Protein conjugates purification and characterization

  • Conan J. Fee
Part of the Milestones in Drug Therapy book series (MDT)


Methods for separation and characterization of PEGylated proteins are reviewed in this chapter. It is explained that these methods are challenging because PEG itself is a relatively inert, neutral, hydrophilic polymer and the starting point for PEGylation is a pure protein. Other than changes to molecular weight and size, differences between the properties of the PEGylated forms of a pure protein are relatively small, since they arise only from the addition to the protein of relatively inert, neutral polymer chains, which tend to shield interactions. Physicochemical properties that are routinely used to characterize and purify proteins are discussed with regard to their applications for PEGylated proteins, including molecular mass, size and shape (mass spectrometry, size exclusion chromatography, membranes, capillary electrophoresis, gel electrophoresis), electrostatic charge (cation and anion exchange chromatography, isoelectric point gel electrophoresis, capillary electrophoresis) and relative hydrophobicity (hydrophobic interaction, reversed phase).


Size Exclusion Chromatography Size Exclusion Chromatography Positional Isomer Nominal Molecular Weight Total Molecular Weight 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.


Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.


  1. 1.
    Fee, C.J., Size comparison between proteins PEGylated with branched and linear poly(ethylene glycol) molecules. Biotechnology and Bioengineering, 2007. 98(4): p. 725–31.CrossRefPubMedGoogle Scholar
  2. 2.
    Watson, E., Shah, B., DePrince, R., Hendren, R.W., and Nelson, R., Matrix-assisted laser desorption mass spectrometric analysis of a pegylated recombinant protein. Biotechniques, 1994. 16(2): p. 278–80.PubMedGoogle Scholar
  3. 3.
    Basu, A., Yang, K., Wang, M., Liu, S., Chintala, R., Palm, T., Zhao, H., Peng, P., Wu, D., Zhang, Z. et al., Structure-function engineering of interferon-α-1b for improving stability, solubility, potency, immunogenicity, and pharmacokinetic properties by site-selective mono-PEGylation. Bioconjugate Chem., 2006. 17(3): p. 618–30.CrossRefGoogle Scholar
  4. 4.
    Foser, S., Schacher, A., Weyer, K.A., Brugger, D., Dietel, E., Marti, S., and Schreitmüller, T., Isolation, structural characterization, and antiviral activity of positional isomers of monopegylated interferon [alpha]-2a (PEGASYS). Protein Expression and Purification, 2003. 30(1): p. 78–87.CrossRefPubMedGoogle Scholar
  5. 5.
    Lee, K.C., Moon, S.C., Park, M.O., Lee, J.T., Na, D.H., Yoo, S.D., Lee, H.S., and DeLuca, P.P., Isolation, characterization, and stability of positional isomers of mono-PEGylated salmon calcitonins. Pharmaceutical Research, 1999. 16(6): p. 813–18.CrossRefPubMedGoogle Scholar
  6. 6.
    Li, X.-Q., Lei, J.-D., Su, Z.-G., and Ma, G.-H., Comparison of bioactivities of monopegylated rhG-CSE with branched and linear mPEG. Process Biochemistry, 2007. 42(12): p. 1625–31.CrossRefGoogle Scholar
  7. 7.
    Youn, Y.S., Na, D.H., Yoo, S.D., Song, S.-C, and Lee, K.C., Chromatographic separation and mass spectrometric identification of positional isomers of polyethylene glycol-modified growth hormone-releasing factor (1–29). Journal of Chromatography A, 2004. 1061(1): p. 45–49.CrossRefPubMedGoogle Scholar
  8. 8.
    Fee, C.J. and Van Alstine, J.M., Prediction of viscosity radius and size exclusion chromatography behavior of PEGylated proteins. Bioconjugate Chemistry, 2004. 15(6): p. 1304–13.CrossRefPubMedGoogle Scholar
  9. 9.
