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Conjugates of Peptides and Proteins to Polyethylene Glycols

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Book cover Bioconjugation Protocols

Part of the book series: Methods in Molecular Biology™ ((MIMB,volume 283))

Abstract

This chapter provides a critical overview of the technology presently available in the field of protein PEGylation. The chemistry of the polymer and of its reactive derivatives is discussed and presented together with several protocols used to obtain PEG-protein conjugates. The coupling protocols are critically discussed on the basis of the properties of the protein to be modified and those desired for the final product. Methods for product purification and characterization are also provided. The overall information provided will guide the reader toward all of the critical steps involved in the preparation of PEG-protein adducts.

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References

  1. Abuchowski, A., McCoy, J. R., Palczuk, N. C., van Es, T., and Davis, F. F. (1977) Effect of covalent attachment of polyethylene glycol on immunogenicity and circulating life of bovine liver catalase. J. Biol. Chem. 252, 3582–3586.

    PubMed  CAS  Google Scholar 

  2. Abuchowski, A., van Es, T., Palczuk, N. C., and Davis, F. F. (1977) Alteration of immunological properties of bovine serum albumin by covalent attachment of polyethylene glycol. J. Biol. Chem. 252, 3578–3581.

    PubMed  CAS  Google Scholar 

  3. Francis, G. E., Fisher, D., Delgado, C., Malik, F., Gardiner, A., and Neale, D. (1998) PEGylation of cytokines and other therapeutic proteins and peptides: the importance of biological optimisation of coupling techniques. Int. J. Hematol. 68, 1–18.

    Article  PubMed  CAS  Google Scholar 

  4. Lee, L. S., Conover, C., Shi, C., Whitlow, M., and Filpula, D. (1999) Prolonged circulating lives of single-chain Fv proteins conjugated with polyethylene glycol: a comparison of conjugation chemistries and compounds. Bioconjug. Chem. 10, 973–981.

    Article  PubMed  CAS  Google Scholar 

  5. Bückmann A. F., Morr, M., and Johansson, G. (1981) Functionalization of poly(ethylene glycol) and monomethoxy-poly(ethylene glycol). Makromol. Chem. 182, 1379–1384

    Article  Google Scholar 

  6. Harris, J. M., Struck, E. C., Case, M. G., Paley, M. S., Yalpani, M., van Alstine, J. M., et al. (1984) Synthesis and characterization of PEG derivatives. J. Polymer Sci: Polymer Chem. Ed. 22, 341–352.

    Article  CAS  Google Scholar 

  7. Zalipsky, S. (1995) Functionalized poly(ethy1ene glycol) for preparation of biologically relevant conjugates. Bioconjug. Chem. 6, 150–165.

    Article  PubMed  CAS  Google Scholar 

  8. Veronese, F. M. and Morpurgo, M. (1999) Bioconjugation in pharmaceutical chemistry. Farmaco 54, 497–516.

    Article  PubMed  CAS  Google Scholar 

  9. Veronese, F. M. (2001) Peptide and protein PEGylation: a review of problems and solutions. Biomaterials 22, 405–417.

    Article  PubMed  CAS  Google Scholar 

  10. Roberts, M. J., Bentley, M. D., and Harris, J. M. (2002) Chemistry for peptide and protein PEGylation. Adv. Drug Deliv. Rev. 54, 459–476.

    Article  PubMed  CAS  Google Scholar 

  11. Veronese, F. M., Largajolli, R., Boccu, E., Benassi, C. A., and Schiavon, O. (1985) Surface modification of proteins. Activation of monomethoxy-polyethylene glycols by phenylchloroformates and modification of ribonuclease and superoxide dismutase. Appl. Biochem. Biotechnol. 11, 141–152.

    Article  PubMed  CAS  Google Scholar 

  12. Jackson, C. J., Charlton, J. L., Kuzminski, K., Lang, G. M., and Sehon, A. H. (1987) Synthesis, isolation, and characterization of conjugates of ovalbumin with monomethoxypolyethylene glycol using cyanuric chloride as the coupling agent. Anal. Biochem. 165, 114–127.

