Mechanical Behavior and Structure of Freeze-Dried Cakes

  • Sarah H. M. Hedberg
  • Sharmila Devi
  • Arnold Duralliu
  • Daryl R. WilliamsEmail author
Part of the Methods in Pharmacology and Toxicology book series (MIPT)


Freeze-drying or the lyophilization of biopharmaceuticals is a standard method for product manufacture in order to increase product shelf-life and minimize the tendency of re-constituted products to aggregate. However, the physical and or mechanical stability of freeze-dried cakes can be problematic, which can directly result in financial losses due to unusable or damaged products. Currently, there is very limited systematic knowledge of the relationship between lyophilization process conditions and the cake-specific physical structure, mechanical performance, and stability. This Chapter reviews the detailed mechanical properties and structure of freeze-dried cakes formed from aqueous solutions with concentrations from 1 to 40% w/v of common excipients, mannitol, sucrose, and trehalose in some detail. In addition, the mechanical properties of commercial freeze-dried products as well as effects of moisture content and ingress into freeze-dried cakes are also reported. Both experimentally measured Young’s moduli and yield stress data scale well with reduced cake density, in line with theoretical predictions from classical cellular solids theory. A novel compressive indentation method is reviewed which can accurately determine a cake’s Young’s modulus and yield stress within 1 min, allowing the potential future use of these mechanical cake attributes as Critical Quality Attributes (CQAs).

Key words

Compressive mechanics Young’s modulus Sucrose Trehalose Mannitol Yield stress Normal indentation 



A.D. acknowledges the support of the EPSRC Centre for Doctoral Training in Emergent Macromolecular Therapies (CDT) at University College London in collaboration with National Institute for Biological Standards and Control (NIBSC, UK). S.H.M.H. acknowledges the financial support of the EPSRC Impact Acceleration Account at Imperial College.


  1. 1.
    Arakawa T, Prestrelski SJ, Kenney WC, Carpenter JF (2001) Factors affecting short-term and long-term stabilities of proteins. Adv Drug Deliv Rev 46:307–326CrossRefGoogle Scholar
  2. 2.
    Carpenter JF, Pikal MJ, Chang BS, Randolph TW (1997) Rational design of stable lyophilized protein formulations: some practical advice. Pharm Res 14:969–975CrossRefGoogle Scholar
  3. 3.
    Pikal MJ (1994) Freeze-drying of proteins: process, formulation, and stability. ACS Publications, Washington, DCCrossRefGoogle Scholar
  4. 4.
    Costantino HR (2004) Excipients for use in lyophilized pharmaceutical peptide, protein and other bioproducts. In: Constantino HR, Pikal MJ (eds) Lyophilization of biopharmaceuticals. AAPS Press, Arlington, VAGoogle Scholar
  5. 5.
    Telikepalli S, Kumru OS, Kim JH, Joshi SB, O’Berry KB, Blake-Haskins AW, Perkins MD, Middaugh CR, Volkin DB (2015) Characterization of the physical stability of a lyophilized IgG1 mAb after accelerated shipping-like stress. J Pharm Sci 104:495–507CrossRefGoogle Scholar
  6. 6.
    Patel SM, Nail SL, Pikal MJ, Geidobler R, Winter G, Hawe A, Davagnino J, Rambhatla Gupta S (2017) Lyophilized drug product cake appearance: what is acceptable? J Pharm Sci 106:1706–1721CrossRefGoogle Scholar
  7. 7.
    Devi S (2014) Mechanical characterisation of freeze-dried biopharmaceuticals. Ph.D. Thesis, Imperial College LondonGoogle Scholar
  8. 8.
    Suzuki Y, Takeda T, Inazu K, Sakamoto T (1990) Influences of physical stress given to supersaturated cephalothin sodium solution upon the freeze-dried product quality, 1. Yakugaku Zasshi 110:849–857CrossRefGoogle Scholar
  9. 9.
    DeLuca P (1976) Research and development of phamaceutical dosage forms. Dev Biol Stand 36:41–50PubMedGoogle Scholar
  10. 10.
