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Applications of Freezing and Freeze-Drying in Pharmaceutical Formulations

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Survival Strategies in Extreme Cold and Desiccation

Part of the book series: Advances in Experimental Medicine and Biology ((AEMB,volume 1081))

Abstract

Freeze-drying is a popular method to prepare pharmaceutical formulations containing structurally complex active ingredients and drug delivery system carriers. The solidification performed at lower temperature significantly improves storage stability of proteins, peptides, antibiotics, vaccines, and liposomes, which are marginally stable in aqueous solutions. Individual components of the freeze-drying process (freezing, primary drying, secondary drying), however, expose proteins to various stresses. Certain excipients, including disaccharides (e.g., sucrose, trehalose) and amino acids, can be added to protect the proteins and supramolecular drug delivery systems against physical stress associated with freezing and storage by substituting the molecular interactions provided by water molecules. Some excipients embed the active ingredients in glass-state solids with low molecular mobility, thereby reducing chemical reactivity. Thus, the use of appropriate excipients and process control is important to protect proteins during freeze-drying. This chapter describes the applications of freeze-drying in the pharmaceutical production process, mainly focusing on formulation and process optimization for protein therapeutics.

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Abbreviations

ADC:

Antibody–drug conjugate

API:

Active pharmaceutical ingredient

BSE:

Bovine spongiform encephalopathy

CJD:

Creutzfeldt-Jakob disease

DDS:

Drug delivery system

DSC:

Differential scanning calorimeter

LiCl:

Lithium chloride

MS:

Mass spectrometry

NaCl:

Sodium chloride

OD:

Orally disintegrating

PAT:

Process analytical technology

PVP:

Polyvinylpyrrolidone

T g :

Glass transition temperature

T g′ :

Glass transition temperature of maximally freeze-concentrated solute

References

  • Allison SD, Manning MC, Randolph TW, Middleton K, Davis A, Carpenter JF (2000) Optimization of storage stability of lyophilized actin using combinations of disaccharides and dextran. J Pharm Sci 89:199–214

    Article  CAS  Google Scholar 

  • Anchordoquy TJ, Izutsu KI, Randolph TW, Carpenter JF (2001) Maintenance of quaternary structure in the frozen state stabilizes lactate dehydrogenase during freeze-drying. Arch Biochem Biophys 390:35–41

    Article  CAS  Google Scholar 

  • Arakawa T, Prestrelski SJ, Kenney WC, Carpenter JF (2001) Factors affecting short-term and long-term stabilities of proteins. Adv Drug Deliv Rev 46:307–326

    Article  CAS  Google Scholar 

  • Beech KE, Biddlecombe JG, van der Walle CF, Stevens LA, Rigby SP, Burley JC, Allen S (2015) Insights into the influence of the cooling profile on the reconstitution times of amorphous lyophilized protein formulations. Eur J Pharm Biopharm 96:247–254

    Article  CAS  Google Scholar 

  • Bellavia G, Paccou L, Guinet Y, Hedoux A (2014) How does glycerol enhance the bioprotective properties of trehalose? Insight from protein-solvent dynamics. J Phys Chem B 118:8928–8934

    Article  CAS  Google Scholar 

  • Cao W, Xie Y, Krishnan S, Lin H, Ricci M (2013) Influence of process conditions on the crystallization and transition of metastable mannitol forms in protein formulations during lyophilization. Pharm Res 30:131–139

    Article  CAS  Google Scholar 

  • Carpenter JF, Chang BS, Garzon-Rodriguez W, Randolph TW (2002) Rational design of stable lyophilized protein formulations: theory and practice. Pharm Biotechnol 13:109–133

    Article  CAS  Google Scholar 

  • Chang BS, Patro SY (2004) Freeze-drying process development for protein pharmaceuticals. In: Costantino HR, Pikal MJ (eds) Lyophilization of biopharmaceuticals. American Association of Pharmaceutical Scientists, Arlington, pp 113–138

    Google Scholar 

  • Chang LL, Pikal MJ (2009) Mechanisms of protein stabilization in the solid state. J Pharm Sci 98:2886–2908

