Pharmaceutical Research

, Volume 27, Issue 2, pp 371–379 | Cite as

Improved Lysozyme Stability and Release Properties of Poly(lactide-co-glycolide) Implants Prepared by Hot-Melt Extrusion

  • Zahra Ghalanbor
  • Martin Körber
  • Roland BodmeierEmail author
Research Paper



To assess the feasibility of hot-melt extrusion (HME) for preparing implants based on protein/poly(lactide-co-glycolide) (PLGA) formulations with special emphasis on protein stability, burst release and release completeness.


Model protein (lysozyme)-loaded PLGA implants were prepared with a screw extruder and a self-built syringe-die device as a rapid screening tool for HME formulation optimization. Lysozyme stability was determined using DSC, FTIR, HPLC and biological activity. The simultaneous effect of lysozyme and PEG loadings was investigated to obtain optimized formulations with high drug loading but low initial release.


Lysozyme was recovered from implants with full biological activity after HME. The release from all implants reached the 100% value in 60–80 days with nearly complete enzymatic activity of the last fraction of released lysozyme. Pure PLGA implants with up to 20% lysozyme loading could be formulated without initial burst. The incorporation of PEG 400 reduced the initial burst at drug loadings in excess of 20%.


A complete lysozyme recovery in active form with a burst-free and complete release from PLGA implants prepared by hot-melt extrusion was obtained. This is in contrast to many reported microparticulate lysozyme-PLGA systems and suggests the great potential of hot-melt extrusion for the preparation of protein-PLGA implants.


biodegradable implant hot-melt extrusion poly(lactide-co-glycolide) protein release protein stability 



The financial support of the German Academic Exchange Service (DAAD) for Zahra Ghalanbor Ph.D. studies and the support of Laszlo David (Department of Mathematics and Informatics, Freie Universität Berlin) with the MATLAB® code are acknowledged.

Supplementary material

11095_2009_33_MOESM1_ESM.pdf (304 kb)
ESM 1 (PDF 303 kb)