    Hagel, L., Gel Filtration, in Protein Purification, J.-C. Janson and Rydén, L., Editors. 1998, John Wiley & Sons: New York.Google Scholar
  10. 10.
    Zheng, C.Y., Ma, G.H., and Su, Z.G., Native PAGE eliminates the problem of PEG-SDS interaction in SDS-PAGE and provides an alternative to HPLC in characterization of protein PEGylation. Electrophoresis, 2007. 28(16): p. 2801–7.CrossRefPubMedGoogle Scholar
  11. 11.
    Fee, C.J. and Van Alstine, J.M., PEG-proteins: Reaction engineering and separation issues. Chemical Engineering Science, 2006. 61(3): p. 924–39.CrossRefGoogle Scholar
  12. 12.
    Bailon, P. and Berthold, W., Polyethylene glycol-conjugated pharmaceutical proteins. Pharmaceutical Science & Technology Today, 1998. 1(8): p. 352–56.CrossRefGoogle Scholar
  13. 13.
    Edwards, CK., Martin, S.W., Seely, J., Kinstler, O.B., Buckel, S., Bendele, A.M., Cosenza, M.E., Feige, U., and Kohno, T., Design of PEGylated soluble tumour necrosis factor receptor type I (PEG STNF-RI) for chronic inflammatory diseases. Advanced Drug Delivery Reviews, 2003. 55: p. 1315–36.CrossRefPubMedGoogle Scholar
  14. 14.
    Maeda, N., Kimura, M., Sasaki, I., Hirose, Y., and Konno, T., Toxicity of bilirubin and detoxification by PEG-bilirubin oxidase conjugate, in Poly(ethylene glycol) chemistry: Biotechical and biomedical applications, J.M. Harris, Editor. 1992, Plenum Press: New York. p. 153–69.Google Scholar
  15. 15.
    Tan, Y., Sun, X., Xu, M., An, Z., Tan, X., Han, Q., Miljkovic, D.A., Yang, M., and Hoffman, R.M., Polyethylene glycol conjugation of recombinant methioninase for cancer therapy. Protein Expression and Purification, 1998. 12(1): p. 45–52.CrossRefPubMedGoogle Scholar
  16. 16.
    Molek, J.R. and Zydney, A.L., Ultrafiltration characteristics of pegylated proteins. Biotechnology and Bioengineering, 2006. 95(3): p. 474–82.CrossRefPubMedGoogle Scholar
  17. 17.
    Molek, J.R. and Zydney, A.L., Separation of PEGylated alpha-lactalbumin from unreacted precursors and byproducts using ultrafiltration. Biotechnology Progress, 2007. 23(6): p. 1417–24.CrossRefPubMedGoogle Scholar
  18. 18.
    Li, W., Zhong, Y., Lin, B., and Su, Z., Characterization of polyethylene glycol-modified proteins by semi-aqueous capillary electrophoresis. Journal of Chromatography A, 2001. 905(1–2): p. 299–307.CrossRefPubMedGoogle Scholar
  19. 19.
    Na, D.H., Park, E.J., Jo, Y.W., and Lee, K.C., Capillary electrophoretic separation of high-molecular-weight poly(ethylene glycol)-modified proteins. Analytical Biochemistry, 2008. 373(2): p. 207–12.CrossRefPubMedGoogle Scholar
  20. 20.
    Na, D.H. and Lee, K.C., Capillary electrophoretic characterization of PEGylated human parathyroid hormone with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Analytical Biochemistry, 2004. 331(2): p. 322–28.CrossRefPubMedGoogle Scholar
  21. 21.
    Brumeanu, T.-D., Zaghouani, H., and Bona, C., Purification of antigenized immunoglobulins derivatized with monomethoxypolyethylene glycol. Journal of Chromatography A, 1995. 696: p. 219–25.CrossRefPubMedGoogle Scholar
  22. 22.
    Esposito, P., Barbero, L., Caccia, P., Caliceti, P., D’Antonio, M., Piquet, G., and Veronese, F., Pegylation of growth hormone-releasing hormone GRF analogues. Advanced Drug Delivery Reviews, 2003. 55(10): p. 1279–91.CrossRefPubMedGoogle Scholar
  23. 23.
    He, X.H., Shaw, P.C., and Tarn, S.C., Reducing the immunogenicity and improving the in vivo activity of trichosanthin by site-directed pegylation. Life Sciences, 1999. 65(4): p. 355–68.CrossRefPubMedGoogle Scholar
  24. 24.
