Skip to main content
SpringerLink
Log in

Interfacial Stress and Proteins Prepared in the Solid State

  • Chapter
  • First Online:
Protein Instability at Interfaces During Drug Product Development

Part of the book series: AAPS Advances in the Pharmaceutical Sciences Series ((AAPS,volume 43))

Abstract

Frozen and freeze-dried biotherapeutical products are exposed to various interfaces during manufacture and use. Three types of interfaces are considered in this chapter, that is, air/solution, ice/solution, and interface between dried protein formulations and the gas phase. Air/solution interfaces, in the form of air bubbles, can be formed both during freezing as the result of expulsion of dissolved air by growing ice crystals and during reconstitution of lyophiles. Protein molecules have a tendency to accumulate at the air/solution interfaces, which could promote unfolding. Ice/solution interface, which protein molecules encounter during both freezing storage and freeze-drying, can destabilize protein either directly, if protein molecules are sorbed on ice crystals or indirectly, when proteins molecules are partitioned in a liquid layer next to growing ice crystals. In spray-dried and freeze-dried formulations, higher protein concentration near the solid surface was reported in many cases; these molecules are expected to be less stable against various degradation mechanisms than those located in the bulk. It is concluded that, while good long-term stability can be expected for many frozen and freeze-dried protein formulations, freezing and drying unit operations also exposes proteins to interfacial stress conditions that can lead to degradation.

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

Access this chapter

Log in via an institution
eBook
USD 16.99
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD 109.99
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD 159.99
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

Abbreviations

AFP:

Anti-freeze proteins

BSA:

Bovine serum albumin

CRM:

Confocal Raman microscopy

DMSO:

Dimethyl sulfoxide

DP:

Drug product

DS:

Drug substance

ESCA:

Electron spectroscopy for chemical analysis

FCS:

Freeze concentrated solutions

FTIR:

Fourier transform infrared spectroscopy

HES:

Hydroxyethyl starch

HIAC:

High accuracy particle counter

Ih:

Hexagonal ice

LDH:

Lactate dehydrogenase

mAb:

Monoclonal antibody

PS20:

Polysorbate 20

PS80:

Polysorbate 80

QLL:

Quasi-liquid layer

rHA:

Recombinant human albumin

rhGH:

Recombinant human growth hormone

SANS:

Small angle neutron scattering

sXRD:

Synchrotron X-ray diffraction

Tg:

Glass transition temperature

References

  1. Authelin J-R, Rodrigues MA, Tchessalov S, Singh S, McCoy T, Wang S, Shalaev E. Freezing of biologicals revisited: scale, stability, excipients, and degradation stresses. J Pharm Sci. 2019; https://doi.org/10.1016/j.xphs.2019.10.062.

  2. Joslyn MA, Supplee J. Solubility of oxygen in solutions of various sugars. Food Res. 1949;14:209–15.

    Article  CAS  PubMed  Google Scholar 

  3. Kanev AN, Kosyakov VI, Malakhov DV, Shalaev EY. An equilibrium phase diagram for the system water-sucrose. Izv. SO AN SSSR, ser. khim. nauk Iss. 1989;2:11–5.

    Google Scholar 

  4. Korber C. Phenomena at the advancing ice-liquid interface: solutes, particles and biological cells. Q Rev Biophys. 1988;21:229–98.

    Article  CAS  PubMed  Google Scholar 

  5. Curtis JE, Nanda H, Khodadadi S, Cicerone M, Lee HJ, McAuley A, Krueger S. Small-angle neutron scattering study of protein crowding in liquid and solid phases: lysozyme in aqueous solution, frozen solution, and carbohydrate powders. J Phys Chem B. 2012;116:9653–76.

    Article  CAS  PubMed  Google Scholar 

  6. Salnikova M, Varshney D, Shalaev E. Heterogeneity of protein environments in frozen solutions and in the dried state. In: Varshney D, Singh M, editors. Lyophilized Biologics and Vaccines: Modality-Based Approaches: Springer; 2015. p. 11–24.

    Google Scholar 

  7. Hauptmann A, Hoelzl G, Loerting T. Distribution of protein content and number of aggregates in monoclonal antibody formulation after large-scale freezing. AAPS PharmSciTech. 2019;20:72.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Barnard J, Shen H, Abiad M, Mohanty P, Chow C, Tran H, Rivers H, Shalaev E. Cloudiness in a reconstituted freeze-dried protein: case study. In: 2016 Workshop on protein aggregation and immunogenicity. Colorados: Breckenridge; 2016.