  1. 1.
    Giteau A, Venier-Julienne MC, Aubert-Pouëssel A, Benoit JP. How to achieve sustained and complete protein release from PLGA-based microparticles? Int J Pharm. 2008;350:14–26.CrossRefPubMedGoogle Scholar
  2. 2.
    Aubert-Pouëssel A, Bibby DC, Venier-Julienne MC, Hindre F, Benoît JP. A novel in vitro delivery system for assessing the biological integrity of protein upon release from PLGA microspheres. Pharm Res. 2002;19:1046–51.CrossRefPubMedGoogle Scholar
  3. 3.
    Ghassemi AH, van Steenbergen MJ, Talsma H, van Nostrum CF, Jiskoot W, Crommelin DJA, et al. Preparation and characterization of protein loaded microspheres based on a hydroxylated aliphatic polyester, poly(lactic-co-hydroxymethyl glycolic acid). J Control Release. 2009;138:57–63.CrossRefPubMedGoogle Scholar
  4. 4.
    Giteau A, Venier-Julienne MC, Marchal S, Courthaudon JL, Sergent M, Montero-Menei C, et al. Reversible protein precipitation to ensure stability during encapsulation within PLGA microspheres. Eur J Pharm Biopharm. 2008;70:127–36.CrossRefPubMedGoogle Scholar
  5. 5.
    Kokai LE, Tan H, Jhunjhunwala S, Little SR, Frank JW, Marra KG. Protein bioactivity and polymer orientation is affected by stabilizer incorporation for double-walled microspheres. J Control Release. In Press, Uncorrected Proof.Google Scholar
  6. 6.
    Lee ES, Park KH, Park IS, Na K. Glycol chitosan as a stabilizer for protein encapsulated into poly(lactide-co-glycolide) microparticle. Int J Pharm. 2007;338:310–6.CrossRefPubMedGoogle Scholar
  7. 7.
    Taluja A, Bae YH. Role of a novel multifunctional excipient poly(ethylene glycol)-block-oligo(vinyl sulfadimethoxine) in controlled release of lysozyme from PLGA microspheres. Int J Pharm. 2008;358:50–9.CrossRefPubMedGoogle Scholar
  8. 8.
    Van De Weert M, Hoechstetter J, Hennink WE, Crommelin DJA. The effect of a water/organic solvent interface on the structural stability of lysozyme. J Control Release. 2000;68:351–9.CrossRefPubMedGoogle Scholar
  9. 9.
    Diwan M, Park TG. Pegylation enhances protein stability during encapsulation in PLGA microspheres. J Control Release. 2001;73:233–44.CrossRefPubMedGoogle Scholar
  10. 10.
    Jain RA. The manufacturing techniques of various drug loaded biodegradable poly(lactide-co-glycolide) (PLGA) devices. Biomaterials. 2000;21:2475–90.CrossRefPubMedGoogle Scholar
  11. 11.
    Pérez C, Griebenow K. Improved activity and stability of lysozyme at the water/CH2Cl2 interface: enzyme unfolding and aggregation and its prevention by polyols. J Pharm Pharmacol. 2001;53:1217–26.CrossRefPubMedGoogle Scholar
  12. 12.
    Van De Weert M, Hennink WE, Jiskoot W. Protein instability in poly(lactic-co-glycolic acid) microparticles. Pharm Res. 2000;17:1159–67.CrossRefPubMedGoogle Scholar
  13. 13.
    Prestrelski SJ, Tedeschi N, Arakawa T, Carpenter JF. Dehydration-induced conformational transitions in proteins and their inhibition by stabilizers. Biophys J. 1993;65:661–71.CrossRefPubMedGoogle Scholar
  14. 14.
    Ghaderi R, Carlfors J. Biological activity of lysozyme after entrapment in poly (d, l-lactide-co-glycolide)-microspheres. Pharm Res. 1997;14:1556–62.CrossRefPubMedGoogle Scholar
  15. 15.
    Pérez C, Castellanos IJ, Costantino HR, Al-Azzam W, Griebenow K. Recent trends in stabilizing protein structure upon encapsulation and release from bioerodible polymers. J Pharm Pharmacol. 2002;54:301–13.CrossRefPubMedGoogle Scholar
  16. 16.
    Hageman MJ. The role of moisture in protein stability. Drug Dev Ind Pharm. 1988;14:2047–70.CrossRefGoogle Scholar
  17. 17.
    Bell LN, Hageman MJ, Muraoka LM. Thermally induced denaturation of lyophilized bovine somatotropin and lysozyme as impacted by moisture and excipients. J Pharm Sci. 1995;84:707–12.CrossRefPubMedGoogle Scholar
  18. 18.
    Chien-Hua N, Yuan-Yuan C. FDA perspective on peptide formulation and stability issues. J Pharm Sci. 1998;87:1331–4.CrossRefGoogle Scholar
  19. 19.
    Rothen-Weinhold A, Oudry N, Schwach-Abdellaoui K, Frutiger-Hughes S, Hughes GJ, Jeannerat D, et al. Formation of peptide impurities in polyester matrices during implant manufacturing. Eur J Pharm Biopharm. 2000;49:253–7.CrossRefPubMedGoogle Scholar