    Kinstler, O.B., Brems, D.N., Lauren, S.L., Paige, A.G., Hamburger, J.B., and Treuheit, M.J., Characterization and stability of N-terminally PEGylated rhG-CSF. Pharmaceutical Research, 1996. 13: p. 996–1002.CrossRefPubMedGoogle Scholar
  25. 25.
    Koumenis, I.L., Shahrokh, Z., Leong, S., Hsei, V., Deforge, L., and Zapata, G., Modulating pharmacokinetics of an anti-interleukin-8 F(ab’)(2) by amine-specific PEGylation with preserved bioactivity. 2000. 198(1): p. 83–95.Google Scholar
  26. 6.
    Manjula, B.N., Tsai, A., Upadhya, R., Perumalsamy, K., Smith, P.K., Malavalli, A., Vandegriff, K., Winslow, R.M., Intaglietta, M., Prabhakaran, M et al., Site-specific PEGylation of hemoglobin at cys-93(b): correlation between the colligative properties of the PEGylated protein and the length of the conjugated PEG chain. Bioconjugate Chem., 2003. 14(2): p. 464–72.CrossRefGoogle Scholar
  27. 27.
    Piquet, G., Gatti, M., Barbero, L., Traversa, S., Caccia, P., and Esposito, P., Set-up of large laboratory-scale chromatographic separations of poly(ethylene glycol) derivatives of the growth hormone-releasing factor 1-29 analogue. Journal of Chromatography A, 2002. 944(1–2): p. 141–48.CrossRefPubMedGoogle Scholar
  28. 28.
    Reddy, K.R., Modi, M., and Pedder, S., Use of PEGinterferon alfa-2a (40 kD) (Pegasys) for the treatment of hepatitis C. Advanced Drug Delivery Reviews, 2002. 54: p. 571–86.CrossRefGoogle Scholar
  29. 29.
    Sato, H., Enzymatic procedure for site-specific pegylation of proteins. Advanced Drug Delivery Reviews, 2002. 54(4): p. 487–504.CrossRefPubMedGoogle Scholar
  30. 30.
    Wang, Y.-S., Youngster, S., Grace, M., Bausch, J., Bordens, R., and Wyss, D.F., Structural and biological characterization of PEGylated interferon alpha-2b and its therapeutic implications. Advanced Drug Delivery Reviews, 2002. 54(4): p. 547–70.CrossRefPubMedGoogle Scholar
  31. 31.
    Pabst, T.M., Buckley, J. J., Ramasubramanyan, N., and Hunter, A.K., Comparison of strong anion-exchangers for the purification of a PEGylated protein. Journal of Chromatography A, 2007. 1147(2): p. 172–82.CrossRefPubMedGoogle Scholar
  32. 32.
    Lee, D.L., Sharif, I., Kodihalli, S., Stewart, D.I.H., and Tsvetnitsky, V., Preparation and characterization of monopegylated human granulocyte-macrophage colony-stimulating factor. Journal of Interferon and Cytokine Research, 2008. 28(2): p. 101–12.CrossRefPubMedGoogle Scholar
  33. 33.
    Yamamoto, S., Fujii, S., Yoshimoto, N., and Akbarzadehlaleh, P., Effects of protein conformational changes on separation performance in electrostatic interaction chromatography: Unfolded proteins and PEGylated proteins. Journal of Biotechnology, 2007. 132(2): p. 196–201.CrossRefPubMedGoogle Scholar
  34. 34.
    Fee, C.J., Bergstrom, J., Stadler, J., Magnusson, R., and Van Alstine, J.M., Challenges related to the processing of PEG-modified proteins, in International Conference on Biopartitioning and Purification (BPP 2005) 2005: Delft, Netherlands.Google Scholar
  35. 35.
    Harris, J.M., ed. Poly(ethylene glycol) Chemistry: Biotechnical and Biomedical Applications. Topics in Applied Chemistry, ed. A.R. Katritzky and Sabongi, G.J. 1992, Plenum Press: New York.Google Scholar
  36. 36.
    Harris, J.M. and Zalipsky, S., eds. Poly(ethylene glycol): Chemistry and Biological Applications. ACS Symposium Series. Vol. 680. 1997, American Chemical Society: Washington D.C.Google Scholar
  37. 37.
    Li, H., Robertson, A.D., and Jensen, J.H., Very fast empirical prediction and rationalization of protein pKa values. Proteins, 2005. 61: p. 704–21.CrossRefPubMedGoogle Scholar
  38. 38.
