Bulk Protein Solution: Freeze–Thaw Process, Storage and Shipping Considerations

  • Parag KolheEmail author
  • Sumit Goswami
Part of the AAPS Advances in the Pharmaceutical Sciences Series book series (AAPS, volume 38)


Protein drug substance is typically frozen to enable manufacturing flexibility through prolonging the shelf life of drug substance and providing better biochemical stability. The process of freezing and thawing of bulk protein solutions poses several challenges. It is important to understand the process and define mitigation strategies to address these challenges. Just not the process but the choice of storage container can have an impact on stability and downstream operation of formulation during drug product process. Therefore, understanding the options currently available in terms of drug substance storage containers and how to balance the need based on specific scenarios is critical. Storage temperature is as important as the container the drug substance is stored at. Furthermore, shipping drug substance in frozen state requires thorough understanding of logistics and dependence of shipping temperature on stability. This chapter fundamentally looks at the freezing and thawing process, discusses phenomenon of cryoconcentration in various containers and mitigation strategies, consequence of not choosing appropriate storage temperature, and provides considerations for storage and shipping of drug substance.


Drug substance Cryoconcentration Freeze–thaw Glass transition temperature Stability Shipping 


  1. 1.
    Singh SK, et al. Frozen state storage instability of a monoclonal antibody: aggregation as a consequence of trehalose crystallization and protein unfolding. Pharm Res. 2011;28(4):873–85.CrossRefPubMedGoogle Scholar
  2. 2.
    Kolhe P, Badkar A. Freeze-thaw of protein bulk drug substance. AAPS Sterile Prod Focus Group Ann Newsl. 2013:2–5.Google Scholar
  3. 3.
    Singh SK, Nema S. Freezing and thawing of protein solutions. In: Formulation and process development strategies for manufacturing biopharmaceuticals. Hoboken: Wiley; 2010.CrossRefGoogle Scholar
  4. 4.
    Cocks FH, Brower WE. Phase diagram relationships in cryobiology. Cryobiology. 1974;11(4):340–58.CrossRefPubMedGoogle Scholar
  5. 5.
    Angell CA. Liquid fragility and the glass transition in water and aqueous solutions. Chem Rev. 2002;102(8):2627–50.CrossRefPubMedGoogle Scholar
  6. 6.
    Gayle FW, Cocks FH, Shepard ML. The H2O-NaCl-sucrose phasediagram and applications in cryobiology. J Appl Chem Biotechnol. 1977;27:599–607.CrossRefGoogle Scholar
  7. 7.
    Shalaev EY, Franks F. Equilibrium phase diagram of the water-sucrose-NaCl system. Thermochim Acta. 1995;255.Google Scholar
  8. 8.
    Green JL, Angell CA. Phase relationships and vitrification in saccharidewater solutions and the trehalose anomaly. J Phys Chem. 1989;93:2880–2.Google Scholar
  9. 9.
    Siniti M, Jabrane S, Letoffe JM. Study of the respective binary phase diagrams of sorbitol with mannitol, maltitol and water. Thermochim Acta. 1999;325:171–80.CrossRefGoogle Scholar
  10. 10.
    Fahy GM. Analysis of “solution effects” injury. Equations for calculating phase diagram information for the ternary systems NaCl-dimethylsulfoxide-water and NaCl-glycerol-water. Biophys J. 1980;32(2):837–50.CrossRefPubMedPubMedCentralGoogle Scholar
  11. 11.
    Shepard ML, Goldston CS, Cocks FH. The H2O-NaCl-glycerol phase diagram and its application in cryobiology. Cryobiology. 1976;13(1):9–23.CrossRefPubMedPubMedCentralGoogle Scholar