  20. 20.
    Park TG, Lee HY, Nam YS. A new preparation method for protein loaded poly(D, L-lactic-co-glycolic acid) microspheres and protein release mechanism study. J Control Release. 1998;55:181–91.CrossRefPubMedGoogle Scholar
  21. 21.
    Van De Weert M, Van’T Hof R, Van Der Weerd J, Heeren RMA, Posthuma G, Hennink WE, et al. Lysozyme distribution and conformation in a biodegradable polymer matrix as determined by FTIR techniques. J Control Release. 2000;68:31–40.CrossRefPubMedGoogle Scholar
  22. 22.
    Kang F, Singh J. Effect of additives on the release of a model protein from PLGA microspheres. AAPS PharmSciTech. 2001;2.Google Scholar
  23. 23.
    Jiang W, Schwendeman SP. Stabilization and controlled release of bovine serum albumin encapsulated in poly(D, L-lactide) and poly(ethylene glycol) microsphere blends. Pharm Res. 2001;18:878–85.CrossRefPubMedGoogle Scholar
  24. 24.
    Malzert A, Boury F, Renard D, Robert P, Lavenant L, Benoît JP, et al. Spectroscopic studies on poly(ethylene glycol)-lysozyme interactions. Int J Pharm. 2003;260:175–86.CrossRefPubMedGoogle Scholar
  25. 25.
    Körber M, Bodmeier R. Development of an in situ forming PLGA drug delivery system. I. Characterization of a non-aqueous protein precipitation. Eur J Pharm Sci. 2008;35:283–92.CrossRefPubMedGoogle Scholar
  26. 26.
    Claudy P, Letoffe JM, Bayol A, Bonnet MC, Maurizot JC. Denaturation versus pH of lysozyme and biosynthetic human growth hormone by differential scanning calorimetry and circular dichroism: a comparative study. Thermochim Acta. 1992;207:227–37.CrossRefGoogle Scholar
  27. 27.
    Shugar D. The measurement of lysozyme activity and the ultra-violet inactivation of lysozyme. BBA–Biochim Biophys Acta. 1952;8:302–9.CrossRefGoogle Scholar
  28. 28.
    Wang J, Chua KM, Wang CH. Stabilization and encapsulation of human immunoglobulin G into biodegradable microspheres. J Colloid Interface Sci. 2004;271:92–101.CrossRefPubMedGoogle Scholar
  29. 29.
    Elkordy AA, Forbes RT, Barry BW. Integrity of crystalline lysozyme exceeds that of a spray-dried form. Int J Pharm. 2002;247:79–90.CrossRefPubMedGoogle Scholar
  30. 30.
    Hill JJ, Shalaev EY, Zografi G. Thermodynamic and dynamic factors involved in the stability of native protein structure in amorphous solids in relation to levels of hydration. J Pharm Sci. 2005;94:1636–67.CrossRefPubMedGoogle Scholar
  31. 31.
    Schwendeman SP, Costantino HR, Gupta RK, Langer R. Peptide, protein and vaccine delivery from implantable polymeric systems: Progress and challenges. Controlled Drug Delivery: Challenges and Strategies; 1997. p. 229–267.Google Scholar
  32. 32.
    Ding QB, Ainsworth P, Plunkett A, Tucker G, Marson H. The effect of extrusion conditions on the functional and physical properties of wheat-based expanded snacks. J Food Eng. 2006;73:142–8.CrossRefGoogle Scholar
  33. 33.
    Akdogan H. Pressure, torque, and energy responses of a twin screw extruder at high moisture contents. Food Res Int. 1996;29:423–9.CrossRefGoogle Scholar
  34. 34.
    Warfield RW. Compressibility of bulk polymers. Polym Eng Sci. 1966;6:176–80.CrossRefGoogle Scholar
  35. 35.
    Tomizawa H, Yamada H, Ueda T, Imoto T. Isolation and characterization of 101-succinimide lysozyme that possesses the cyclic imide at Asp101-Gly102. Biochemistry. 1994;33:8770–4.CrossRefPubMedGoogle Scholar
  36. 36.
    Ritger P, Peppas N. A simple equation for description of solute release I. Fickian and non-fickian release from non-swellable devices in the form of slabs, spheres, cylinders or discs. J Control Release. 1987;5:23–36.CrossRefGoogle Scholar
  37. 37.
    Bezemer JM, Radersma R, Grijpma DW, Dijkstra PJ, Van Blitterswijk CA, Feijen J. Microspheres for protein delivery prepared from amphiphilic multiblock copolymers 2. Modulation of release rate. J Control Release. 2000;67:249–60.CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2009

Authors and Affiliations

  • Zahra Ghalanbor
    • 1
  • Martin Körber
    • 1
  • Roland Bodmeier
    • 1
    Email author
  1. 1.College of PharmacyFreie Universität BerlinBerlinGermany

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