    Google Scholar 

  9. Webb SD, Cleland JL, Carpenter JF, Randolph TW. Effects of annealing lyophilized and spray-lyophilized formulations of recombinant human interferon-gamma. J Pharm Sci. 2003;92(4):715–29.

    Article  CAS  PubMed  Google Scholar 

  10. Zhou C, Cleland D, Snell J, Qi W, Randolph TW, Carpenter JF. Formation of stable nanobubbles on reconstituting lyophilized formulations containing trehalose. J Pharm Sci. 2016;105(7):2249–53.

    Article  CAS  PubMed  Google Scholar 

  11. Chou SG, Soper AK, Khodadadi S, Curtis JE, Krueger S, Cicerone MT, Fitch A, Shalaev EY. Pronounced micro-heterogeneity in a sorbitol-water mixture observed through variable temperature neutron scattering. J Phys Chem B. 2012;116(15):4439–47.

    Article  CAS  PubMed  Google Scholar 

  12. Snell JR, Zhou C, Carpenter JF, Randolph TW. Particle formation and aggregation of a therapeutic protein in nanobubble suspensions. J Pharm Sci. 2016;105(10):3057–63.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Webb SD, Cleland JL, Carpenter JF, Randolph TW. A new mechanism for decreasing aggregation of recombinant human interferon-γ by a surfactant: slowed dissolution of lyophilized formulations in a solution containing 0.03% polysorbate 20. J Pharm Sci. 2002;91:543–58.

    Article  CAS  PubMed  Google Scholar 

  14. Piedmonte DM, Sumers C, McAuley A, Karamuji L, Ratnaswamy G. Sorbitol crystallization can lead to protein aggregation in frozen protein formulations. Pharm Res. 2007;24:136–46.

    Article  CAS  PubMed  Google Scholar 

  15. Singh SK, Kolhe P, Mehta AP, Chico SC, Lary AL, Huang M. Frozen state storage instability of a monoclonal antibody: aggregation as a consequence of trehalose crystallization and protein unfolding. Pharm Res. 2011;28:873–85.

    Article  CAS  PubMed  Google Scholar 

  16. Connolly BD, Le L, Patapoff TW, Cromwell MEME, Moore JMR. Protein aggregation in frozen trehalose formulations: effects of composition, cooling rate and storage temperature. J Pharm Sci. 2015;104:4170–84.

    Article  CAS  PubMed  Google Scholar 

  17. Gu JH, Beekman A, Wu T, Piedmonte DM, Baker P, Eschenberg M, Hale M, Goldenbergh M. Beyond glass transitions: studying the highly viscous and elastic behavior of frozen protein formulations using low temperature rheology and its potential implications on protein stability. Pharm Res. 2013;30:387–401.

    Article  CAS  PubMed  Google Scholar 

  18. Franks F, Hatley RHM, Friedman HL. The thermodynamics of protein stability: cold destabilization as a general phenomenon. Biophvs Chem. 1988;31:307–15.

    Article  CAS  Google Scholar 

  19. Franks F, Hatley RHM. Stability of proteins at subzero temperatures: thermodynamics and some ecological consequences. Pure Appl Chem. 1991;63:1367–80.

    Article  CAS  Google Scholar 

  20. Rosa M, Lopes C, Melo EP, Singh SK, Geraldes V, Rodrigues MA. Measuring and Modelling hemoglobin aggregation below the freezing temperature. J Phys Chem B. 2013;117:8939–46.

    Article  CAS  PubMed  Google Scholar 

  21. Tang C, Pikal MJ. The effect of stabilizers and denaturants on the cold denaturation temperatures of proteins and implications for freeze-drying. Pharm Res. 2005;22:1167–75.

    Article  CAS  PubMed  Google Scholar 

  22. Strambini GB, Gabellieri E. Proteins in frozen solutions: evidence of ice-induced partial unfolding. Biophys J. 1996;70:971–6.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Bhatnagar BS, Pikal MJ, Bogner RH. Study of the individual contributions of ice formation and freeze-concentration on isothermal stability of lactate dehydrogenase during freezing. J Pharm Sci. 2008;97:798–814.

    Article  CAS  PubMed  Google Scholar 

  24. Anzo K, Harada M, Okada T. Enhanced kinetics of pseudo first-order hydrolysis in liquid phase coexistent with ice. J Phys Chem A. 2013;117:10619–25.

    Article  CAS  PubMed  Google Scholar 

  25. Twomey A, Less R, Kurata K, Takamatsu H, Aksan A. In situ spectroscopic quantification of protein–ice interactions. J Phys Chem B. 2013;117(26):7889–97.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. Patel MR, Baer RJ, Acharya MR. Increasing the protein content of ice cream. J Dairy Sci. 2006;89:1400–6.

