KSBB Journal

The Korean Society for Biotechnology and Bioengineering (한국생물공학회)
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Solid-phase PEGylation for Site-Specific Modification of Recombinant Interferon ${\alpha}$-2a : Process Performance, Characterization, and In-vitro Bioactivity

재조합 인터페론 알파-2a의 부위 특이적 수식을 위한 고체상 PEGylation : 공정 성능, 특성화 및 생물학적 활성

  • Lee, Byung-Kook (Meditox, Inc.) ;
  • Kwon, Jin-Sook (Bioprocessing Research Laboratory, Department of Chemical Engineering, Hanyang University) ;
  • Lee, E.K. (Bioprocessing Research Laboratory, Department of Chemical Engineering, Hanyang University)
  • 이병국 ;
  • 권진숙 (한양대학교 화학공학과 생물공정연구실) ;
  • 이은규 (한양대학교 화학공학과 생물공정연구실)
  • Published : 2006.04.28
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Abstract

In 'solid-phase' PEGylation, the conjugation reaction occurs as the proteins are attached to a solid matrix, and thus it can have distinct advantages over the conventional, solution-phase process. We report a case study: rhIFN-${\alpha}$-2a was first adsorbed to cation exchange resin and then N-terminally PEGylated by aldehyde mPEG of 5, 10, and 20 kD through reductive alkylation. After the PEGylation, salt gradient elution efficiently recovered the mono-PEGylate in a purified form from the unwanted species such as unmodified IFN, unreacted PEG, and others. The mono-PEGylation and its purification were integrated in a single chromatographic step. Depending on the molecular weight of the mPEG aldehyde used, the mono-PEGylation yield ranged 50-64%. We could overcome the major problems of random, or uncontrollable, multi-PEGylation and the post-PEGylation purification difficulties associated with the solution-phase process. N-terminal sequencing and MALDI-TOF MS confirmed that a PEG molecule was conjugated only to the N-terminus. Compared with the unmodified IFN, the mono-PEGylate showed the reduced anti-viral activity as measured by the cell proliferation assay. The bioactivity was reduced more as the higher molecular weight PEG was conjugated. Immunoreactivity, evaluated indirectly by antibody binding activity using a surface plasmon resonance biosensor, also decreased. Nevertheless, trypsin resistance as well as thermal stability was considerably improved.

혈액 내 순환시 안정성 향상과 면역원성의 감소를 위해, rhIFN-${\alpha}$-2a은 N-terminus의 ${\alpha}$-아민기에 mPEG aldehyde를 solid-phase PEGylation 시킨다. CM-Sepharose와 같은 양이온 교환수지가 고체 지지체로 사용되었다. Mono-PEGylate는 양이온 교환 수지에서 unmodified 단백질과 분리되어 용출된다. Site-srecific PEGylation과 mono-PEGylate의 분리가 한 단계의 공정으로 얻어진다는 점은 solid-phase PEGylation의 이점을 뒷받침해준다. 위치 특이성은 peptide digest의 질량 분석과 Edman degradation을 이용한 N-terminal sequencing에 의해 확인하였다. Mono-PEGylate는 항바이러스 활성과 면역원성의 감소를 나타내고, 감소 정도는 결합되는 mPEG의 분자량에 비례한다. Trypsin 저항성과 온도 안정성은 mono-PEGylation에 의해 두드러지게 개선되었다. Solid-phase PEGylation을 통해 종래의 액상 반응에서 나타날 수 있는 재현성 낮은 반응, 부 반응물 생성, 부 반응물 제거 공정 등의 단점을 극복할 수 있었다. 그러나 solid-phase PEGylation의 문제점인 액상 반응에 비교하여 많은 양의 PEG를 사용하여야 한다는 점은 개선되어야 한다.

