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Bioprocess and Biosystems Engineering

, Volume 43, Issue 2, pp 179–192 | Cite as

A phase diagram-based toolbox to assess the impact of freeze/thaw ramps on the phase behavior of proteins

  • Anna Katharina Wöll
  • Monika Desombre
  • Lena Enghauser
  • Jürgen HubbuchEmail author
Research Paper

Abstract

The influence of process parameters during freeze/thaw (FT) operations is essential for the preservation of the protein stability/activity during production and storage processes in the biopharmaceutical industry. Process parameters, such as FT ramps, the final storage time and temperature, affect the occurring FT stress onto the target protein in different ways. FT stress includes cold denaturation, freeze concentration, and ice crystal formation which can result in protein aggregation. To visualize the impact of variations in FT ramps, descriptors such as solubility, phase behavior and crystal morphology were evaluated. The phase diagram-based toolbox in combination with an HTS-compatible cryo-device allowed the identification of suitable ramping schemes during FT operations. It could be clearly shown that rapid operations are needed above the glass transition temperature of the target protein to circumvent precipitation during FT cycles. Finally, a stability index is introduced which allows ranking of the systems investigated.

Graphic abstract

Keywords

Temperature ramps Phase diagram Stability index Lysozyme 

Notes

Compliance with ethical standards

Conflict of interest

The authors report no conflict of interest.

Supplementary material

449_2019_2215_MOESM1_ESM.docx (489 kb)
Supplementary file1 (DOCX 489 kb)