    Di Tommaso C, Como C, Gurny R, Möller M (2010) Investigations on the lyophilisation of MPEG–hexPLA micelle based pharmaceutical formulations. Eur J Pharm Sci 40:38–47CrossRefGoogle Scholar
  11. 11.
    Schersch K, Betz O, Garidel P, Muehlau S, Bassarab S, Winter G (2012) Systematic investigation of the effect of lyophilizate collapse on pharmaceutically relevant proteins, Part 2: Stability during storage at elevated temperatures. J Pharm Sci 101:2288–2306CrossRefGoogle Scholar
  12. 12.
    Wekx J, De Kleijn J (1990) The determination of water in freeze dried pharmaceutical products by performing the Karl Fischer titration in the glass container itself. Drug Dev Ind Pharm 16:1465–1472CrossRefGoogle Scholar
  13. 13.
    Parker A, Rigby-Singleton S, Perkins M, Bates D, Le Roux D, Roberts CJ, Madden-Smith C, Lewis L, Teagarden DL, Johnson RE (2010) Determination of the influence of primary drying rates on the microscale structural attributes and physicochemical properties of protein containing lyophilized products. J Pharm Sci 99:4616–4629CrossRefGoogle Scholar
  14. 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 74:153–164PubMedGoogle Scholar
  15. 15.
    Duncan DI, Veale JR, Cook I, Ward K (2010) Using laser-based headspace moisture analysis for rapid nondestructive moisture determination of sterile freeze-dried product. Lighthouse Instruments White Paper, 1–10Google Scholar
  16. 16.
    Schneid SC, Stärtzel PM, Lettner P, Gieseler H (2011) Robustness testing in pharmaceutical freeze-drying: inter-relation of process conditions and product quality attributes studied for a vaccine formulation. Pharm Dev Technol 16:583–590CrossRefGoogle Scholar
  17. 17.
    Jovanović N, Bouchard A, Hofland GW, Witkamp G-J, Crommelin DJ, Jiskoot W (2006) Distinct effects of sucrose and trehalose on protein stability during supercritical fluid drying and freeze-drying. Eur J Pharm Sci 27:336–345CrossRefGoogle Scholar
  18. 18.
    FDA (2009) Inspection guide lyophilization of parenterals 7/93. US FDAGoogle Scholar
  19. 19.
    Hatley R, Franks F (1991) Applications of DSC in the development of improved freeze-drying processes for labile biologicals. J Therm Anal Calorim 37:1905–1914CrossRefGoogle Scholar
  20. 20.
    Pikal M (1995) Modulated DSC studies on proteins in the solid state: relaxation enthalpy, glass transitions and denaturation. Pharm Res 135:13–29Google Scholar
  21. 21.
    Chang BS, Randall CS (1992) Use of subambient thermal analysis to optimize protein lyophilization. Cryobiology 29:632–656CrossRefGoogle Scholar
  22. 22.
    Overcashier DE, Patapoff TW, Hsu CC (1999) Lyophilization of protein formulations in vials: investigation of the relationship between resistance to vapor flow during primary drying and small-scale product collapse. J Pharm Sci 88:688–695CrossRefGoogle Scholar
  23. 23.
    Shamblin SL, Tang X, Chang L, Hancock BC, Pikal MJ (1999) Characterization of the time scales of molecular motion in pharmaceutically important glasses. J Phys Chem B 103:4113–4121CrossRefGoogle Scholar
  24. 24.
    Chongprasert S, Knopp SA, Nail SL (2001) Characterization of frozen solutions of glycine. J Pharm Sci 90:1720–1728CrossRefGoogle Scholar
  25. 25.
    Ma X, Wang D, Bouffard R, MacKenzie A (2001) Characterization of murine monoclonal antibody to tumor necrosis factor (TNF-MAb) formulation for freeze-drying cycle development. Pharm Res 18:196–202CrossRefGoogle Scholar
  26. 26.
