Browse > Article

Solid-phase PEGylation for Site-Specific Modification of Recombinant Interferon ${\alpha}$-2a : Process Performance, Characterization, and In-vitro Bioactivity  

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)
Publication Information
KSBB Journal / v.21, no.2, 2006 , pp. 133-139 More about this Journal
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.
Keywords
Solid-phase PEGylation; protein modification; bioconjugation;
Citations & Related Records
연도 인용수 순위
  • Reference
1 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   DOI   ScienceOn
2 Harris, J. M., N. E. Martin and M. Modi (2001), PEGylation: A novel process for modifying pharmacokinetics, Clinical Pharmacokinetics 40, 539-551   DOI   ScienceOn
3 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
4 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   DOI   ScienceOn
5 Kinstler, O. B., US patent, 19, 5824784 (1998)
6 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   DOI   ScienceOn
7 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   DOI   ScienceOn
8 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   DOI   ScienceOn
9 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   DOI   ScienceOn
10 Perry, C. M. and Blair, J. (2001),Peginterferon-alpha-2a (40kD), Drugs. 61, 2263-2288   DOI
11 Rubinstein, S., P. C. Familletti, and S. Pestaka (1981), Convenient assay for interferons, J. Virology 37, 755-758
12 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
13 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   DOI   ScienceOn
14 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)   DOI
15 Kinstler, O., G. Molineux, M. Treuheit, D. Ladd, C. Gegg(2002), Mono-N-terminal poly(ethylene glycol)-proteinconjugates, Advaced Durg Delivery 54, 477-485   DOI   ScienceOn
16 Liptakova, H., P. Kontsek (1990), Problem of recombinant interferon alpha 2-immunogenicity in theraphy, Do Redakcie Doslo 100, 139-143
17 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
18 Lee, B.K.,, Korean patent #0453185(2004)
19 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   DOI   ScienceOn
20 Means, G. E., and R. E. Feeney (1995), Reductive alkylation of proteins, Analytical Biochemistry 224, 1-16   DOI   ScienceOn
21 Hinds, K. D. and S. W. Kim (2002), Effect of PEGconjugation on insulin properties, Advanced Drug Delivery Reviews 54, 505-530   DOI   ScienceOn
22 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   DOI   ScienceOn
23 Zalipsky, S. (1995), Chemistry of polyethylene glycolconjugates with biologically active molecules, AdvancedDrug Delivery Reviews 16, 157-182   DOI   ScienceOn
24 Potera, C. (2003), Pegylation for improving polypeptidedrugs, Genetic Engineering News. 23, 58-60
25 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   DOI   ScienceOn
26 Ghorbel, B., A. S. Kamoun, M. Nasri (2003), Stabilitystudies of protease from bacillus cereus BG1, Enzyme and Microbial Technology 6261, 1-6
27 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   DOI   ScienceOn
28 Lofas, S., M. Malmqvist, I. Ronnberg and E. Stenberg (1991), Bioanalysis with surface plasmon resonance, Sensors and Actuators B 5, 79-84   DOI   ScienceOn
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   DOI   ScienceOn
30 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
31 You, C. H (2001), Solid-phase mono-PEGylation forfunctionally improved recombinant interferon-alpha-2a, M. S. Thesis, Dep. of Chemical Engineering, Hanyang University, Korea
32 Bailon, P., and W. Berthold (1998), Polyethylene glycol- conjugated pharmaceutical proteins, P. S. T. T. 1,352-356