    Petitpas, I., Petersen, C.E., Ha, C.E., Bhattacharya, A.A., Zunszain, P.A., Ghuman, J., Bhagavan, N.V., and Curry, S., Structural basis of albumin-thyroxine interactions and familial dysalbuminemic hyperthyroxinemia. Proceedings of National Academic Science, USA, 2003. 100: p. 6440–45.CrossRefGoogle Scholar
  39. 39.
    Murgolo, N.J., Windsor, W.T., Hruza, A., TReichert, P., Tsarbopoulos, A., Baldwin, S., Huang, E., Pramanik, S., Ealick, P., and Trotta, P., A homology model of human interferon alpha-2. Proteins, 1993. 17: p. 62–74.CrossRefPubMedGoogle Scholar
  40. 40.
    Clark, R., Olson, K., Fuh, G., Marian, M., Mortensen, D., Teshima, G., Chang, S., Chu, H., Mukku, V., Canova-Davis, E et al., Long-acting growth hormones produced by conjugation with poly(ethylene glycol). Journal of Biological Chemistry, 1996. 271(21): p. 969–77.Google Scholar
  41. 41.
    Nijs, M., Azarkan, M., Smolders, N., Brygier, J., Vincentelli, J., Vries, G.M.P., Duchateau, J., and Looze, Y., Preliminary characterization of poly(ethylene glycol)ylated human growth hormone antagonist, in Poly(ethylene glycol): Chemistry and Biological Applications, J.M. Harris and Zalipsky, S., Editors. 1997, American Chemical Society: Washington, D.C. p. 170–81.Google Scholar
  42. 42.
    Vincentelli, J., Paul, C., Azarkan, M., Guermant, C., El Moussaoui, A., and Looze, Y., Evaluation of the polyethylene glycol-KF-water system in the context of purifying PEG-protein adducts. International Journal of Pharmaceutics, 1999. 176(2): p. 241–49.CrossRefGoogle Scholar
  43. 43.
    Azarkan, M., El Moussaoui, A., van Wuytswinkel, D., Dehon, G., and Looze, Y., Fractionation and purification of the enzymes stored in the latex of carica papaya. Journal of Chromtography B, 2003. 790(1–2): p. 229–38.CrossRefGoogle Scholar
  44. 44.
    Azarkan, M., Maes, D., Bouckaert, J., Thi, M.-H.D., Wyns, L., and Looze, Y, Thiol PEGylation facilitates purification of chymopapain leading to diffraction studies at 1.4 A resolution. Journal of Chromatography A, 1996. 749(1–2): p. 69–72.CrossRefGoogle Scholar
  45. 45.
    Lee, H.S. and Park, T.G., Preparation and characterization of mono-PEGylated epidermal growth factor: evaluation of in vitro biologic activity. Pharmaceutical Research, 2002. 19(6): p. 845–51.CrossRefPubMedGoogle Scholar
  46. 46.
    Lee, L.S., Conover, C., Shi, C., Whitlow, M., and Filpula, D., Prolonged circulating lives of single-chain Fv proteins conjugated with polyethylene glycol: a comparison of conjugation chemistries and compounds. Bioconjugate Chemistry, 1999. 10(6): p. 973–81.CrossRefPubMedGoogle Scholar
  47. 47.
    Veronese, F., Sacca, B., Laureto, P.P.d., Sergi, M., Caliceti, P., Schiavon, O., and Orsolini, P., New PEGs for peptide and protein modification, suitable for identification of the PEGylation site. Bioconjugate Chemistry, 2001. 12(1): p. 62–70.CrossRefPubMedGoogle Scholar
  48. 48.
    Lee, K.C., Tak, K.K., Park, M.O., Lee, J.T., Woo, B.H., Yoo, S.D., Lee, H.S., and DeLuca, P.P., Preparation and characterization of polyethylene-glycol-modified salmon calcitonins. 1999. 4(2): p. 269–75.Google Scholar
  49. 49.
    Johnson, C, Royal, M., Moreadith, R., Bedu-Addo, F., Advant, S., Wan, M., and Conn, G., Monitoring manufacturing process yields, purity and stability of structural variants of PEGylated staphylokinase mutant SY161 by quantitative reverse-phase chromatography. Biomedical Chromatography, 2003. 17(5): p. 335–44.CrossRefPubMedGoogle Scholar

Copyright information

© Birkhäuser Verlag/Switzerland 2009

Authors and Affiliations

  • Conan J. Fee
    • 1
  1. 1.Department of Chemical & Process EngineeringUniversity of CanterburyChristchurchNew Zealand

Personalised recommendations