Skip to main content
SpringerLink
Log in

Lyophilization of Synthetic Gene Carriers

  • Protocol
  • First Online:
Book cover Nanotechnology for Nucleic Acid Delivery

Part of the book series: Methods in Molecular Biology ((MIMB,volume 948))

Abstract

Lyophilization, also known as freeze-drying, is a widely used method for stabilization, improvement of long-term storage stability, and simplification of the handling of drugs and/or carrier systems. Lyophilization is time- and energy-consuming; hence, optimized processes are required to avoid time loss and higher costs without compromising product stability. Since the last decade nonviral, synthetic carriers for gene delivery are of increasing interest. However, these systems suffer from poor physical stability in aqueous solution or suspension. Hence, to ensure long-term storage stability lyophilization of the gene carrier systems is favored. Though, lyophilized products retrieving original carrier size and transfection efficiency after reconstitution are mandatory. This chapter gives an overview of the basic steps and troubleshooting for successful lyophilization of synthetic gene carriers. Furthermore the required excipients and their mechanism of action are summarized.

This is a preview of subscription content, log in via an institution to check access.

Access this chapter

Log in via an institution
Protocol
USD 49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD 84.99
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD 119.99
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD 109.99
Price excludes VAT (USA)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Institutional subscriptions

References

  1. Talsma H, Cherng J-Y, Lehrmann H, Kursa M, Ogris M, Hennink WE, Cotten M, Wagner E (1997) Stabilization of gene delivery systems by freeze-drying. Int J Pharm 157:233–238

    Article  CAS  Google Scholar 

  2. Brus C, Kleemann E, Aigner A, Czubayko F, Kissel T (2004) Stabilization of oligonucleotide-polyethylenimine complexes by freeze-drying: physicochemical and biological characterization. J Control Release 95:119–131

    Article  CAS  Google Scholar 

  3. Kasper JC, Schaffert D, Ogris M, Wagner E, Friess W (2011) Development of a lyophilized plasmid/LPEI polyplex formulation with long-term stability—a step closer from promising technology to application. J Control Release 151:246–255

    Google Scholar 

  4. Tamura A, Nagasaki Y (2010) Smart siRNA delivery systems based on polymeric nanoassemblies and nanoparticles. Nanomedicine-Uk 5:1089–1102

    Article  CAS  Google Scholar 

  5. Anchordoquy TJ, Koe GS (2000) Physical ­stability of nonviral plasmid-based therapeutics. J Pharm Sci 89:289–296

    Article  CAS  Google Scholar 

  6. Abdelwahed W, Degobert G, Stainmesse S, Fessi H (2006) Freeze-drying of nanoparticles: formulation, process and storage considerations. Adv Drug Deliv Rev 58:1688–1713

    Article  CAS  Google Scholar 

  7. Schwegman JJ, Hardwick LM, Akers MJ (2005) Practical formulation and process development of freeze-dried products. Pharm Dev Technol 10:151–173

    CAS  Google Scholar 

  8. Wang W (2000) Lyophilization and development of solid protein pharmaceuticals. Int J Pharm 203:1–60

    Article  CAS  Google Scholar 

  9. Anchordoquy TJ, Armstrong TK, MdC M, Allison SD, Zhang Y, Patel MM, Lentz YK, Koe GS (2004) Physical stabilization of plasmid DNA-based therapeutics during freezing and drying. In: Constantino HR, Pikal MJ (eds) Lyophilization of biopharmaceuticals. AAPS Press, Arlington, TX

    Google Scholar 

  10. Tang X, Pikal MJ (2004) Design of freeze-drying processes for pharmaceuticals: practical advice. Pharm Res 21:191–200