    Article  CAS  Google Scholar 

  • Chen C, Han D, Cai C, Tang X (2010) An overview of liposome lyophilization and its future potential. J Control Release 142:299–311

    Article  CAS  Google Scholar 

  • Clavaud M, Roggo Y, Dégardin K, Sacré PY, Hubert P, Ziemons E (2016) Moisture content determination in an antibody-drug conjugate freeze-dried medicine by near-infrared spectroscopy: a case study for release testing. J Pharm Biomed Anal 131:380–390

    Article  CAS  Google Scholar 

  • Cleland JL et al (2001) A specific molar ratio of stabilizer to protein is required for storage stability of a lyophilized monoclonal antibody. J Pharm Sci 90:310–321

    Article  CAS  Google Scholar 

  • Costantino HR, Langer R, Klibanov AM (1994) Moisture-induced aggregation of lyophilized insulin. Pharm Res 11:21–29

    Article  CAS  Google Scholar 

  • Costantino HR, Schwendeman SP, Langer R, Klibanov AM (1998) Deterioration of lyophilized pharmaceutical proteins. Biochemistry 63:357–363

    CAS  PubMed  Google Scholar 

  • Devineni D, Gonschorek C, Cicerone MT, Xu Y, Carpenter JF, Randolph TW (2014) Storage stability of keratinocyte growth factor-2 in lyophilized formulations: effects of formulation physical properties and protein fraction at the solid-air interface. Eur J Pharm Biopharm 88:332–341

    Article  CAS  Google Scholar 

  • Ejima D, Tsumoto K, Fukada H, Yumioka R, Nagase K, Arakawa T, Philo JS (2007) Effects of acid exposure on the conformation, stability, and aggregation of monoclonal antibodies. Proteins 66:954–962

    Article  CAS  Google Scholar 

  • Fonte P, Reis S, Sarmento B (2016) Facts and evidences on the lyophilization of polymeric nanoparticles for drug delivery. J Control Release 225:75–86

    Article  CAS  Google Scholar 

  • Forney-Stevens KM, Bogner RH, Pikal MJ (2016) Addition of amino acids to further stabilize lyophilized sucrose-based protein formulations: I. Screening of 15 amino acids in two model proteins. J Pharm Sci 105:697–704

    Article  CAS  Google Scholar 

  • Furman JL, Chiu M, Hunter MJ (2015) Early engineering approaches to improve peptide developability and manufacturability. AAPS J 17:111–120

    Article  CAS  Google Scholar 

  • Geidobler R, Winter G (2013) Controlled ice nucleation in the field of freeze-drying: fundamentals and technology review. Eur J Pharm Biopharm 85:214–222

    Article  CAS  Google Scholar 

  • Goshima H, Do G, Nakagawa K (2016a) Impact of ice morphology on design space of pharmaceutical freeze-drying. J Pharm Sci 105:1920–1933

    Article  CAS  Google Scholar 

  • Goshima H, Forney-Stevens KM, Liu M, Qian KK, Tyagi M, Cicerone MT, Pikal MJ (2016b) Addition of monovalent electrolytes to improve storage stability of freeze-dried protein formulations. J Pharm Sci 105:530–541

    Article  CAS  Google Scholar 

  • Hansen LJJ, Daoussi R, Vervaet C, Remon JP, De Beer TRM (2015) Freeze-drying of live virus vaccines: a review. Vaccine 33:5507–5519

    Article  CAS  Google Scholar 

  • Iyer LK, Sacha GA, Moorthy BS, Nail SL, Topp EM (2016) Process and formulation effects on protein structure in lyophilized solids using mass spectrometric methods. J Pharm Sci 105:1684–1692

    Article  CAS  Google Scholar 

  • Izutsu K, Kojima S (2000) Freeze-concentration separates proteins and polymer excipients into different amorphous phases. Pharm Res 17:1316–1322

    Article  CAS  Google Scholar 

  • Izutsu K, Yoshioka S, Terao T (1993) Decreased protein-stabilizing effects of cryoprotectants due to crystallization. Pharm Res 10:1232–1237