    Article  PubMed  CAS  Google Scholar 

  13. Woghiren, C., Sharma, B., and Stein, S. (1993) Protected thiol-polyethylene glycol: a new activated polymer for reversible protein modification. Bioconjug. Chem. 4, 314–318.

    Article  PubMed  CAS  Google Scholar 

  14. Miron, T. and Wilchek, M. (1991) A simplified method for the preparation of succinimidyl carbonate polyethylene glycol for coupling to proteins. Bioconjug. Chem. 4, 568–569.

    Article  Google Scholar 

  15. Chamow, S. M., Kogant, T. P., Venuti, M., Gadek, T., Harris, R. J., Peers, D. H., et al. (1994) Modification of CD4 immunoadhesin with monomethoxypoly(ethy1eneglycol) aldehyde via reductive alkylation. Bioconjug. Chem. 5, 133–140.

    Article  PubMed  CAS  Google Scholar 

  16. Monfardini, C., Schiavon, O., Caliceti, P., Morpurgo, M., Harris, J. M., and Veronese, F. M. (1995) A branched monomethoxypoly(ethylene glycol) for protein modification. Bioconjug Chem. 6, 62–69.

    Article  PubMed  CAS  Google Scholar 

  17. Morpurgo, M., Veronese, F. M., Kachensky, D., and Harris, J. M. (1996) Preparation and Characterization of Poly(ethylene glycol) Vinyl Sulfone. Bioconjug. Chem. 7, 363–368.

    Article  PubMed  CAS  Google Scholar 

  18. Dolence, E. K., Hu, C., Tsang R., Sanders C. G., and Osaki, S. (1997) Electrophilic polyethylene oxides for the modification of polysaccharides, polypeptides (proteins) and surfaces, US Patent no. 5,650,234.

    Google Scholar 

  19. Guiotto, A., Pozzobon, M., Sanavio, C., Schiavon, O., Orsolini, P., and Veronese, F. M. (2002) An improved procedure for the synthesis of branched polyethylene glycols (PEGs) with the reporter dipeptide Met-beta Ala for protein conjugation. Bioorg. Med. Chem. Lett. 12, 177–180.

    Article  PubMed  CAS  Google Scholar 

  20. Manjula, B. N., Tsai, A., Upadhya, R., Perumalsamy, K., Smith, P. K., Malavalli, A., et al. (2003) Site-specific PEGylation of hemoglobin at cys-93(beta): correlation between the colligative properties of the PEGylated protein and the length of the conjugated PEG chain. Bioconjug Chem. 14, 464–472.

    Article  PubMed  CAS  Google Scholar 

  21. Riddles, P. W., Blakeley, R. L., and Zerner, B. (1979) Ellman’s reagent: 5, 5′-dithiobis(2-nitrobenzoic acid)—a reexamination. Anal. Biochem. 94, 75–81.

    Article  PubMed  CAS  Google Scholar 

  22. Riddles, P. W., Blakeley, R. L., and Zerner, B. (1983) Reassessment of Ellman’s reagent. Methods Enzymol. 91, 49–60.

    Article  PubMed  CAS  Google Scholar 

  23. Snyder, S. L. and Sobocinski, P. Z. (1975) An improved 2,4,6-trinitrobenzenesulfonic acid method for the determination of amines. Anal Biochem. 64, 284–288.

    Article  PubMed  CAS  Google Scholar 

  24. Stoscheck, C. M. (1990) Quantitation of Protein. Methods Enzymol. 182, 50–69.

    Article  PubMed  CAS  Google Scholar 

  25. Lehninger, A. L. (1970) Biochemistry: the Molecular Basis of Cell Structure and Function. Worth Publishers Inc., New York, NY.

