Pharmaceutical Research

, Volume 24, Issue 8, pp 1527–1537 | Cite as

New Insight into the Role of Polyethylene Glycol Acting as Protein Release Modifier in Lipidic Implants

  • Sandra Herrmann
  • Silke Mohl
  • Florence Siepmann
  • Juergen Siepmann
  • Gerhard Winter
Research Paper



It has recently been shown that the addition of polyethylene glycol 6000 (PEG) to lipidic implants fundamentally affects the resulting protein release kinetics and moreover, the underlying mass transport mechanisms (Herrmann, Winter, Mohl, F. Siepmann, & J. Siepmann, J. Control. Release, 2007). However, it is yet unclear in which way PEG acts. It was the aim of this study to elucidate the effect of PEG in a mechanistic manner.

Materials and Methods

rh-interferon α-2a (IFN-α)-loaded, tristearin-based implants containing various amounts of PEG were prepared by compression. Protein and PEG release was monitored in phosphate buffer pH 4.0 and pH 7.4. IFN-α solubility and stability were assessed by reverse phase and size exclusion HPLC, SDS PAGE, fluorescence and FTIR.


Importantly, in presence of PEG IFN-α was drastically precipitated at pH 7.4. In contrast, at pH 4.0 up to a PEG concentration of 20% no precipitation occurred. These fundamental effects of PEG on protein solubility were reflected in the release kinetics of IFN-α from the tristearin implants: At pH 7.4 the protein release rates remained nearly constant over prolonged periods of time, whereas at pH 4.0 high initial bursts and continuously decreasing release rates were observed. Interestingly, it could be shown that IFN-α release was governed by pure diffusion at pH 4.0, irrespective of the PEG content of the matrices. In contrast, at pH 7.4 both—the limited solubility of the protein as well as diffusion through tortuous liquid-filled pores—are dominating.


For the first time it is shown that the release of pharmaceutical proteins can be controlled by an in-situ precipitation within inert matrices.

Key words

lipid polyethylene glycol protein release mechanism solubility 



This study was financially supported by the Centre de Coopération Franco-Bavarois. We further express our grateful thanks to Roche Diagnostics (Penzberg, Germany) for the supply of interferon α-2a and Sasol (Witten, Germany) for the donation of the lipids.