Keywords

References

  1. Corssmit, E. P. M., J. D. Metz, H. P. Sauerwein, and J. A.Romun (2000), Biologic responses to IFN-a administration in humans, J. Interferon and Cytokine Research 20, 1039-1047 https://doi.org/10.1089/107999000750053690
  2. Potera, C. (2003), Pegylation for improving polypeptidedrugs, Genetic Engineering News. 23, 58-60
  3. Perry, C. M. and Blair, J. (2001),Peginterferon-alpha-2a (40kD), Drugs. 61, 2263-2288 https://doi.org/10.2165/00003495-200161150-00013
  4. Bailon, P., A. Palleroni, C. A. Schaffer, C. L. Spence, W. J. Fung, J. E. Porter, G. K. Ehrlich, W. Pan, Z. X. Xu, M. W. Modi, A. Farid, and W. Berthold (2001) Rational design of a potent, long-lasting form of interferon: A 40 kDa branched polyethylene glycol-conjugated interferon alpha 2a for the treatment of Hepatitis C, Bioconjugate Chemistry 12, 195-202 https://doi.org/10.1021/bc000082g
  5. Harris, J. M., N. E. Martin and M. Modi (2001), PEGylation: A novel process for modifying pharmacokinetics, Clinical Pharmacokinetics 40, 539-551 https://doi.org/10.2165/00003088-200140070-00005
  6. Liptakova, H., P. Kontsek (1990), Problem of recombinant interferon alpha 2-immunogenicity in theraphy, Do Redakcie Doslo 100, 139-143
  7. Reddy, K. R., T. L. Wright, P. J. Pockros, M. Shiffman, G. Eversom, R. Reindollar, M. W. Fride, P. P. Purdum III, D. Jensen, C. Smith, W. M. Lee, T. D. Boyer, A. Lin, S. Pedder, and J. Depamphilis (2001), Efficacy and safety of PEGylated(40 kDa) interferon alpha 2a compared with interferon alpha 2a in noncirrhotic patients with chronic Hepatitis C, Hepatology 33, 433-438 https://doi.org/10.1053/jhep.2001.21747
  8. Pepinsky, R. B., J. L. Doreen, G. Alan, and etc. (2001),Improved pharmacokinetic properties of a polyethyleneglycol modified form of interferon-beta-1a with preserved in vitro bioactivity, The J. pharmacology and experimental therapeutics 297, 1059-1066
  9. Bailon, P., and W. Berthold (1998), Polyethylene glycol- conjugated pharmaceutical proteins, P. S. T. T. 1,352-356
  10. Hinds, K. D. and S. W. Kim (2002), Effect of PEGconjugation on insulin properties, Advanced Drug Delivery Reviews 54, 505-530 https://doi.org/10.1016/S0169-409X(02)00025-X
  11. Kodera, Y., A. Matsushima, M. Hiroto, H. Nishimura, A.Ishii, T. Ueno, and Y. Inada (1998), PEGylation of proteins and bioactive substances for medical and technical applications, Progress Polymer Science 23, 1233-1271 https://doi.org/10.1016/S0079-6700(97)00033-6
  12. Zalipsky, S. (1995), Chemistry of polyethylene glycolconjugates with biologically active molecules, AdvancedDrug Delivery Reviews 16, 157-182 https://doi.org/10.1016/0169-409X(95)00023-Z
  13. Conover, C. D., R. B. Greenwald, A. Pendri and K. L.Shum (1999), Camptothecin delivery systems: the utilityof amino acid spacers for the conjugation of camptothecin with polyethylene glycol to create prodrugs, Anti-cancer Drug Design 14, 499-506
  14. Means, G. E., and R. E. Feeney (1995), Reductive alkylation of proteins, Analytical Biochemistry 224, 1-16 https://doi.org/10.1006/abio.1995.1001
  15. Kinstler, O.B., Brems, D.N., Lauren, S.L., Paige, A.G.,Hamburger, J.B. and Treuheit, M.J. Charqacterization and stability of N-terminally PEGylated rhG-CSF. Pharm. Res. 13, 996-1002 (1996) https://doi.org/10.1023/A:1016042220817
  16. Kinstler, O. B., US patent, 19, 5824784 (1998)