    Google Scholar 

  26. Harris, J. M., Guo, Z., Fang, L., and Morpurgo, M. (1995) PEG-protein tethering for pharmaceutical applications. Proceeding s of the 7th International Symposium on Recent advances in drug delivery systems. Salt Lake City, Utah, February 27th–March 2nd, 1995.

    Google Scholar 

  27. Veronese, F. M. Sacca, B., de Laureto, P. P., Sergi, M., Caliceti, P., Schiavon, O., et al. (2001) New PEGs for peptide and protein modification, suitable for identification of the PEGylation site. Bioconjug. Chem. 12, 62–70.

    Article  PubMed  CAS  Google Scholar 

  28. Orsatti, L. and Veronese, F. M. (1999) An unusual coupling of poly(ethylene glycol) to tyrosine residues in epidermal growth factor. J. Bioactive Compatible Polymers 14, 429–436.

    CAS  Google Scholar 

  29. El-Tayar, N., Zhao, X., and Bentley, M (2002). PEG-LHRH analog conjugates. US Patent Application no. 20020183257.

    Google Scholar 

  30. Holmquist, B., Blumberg, S., and Vallee, B. L. (1976) Superactivation of neutral proteases: acylation with N-hydroxysuccinimide esters. Biochemistry 15, 4675–4680.

    Article  PubMed  CAS  Google Scholar 

  31. Wang, Y. S., Youngster, S., Bausch, J., Zhang, R., McNemar, C., and Wyss, D. F. (2000) Identification of the major positional isomer of pegylated interferon alpha-2b. Biochemistry 39, 10,634–10,640.

    Article  PubMed  CAS  Google Scholar 

  32. Wylie, D. C., Voloch, M., Lee, S., Liu, Y. H., Cannon-Carlson, S., Cutler, C., et al. (2001) Carboxyalkylated histidine is a pH-dependent product of pegylation with SC-PEG. Pharm. Res. 18, 1354–1360.

    Article  PubMed  CAS  Google Scholar 

  33. Sivakolundu, S. G. and Mabrouk, P. A. (2003) Proton NMR study of chemically modified horse heart ferricytochrome c confirms the presence of histidine and lysine-ligated conformers in 30% acetonitrile solution. J. Inorg. Biochem. 94, 381–385.

    Article  PubMed  CAS  Google Scholar 

  34. Riordan, J. F. and Vallee, B. L. (1972) O-acetyl tyrosine. Methods Enzymol. 25, 500–506.

    Article  PubMed  CAS  Google Scholar 

  35. Bentley, M. D., Roberts, M. J., and Harris, J. M. (1998) Reductive amination using poly(ethylene glycol) acetaldehyde hydrate generated in situ: applications to chitosan and lysozyme. J. Pharm. Sci. 87, 1446–1449.

    Article  PubMed  CAS  Google Scholar 

  36. Sherman, M. R., Williams, L. D., Saifer M. G.P., French, J. A., Kwak, L. W., and Oppenheim, J. J. (1997) Poly(ethylene glycol) Chemistry and Biological Applications (Harris, J. M., and Zalipsky, S., eds.), ACS, Washington, DC.

    Google Scholar 

  37. Kinstler, O. B., Brems, D. N., Lauren, S. L., Paige, A. G., Hamburger, J. B., and Treuheit, M. J. (1996) Characterization and stability of N-terminally PEGylated rhG-CSF. Pharm. Res. 13, 996–1002.

    Article  PubMed  CAS  Google Scholar 

  38. Kinstler, O., Molineux, G., Treuheit, M., Ladd, D., and Gegg, C. (2002) Mono-N-terminal poly(ethylene glycol)-protein conjugates Adv. Drug Deliv. Rev. 54, 477–485.

    Article  PubMed  CAS  Google Scholar 

  39. Lee, H., Jang, I. H., Ryu, S. H., and Park, T. G. (2003) N-terminal site-specific mono-PEGylation of epidermal growth factor. Pharm. Res. 20, 818–825.