  1. 1.
    S. Herrmann, G. Winter, S. Mohl, F. Siepmann, and J. Siepmann. Mechanisms controlling protein release from lipidic implants: effects of PEG addition. J. Control. Release (2007).
  2. 2.
    J. L. Cleland. Protein delivery from biodegradable microspheres. Pharm. Biotechnol. 10:1–43 (1997).PubMedCrossRefGoogle Scholar
  3. 3.
    W. R. Gombotz and D. K. Pettit. Biodegradable polymers for protein and peptide drug delivery. Bioconjug. Chem. 6:332–351 (1995).PubMedCrossRefGoogle Scholar
  4. 4.
    J. Siepmann and A. Goepferich. Mathematical modeling of bioerodible, polymeric drug delivery systems. Adv. Drug Deliv. Rev. 48:229–247 (2001).PubMedCrossRefGoogle Scholar
  5. 5.
    V. R. Sinha and A. Trehan. Biodegradable microspheres for protein delivery. J. Control. Release 90:261–280 (2003).PubMedCrossRefGoogle Scholar
  6. 6.
    R. Bawa, R. A. Siegel, B. Marasca, M. Karel, and R. Langer. An explanation for the controlled release of macromolecules from polymers. J. Control. Release 1:259–267 (1985).CrossRefGoogle Scholar
  7. 7.
    R. A. Siegel and R. Langer. Controlled release of polypeptides and other macromolecules. Pharm. Res. 1:2–10 (1984).CrossRefGoogle Scholar
  8. 8.
    R. A. Siegel, J. Kost, and R. Langer. Mechanistic studies of macromolecular drug release from macroporous polymers. I. Experiments and preliminary theory concerning completeness of drug release. J. Control. Release 8:223–236 (1989).CrossRefGoogle Scholar
  9. 9.
    K. Fu, A. M. Klibanov, and R. Langer. Protein stability in controlled-release systems. Nat. Biotech. 18:24–25 (2000).CrossRefGoogle Scholar
  10. 10.
    M. Morlock, H. Koll, G. Winter, and T. Kissel. Microencapsulation of rh-erythropoietin, using biodegradable poly(D, L-lactide-co-glycolide). Protein stability and the effects of stabilizing excipients. Eur. J. Pharm. Biopharm. 43:29–36 (1997).CrossRefGoogle Scholar
  11. 11.
    S. P. Schwendeman, M. Cardamone, A. Klibanov, R. Langer, and M. R. Brandon. Stability of proteins and their delivery from biodegradable polymer microspheres. Drugs Pharm. Sci. 77:1–49 (1996).Google Scholar
  12. 12.
    M. van de Weert, W. E. Hennink, and W. Jiskoot. Protein instability in poly(lactic-co-glycolic acid) microparticles. Pharm. Res. 17:1159–1167 (2000).PubMedCrossRefGoogle Scholar
  13. 13.
    A. Maschke, A. Lucke, W. Vogelhuber, C. Fischbach, T. Appel, T. Blunk, and A. Goepferich. Lipids: an alternative material for protein and peptide release. In A.C.S. Symp. Ser. 79 (Carrier-Based Drug Delivery), 2004, pp. 176–196.Google Scholar
  14. 14.
    T. Pongjanyakul, N. J. Medlicott, and I. G. Tucker. Melted glyceryl palmitostearate (GPS) pellets for protein delivery. Int. J. Pharm. 271:53–62 (2004).PubMedCrossRefGoogle Scholar
  15. 15.
    H. Reithmeier, J. Herrmann, and A. Goepferich. Lipid microparticles as a parenteral controlled release device for peptides. J. Control. Release 73:339–350 (2001).PubMedCrossRefGoogle Scholar
  16. 16.
    W. Vogelhuber, E. Magni, M. Mouro, T. Spruss, C. Guse, A. Gazzaniga, and A. Goepferich. Monolithic triglyceride matrices: a controlled-release system for proteins. Pharm. Dev. Technol. 8:71–79 (2003).PubMedCrossRefGoogle Scholar
  17. 17.
    P. Y. Wang. Lipids as excipient in sustained release insulin implants. Int. J. Pharm. 54:223–230 (1989).CrossRefGoogle Scholar
  18. 18.
    S. Mohl and G. Winter. Continuous release of rh-interferon alpha-2a from triglyceride matrices. J. Control. Release 97:67–78 (2004).PubMedCrossRefGoogle Scholar
  19. 19.
    R. L. Cleek, K. C. Ting, S. G. Eskin, and A. G. Mikos. Microparticles of poly(DL-lactic-co-glycolic acid)/poly(ethylene glycol) blends for controlled drug delivery. J. Control. Release 48:259–268 (1997).CrossRefGoogle Scholar
  20. 20.
    W. J. Lin and C. C. Yu. Comparison of protein loaded poly(γ-caprolactone) microparticles prepared by the hot-melt technique. J. Microencapsul. 18:585–592 (2001).PubMedCrossRefGoogle Scholar
  21. 21.
    J. W. Jiang and S. P. Schwendeman. Stabilization and controlled release of bovine serum albumin encapsulated in poly(D, L-lactide) and poly(ethylene glycol) microsphere blends. Pharm. Res. 18:878–885 (2001).PubMedCrossRefGoogle Scholar
  22. 22.
    J. M. Péan, F. Boury, M. C. Venier-Julienne, P. Menei, J. E. Proust, and J. P. Benoit. Why does PEG 400 co-encapsulation improve NGF stability and release from PLGA biodegradable microspheres? Pharm. Res. 16:1294–1299 (1999).PubMedCrossRefGoogle Scholar
  23. 23.
