharmacokinetics of a novel recombinant fusion protein linking coagulation factor IX with albumin (rIX-FP) in hemophilia B patients. Blood 2012, 120, 2405-2411.
88. Collins, P. W., Young, G., Knobe, K., Karim, F. A. et al., Recombinant long-acting glycoPEGylated factor IX in hemophilia B: A multinational randomized phase 3 trial. Blood 2014, 124, 3880-3886.
89. Gutierrez, A. H., Moise, L., De Groot, A. S., Of [Hamsters] and men: A new perspective on host cell proteins. Hum. Vaccin. Immunother. 2012, 8, 1172-1174.
90. Hart, G., Monahan, P., Seligsohn, U., Zakar, M. et al., FVIIa-CTP and FIX-CTP are novel long-acting coagulation factors with prolonged hemostatic activity in hemophilic animal models. Haemophilia 2012, 18, 1-208 Abstract PO-TU-025.
91. Mahlangu, J. N., Coetzee, M. J., Laffan, M., Windyga, J. et al., Phase I, randomized, double-blind, placebo-controlled, single-dose escalation study of the recombinant factor VIIa variant BAY 86-6150 in hemophilia. J. Thromb. Haemost. 2012, 10, 773-780.
92. Pittman, D., Weston, S., Shields, K., Parng, C. et al., A novel FVIIa variant with increased potency and duration of effect compared to wildtype FVIIa. A study in a dog model of hemophilia A. Blood 2011, 118, 2252 Abstract.
93. Zollner, S., Schuermann, D., Raquet, E., Mueller-Cohrs, J. et al., Pharmacological characteristics of a novel, recombinant fusion protein linking coagulation factor VIIa with albumin (rVIIa-FP). J- Thromb. Haemost. 2014, 12, 220-228.
94. Hart, G., Hershkovitz, O., Iilan, A. B., Zakar, M. et al., Mod-5014, a novel long-acting FVIIa proposing an improved prophylactic and on demand treatment for hemophilic patients following SC and IV administration - comprehensive in -vitro and in -vivo evaluation in preparation for clinical studies. Blood 2013, 122, 3578.
95. Reema, J., Patel-Hett, S., Camire, R. M., Fruebis, J., Pittman, D., A zymogen-like factor Xa improves hemostasis in a murine bleeding model. Blood 2014, 124, 1476 Abstract.
96. Walsh, G., Proteins: Biochemistry and Biotechnology, John Wiley & Sons, Ltd. 2014.
97. Durocher, Y., Butler, M., Expression systems for therapeutic glycoprotein production. Curr. Opin. Biotechnol. 2009, 20, 700-707.
98. Coyle, T. E., Reding, M. T., Lin, J. C., Michaels, L. A. et al., Phase I study of BAY 94-9027, a PEGylated B-domain-deleted recombinant factor VIII with an extended half-life, in subjects with hemophilia A. J. Thromb. Haemostasis 2014, 12, 488-496.
99. Mei, B., Pan, C., Jiang, H., Tjandra, H. et al., Rational design of a fully active, long-acting PEGylated factor VIII for hemophilia A treatment. Blood 2010, 116, 270-279.
100. Giangrande, P. L., Porcine factor VIII. Haemophilia 2012, 18, 305-309.
101. Schmidbauer, S., Characterization of recombinant single chain factor VIII, rVIII-singlechain (CSL627). Haemophilia 2012, 18, 1-208 Abstract.
102. Orlova, N. A., Kovnir, S. V., Vorobiev, II., Gabibov, A. G., Coagulation factor IX for hemophilia B therapy. Acta Naturae 2012, 4, 62-73.
103. Yoshitake, S., Schach, B. G., Foster, D. C., Davie, E. W., Kurachi, K., Nucleotide sequence of the gene for human factor IX (antihemophilic factor B). Biochemistry 1985, 24, 3736-3750.
104. Zhou, Y. F., Eng, E. T., Zhu, J., Lu, C. et al., Sequence and structure relationships within von Willebrand factor. Blood 2012, 120, 449-458.