    Pikal M, Shah S, Senior D, Lang J (1983) Physical chemistry of freeze-drying: measurement of sublimation rates for frozen aqueous solutions by a microbalance technique. J Pharm Sci 72:635–650CrossRefGoogle Scholar
  27. 27.
    Nail SL, Her L-M, Proffitt CP, Nail LL (1994) An improved microscope stage for direct observation of freezing and freeze drying. Pharm Res 11:1098–1100CrossRefGoogle Scholar
  28. 28.
    Cavatur RK, Suryanarayanan R (1998) Characterization of frozen aqueous solutions by low temperature X-ray powder diffractometry. Pharm Res 15:194–199CrossRefGoogle Scholar
  29. 29.
    Chatterjee K, Shalaev EY, Suryanarayanan R (2005) Raffinose crystallization during freeze-drying and its impact on recovery of protein activity. Pharm Res 22:303–309CrossRefGoogle Scholar
  30. 30.
    Meredith P, Donald AM, Payne RS (1996) Freeze-drying: in situ observations using cryoenvironmental scanning electron microscopy and differential scanning calorimetry. J Pharm Sci 85:631–637CrossRefGoogle Scholar
  31. 31.
    Her L-M, Jefferis RP, Gatlin LA, Braxton B, Nail SL (1994) Measurement of glass transition temperatures in freeze concentrated solutions of non-electrolytes by electrical thermal analysis. Pharm Res 11:1023–1029CrossRefGoogle Scholar
  32. 32.
    Le Meste M, Huang V (1992) Thermomechanical properties of frozen sucrose solutions. J Food Sci 57:1230–1233CrossRefGoogle Scholar
  33. 33.
    Kararli TT, Hurlbut JB, Needham TE (1990) Glass–rubber transitions of cellulosic polymers by dynamic mechanical analysis. J Pharm Sci 79:845–848CrossRefGoogle Scholar
  34. 34.
    Blond G (1994) Mechanical properties of frozen model solutions. In: Fito P, Mulet A, Mckenna B (eds) Water in foods. Pergamon, AmsterdamGoogle Scholar
  35. 35.
    Morris KR, Evans SA, Mackenzie AP, Scheule D, Lordi NG (1994) Prediction of lyophile collapse temperature by dielectric analysis. PDA J Pharm Sci Technol 48:318–329Google Scholar
  36. 36.
    Marques JM, Loch C, Wolff E, Rutledge D (1991) Monitoring freeze-drying by low resolution pulse NMR: determination of sublimation endpoint. J Food Sci 56:1707–1710CrossRefGoogle Scholar
  37. 37.
    Izutsu K, Yoshioka S, Kojima S (1995) Effect of cryoprotectants on the eutectic crystallization of NaCl in frozen solutions studied by differential scanning calorimetry (DSC) and broad-line pulsed NMR. Chem Pharm Bull 43:1804–1806CrossRefGoogle Scholar
  38. 38.
    Lin TP, Hsu CC (2002) Determination of residual moisture in lyophilized protein pharmaceuticals using a rapid and non-invasive method: near infrared spectroscopy. PDA J Pharm Sci Technol 56:196–205Google Scholar
  39. 39.
    Kamat MS, Lodder RA, DeLuca PP (1989) Near-infrared spectroscopic determination of residual moisture in lyophilized sucrose through intact glass vials. Pharm Res 6:961–965CrossRefGoogle Scholar
  40. 40.
    Brülls M, Folestad S, Sparén A, Rasmuson A (2003) In-situ near-infrared spectroscopy monitoring of the lyophilization process. Pharm Res 20:494–499CrossRefGoogle Scholar
  41. 41.
    Chan H-K, Au-Yeung K-L, Gonda I (1999) Development of a mathematical model for the water distribution in freeze-dried solids. Pharm Res 16:660–665CrossRefGoogle Scholar
  42. 42.
    Costantino HR, Curley JG, Wu S, Hsu CC (1998) Water sorption behavior of lyophilized protein–sugar systems and implications for solid-state interactions. Int J Pharm 166:211–221CrossRefGoogle Scholar
  43. 43.