  12. 12.
    Jochem M, Korber C. Extended phase diagrams for the ternary solutions H2O-NaCl-glycerol and H2O-NaCl-hydroxyethylstarch (HES) determined by DSC. Cryobiology. 1987;24:513–36.CrossRefGoogle Scholar
  13. 13.
    Shalaev EY, Kanev AN. Study of the solid-liquid state diagram of the water-glycine-sucrose system. Cryobiology, 1994;31:374–82.CrossRefGoogle Scholar
  14. 14.
    Suzuki T, Franks F. Solid-liquid phase transitions and amorphous states in ternary sucrose-glycine-water systems. J Chem Soc Faraday Trans. 1993;89(17).CrossRefGoogle Scholar
  15. 15.
    Chatterjee K, Shalaev EY, Suryanarayanan R. Partially crystalline systems in lyophilization: I. Use of ternary state diagrams to determine extent of crystallization of bulking agent. J Pharm Sci. 2005;94(4):798–808.CrossRefPubMedGoogle Scholar
  16. 16.
    Akyurt M, Zaki G, Habeebullah B. Freezing phenomena in ice-water systems. Energy Conserv Manage. 2002;43:1773–89.CrossRefGoogle Scholar
  17. 17.
    Bhatnagar BS, Bogner RH, Pikal MJ. Protein stability during freezing: separation of stresses and mechanisms of protein stabilization. Pharm Dev Technol. 2007;12(5):505–23.CrossRefPubMedGoogle Scholar
  18. 18.
    Schenz TW. Glass transitions and product stability—an overview. Food Hydrocolloids. 1995;9:307–15.CrossRefGoogle Scholar
  19. 19.
    Singh SK, et al. Large-scale freezing of biologics: a practitioner’s review, Part 1—freezing mechanisms. BioProcess Int. 2009;7(9):32–44.Google Scholar
  20. 20.
    Franks F. Protein stability: the value of ‘old literature’. Biophys Chem. 2002;96(2–3):117–27.CrossRefPubMedGoogle Scholar
  21. 21.
    Chaplin M. Do we underestimate the importance of water in cell biology? Nat Rev Mol Cell Biol. 2006;7(11):861–6.CrossRefPubMedGoogle Scholar
  22. 22.
    Carpenter JF, Crowe JH. The mechanism of cryoprotection of proteins by solutes. Cryobiology. 1988;25(3):244–55.CrossRefPubMedGoogle Scholar
  23. 23.
    Franks F. Biophysics and biochemistry at low temperatures. 2012.Google Scholar
  24. 24.
    Piedmonte DM, et al. Sorbitol crystallization can lead to protein aggregation in frozen protein formulations. Pharm Res. 2007;24(1):136–46.CrossRefPubMedGoogle Scholar
  25. 25.
    Levine H, Slade L. Thermomechanical properties of small carbohydrate-water glasses and ‘rubbers’: kinetically metastable systems at sub-zero temperatures. J Chem Soc Faraday Trans. 1988;84(8):2619–33.CrossRefGoogle Scholar
  26. 26.
    Costantino H. Excipients for use in lyophilized pharmaceutical peptide, protein and other bioproducts. Lyophilization Biopharm, AAPS. 2004.Google Scholar
  27. 27.
    Williams ML, Landel RF, Ferry JD. The temperature dependence of relaxation mechanisms in amorphous polymers and other glass-forming liquids. JACS. 1955;77:3701–7.CrossRefGoogle Scholar
  28. 28.
    Strambini GB, Gabellieri E. Proteins in frozen solutions: evidence of ice-induced partial unfolding. Biophys J. 1996;70(2):971–6.CrossRefPubMedPubMedCentralGoogle Scholar
  29. 29.
    Strambini GB, Gonnelli M. Protein stability in ice. Biophys J. 2007;92(6):2131–8.CrossRefPubMedGoogle Scholar
  30. 30.
    Dong J, et al. Freezing-induced phase separation and spatial microheterogeneity in protein solutions. J Phys Chem B. 2009;113(30):10081–7.CrossRefPubMedGoogle Scholar
  31. 31.