    Article  CAS  Google Scholar 

  11. Franks F (1998) Freeze-drying of bioproducts: putting principles into practice. Eur J Pharm Biopharm 45:221–229

    Article  CAS  Google Scholar 

  12. Palmer CNA, Irvine AD, Terron-Kwiatkowski A, Zhao YW, Liao HH, Lee SP, Goudie DR, Sandilands A, Campbell LE, Smith FJD, O’Regan GM, Watson RM, Cecil JE, Bale SJ, Compton JG, DiGiovanna JJ, Fleckman P, Lewis-Jones S, Arseculeratne G, Sergeant A, Munro CS, El Houate B, McElreavey K, Halkjaer LB, Bisgaard H, Mukhopadhyay S, McLean WHI (2006) Common loss-of-function variants of the epidermal barrier protein filaggrin are a major predisposing factor for atopic dermatitis. Nat Genet 38:441–446

    Article  CAS  Google Scholar 

  13. Weidinger S, Illig T, Baurecht H, Irvine AD, Rodriguez E, Diaz-Lacava A, Klopp N, Wagenpfeil S, Zhao YW, Liao HH, Lee SP, Palmer CNA, Jenneck C, Maintz L, Hagemann T, Behrendt M, Ring J, Nothen MM, McLean WHI, Novak N (2006) Loss-of-function variations within the filaggrin gene predispose for atopic dermatitis with allergic sensitizations. J Allergy Clin Immunol 118:214–219

    Article  CAS  Google Scholar 

  14. May JC, Wheeler RM, Etz N, Del Grosso A (1992) Measurement of final container residual moisture in freeze-dried biological products. Dev Biol Stand 74:153–164

    CAS  Google Scholar 

  15. May JC (2010) Regulatory control of freeze-dried products: importance and evaluation of residual moisture. In: Rey L, May JC (eds) Freeze drying/lyophilization of pharmaceutical and biological products, 3rd edn. Informa Healthcare, New York, NY

    Google Scholar 

  16. Oetjen G-W, Haseley P (2004) Measurement of the residual moisture content (RM). In: Oetjen G-W, Haseley P (eds) Freeze-drying, 2nd edn. Viley-VHC, Weinheim

    Google Scholar 

  17. ICH Guidance for Industry (2003) Q1A(R2) stability testing of new drug substances and products

    Google Scholar 

  18. Dietrich C, Maurer F, Roehl H, Friess W (2010) Pharmaceutical packaging for lyophilization applications. In: Rey L, May JC (eds) Freeze drying/lyophilization of pharmaceutical and biological products, 3rd edn. Informa Healthcare, New York, NY

    Google Scholar 

  19. DeGrazio FL (2010) Closure and container considerations in lyophilization. In: Rey L, May JC (eds) Freeze drying/lyophilization of pharmaceutical and biological products, 3rd edn. Informa Healthcare, New York, NY

    Google Scholar 

  20. Ogris M (2005) Gene delivery using polyethylenimine and copolymers. In: Amiji MM (ed) Polymeric gene delivery, principles and applications. CRC Press LLC, Boca Raton, FL, pp 97–106

    Google Scholar 

  21. Pikal-Cleland KA, Rodríguez-Hornedo N, Amidon GL, Carpenter JF (2000) Protein denaturation during freezing and thawing in phosphate buffer systems: monomeric and tetrameric [beta]-galactosidase. Arch Biochem Biophys 384:398–406

    Article  CAS  Google Scholar 

  22. Kasper JC, Schaffert D, Ogris M, Wagner E, Friess W (2011) The establishment of an up-scaled micro-mixer method allows the standardized and reproducible preparation of well-defined plasmid/LPEI polyplexes. Eur J Pharm Biopharm 77:182–185

    Article  CAS  Google Scholar 

  23. Davies LA, Nunez-Alonso GA, Hebel HL, Scheule RK, Cheng SH, Hyde SC, Gill DR (2010) A novel mixing device for the reproducible generation of nonviral gene therapy formulations. BioTech 49:666–668

    Article  CAS  Google Scholar 

  24. Cherng J-Y, van de Wetering P, Talsma H, Crommelin DJA, Hennink WE (1997) Freeze-drying of poly((2-dimethylamino)ethyl methacrylate)-based gene delivery systems. Pharm Res 14:1838–1841