    Article  CAS  PubMed  Google Scholar 

  28. Regand A, Goff HD. Effect of biopolymers on structure and ice recrystallization in dynamically-frozen ice cream model systems. J Dairy Sci. 2002;85:2722–32.

    Article  CAS  PubMed  Google Scholar 

  29. Kuiper MJ, Lankin C, Gauthier SY, Walker VK, Davies PL. Purification of antifreeze proteins by adsorption to ice. Biochem Biophys Res Commun. 2003;300:645–8.

    Article  CAS  PubMed  Google Scholar 

  30. Olijve LLC, Meister K, DeVries AL, Duman JG, Guo S, Bakker HJ, Voets IK. Blocking rapid ice crystal growth through nonbasal plane adsorption of antifreeze proteins. Proc Natl Acad Sci U S A. 2016;113:3740–5.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. Devries AL, Lin Y. Structure of a peptide antifreeze and mechanism of adsorption to ice. Biochim Biophys Acta. 1977;495:388–92.

    Article  CAS  PubMed  Google Scholar 

  32. Chao H, Houston ME, Hodges RS, Kay CM, Sykes BD, Loewen MC, Davies PL, Sönnichsen FD. A diminished role for hydrogen bonds in antifreeze protein binding to ice. Biochemistry. 1997;36:14652–60.

    Article  CAS  PubMed  Google Scholar 

  33. Baardsnes J, Kondejewski LH, Hodges RS, Chao H, Kay C, Davies PL. New ice-binding face for type I antifreeze protein. FEBS Lett. 1999;463:87–91.

    Article  CAS  PubMed  Google Scholar 

  34. Garnham CP, Campbell RL, Davies PL. Anchored clathrate waters bind antifreeze proteins to ice. Proc Natl Acad Sci U S A. 2011;108:7363–7.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  35. Yagci YE, Antonietti M, Börner HG. Synthesis of poly(tartar amides) as bio-inspired antifreeze additives. Macromol Rapid Commun. 2006;27:1660–4.

    Article  CAS  Google Scholar 

  36. Baruch E, Mastai Y. Antifreeze properties of polyglycidol block copolymers. Macromol Rapid Commun. 2007;28:2256–61.

    Article  CAS  Google Scholar 

  37. Huang ML, Ehre D, Jiang Q, Hu C, Kirshenbaum K, Ward MD. Biomimetic peptoid oligomers as dual-action antifreeze agents. Proc Natl Acad Sci U S A. 2012;109:19922–7.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  38. Zakharov B, Fisyuk A, Fitch A, Watier Y, Kostyuchenko A, Varshney D, Sztucki M, Boldyreva E, Shalaev E. Ice recrystallization in a solution of a cryoprotector and its inhibition by a protein: synchrotron X-ray diffraction study. J Pharm Sci. 2016;105:2129–38.

    Article  CAS  PubMed  Google Scholar 

  39. Bhatnagar B, Zakharov B, Fisyuk A, Wen X, Karim F, Lee K, Seryotkin Y, Mogodi M, Fitch A, Boldyreva E, Kostyuchenko A, Shalaev E. Protein/ice interaction: high-resolution synchrotron X-ray diffraction differentiates pharmaceutical proteins from lysozyme. J Phys Chem B. 2019; https://doi.org/10.1021/acs.jpcb.9b02443.

  40. Petrenko VF, Whitworth RW. Physics of ice. Oxford University Press: Oxford; 1999. p. 227–51.

    Google Scholar 

  41. Sanchez MA, Kling T, Ishiyama T, van Zadel M-J, Bisson PJ, Mezger M, Jochum MN, Cyran JD, Smit WJ, Bakker HJ, Shultz MJ, Morita A, Donadio D, Nagata Y, Bonn M, Backus EHG. Experimental and theoretical evidence for bilayer-by-bilayer surface melting of crystalline ice. Proc Natl Acad Sci U S A. 2017;114:227–32.

    Article  CAS  PubMed  Google Scholar 

  42. Jeffrey GA. An introduction to hydrogen bonding. New York: Oxford University Press; 1997. p. 191.

    Google Scholar 

  43. Israelachvili J, Pashley R. The hydrophobic interaction is long range, decaying exponentially with distance. Nature. 1982;300:341–2.

    Article  CAS  PubMed  Google Scholar 

  44. Elbaum M, Lipson SG, Dash JG. Optical study of surface melting on ice. J Cryst Growth. 1993;129:491–505.

    Article  CAS  Google Scholar 

  45. Adler M, Lee G. Stability and surface activity of lactate dehydrogenase in spray-dried trehalose. J Pharm Sci. 1999;88:199–208.