  17. Kinstler, O., G. Molineux, M. Treuheit, D. Ladd, C. Gegg(2002), Mono-N-terminal poly(ethylene glycol)-proteinconjugates, Advaced Durg Delivery 54, 477-485 https://doi.org/10.1016/S0169-409X(02)00023-6
  18. Wang, Y. S., S. youngster, M. Grace, J. Bausch, R. Bordens, and D. F. Wyss (2002), Structural and biological characterization of pegylated recombinant interferon alpha 2b and its therapeutic implications, Advanced Drug Delivery Reviews 54, 547-570 https://doi.org/10.1016/S0169-409X(02)00027-3
  19. Lee, H. S., I. H. Jang, S. H. Ryu, and T. G. Park (2003), N-Terminal site-specific mono-PEGylation of epithermal growth factor, Pharmaceutical Research 20, 818-825 https://doi.org/10.1023/A:1023402123119
  20. Yamamoto, Y., Y. Tsutsumi, Y. Yoshioka, T. Nishibata, K. Kobayashi, T. Okamoto, Y. Mukai, T. Shimizu, S. Nakagawa, S. Nagata, and T. Mayumi (2003), Site-specific PEGylation of a lysine-deficient TNF-alpha with full bioactivity, Nature Biotechnology 21, 546-552 https://doi.org/10.1038/nbt812
  21. You, C. H (2001), Solid-phase mono-PEGylation forfunctionally improved recombinant interferon-alpha-2a, M. S. Thesis, Dep. of Chemical Engineering, Hanyang University, Korea
  22. Lee, B.K., Lee, J.D. and Lee, E.K. (2004), Solid-phase, N-terminus-specific, mono-PEGylation of Recombinant Interferon-$\alpha$-2a: Purification, Characterization, and Bioactivity, presented at ISPPP 2004, Aachen, Germany
  23. Lee, B.K.,, Korean patent #0453185(2004)
  24. Allen, D., R. Baffi, J. Bausch, J. Bongers, M. Costello, J.Dougherty, Jr. M. Federici, R. Garnick, S. Peterson, R.Riggins, K. Sewerin, and J. Tuls (1996), Validation ofpeptide mapping for protein identity and genetic stability, Biologicals 24, 255-275 https://doi.org/10.1006/biol.1996.0034
  25. Takacs, M. A., S. J. Jacobs, R. M. Bordens, and S. J.Swanson. (1999), Detention and characterization ofantibodies to PEG-IFN-alpha 2b using surface plasmonresonance, J. Interferon and Cytokine Research 19,781-789 https://doi.org/10.1089/107999099313631
  26. Rubinstein, S., P. C. Familletti, and S. Pestaka (1981), Convenient assay for interferons, J. Virology 37, 755-758
  27. Gillis, E. H., J. P. Gosling, J. M. Sreenan, and M. Kane (2002), Development and validation of a biosensor-based immunoassay for progesterone in bovine milk, J. Immunological Methods 267, 131-138 https://doi.org/10.1016/S0022-1759(02)00166-7
  28. Lofas, S., M. Malmqvist, I. Ronnberg and E. Stenberg (1991), Bioanalysis with surface plasmon resonance, Sensors and Actuators B 5, 79-84 https://doi.org/10.1016/0925-4005(91)80224-8
  29. Hellman, U., C. Wernstedt, J. Gonez, and C. Heldin (1995), Improvement of an 'In-Gel' digestion procedure for the micropreparation of internal protein fragments for amino acid sequencing, Analytical Biochemistry 224, 451-455 https://doi.org/10.1006/abio.1995.1070
  30. Ghorbel, B., A. S. Kamoun, M. Nasri (2003), Stabilitystudies of protease from bacillus cereus BG1, Enzyme and Microbial Technology 6261, 1-6
  31. Seely, J. E., Buckel, S. D., Green, P. D. and Richey, C. W. (2005), Making site-specific PEGylation work, BioPharm International, March 2005, p.30-41
  32. Kim, C.S. and Lee, E.K. (2000), Effect of operating parameters in in-vitro renaturation of a fusion protein of human growth hormon and glutathione S transferase from inclusion body, Process Biochemistry, 36, 111-117 https://doi.org/10.1016/S0032-9592(00)00185-0