    Sundaramurthi P, Suryanarayanan R. Trehalose crystallization during freeze-drying: implications on lyoprotection. J Phys Chem Lett. 2010;1:510–4.CrossRefGoogle Scholar
  32. 32.
    Levine H, Slade L. Principles of “cryostabilization” technology from structure/property relationships of carbohydrate/water systems: a review. Cryoletters. 1988;9:21–63.Google Scholar
  33. 33.
    Ablett S, Izzard MJ, Lillford PJ. Differential scanning calorimetric study of frozen sucrose and glycerol solutions. J Chem Soc, Faraday Trans. 1992;88(6):789–94.CrossRefGoogle Scholar
  34. 34.
    Chang BS, Randall CS. Use of subambient thermal analysis to optimize protein lyophilization. Cryobiology. 1992;29:632–56.CrossRefGoogle Scholar
  35. 35.
    Colandene JD, et al. Lyophilization cycle development for a high-concentration monoclonal antibody formulation lacking a crystalline bulking agent. J Pharm Sci. 2007;96(6):1598–608.CrossRefPubMedGoogle Scholar
  36. 36.
    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.CrossRefPubMedGoogle Scholar
  37. 37.
    Hsu CC, et al. Surface denaturation at solid-void interface—a possible pathway by which opalescent particulates form during the storage of lyophilized tissue-type plasminogen activator at high temperatures. Pharm Res. 1995;12(1):69–77.CrossRefPubMedGoogle Scholar
  38. 38.
    Nema S, Avis KE. Freeze-thaw studies of a model protein, lactate dehydrogenase, in the presence of cryoprotectants. J Parenter Sci Technol. 1993;47(2):76–83.PubMedGoogle Scholar
  39. 39.
    Norde W. Adsorption of proteins from solution at the solid-liquid interface. Adv Colloid Interface Sci. 1986;25(4):267–340.CrossRefPubMedGoogle Scholar
  40. 40.
    Sonnichsen FD, et al. Refined solution structure of type III antifreeze protein: hydrophobic groups may be involved in the energetics of the protein-ice interaction. Structure. 1996;4(11):1325–37.CrossRefPubMedGoogle Scholar
  41. 41.
    Griko YV, et al. Cold denaturation of staphylococcal nuclease. Proc Natl Acad Sci U S A. 1988;85(10):3343–7.CrossRefPubMedPubMedCentralGoogle Scholar
  42. 42.
    Franks F. Protein destabilization at low temperatures. Adv Protein Chem. 1995;46:105–39.CrossRefPubMedGoogle Scholar
  43. 43.
    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.Google Scholar
  44. 44.
    Privalov PL. Cold denaturation of proteins. Crit Rev Biochem Mol Biol. 1990;25(4):281–305.CrossRefPubMedGoogle Scholar
  45. 45.
    Brandts JF, Fu J, Nordin JH. Low temperature denaturation of chymotrypsinogen in aqueous solution and in frozen aqueous solution. In: Wolstenholme GEW, O’Connor M, editors. The frozen cell, A Ciba foundation symposium. London: J & A Churchill; 1970. p. 189–212.CrossRefGoogle Scholar
  46. 46.
    Franks F, Hatley RHM. Low temperature unfolding of chymotrypsinogen. Cryoltters. 1985;6(3).Google Scholar
  47. 47.
    Privalov PL, et al. Cold denaturation of myoglobin. J Mol Biol. 1986;190(3):487–98.CrossRefPubMedGoogle Scholar
  48. 48.
    Griko YV, Privalov PL. Calorimetric study of the heat and cold denaturation of beta-lactoglobulin. Biochemistry. 1992;31(37):8810–5.CrossRefPubMedGoogle Scholar
  49. 49.
    Zhang J, et al. NMR study of the cold, heat, and pressure unfolding of ribonuclease A. Biochemistry. 1995;34(27):8631–41.CrossRefPubMedGoogle Scholar
  50. 50.
    Tang XC, Pikal MJ. Measurement of the kinetics of protein unfolding in viscous systems and implications for protein stability in freeze-drying. Pharm Res. 2005;22(7):1176–85.CrossRefPubMedGoogle Scholar