    Article  CAS  Google Scholar 

  • Izutsu K, Fujimaki Y, Kuwabara A, Aoyagi N (2005) Effect of counterions on the physical properties of l-arginine in frozen solutions and freeze-dried solids. Int J Pharm 301:161–169

    Article  CAS  Google Scholar 

  • Jena S, Suryanarayanan R, Aksan A (2016) Mutual influence of mannitol and trehalose on crystallization behavior in frozen solutions. Pharm Res 33:1413–1425

    Article  CAS  Google Scholar 

  • Kasper JC, Winter G, Friess W (2013) Recent advances and further challenges in lyophilization. Eur J Pharm Biopharm 85:162–169

    Article  CAS  Google Scholar 

  • Kawai H, Sakurai M, Inoue Y, Chûjô R, Kobayashi S (1992) Hydration of oligosaccharides: anomalous hydration ability of trehalose. Cryobiology 29:599–606

    Article  CAS  Google Scholar 

  • Kishore D, Kundu S, Kayastha AM (2012) Thermal, chemical and pH induced denaturation of a multimeric β-galactosidase reveals multiple unfolding pathways. PLoS One 7:e50380

    Article  CAS  Google Scholar 

  • Korpus C, Pikal M, Friess W (2016) Heat transfer analysis of an optimized, flexible holder system for freeze-drying in dual chamber cartridges using different state-of-the-art PAT tools. J Pharm Sci 105:3304–3313

    Article  CAS  Google Scholar 

  • Lai MC, Topp EM (1999) Solid-state chemical stability of proteins and peptides. J Pharm Sci 88:489–500

    Article  CAS  Google Scholar 

  • Lai F, Pini E, Corrias F, Perricci J, Manconi M, Fadda AM, Sinico C (2014) Formulation strategy and evaluation of nanocrystal piroxicam orally disintegrating tablets manufacturing by freeze-drying. Int J Pharm 467:27–33

    Article  CAS  Google Scholar 

  • Lim JY, Lim DG, Kim KH, Park SK, Jeong SH (2018) Effects of annealing on the physical properties of therapeutic proteins during freeze drying process. Int J Biol Macromol 107(Pt A):730–740

    Article  CAS  Google Scholar 

  • Liu B, Zhou X (2015) Freeze-drying of proteins. Methods Mol Biol 1257:459–476

    Article  CAS  Google Scholar 

  • Manning MC, Patel K, Borchardt RT (1989) Stability of protein pharmaceuticals. Pharm Res 6:903–918

    Article  CAS  Google Scholar 

  • Mensink MA, Frijlink HW, van der Voort Maarschalk K, Hinrichs WL (2017) How sugars protect proteins in the solid state and during drying (review): mechanisms of stabilization in relation to stress conditions. Eur J Pharm Biopharm 114:288–295

    Article  CAS  Google Scholar 

  • Moussa EM, Panchal JP, Moorthy BS, Blum JS, Joubert MK, Narhi LO, Topp EM (2016) Immunogenicity of therapeutic protein aggregates. J Pharm Sci 105:417–430

    Article  CAS  Google Scholar 

  • Murase N, Franks F (1989) Salt precipitation during the freeze-concentration of phosphate buffer solutions. Biophys Chem 34:293–300

    Article  CAS  Google Scholar 

  • Nail SL, Jiang S, Chongprasert S, Knopp SA (2002) Fundamentals of freeze-drying. Pharm Biotechnol 14:281–360

    Article  CAS  Google Scholar 

  • Pansare SK, Patel SM (2016) Practical considerations for determination of glass transition temperature of a maximally freeze concentrated solution. AAPS PharmSciTech 17:805–819

    Article  CAS  Google Scholar 

  • Patel SM, Pikal M (2009) Process analytical technologies (PAT) in freeze-drying of parenteral products. Pharm Dev Technol 14:567–587

    Article  CAS  Google Scholar 

  • Patel SM et al (2017) Lyophilized drug product cake appearance: what is acceptable? J Pharm Sci 106:1706–1721