    Article  PubMed  CAS  Google Scholar 

  40. Habeeb, A. F. S. A. (1966) Determination of free amino groups in proteins by trinitrobenzenesulphonic acid. Anal. Biochem. 14, 328–336.

    Article  PubMed  CAS  Google Scholar 

  41. Goodson, R. J. and Katre, N. V. (1990) Site-directed pegylation of recombinant interleukin-2 at its glycosylation site. Biotechnology 8, 343–346.

    Article  PubMed  CAS  Google Scholar 

  42. Benhar, I., Wang, Q. C., FitzGerald, D., and Pastan, I. (1994) Pseudomonas exotoxin A mutants. Replacement of surface-exposed residues in domain III with cysteine residues that can be modified with polyethylene glycol in a site-specific manner. J. Biol. Chem. 269, 13,398–13,404.

    PubMed  CAS  Google Scholar 

  43. Pepinsky, R. B., Shapiro, R. I., Wang, S., Chakraborty, A., Gill, A., Lepage, D. J., et al. (2002) Long-acting forms of Sonic hedgehog with improved pharmacokinetic and pharmacodynamic properties are efficacious in a nerve injury model. J. Pharm. Sci. 91, 371–387.

    Article  PubMed  CAS  Google Scholar 

  44. Zalipsky, S. and Menon-Rudolph, S. (1997) Hydrazide derivatives of polyethylene glycols) and their bioconjugates, in Poly(ethyleneglycol) Chemistry and Biological Applications (Harris, J. M., and Zalipsky, S., eds.), ACS, Washington, DC, pp. 318–341.

    Chapter  Google Scholar 

  45. Sakane, T. and Pardridge, W. M. (1997) Carboxyl-directed pegylation of brainderived neurotrophic factor markedly reduces systemic clearance with minimal loss of biologic activity. Pharm. Res. 14, 1085–1091.

    Article  PubMed  CAS  Google Scholar 

  46. Larson, R. S., Menard, V., Jacobs, H., and Kim, S. W. (2001) Physicochemical characterization of poly(ethylene glycol)-modified anti-GAD antibodies. Bioconjug. Chem. 12, 861–869.

    Article  PubMed  CAS  Google Scholar 

  47. Sato, H., Yamamoto, K., Hayashi, E., and Takahara, Y. (2000) Transglutaminase-mediated dual and site-specific incorporation of poly(ethylene glycol) derivatives into a chimeric interleukin-2. Bioconjug. Chem. 11, 502–509.

    Article  PubMed  CAS  Google Scholar 

  48. Sato, H. (2002) Enzymatic procedure for site-specific pegylation of proteins. Adv. Drug Deliv. Rev. 54, 487–504.

    Article  PubMed  CAS  Google Scholar 

  49. Uchio, T., Baudys, M., Liu, F. Song, S. C., and Kim, S. W. (1999) Site-specific insulin conjugates with enhanced stability and extended action profile. Adv. Drug Deliv. Rev. 35, 289–306.

    Article  PubMed  CAS  Google Scholar 

  50. Digilio, G., Barbero, L., Bracco, C., Corpillo, D., Esposito, P., Piquet, G., et al. (2003) NMR structure of two novel polyethylene glycol conjugates of the human growth hormone-releasing factor, hGRF(1–29)-NH2. J. Am. Chem. Soc. 125, 3458–3470.

    Article  PubMed  CAS  Google Scholar 

  51. Caliceti, P., Schiavon, O., Sartore, L., Monfardini, C., and Veronese, F. M. (1993) Active site protection of proteolytic enzymes by poly(ethylene glycol) surface modification J. Bioactive Compatible Polymers 8, 41–50.

    Article  CAS  Google Scholar 

  52. Caliceti, P., Morpurgo, M., Schiavon, O., Monfardini, C., and Verronese, F. M. (1994) Preservation of thrombolytic activity of urokinase modified with monomethoxypoly(ethylene glycol) J. Bioactive Compatible Polymers 9, 252–266.