    Article  CAS  PubMed  Google Scholar 

  46. Millqvist-Fureby A, Malmsten M, Bergenstahl B. Surface characterisation of freeze-dried protein:carbohydrate mixtures. Int J Pharm. 1999;191:103–14.

    Article  CAS  PubMed  Google Scholar 

  47. Faldt P, Bergenstahl B. The surface composition of spray-dried protein-lactose powders. Colloids Surf A Physicochem Eng Asp. 1994;90:183–90.

    Article  Google Scholar 

  48. Webb SD, Golledge SL, Cleland JL, Carpenter JF, Randolph TW. Surface adsorption of recombinant human interferon-gamma in lyophilized and spray-lyophilized formulations. J Pharm Sci. 2002;91(6):1474–87.

    Article  CAS  PubMed  Google Scholar 

  49. Xu Y, Grobelny P, Von Allmen A, Knudson K, Pikal MJ, Carpenter JF, Randolph TW. Protein quantity on the air–solid interface determines degradation rates of human growth hormone in lyophilized samples. Pharm Biotech. 2014; https://doi.org/10.1002/jps.23926.

  50. Abdul-Fattah AM, Lechuga-Ballesteros D, Kalonia DS, Pikal MJ. The impact of drying method and formulation on the physical properties and stability of methionyl human growth hormone in the amorphous solid state. J Pharm Sci. 2008;97(1):163–84.

    Article  CAS  PubMed  Google Scholar 

  51. Zhu L, Brian CW, Swallen SF, Straus PT, Ediger MD, Yu L. Surface self-diffusion of an organic glass. Phys Rev Lett. 2011;106(25):256103/1–4.

    Article  CAS  Google Scholar 

  52. Arsiccio A, McCarty J, Pisano R, Shea J-E. Effect of surfactants on surface-induced denaturation of proteins: evidence of an orientation-dependent mechanism. J Phys Chem B. 2018;122(49):11390–9.

    Article  CAS  PubMed  Google Scholar 

  53. Jia Z, Davies PL. Antifreeze proteins: an unusual receptor-ligand interaction. Trends Biochem Sci. 2002;27(2):101–6.

    Article  CAS  PubMed  Google Scholar 

  54. Kallay N, Cakara D. Reversible charging of ice-water interface. I Measurement of the surface potential. J Colloid Interface Sci. 2000;232:81–5.

    Article  CAS  PubMed  Google Scholar 

  55. Takahashi M. ζ potential of microbubbles in aqueous solutions: electrical properties of the gas−water Interface. J Phys Chem B. 2005;109:21858–64.

    Article  CAS  PubMed  Google Scholar 

  56. Kreilgaard L, Jones LS, Randolph TW, Frokjaer S, Flink JM, Manning MC, Carpenter JF. Effect of tween 20 on freeze-thawing- and agitation-induced aggregation of recombinant human factor XIII. J Pharm Sci. 1998;87(12):1597–603.

    Article  CAS  PubMed  Google Scholar 

  57. Krausková L, Procházková J, Klašková M, Filipová L, Chaloupková R, Malý S, Damborský J, Heger D. Suppression of protein inactivation during freezing by minimizing pH changes using ionic cryoprotectants. I J Pharm. 2016;509:41–9.

    Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Pharmaceutical Sciences, Abbvie, Irvine, CA, USA

    Evgenyi Shalaev

  2. Dept of Bioengineering, University of Washington, Seattle, WA, USA

    John J. Hill

  3. BioProcess Technology Consultants, Woburn, MA, USA

    John J. Hill

Authors
  1. Evgenyi Shalaev
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. John J. Hill
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Evgenyi Shalaev .

Editor information

Editors and Affiliations

  1. Department of Formulation Development Nevakar Inc., Bridgewater, NJ, USA

    Jinjiang Li

  2. Drug Product Development, Bristol Myers Squibb, New Brunswick, NJ, USA

    Mary E. Krause

  3. Chemical Engineering, The City College of New York - CUNY, New York, NY, USA

    Raymond Tu

Rights and permissions

Reprints and permissions

Copyright information

© 2021 American Association of Pharmaceutical Scientists

About this chapter

Check for updates. Verify currency and authenticity via CrossMark

Cite this chapter

Shalaev, E., Hill, J.J. (2021). Interfacial Stress and Proteins Prepared in the Solid State. In: Li, J., Krause, M.E., Tu, R. (eds) Protein Instability at Interfaces During Drug Product Development. AAPS Advances in the Pharmaceutical Sciences Series, vol 43. Springer, Cham. https://doi.org/10.1007/978-3-030-57177-1_11

Download citation

Publish with us

Policies and ethics

Search

Navigation