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The Korean Society for Biotechnology and Bioengineering (한국생물공학회)
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Industrial-Scale Production of High-Purity Antihemophilic Factor IX from Human Plasma

사람 혈장으로부터 고순도 혈액응고 제9인자의 산업적 생산

  • Kang, Yong (Central Research Center, Green Cross Corp.) ;
  • Choi, Yong-Woon (Central Research Center, Green Cross Corp.) ;
  • Sung, Hark-Mo (Central Research Center, Green Cross Corp.) ;
  • Sohn, Ki-Whan (Central Research Center, Green Cross Corp.) ;
  • Shin, Jeong-Sup (Central Research Center, Green Cross Corp.) ;
  • Kim, In-Seop (Department of Biological Sciences, College of Life Science and Nanotechnology, Hannam University)
  • 강용 ((주)녹십자 종합연구소) ;
  • 최용운 ((주)녹십자 종합연구소) ;
  • 성학모 ((주)녹십자 종합연구소) ;
  • 손기환 ((주)녹십자 종합연구소) ;
  • 신정섭 ((주)녹십자 종합연구소) ;
  • 김인섭 (한남대학교 생명과학과)
  • Published : 2008.02.29
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Abstract

The use of antihemophilic factor IX complex has been associated with a variety of thrombotic complications, the major cause of which was the contamination of thrombogenic proteins such as vitamin K-dependent clotting factors II, VII, and X. In order to produce a commercial factor IX (GreenNine VF) free from thrombogenic potential, industrial-scale production process for high-purity factor IX from human plasma has been developed. The purification process contains cryo-precipitation, DEAE-sephadex A-50 anion-exchange chromatography, DEAE-toyopearl 650M anion-exchange column chromatography, heparin-sepharose 6FF affinity column chromatography, and CM-sepharose FF cation-exchange column chromatography. Also the process includes two viral inactivation and removal procedures, solvent/detergent treatment and nanofiltration using Viresolve NFP filter. The purification yield was 35.4%. The specific activity in the purified concentrate was 190.8 IU/mg which exceeded that in the factor IX complex (FacNine) by a factor of 48. The activities of factor II, VII, and X were not detected in GreenNine VF. SDS-PAGE analysis showed that GreenNine VF had the highest purity in comparison with commercially available high purity factor IX concentrates, Mononine, Octanyne, Berinin HS, and Immunine STIM plus 600. One batch size of the production was 2,400 vials of 250 IU product or 1,200 vials of 500 IU product from 1,600 L cryo-poor plasma.

(주)녹십자는 1986년 "훽나인"을 B형 혈우병 치료제로 제조품목허가를 받아 B형 혈우병치료제 공급을 시작하였다. 또한 1991년 New York Blood Center에서 selvent/detergent 바이러스 불활화 방법을 도입하여 제조공정에 추가한 후 혈액유래 바이러스로부터 안전한 제품을 생산하여 왔다. 하지만 이 제품은 혈액응고 제2인자, 제7인자, 제10인자가 함유된 제9인자 복합체로, 정맥 혈전증과 파종성 혈관내응고병증 같은 혈전형성 부작용이 일어날 가능성이 있어, 훽나인보다 순도, 유효성, 바이러스 안전성이 우수한 제품의 개발이 필요하였다. 이를 위해 고순도 제9인자 제제인 "GreenNine VF" 제조공정을 개발하였다. GreenNine VF 제조공정은 기존의 훽나인 생산 공정에 heparin 친화성 크로마토그래피와 양이온 크로마토그래피가 추가된 공정으로, 바이러스 안전성을 증진시키기 위한 바이러스 필터 공정도 포함하고 있다. 이러한 공정에 의해서 산업적 규모로 생산된 GreenNine VF는 훽나인에 비해 순도와 바이러스 안전성이 월등히 높은 것으로 확인되었다. 또한 고순도 혈액응고 제9인자 제제인 Mononine, Octanyne, Berinin HS, Immunine STIM plus 600보다 순도가 더 높았다. Cryo-poor plasma 1,600 L를 원료로 사용했을 때 1 batch 당 250IU 분병제품 2,400병 이상, 500 IU 분병제품 1,200병 이상을 생산할 수 있었다.