    Article  CAS  Google Scholar 

  25. Molina MC, Allison SD, Anchordoquy TJ (2001) Maintenance of nonviral vector particle size during the freezing step of the lyophilization process is insufficient for preservation of activity: insight from other structural indicators. J Pharm Sci 90:1445–1455

    Article  CAS  Google Scholar 

  26. Hinrichs WLJ, Manceñido FA, Sanders NN, Braeckmans K, De Smedt SC, Demeester J, Frijlink HW (2006) The choice of a suitable oligosaccharide to prevent aggregation of PEGylated nanoparticles during freeze thawing and freeze drying. Int J Pharm 311:237–244

    Article  CAS  Google Scholar 

  27. Anchordoquy TJ, Armstrong TK, Molina MC (2005) Low molecular weight dextrans stabilize nonviral vectors during lyophilization at low osmolalities: concentrating suspensions by rehydration to reduced volumes. J Pharm Sci 94:1226–1236

    Article  CAS  Google Scholar 

  28. Anchordoquy TJ, Armstrong TK, Molina MDC, Allison SD, Zhang Y, Patel MM, Lentz YK, Koe GS (2004) Formulation considerations for DNA-based therapeutics. In: Lu DR, Oeie S (eds) Cellular drug delivery. Humana Press Inc., Totowa, NJ, pp 237–263

    Google Scholar 

  29. Kett V, McMahon D, Ward K (2005) Thermoanalytical techniques for the investigation of the freeze drying process and freeze-dried products. Curr Pharm Biotechnol 6:239–250

    Article  CAS  Google Scholar 

  30. Ward KR, Mateijtschuk P (2010) The use of microscopy, thermal analysis, and impedance measurements to establish critical formulation parameters for freeze-drying cycle development. In: Rey L, May JC (eds) Freeze drying/lyophilization of pharmaceutical and biological products, 3rd edn. Informa Healthcare, New York, NY

    Google Scholar 

  31. Passot S, Tréléa IC, Marin M, Fonseca F (2010) The relevance of thermal properties for improving formulation and cycle development: application to freeze-drying of proteins. In: Rey L, May JC (eds) Freeze drying/lyophilization of pharmaceutical and biological products, 2nd edn. Informa Healthcare, New York, NY

    Google Scholar 

  32. Yu J, Anchordoquy TJ (2009) Synergistic effects of surfactants and sugars on lipoplex ­stability during freeze-drying and rehydration. J Pharm Sci 98:3319–3328

    Article  CAS  Google Scholar 

  33. Hsu CC, Ward CA, Pearlman R, Nguyen HM, Yeung DA, Curley JG (1992) Determining the optimum residual moisture in lyophilized protein pharmaceuticals. Dev Biol Stand 74:255–270

    CAS  Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Department of Pharmacy, Pharmaceutical Technology and Biopharmaceutics, Ludwig-Maximilians-Universität, Munich, Germany

    Julia Christina Kasper, Sarah Küchler & Wolfgang Friess

Authors
  1. Julia Christina Kasper
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Sarah Küchler
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Wolfgang Friess
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Wolfgang Friess .

Editor information

Editors and Affiliations

  1. , Department of Pharmacy, Ludwig-Maximilians-University Munich, Butenandstraße 5-13, Munich, 81377, Germany

    Manfred Ogris

  2. , Department of Pharmaceutical Sciences, Wayne State University, Mack Avenue 259, Detroit, 48201, USA

    David Oupicky

Rights and permissions

Reprints and permissions

Copyright information

© 2013 Springer Science+Business Media, LLC

About this protocol

Cite this protocol

Kasper, J.C., Küchler, S., Friess, W. (2013). Lyophilization of Synthetic Gene Carriers. In: Ogris, M., Oupicky, D. (eds) Nanotechnology for Nucleic Acid Delivery. Methods in Molecular Biology, vol 948. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-62703-140-0_10

Download citation

  • DOI: https://doi.org/10.1007/978-1-62703-140-0_10

  • Published:

  • Publisher Name: Humana Press, Totowa, NJ

  • Print ISBN: 978-1-62703-139-4

  • Online ISBN: 978-1-62703-140-0

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation