Chapter 19: Design of a Bulk Freeze-Thaw Process for Biologics

  • Feroz JameelEmail author
  • Tong Zhu
  • Brittney J. Mills
Part of the AAPS Advances in the Pharmaceutical Sciences Series book series (AAPS, volume 35)


Because of their marginal stability in the liquid state, biologic solutions are stored and shipped frozen to enhance stability and operational flexibility for further processing. During the freezing process, protein solutions can undergo denaturation through various pathways depending upon the scale and technologies used. In this chapter, the physics of freezing, various modes of denaturation, and technologies available for large-scale freeze-thaw process are discussed. Practical solutions to address and mitigate the problems are also recommended with illustrations.


Freezing Thawing Cryoconcentration Cold denaturation Proteins Controlled rate freeze-thaw Uncontrolled rate freeze-thaw Carboys Celsius bags CryoVessel 


  1. 1.
    Schmidt DJ, Akers MJ. Cryogranulation: a potential new final process for bulk drug substances. Biopharm. 1997;10:28–32.Google Scholar
  2. 2.
    Jaenicke R. Protein structure and function at low temperatures. Philos Trans R Soc Lond B Biol Sci. 1990;326(1237):535–51; discussion 551–3.CrossRefGoogle Scholar
  3. 3.
    Privalov PL. Cold denaturation of protein. Crit Rev Biochem Mol Biol. 1990;25(4):281–306.CrossRefGoogle Scholar
  4. 4.
    Franks F. Protein destabilization at low temperatures. Adv Protein Chem. 1995;46:105–39.CrossRefGoogle Scholar
  5. 5.
    Chang BS, Kendrick BS, Carpenter JF. Surface-induced denaturation of proteins during freezing and its inhibition by surfactants. J Pharm Sci. 1996;85(12):1325–30.CrossRefGoogle Scholar
  6. 6.
    Strambini GB, Gabellieri E. Proteins in frozen solutions: evidence of ice-induced partial unfolding. Biophys J. 1996;70(2):971–6.CrossRefGoogle Scholar
  7. 7.
    Kreilgaard L, et al. Effect of Tween 20 on freeze-thawing- and agitation-induced aggregation of recombinant human factor XIII. J Pharm Sci. 1998;87(12):1597–603.CrossRefGoogle Scholar
  8. 8.
    Gabellieri E, Strambini GB. Perturbation of protein tertiary structure in frozen solutions revealed by 1-anilino-8-naphthalene sulfonate fluorescence. Biophys J. 2003;85(5):3214–20.CrossRefGoogle Scholar
  9. 9.
    Gabellieri E, Strambini GB. ANS fluorescence detects widespread perturbations of protein tertiary structure in ice. Biophys J. 2006;90(9):3239–45.CrossRefGoogle Scholar
  10. 10.
    Strambini GB, Gonnelli M. Protein stability in ice. Biophys J. 2007;92(6):2131–8.CrossRefGoogle Scholar
  11. 11.
    Franks F, Auffret T. Freeze-drying of pharmaceuticals and biopharmaceuticals: principles and practice. London: The Royal Society of Chemistry; 2008.Google Scholar
  12. 12.
    Padala C, et al. Impact of uncontrolled vs controlled rate freeze-thaw technologies on process performance and product quality. PDA J Pharm Sci Technol. 2010;64(4):290–8.PubMedGoogle Scholar
  13. 13.
    Kapil Gupta NR, Barron L, Ji W, Jameel F, Murphy K. Assessment of protein stability during freeze-thaw process. In: ACS Fall National Meeting. Boston; 2007.Google Scholar
  14. 14.
    Cao E, et al. Effect of freezing and thawing rates on denaturation of proteins in aqueous solutions. Biotechnol Bioeng. 2003;82(6):684–90.CrossRefGoogle Scholar
  15. 15.
    Bradley R. Plotting freezing curves for frozen desserts. Dairy Rec. 1984;85:114–5.Google Scholar
  16. 16.
    Franks F. Freeze-drying: from empiricism to predictability. The significance of glass transitions. Dev Biol Stand. 1992;74:9–18; discussion 19.PubMedGoogle Scholar
  17. 17.
    Jameel F, Searles J. Development and optimization of the freeze-drying processes. In Formulation and process development strategies for manufacturing biopharmaceuticals; 2010. John Wiley & Sons, Ltd: Hoboken, New Jersey.Google Scholar
  18. 18.
    Pikal MJ. Freeze-drying of proteins. Part II: Formulation selection. BioPharm. 1990;3(9):26–30.Google Scholar
  19. 19.
    Gomez G, Pikal MJ, Rodriguez-Hornedo N. Effect of initial buffer composition on pH changes during far-from-equilibrium freezing of sodium phosphate buffer solutions. Pharm Res. 2001;18(1):90–7.CrossRefGoogle Scholar
  20. 20.
    Becktel, WJ, Schellman JA. Protein stability curves. Biopolymers. 1987; 26:1859–1877.
  21. 21.
    Carpenter JF, Crowe JH. The mechanism of cryoprotection of proteins by solutes. Cryobiology. 1988;25(3):244–55.CrossRefGoogle Scholar
  22. 22.
    Lee JC, Timasheff SN. The stabilization of proteins by sucrose. J Biol Chem. 1981;256(14):7193–201.PubMedGoogle Scholar
  23. 23.
    Arakawa T, Timasheff SN. Stabilization of protein structure by sugars. Biochemistry. 1982;21(25):6536–44.CrossRefGoogle Scholar
  24. 24.
    Arakawa T, Timasheff SN. Preferential interactions of proteins with salts in concentrated solutions. Biochemistry. 1982;21(25):6545–52.CrossRefGoogle Scholar
  25. 25.
    Arakawa T, Timasheff SN. Mechanism of poly(ethylene glycol) interaction with proteins. Biochemistry. 1985;24(24):6756–62.CrossRefGoogle Scholar
  26. 26.
    Arakawa T, Timasheff SN. Protein stabilization and destabilization by guanidinium salts. Biochemistry. 1984;23(25):5924–9.CrossRefGoogle Scholar
  27. 27.
    Arakawa T, Kita Y, Carpenter JF. Protein – solvent interactions in pharmaceutical formulations. Pharm Res. 1991;8(3):285–91.CrossRefGoogle Scholar
  28. 28.
    Arakawa T, et al. Factors affecting short-term and long-term stabilities of proteins. Adv Drug Deliv Rev. 2001;46(1-3):307–26.CrossRefGoogle Scholar
  29. 29.
    Carpenter JF, Izutsu K, Randolph TW. Freezing and drying-induced perturbations of protein structure and mechanisms of protein protection by stabilizing additives. In: Rey L, May JC, editors. Freeze-drying/lyophilization of pharmaceutical and biological products, vol. 96. New York: Marcel Dekker, Inc; 1999.Google Scholar
  30. 30.
    Carpenter JF, et al. Rational design of stable lyophilized protein formulations: theory and practice. In: Carpenter JF, Manning MC, editors. Rational design of stable protein formulations, Pharmaceutical biotechnology, vol. 13: Springer; 2002.Google Scholar
  31. 31.
    Wilkins J, Sesin D, Wisniewski R. Large-scale cryopreservation of biotherapeutic products. Innov Pharm Technol. 2001;1:174–80.Google Scholar
  32. 32.
    Hill JP, Buckley PD. The use of pH indicators to identify suitable environments for freezing samples in aqueous and mixed aqueous/nonaqueous solutions. Anal Biochem. 1991;192(2):358–61.CrossRefGoogle Scholar
  33. 33.
    Shalaev EY, et al. Thermophysical properties of pharmaceutically compatible buffers at sub-zero temperatures: Implications for freeze-drying. Pharmaceutical Research. 2002;19(2):195–201.CrossRefGoogle Scholar
  34. 34.
    Kerwin BA, et al. Effects of Tween 80 and sucrose on acute short-term stability and long-term storage at -20 degrees C of a recombinant hemoglobin. J Pharm Sci. 1998;87(9):1062–8.CrossRefGoogle Scholar
  35. 35.
    Kim HL, et al. Modulation of protein adsorption by poloxamer 188 in relation to polysorbates 80 and 20 at solid surfaces. J Pharm Sci. 2014;103(4):1043–9.CrossRefGoogle Scholar
  36. 36.
    Yin J, et al. Effects of excipients on the hydrogen peroxide-induced oxidation of methionine residues in granulocyte colony-stimulating factor. Pharm Res. 2005;22(1):141–7.CrossRefGoogle Scholar
  37. 37.
    MG Scientific supplying laboratories.
  38. 38.
    Nail SL, et al. Fundamentals of freeze-drying in development and manufacture of protein pharmaceuticals, ed. S.L. Nail and M.J. Akers. 2002:281–360.Google Scholar

Copyright information

© American Association of Pharmaceutical Scientists 2020

Authors and Affiliations

  1. 1.Formulation Development, New Biological EntitiesAbbVie (United States)North ChicagoUSA

Personalised recommendations