    Article  CAS  Google Scholar 

  53. Clark, R., Olson, K., Fuh, G., Marian, M., Mortensen, D., Teshima, G., et al. (1996) Long-acting growth hormones produced by conjugation with polyethylene glycol. J. Biol. Chem. 271, 21,969–21,977.

    Article  PubMed  CAS  Google Scholar 

  54. Guerra, P. I., Acklin, C., Kosky, A. A., Davis, J. M., Treuheit, M. J., and Brems, D. N. (1998) PEGylation prevents the N-terminal degradation of megakaryocyte growth and development factor. Pharm. Res. 15, 1822–1827.

    Article  PubMed  CAS  Google Scholar 

  55. Snider, J., Neville, C., Yuan, L. C., and Bullock, J. (1992) Characterization of the heterogeneity of polyethylene glycol-modified superoxide dismutase by chromatographic and electrophoretic techniques. J. Chromatogr. 599, 141–155.

    Article  PubMed  CAS  Google Scholar 

  56. Bullock, J., Chowdhury, S., Severdia, A., Sweeney, J., Johnston, D., and Pachla, L. (1997) Comparison of results of various methods used to determine the extent of modification of methoxy polyethylene glycol 5000-modified bovine cupri-zinc superoxide dismutase. Anal. Biochem. 254, 254–262.

    Article  PubMed  CAS  Google Scholar 

  57. Zimmerman, S. B. and Murphy, L. D. (1996) Electrophoresis of polyethylene glycols and related materials as sodium dodecyl sulfate complexes. Anal. Biochem. 234, 190–193.

    Article  PubMed  CAS  Google Scholar 

  58. Na, D. H., Park, M. O., Choi, S. Y., Kim, Y. S., Lee, S. S., Yoo, S. D., et al. (2001) Identification of the modifying sites of mono-PEGylated salmon calcitonins by capillary electrophoresis and MALDI-TOF mass spectrometry. J. Chromatogr. B Biomed. Sci. Appl. 754, 259–263.

    Article  PubMed  CAS  Google Scholar 

  59. Li, W., Wang, Y., Zhu, X., Li, M., and Su, Z. (2002) Preparation and characterization of PEGylated adducts of recombinant human tumor necrosis factor-alpha from Escherichia coli. J. Biotechnol. 92, 251–258.

    Article  PubMed  CAS  Google Scholar 

  60. Vestling, M. M., Murphy, C. M., Keller, D. A., Fenselau, C., Dedinas, J., Ladd, D. L., and Olsen, M. A. (1993) A strategy for characterization of polyethylene glycol-derivatized proteins. A mass spectrometric analysis of the attachment sites in polyethylene glycol-derivatized superoxide dismutase. Drug Metab. Dispos. 21, 911–917.

    PubMed  CAS  Google Scholar 

  61. Watson, E., Shah, B., DePrince, R., Hendren, R. W., and Nelson, R. (1994) Matrix-assisted laser desorption mass spectrometric analysis of a pegylated recombinant protein. Biotechniques 16, 278–281.

    PubMed  CAS  Google Scholar 

  62. Chowdhury, S. K., Doleman, M., and Johnston, D. (1995) Fingerprinting proteins coupled with polymers by mass spectrometry: Investigation of polyethylene glycol-conjugated superoxide dismutase. J. Am. Soc. Mass. Spectrom. 6, 478–487.

    Article  CAS  Google Scholar 

  63. Sims, G. E., and Snape, T. J. (1980) A method for the estimation of polyethylene glycol in plasma protein fractions. Anal. Biochem. 107, 60–63.

    Article  PubMed  CAS  Google Scholar 

  64. Nag, A., Mitra, G., and Ghosh, P. C. (1996) A colorimetric assay for estimation of polyethylene glycol and polyethylene glycolated protein using ammonium ferrothiocyanate. Anal. Biochem. 237, 224–231.