  51. 51.
    Adrover M, et al. The role of hydration in protein stability: comparison of the cold and heat unfolded states of Yfh1. J Mol Biol. 2012;417(5):413–24.CrossRefPubMedGoogle Scholar
  52. 52.
    Zhang L, et al. Mapping hydration dynamics around a protein surface. Proc Natl Acad Sci U S A. 2007;104(47):18461–6.CrossRefPubMedPubMedCentralGoogle Scholar
  53. 53.
    Remmele RL Jr, Stushnoff C, Carpenter JF. Real-time in situ monitoring of lysozyme during lyophilization using infrared spectroscopy: dehydration stress in the presence of sucrose. Pharm Res. 1997;14(11):1548–55.CrossRefPubMedGoogle Scholar
  54. 54.
    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.CrossRefPubMedGoogle Scholar
  55. 55.
    Murase N, Franks F. Salt precipitation during the freeze-concentration of phosphate buffer solutions. Biophys Chem. 1989;34(3):293–300.CrossRefPubMedGoogle Scholar
  56. 56.
    Van Den Berg L. The effect of addition of sodium and potassium chloride to the reciprocal system: KH2PO4-Na2HPO4-H2O on pH and composition during freezing. Arch Biochem Biophys. 1959;84:305–15.CrossRefGoogle Scholar
  57. 57.
    Van Den Berg L, Rose D. Effect of freezing on the pH and composition of sodium and potassium phosphate solutions; the reciprocal system KH2PO4-Na2-HPO4-H2O. Arch Biochem Biophys. 1959;81(2):319–29.CrossRefGoogle Scholar
  58. 58.
    Pikal-Cleland KA, et al. Protein denaturation during freezing and thawing in phosphate buffer systems: monomeric and tetrameric beta-galactosidase. Arch Biochem Biophys. 2000;384(2):398–406.CrossRefPubMedGoogle Scholar
  59. 59.
    Kolhe P, Amend E, Singh SK. Impact of freezing on pH of buffered solutions and consequences for monoclonal antibody aggregation. Biotechnol Prog. 2010;26(3):727–33.CrossRefPubMedGoogle Scholar
  60. 60.
    Pikal MJ, et al. The effects of formulation variables on the stability of freeze-dried human growth hormone. Pharm Res. 1991;8(4):427–36.CrossRefPubMedGoogle Scholar
  61. 61.
    Sarciaux JM, et al. Effects of buffer composition and processing conditions on aggregation of bovine IgG during freeze-drying. J Pharm Sci. 1999;88(12):1354–61.CrossRefPubMedGoogle Scholar
  62. 62.
    Shalaev EY, et al. Thermophysical properties of pharmaceutically compatible buffers at sub-zero temperatures: implications for freeze-drying. Pharm Res. 2002;19(2):195–201.CrossRefPubMedGoogle Scholar
  63. 63.
    Heller MC, Carpenter JF, Randolph TW. Application of a thermodynamic model to the prediction of phase separations in freeze-concentrated formulations for protein lyophilization. Arch Biochem Biophys. 1999;363(2):191–201.CrossRefPubMedGoogle Scholar
  64. 64.
    Heller MC, Carpenter JF, Randolph TW. Protein formulation and lyophilization cycle design: prevention of damage due to freeze-concentration induced phase separation. Biotechnol Bioeng. 1999;63(2):166–74.CrossRefPubMedGoogle Scholar
  65. 65.
    Butler MF. Freeze concentration of solutes at the ice/solution interface studied by optical interferometry. Crystal Growth Des. 2002;2(6):541–8.CrossRefGoogle Scholar
  66. 66.
    Korber C. Phenomena at the advancing ice-liquid interface: solutes, particles and biological cells. Q Rev Biophys. 1988;21(2):229–98.CrossRefPubMedGoogle Scholar
  67. 67.
    Chen YH, Cao E, Cui ZF. An experimental study of freeze concentration in biological media. Food Bioprod Process. 2001;79(1):35–40.CrossRefGoogle Scholar
  68. 68.