    I. J. Castellanos, R. Crespo, and K. Griebenow. Poly(ethylene glycol) as stabilizer and emulsifying agent: a novel stabilization approach preventing aggregation and inactivation of proteins upon encapsulation in bioerodible polyester microspheres. J. Control. Release 88:135–145 (2003).PubMedCrossRefGoogle Scholar
  24. 24.
    S. Mohl. The development of a sustained and controlled release device for pharmaceutical proteins based on lipid implants. PhD thesis, LMU Munich, Munich (2004). Document available from
  25. 25.
    J. M. Vergnaud. Controlled Drug Release of Oral Dosage Forms. Ellis Horwood Limited, Chichester (1993).Google Scholar
  26. 26.
    J. M. Harris. Poly(ethylene glycol) Chemistry: Biotechnical and Biomedical Applications. Plenum, New York (1992).Google Scholar
  27. 27.
    A. McPherson. Introduction to protein crystallization. Methods 34:254–265 (2004).PubMedCrossRefGoogle Scholar
  28. 28.
    T. Arakawa and S. N. Timasheff. Mechanism of poly(ethylene glycol) interaction with proteins. Biochem. 24:6756–6761 (1985).CrossRefGoogle Scholar
  29. 29.
    D. H. Atha and K. C. Ingham. Mechanism of precipitation of proteins by polyethylene glycols, analysis in terms of excluded volume. J. Biol. Chem. 256:12108–12117 (1981).PubMedGoogle Scholar
  30. 30.
    I. L. Shulgin and E. Ruckenstein. Preferential hydration and solubility of proteins in aqueous solutions of polyethylene glycol. Biophys. Chem. 120:188–198 (2006).PubMedCrossRefGoogle Scholar
  31. 31.
    B. Shenoy, Y. Wang, W. Shan, A. L. and Margolin. Stability of crystalline proteins. Biotechnol. Bioeng. 73:358–369 (2004).CrossRefGoogle Scholar
  32. 32.
    J. C. Falkner, A. M. Al-Somali, J. A. Jamison, J. Zhang, S. L. Adrianse, R. L. Simpson, M. K. Calabretta, W. Radding, G. N. Phillips, Jr., and V. L. Colvin. Generation of size-controlled, submicrometer protein crystals. Chem. Mater. 17:2679–2686 (2005).CrossRefGoogle Scholar
  33. 33.
    R. T. Bartus, M. A. Tracy, D. F. Emerich, and S. E. Zale. Sustained delivery of proteins for novel therapeutic proteins. Science 281:1161–1162 (1998).PubMedCrossRefGoogle Scholar
  34. 34.
    O. L. Johnson, W. Jaworowicz, J. L. Cleland, L. Bailey, M. Charnis, E. Duenas, C. Wu, D. Shepard, S. Magil, T. Last, A. J. S. Jones, and S. D. Putney. The stabilization and encapsulation of human growth hormone into biodegradable microspheres. Pharm. Res. 14:730–735 (1997).PubMedCrossRefGoogle Scholar
  35. 35.
    O. L. Johnson, J. L. Cleland, H. J. Lee, M. Charnis, E. Duenas, W. Jaworowicz, D. Shepard, A. Shihzamani, A. J. S. Jones, and S. D. Putney. A month-long effect from a single injection of microencapsulated human growth hormone. Nature Med. 2:795–799 (1996).PubMedCrossRefGoogle Scholar
  36. 36.
    P. van de Wetering, A. T. Metters, R. G. Schoenmakers, and J. A. Jeffrey. Poly(ethylene glycol) hydrogels formed by conjugate addition with controllable swelling, degradation, and release of pharmaceutically active proteins. J. Control. Release 102:619–627 (2005).PubMedCrossRefGoogle Scholar
  37. 37.
    L. P. Stratton, A. Dong, M. C. Manning, and J. F. Carpenter. Drug delivery matrix containing native protein precipitates suspended in a poloxamer gel. J. Pharm. Sci. 86:1006–1010 (1997).PubMedCrossRefGoogle Scholar
  38. 38.
    A. Dong, P. Huang, and W. S. Caughey. Protein secondary structures in water from second derivative amid I infrared spectra. Biochemistry 29:3303–3308 (1990).PubMedCrossRefGoogle Scholar
  39. 39.
    V. K. Sharma and D. S. Kalonia. Temperature- and pH-induced multiple partially unfolded states of recombinant human Interferon-α2a: Possible Implications in protein stability. Pharm. Res. 20:1721–1729 (2003).PubMedCrossRefGoogle Scholar
  40. 40.
    W. Wang. Protein aggregation and its inhibition in biopharmaceutics. Int. J. Pharm. 289:1–30 (2005).PubMedCrossRefGoogle Scholar
  41. 41.
    Dustrup. Interferon formulations. US Pat. Appl. Publ. 41 (2003).Google Scholar
  42. 42.
    E. Maeyer and P. Somer. Influence of pH on interferon production and activity. Nature 194:1252–1253 (1962).CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2007

Authors and Affiliations

  • Sandra Herrmann
    • 1
  • Silke Mohl
    • 2
  • Florence Siepmann
    • 3
  • Juergen Siepmann
    • 3
  • Gerhard Winter
    • 1
  1. 1.Ludwig-Maximilians-University MunichMunichGermany
  2. 2.Roche Diagnostics GmbHPenzbergGermany
  3. 3.College of Pharmacy, JE 2491University of LilleLilleFrance

Personalised recommendations