    Duralliu A, Matejtschuk P, Williams DR (2018) Humidity induced collapse in freeze dried cakes: a direct visualization study using DVS. Eur J Pharm Biopharm 127:29–36CrossRefGoogle Scholar
  44. 44.
    Suryanarayanan R, Rastogi S (1995) X-ray powder diffractometry. In: Brittain HG, Bogdanowich SJ, Bugay DE, DeVincentis J, Lewen G, Newman AW (eds) Physical characterization of pharmaceutical solids, Drugs and the pharmaceutical sciences. Marcel Dekker, Inc., New YorkGoogle Scholar
  45. 45.
    Guo Y, Byrn SR, Zografi G (2000) Effects of lyophilization on the physical characteristics and chemical stability of amorphous quinapril hydrochloride. Pharm Res 17:930–936CrossRefGoogle Scholar
  46. 46.
    Overcashier DE (2004) Microscopy of lyophilized proteins. In: Costantino HR, Pikal MJ (eds) Lyophilization of biopharmaceuticals. AAPS press, Arlington, VAGoogle Scholar
  47. 47.
    Mosharraf M (2004) Assessment of degree of disorder (Amorphicity) of lyophilized formulations of growth hormone using isothermal microcalorimetry. Drug Dev Ind Pharm 30:461–472CrossRefGoogle Scholar
  48. 48.
    Reddy R, Chang LL, Luthra S, Collins G, Lopez C, Shamblin SL, Pikal MJ, Gatlin LA, Shalaev EY (2009) The glass transition and sub-Tg-relaxation in pharmaceutical powders and dried proteins by thermally stimulated current. J Pharm Sci 98:81–93CrossRefGoogle Scholar
  49. 49.
    Pikal MJ, Shah S (1990) The collapse temperature in freeze drying: dependence on measurement methodology and rate of water removal from the glassy phase. Int J Pharm 62:165–186CrossRefGoogle Scholar
  50. 50.
    Konstantinidis AK, Kuu W, Otten L, Nail SL, Sever RR (2011) Controlled nucleation in freeze-drying: effects on pore size in the dried product layer, mass transfer resistance, and primary drying rate. J Pharm Sci 100:3453–3470CrossRefGoogle Scholar
  51. 51.
    Harnkarnsujarit N, Charoenrein S, Roos YH (2012) Microstructure formation of maltodextrin and sugar matrices in freeze-dried systems. Carbohydr Polym 88:734–742CrossRefGoogle Scholar
  52. 52.
    Bashir JA, Avis K (1973) Evaluation of excipients in freeze-dried products for injection. Bull Parenter Drug Assoc 27:68–83PubMedGoogle Scholar
  53. 53.
    Devi S, Williams D (2013) Morphological and compressional mechanical properties of freeze-dried mannitol, sucrose, and trehalose cakes. J Pharm Sci 102:4246–4255CrossRefGoogle Scholar
  54. 54.
    Devi S, Williams DR (2014) Density dependent mechanical properties and structures of a freeze dried biopharmaceutical excipient–Sucrose. Eur J Pharm Biopharm 88:492–501CrossRefGoogle Scholar
  55. 55.
    Bedu-Addo FK (2004) Understanding lyophilization formulation development. Pharm Technol 20:10–19Google Scholar
  56. 56.
    Pikal-Cleland KA, Rodríguez-Hornedo N, Amidon GL, Carpenter JF (2000) Protein denaturation during freezing and thawing in phosphate buffer systems: monomeric and tetrameric β-galactosidase. Arch Biochem Biophys 384:398–406CrossRefGoogle Scholar
  57. 57.
    Williams RO, Watts AB, Miller DA (2016) Formulating poorly water soluble drugs. Springer International Publishing, New YorkCrossRefGoogle Scholar
  58. 58.
    Kang J, Lin X, Penera J (2016) Rapid formulation development for monoclonal antibodies BioProcess Int 14(4):40Google Scholar
  59. 59.
    Gibson LJ, Ashby MF (1999) Cellular solids: structure and properties. Cambridge University Press, CambridgeGoogle Scholar
  60. 60.