    Rodrigues MA, et al. Effect of freezing rate and dendritic ice formation on concentration profiles of proteins frozen in cylindrical vessels. J Pharm Sci. 2011;100(4):1316–29.CrossRefPubMedGoogle Scholar
  69. 69.
    Ayel V, et al. Crystallisation of undercooled aqueous solutions: Experimental study of free dendritic growth in cylindrical geometry. Int J Heat Mass Transf. 2006;49:1876–84.CrossRefGoogle Scholar
  70. 70.
    Miller MA, et al. Frozen-state storage stability of a monoclonal antibody: aggregation is impacted by freezing rate and solute distribution. J Pharm Sci. 2013;102(4):1194–208.CrossRefPubMedGoogle Scholar
  71. 71.
    Wilkins J, Sesin SD, Wisniewski R. Large-scale cryopreservation of biotherapeutic products. Innov Pharm Technol. 2001;8:174–80.Google Scholar
  72. 72.
    Miyawaki O, Liu L, Nakamura K. Effective partition constant of solute between ice and liquid phases in progressive freeze-concentration. J Food Sci. 1998;63(5):756–8.CrossRefGoogle Scholar
  73. 73.
    Wisniewski R, Wu V. Large-scale freezing and thawing of pharmaceutical products. In Biotechnology and biopharmaceutical manufacturing, processing preservation. Boca Raton: CRC Press;1996. p. 7–59.Google Scholar
  74. 74.
    Butler MF. Instability formation and directional dendritic growth of ice studied by optical interferometry. Cryst Growth Des. 2000;1(3):213–23.CrossRefGoogle Scholar
  75. 75.
    Wisniewski R. Developing large-scale cryopreservation systems for biopharmaceutical products. BioPharm. 1998;11:50–6.Google Scholar
  76. 76.
    Kolhe P, Holding E, Lary A, Chico S, Singh SK. Large scale freezing of biologics: understanding protein and solute concentration changes in a cryovessel—Part II. Biopharm Int. 2010;23(7):40–9.Google Scholar
  77. 77.
    Kolhe P, Badkar A. Protein and solute distribution in drug substance containers during frozen storage as well as post-thawing: a tool to understand and define freezing-thawing parameters in biotechnology process development. Biotechnol Bioeng. 2009;27(2):494–504.Google Scholar
  78. 78.
    Kolhe P, Holding E, Lary A, Chico S, Singh SK. Large scale freezing of biologics: understanding protein and solute concentration changes in a cryovessel—Part I. Biopharm Int. 2010;23(6):53–60.Google Scholar
  79. 79.
    Maity H, Karkaria C, Davagnino J. Mapping of solution components, pH changes, protein stability and the elimination of protein precipitation during freeze-thawing of fibroblast growth factor 20. Int J Pharm. 2009;378(1–2):122–35.CrossRefPubMedGoogle Scholar
  80. 80.
    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
  81. 81.
    Tschoepe M, Schmidt R. Impact of freeze/thaw processing on monoclonal antibody stability. In: Bioprocess International conference. 2008.Google Scholar
  82. 82.
    Webb SDW, Webb JN, Hughes TG, Sesin DF, Kincaid AC. Freezing biopharmaceutical using common techniques and the magnitude of bulk scale freeze-concentration. Bioprocess Int. 2002;15(5):22–34.Google Scholar
  83. 83.
    Wisniewski R, Wu VL. Large-scale freezing and thawing of biopharmaceutical products. In: Wu VL, Avis KE, editors. Biotechnology and biopharmaceutical manufacturing, processing, and preservation. Boca Raton: CRC Press; 1996. p. 7–60.Google Scholar

Copyright information

© American Association of Pharmaceutical Scientists 2018

Authors and Affiliations

  1. 1.BioTherapeutics Pharmaceutical Sciences, Pharmaceutical R&DPfizer Inc.St. LouisUSA

Personalised recommendations