    Article  CAS  Google Scholar 

  • Peters BH, Staels L, Rantanen J, Molnár F, De Beer T, Lehto VP, Ketolainen J (2016) Effects of cooling rate in microscale and pilot scale freeze-drying – variations in excipient polymorphs and protein secondary structure. Eur J Pharm Sci 95:72–81

    Article  CAS  Google Scholar 

  • Pikal-Cleland KA, Cleland JL, Anchordoquy TJ, Carpenter JF (2002) Effect of glycine on pH changes and protein stability during freeze-thawing in phosphate buffer systems. J Pharm Sci 91:1969–1979

    Article  CAS  Google Scholar 

  • Privalov PL (1990) Cold denaturation of proteins. Crit Rev Biochem Mol Biol 25:281–305

    Article  CAS  Google Scholar 

  • Remmele RL, Krishnan S, Callahan WJ (2012) Development of stable lyophilized protein drug products. Curr Pharm Biotechnol 13:471–496

    Article  CAS  Google Scholar 

  • Richardson JM, Lemaire SD, Jacquot JP, Makhatadze GI (2000) Difference in the mechanisms of the cold and heat induced unfolding of thioredoxin h from Chlamydomonas reinhardtii: spectroscopic and calorimetric studies. Biochemistry 39:11154–11162

    Article  CAS  Google Scholar 

  • Shieh IC, Patel AR (2015) Predicting the agitation-induced aggregation of monoclonal antibodies using surface tensiometry. Mol Pharm 12:3184–3193

    Article  CAS  Google Scholar 

  • Shire SJ, Shahrokh Z, Liu J (2004) Challenges in the development of high protein concentration formulations. J Pharm Sci 93:1390–1402

    Article  CAS  Google Scholar 

  • Stärtzel P (2016) The application of amino acids in freeze dried protein formulations. PDA J Pharm Sci Technol:pii

    Google Scholar 

  • Strambini GB, Gonnelli M (2007) Protein stability in ice. Biophys J 92:2131–2138

    Article  CAS  Google Scholar 

  • Sundaramurthi P, Suryanarayanan R (2010) Influence of crystallizing and non-crystallizing cosolutes on trehalose crystallization during freeze-drying. Pharm Res 27:2384–2393

    Article  CAS  Google Scholar 

  • Sylvester B, Porfire A, Achim M, Rus L, Tomuţă I (2018) A step forward towards the development of stable freeze-dried liposomes: a quality by design approach (QbD). Drug Dev Ind Pharm 44:385–397

    Article  CAS  Google Scholar 

  • Tonnis WF, Mensink MA, de Jager A, van der Voort Maarschalk K, Frijlink HW, Hinrichs WL (2015) Size and molecular flexibility of sugars determine the storage stability of freeze-dried proteins. Mol Pharm 12:684–694

    Article  CAS  Google Scholar 

  • Twomey A, Kurata K, Nagare Y, Takamatsu H, Aksan A (2015) Microheterogeneity in frozen protein solutions. Int J Pharm 487:91–100

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  • Zbacnik TJ et al (2017) Role of buffers in protein formulations. J Pharm Sci 106:713–733

    Article  CAS  Google Scholar 

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Authors and Affiliations

  1. National Institute of Health Sciences, Kawasaki, Kanagawa, Japan

    Ken-ichi Izutsu

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  1. Ken-ichi Izutsu
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Correspondence to Ken-ichi Izutsu .

Editor information

Editors and Affiliations

  1. Faculty of Agriculture, Kyushu University, Fukuoka, Japan

    Mari Iwaya-Inoue

  2. Center for Biological Resources and Informatics, Tokyo Institute of Technology, Yokohama, Japan

    Minoru Sakurai

  3. United Graduate School of Agricultural Sciences and Department of Plant-biosciences, Faculty of Agriculture, Iwate University, Morioka, Japan

    Matsuo Uemura

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Izutsu, Ki. (2018). Applications of Freezing and Freeze-Drying in Pharmaceutical Formulations. In: Iwaya-Inoue, M., Sakurai, M., Uemura, M. (eds) Survival Strategies in Extreme Cold and Desiccation. Advances in Experimental Medicine and Biology, vol 1081. Springer, Singapore. https://doi.org/10.1007/978-981-13-1244-1_20

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