    Deville S, Saiz E, Nalla RK, Tomsia AP (2006) Freezing as a path to build complex composites. Science 311:515–518CrossRefGoogle Scholar
  61. 61.
    Ashby MF, Medalist RM (1983) The mechanical properties of cellular solids. Metall Trans A 14:1755–1769CrossRefGoogle Scholar
  62. 62.
    Gibson LJ, Ashby MF (1982) The mechanics of three-dimensional cellular materials. Proc R Soc Lond A 382:43–59CrossRefGoogle Scholar
  63. 63.
    De Beer T, Wiggenhorn M, Hawe A, Kasper J, Almeida A, Quinten T, Friess W, Winter G, Vervaet C, Remon JP (2011) Optimization of a pharmaceutical freeze-dried product and its process using an experimental design approach and innovative process analyzers. Talanta 83:1623–1633CrossRefGoogle Scholar
  64. 64.
    Koganti VR, Shalaev EY, Berry MR, Osterberg T, Youssef M, Hiebert DN, Kanka FA, Nolan M, Barrett R, Scalzo G (2011) Investigation of design space for freeze-drying: use of modeling for primary drying segment of a freeze-drying cycle. AAPS PharmSciTech 12:854–861CrossRefGoogle Scholar
  65. 65.
    Mockus L, LeBlond D, Basu PK, Shah RB, Khan MA (2011) A QbD case study: Bayesian prediction of lyophilization cycle parameters. AAPS PharmSciTech 12:442–448CrossRefGoogle Scholar
  66. 66.
    Kremer D, Pikal M, Petre W, Shalaev E, Gatlin L, Kramer T (2009) A procedure to optimize scale-up for the primary drying phase of lyophilization. J Pharm Sci 98:307–318CrossRefGoogle Scholar
  67. 67.
    Cannon A, Shemeley K (2004) Statistical evaluation of vial design features that influence sublimation rates during primary drying. Pharm Res 21:536–542CrossRefGoogle Scholar
  68. 68.
    Grant Y, Matejtschuk P, Dalby PA (2009) Rapid optimization of protein freeze-drying formulations using ultra scale-down and factorial design of experiment in microplates. Biotechnol Bioeng 104:957–964CrossRefGoogle Scholar
  69. 69.
    Sunderland W (2012) 5th International conference on lyophilisation and freeze drying, Bologna, ItalyGoogle Scholar
  70. 70.
    Sneddon IN (1965) The relation between load and penetration in the axisymmetric boussinesq problem for a punch of arbitrary profile. Int J Eng Sci 3:47–57CrossRefGoogle Scholar
  71. 71.
    Chen J, Kimura Y, Adachi S (2007) Surface activities of monoacyl trehaloses in aqueous solution. LWT Food Sci Technol 40:412–417CrossRefGoogle Scholar
  72. 72.
    Qiu L, Liu JZ, Chang SL, Wu Y, Li D (2012) Biomimetic superelastic graphene-based cellular monoliths. Nat Commun 3:1241CrossRefGoogle Scholar
  73. 73.
    Kanungo BP, Gibson LJ (2010) Density–property relationships in collagen–glycosaminoglycan scaffolds. Acta Biomater 6:344–353CrossRefGoogle Scholar
  74. 74.
    Mao JS, Zhao LG, Yin YJ, De Yao K (2003) Structure and properties of bilayer chitosan–gelatin scaffolds. Biomaterials 24:1067–1074CrossRefGoogle Scholar
  75. 75.
    Roberts AP, Garboczi EJ (2001) Elastic moduli of model random three-dimensional closed-cell cellular solids. Acta Mater 49:189–197CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  • Sarah H. M. Hedberg
    • 1
  • Sharmila Devi
    • 1
  • Arnold Duralliu
    • 1
  • Daryl R. Williams
    • 1
    Email author
  1. 1.Surfaces and Particle Engineering Group, Department of Chemical EngineeringImperial College LondonLondonUK

Personalised recommendations