    Article  PubMed  CAS  Google Scholar 

  65. Sartore, L., Caliceti, P., Schiavon, O., and Veronese, F. M. (1991-a). Enzyme modification by MPEG with an amino acid or peptide as spacer arms. Appl. Biochem. Biotechnol. 27, 45–54.

    Article  PubMed  CAS  Google Scholar 

  66. Sartore, L., Caliceti, P., Schiavon, O., Monfardini, C., and Veronese, F. M. (1991) Accurate evaluation method of the polymer content in monomethoxy-(polyethylene glycol) modified proteins based on amino acid analysis. Appl. Biochem. Biotechnol 31, 213–222.

    Article  PubMed  CAS  Google Scholar 

  67. Udenfriend, S., Stein, S., Bohlen, P., Dairman, W., Leimgruber, W., and Weigele, M. (1972) Fluorescamine: a reagent for assay of amino acids, peptides, proteins, and primary amines in the picomole range. Science 178, 871–872.

    Article  PubMed  CAS  Google Scholar 

  68. Stocks, S. J., Jones, A. J., Ramey, C. W., and Brooks, D. E. (1986) A fluorometric assay of the degree of modification of protein primary amines with polyethylene glycol. Anal. Biochem. 154, 232–234.

    Article  PubMed  CAS  Google Scholar 

  69. Mabrouk, P. A. (1994) Effect of pegylation on the structure and function of horse cytochrome c. Bioconjug. Chem. 5, 236–241.

    Article  PubMed  CAS  Google Scholar 

  70. Lee, K. C., Moon, S. C., Park, M. O., Lee, J. T., Na, D. H., Yoo, S. D., et al. (1999) Isolation, characterization, and stability of positional isomers of mono-PEGylated salmon calcitonins. Pharm. Res. 16, 813–818.

    Article  PubMed  CAS  Google Scholar 

  71. Fang, J., Sawa, T., Akaike, T., and Maeda, H. (2002) Tumor-targeted delivery of polyethylene glycol-conjugated D-amino acid oxidase for antitumor therapy via enzymatic generation of hydrogen peroxide. Cancer Res. 62, 3138–3143.

    PubMed  CAS  Google Scholar 

  72. Lee, K. C., Tak, K. K., Park, M. O., Lee, J. T., Woo, B. H., Yoo, S. D., et al. (1999b) Preparation and characterization of polyethylene-glycol-modified salmon calcitonins. Pharm. Dev. Technol. 4, 269–275.

    Article  PubMed  CAS  Google Scholar 

  73. Morpurgo, M., Monfardini, C., Hofland, L. J., Sergi, M., Orsolini, P., Dumont, J. M., et al. (2002) Selective Alkylation and Acylation of alpha and epsilon Amino Groups with PEG in a Somatostatin Analogue: Tailored Chemistry for Optimized Bioconjugates. Bioconjug. Chem. 13, 1238–1243.

    Article  PubMed  CAS  Google Scholar 

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Authors and Affiliations

  1. Pharmaceutical Sciences Department, University of Padova, Padova, Italy

    Margherita Morpurgo & Francesco M. Veronese

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  1. Margherita Morpurgo
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  2. Francesco M. Veronese
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Editors and Affiliations

  1. Fachbereich Chemie der Universität Dortmund, Dortmund, Germany

    Christof M. Niemeyer

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Morpurgo, M., Veronese, F.M. (2004). Conjugates of Peptides and Proteins to Polyethylene Glycols. In: Niemeyer, C.M. (eds) Bioconjugation Protocols. Methods in Molecular Biology™, vol 283. Humana Press, Totowa, NJ. https://doi.org/10.1385/1-59259-813-7:045

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  • DOI: https://doi.org/10.1385/1-59259-813-7:045

  • Publisher Name: Humana Press, Totowa, NJ

  • Print ISBN: 978-1-58829-098-4

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