Keywords

References

  1. O'Connell, N. M. (2003), Factor XI deficiency-from molecular genetics to clinical management, Blood Coagul. Fibrinolysis 14(Suppl 1), 59-64 https://doi.org/10.1097/00001721-200306001-00014
  2. Valentino, L. A. and V. M. Oza (2006), Blood safety and the choice of anti-hemophilic factor concentrate, Pedatr. Blood Cancer 47, 245-254 https://doi.org/10.1002/pbc.20895
  3. Kasper , C. L. (1975), Clinical use of prothromb in complex concentrate: Report on thrombiembolic complications, Thromb. Diath. Haemorrh. 33, 640-644
  4. Lusher, J. M. (1991), Tnrombogenicity associated with factor IX complex concentrates, Semin. Hematol. 28(suppl 6), 3-5
  5. Kohler, M., P. Hellstern, E. Lechler, P. Uberfuhr, and G. Muller-Berghaus (1998), Thromboembolic complications associated with the use of prothrombin complex and factor IX concentrates, Thrombo. Haemost. 80, 399-402 https://doi.org/10.1055/s-0037-1615219
  6. Limentani, S. A., K. P. Gowell, and S. R. Deitcher (1995), High-purity factor IX concentrates for treatment of hemophilia B: relative purity and thrombogenic potential, Acta Haematol. 94(suppl 1), 12-17 https://doi.org/10.1159/000204023
  7. Hoots, W. K. (2001), Safety issues affecting hemophilia products, Transfus. Med. Rev. 15(Suppl 1), 11-19 https://doi.org/10.1053/tm.2001.25379
  8. Tagariello G, P. G. Davoli, G. B. Gajo, E. De Biasi, R. Risato, R. Baggio, and A. Traldi (1999), Safety and efficacy of high-purity concentrates in haemophiliac patients undergoing surgery by continuous infusion, Haemophilia. 5, 426-30 https://doi.org/10.1046/j.1365-2516.1999.00335.x
  9. Lambert, T., M. Recht, L. A. Valentino, J. S. Powell, C. Udata, S. T. Sullivan, and D. A. Roth (2007), Reformulated BeneFix: efficacy and safety in previously treated patients with moderately severe to severe haemophilia B, Haemophilia. 13, 233-243 https://doi.org/10.1111/j.1365-2516.2007.01458.x
  10. Yee, T. T. and C. A. Lee (2005), Transfusion-transmitted infection in hemophilia in developing countries, Semin. Thromb. Hemost. 31, 527-537 https://doi.org/10.1055/s-2005-922224
  11. Berkman, S. A. (1988), Infectious complications of blood transfusion, Blood Rev. 2, 206-210 https://doi.org/10.1016/0268-960X(88)90026-4
  12. Mosley, J. W. and J. Rakela (1999), Foundling viruses and transfusion medicine, Transfusion 39, 1041-1044 https://doi.org/10.1046/j.1537-2995.1999.39101041.x
  13. Roberts, P. (1996), Virus safety of plasma products, Rev. Med. Virol. 6, 25-38 https://doi.org/10.1002/(SICI)1099-1654(199603)6:1<25::AID-RMV162>3.0.CO;2-2
  14. Schneider, B., M. Becker, H. H. Brackman, and A. M. Eis-Hubinger (2004), Contantination of coagulation factor concentrates with human parvovirus B19 genotype 1 and 2, Thromb. Haemost. 92, 838-845
  15. Klein, H. G. (2005), Pathogen inactivation technology: cleansing the blood supply, J. Intern. Med. 257, 224-237 https://doi.org/10.1111/j.1365-2796.2005.01451.x
  16. Shin, J. S., Y. W. Choi, H. M. Sung, Y. -W. Ryu, and I. S. Kim (2006), Enhanced virus safety of a solvent/detergent-treated antihemophilic factor IX concentrate by dry-heat treatment, Biotechnol. Bioprocess Eng. 11, 19-25 https://doi.org/10.1007/BF02931863
  17. Kim, I. S., Y. W. Choi, S. R. Lee, and H. M. Sung (2004), Cold ethanol fractionation and heat inactivation of hepatitis A virus during manufacture of albumin from human plasma, Biotechnol. Bioprocess Eng. 9, 57-60
  18. Kim, I. S., Y. W. Choi, and S. R. Lee (2004), Optimization and validation of a virus filtration process for efficient removal of viruses from urokinase solution prepared from human urine, J. Microbiol. Biotechnol. 14, 140-147
  19. Bradford, M. M. (1976), A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding, Anal. Biochem. 72, 248-254 https://doi.org/10.1016/0003-2697(76)90527-3
  20. Grinde, B., K. Stene-Johansen, B. Sharma, T. Hoel, M. Jensenius, and K. Skaug (1997), Characterization of an epidemic of hepatitis A virus involving intravenous drug abusers-infection by needle sharing?, J. Med. Virol. 53, 69-75 https://doi.org/10.1002/(SICI)1096-9071(199709)53:1<69::AID-JMV12>3.0.CO;2-S
  21. Horowitz, M. S., C. Rooks, B. Horowitz, and M. W. Hilgartner (1986), Virus safety of solvent/detergent treated antihaemophiliac factor concentrates, Lancet 2, 186-189
  22. Kim, I. S., Y. W. Choi, H. S. Woo, C. E. Chang, and S. Lee (2000), Solvent/detergent inactivation and chromatographic removal of human immunodeficiency virus during the manufacturing of a high purity antihemophilic factor VllI concentrate, J. Microbiol. 38, 187-191
  23. Suomela, H. (1976), Human coagulation factor IX isolation and characterization, Eur. J. Biochem. 71, 145-154 https://doi.org/10.1111/j.1432-1033.1976.tb11100.x
  24. Kim, J. S., Y. W. Choi, S. R. Lee, Y. Kang, K. M. Lee, D. H. Park, H. S. Woo, and S. Lee (2002), Removal and inactivation of hepatitis A virus during manufacture of urokinase from human urine. Biotechnol. Bioprocess Eng. 7, 340-346 https://doi.org/10.1007/BF02933518
  25. Burnouf, T. and M. Radosevich (2003), Nanofiltration of plasma-derived biopharmaceutical products, Haemophilia. 9, 24-37 https://doi.org/10.1046/j.1365-2516.2003.00701.x
  26. Oshima, K. H., T. T. Evans-strickfaden, A. K. Highsmith, and E. W. Ades (1996), The use of a microporous polyvinylidene fluoride (PVDF) membrane filter to separate contaminating viral particles from biologically important proteins, Biologicals 24, 137-145 https://doi.org/10.1006/biol.1996.0018
  27. World Federation of Hemophilia. (2004), Registry of clotting factor concentrates
  28. Feldman, P. A., P. I. Bradbury, J. D. Williams, G. E. Sims, J. W. McPhee, M. A. Pinnell, L. Harris, G. I. Crombie, and D. R. Evans (1994), Large-scale preparation and biochemical characterization of a new high purity factor IX concentrate prepared by metal chelate affinity chromatography, Blood Coagul. Fibrinolysis 5, 939-948 https://doi.org/10.1097/00001721-199412000-00010