References

  1. 1.
    Shamlou PA, Breen LH, Bell WV et al (2007) A new scaleable freeze–thaw technology for bulk protein solutions. Biotechnol Appl Biochem 46:13–26.  https://doi.org/10.1042/BA20060075 CrossRefPubMedGoogle Scholar
  2. 2.
    Ho K, Tchessalov S, Kantor A, Warne N (2008) Development of freeze and thaw processes for bulk biologics in disposable bags. Am Pharm Rev 11:1–6Google Scholar
  3. 3.
    Singh SK, Kolhe P, Mehta AP et al (2011) Frozen state storage instability of a monoclonal antibody: aggregation as a consequence of trehalose crystallization and protein unfolding. Pharm Res 28:873–885.  https://doi.org/10.1007/s11095-010-0343-z CrossRefPubMedGoogle Scholar
  4. 4.
    Wang W (2005) Protein aggregation and its inhibition in biopharmaceutics. Int J Pharm 289:1–30.  https://doi.org/10.1016/j.ijpharm.2004.11.014 CrossRefPubMedGoogle Scholar
  5. 5.
    Bhatnagar BS, Bogner RH, Pikal MJ (2007) Protein stability during freezing: separation of stresses and mechanisms of protein stabilization. Pharm Dev Technol 12:505–523.  https://doi.org/10.1080/10837450701481157 CrossRefPubMedGoogle Scholar
  6. 6.
    Kueltzo LA, Wang W, Randoplph TW, Carpenter JF (2008) Effects of solution conditions, processing parameters, and container materials on aggregation of a monoclonal antibody during freeze–thawing. J Pharm Sci 99:4215–4227.  https://doi.org/10.1002/jps.21110 CrossRefGoogle Scholar
  7. 7.
    Strambini GB, Gabellieri E (1996) Proteins in frozen solutions: evidence of ice-induced partial unfolding. Biophys J 70:971–976.  https://doi.org/10.1016/S0006-3495(96)79640-6 CrossRefPubMedPubMedCentralGoogle Scholar
  8. 8.
    Hauptmann A, Podgoršek K, Kuzman D et al (2018) Impact of buffer, protein concentration and sucrose addition on the aggregation and particle formation during freezing and thawing. Pharm Res.  https://doi.org/10.1007/s11095-018-2378-5 CrossRefPubMedPubMedCentralGoogle Scholar
  9. 9.
    Wang W, Nema S, Teagarden D (2010) Protein aggregation—pathways and influencing factors. Int J Pharm 390:89–99.  https://doi.org/10.1016/j.ijpharm.2010.02.025 CrossRefPubMedGoogle Scholar
  10. 10.
    Somero GN (1995) Proteins and temperature. Annu Rev Physiol 57:43–68.  https://doi.org/10.1146/annurev.ph.57.030195.000355 CrossRefPubMedGoogle Scholar
  11. 11.
    Liu L, Braun LJ, Wang W et al (2014) Freezing-induced perturbation of tertiary structure of a monoclonal antibody. J Pharm Sci 103:1979–1986.  https://doi.org/10.1002/jps.24013 CrossRefPubMedPubMedCentralGoogle Scholar
  12. 12.
    Pikal MJ (2004) Mechanisms of protein stabilization during freeze-drying storage: the relative importance of thermodynamic stabilization and glassy state relaxation dynamics. In: Freeze drying/lyophilization of pharmaceutical and biological products, Chapt 8. pp 198–232Google Scholar
  13. 13.
    Privalov PL (1990) Cold denaturation of proteins. Crit Rev Biochem Mol Biol 25:281–306.  https://doi.org/10.3109/10409239009090613 CrossRefPubMedGoogle Scholar
  14. 14.
    Her L-M, Deras M, Nail SL (1995) Electrolyte-induced changes in glass transition temperatures of freeze-concentrated solutes. Pharm Res 12:768–772.  https://doi.org/10.1023/A:1016280113800 CrossRefPubMedGoogle Scholar
  15. 15.
    Heller MC, Carpenter JF, Randolph TW (1996) Effects of phase separating systems on lyophilized hemoglobin. J Pharm Sci 85:1358–1362.  https://doi.org/10.1021/js960019t CrossRefPubMedGoogle Scholar
  16. 16.
    Heller MC, Carpenter JF, Randolph TW (1997) Manipulation of lyophilization-induced phase separation: implications for pharmaceutical proteins. Biotechnol Prog 13:590–596.  https://doi.org/10.1021/bp970081b CrossRefPubMedGoogle Scholar
  17. 17.
    Heller MC, Carpenter JF, Randolph TW (1999) Protein formulation and lyophilization cycle design: prevention of damage due to freeze-concentration induced phase separation. Biotechnol Bioeng 63:166–174.  https://doi.org/10.1002/(SICI)1097-0290(19990420)63:2%3c166:AID-BIT5%3e3.0.CO;2-H CrossRefPubMedGoogle Scholar
  18. 18.
    Chang BS, Kendrick BS, Carpenter JF (1996) Surface-induced denaturation of proteins during freezing and its inhibition by surfactants. J Pharm Sci 85:1325–1330.  https://doi.org/10.1021/js960080y CrossRefPubMedGoogle Scholar
  19. 19.
    Miller MA, Rodrigues MA, Glass MA et al (2013) Frozen-state storage stability of a monoclonal antibody: aggregation is impacted by freezing rate and solute distribution. J Pharm Sci 102:1194–1208.  https://doi.org/10.1002/jps.23473 CrossRefPubMedGoogle Scholar
  20. 20.
    Maity H, Karkaria C, Davagnino J (2009) 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 378:122–135.  https://doi.org/10.1016/j.ijpharm.2009.05.063 CrossRefPubMedGoogle Scholar
  21. 21.
    Cao E, Chen Y, Cui Z, Foster PR (2003) Effect of freezing and thawing rates on denaturation of proteins in aqueous solutions. Biotechnol Bioeng 82:684–690.  https://doi.org/10.1002/bit.10612 CrossRefPubMedGoogle Scholar
  22. 22.
    Anchordoquy TJ, Carpenter JF (1996) Polymers protect lactate dehydrogenase during freeze-drying by inhibiting dissociation in the frozen state. Arch Biochem Biophys 332:231–238.  https://doi.org/10.1006/abbi.1996.0337 CrossRefPubMedGoogle Scholar
  23. 23.
    Pikal-Cleland KA, Rodríguez-Hornedo N, Amidon GL, Carpenter JF (2000) Protein denaturation during freezing and thawing in phosphate buffer systems: monomeric and tetrameric beta-galactosidase. Arch Biochem Biophys 384:398–406.  https://doi.org/10.1006/abbi.2000.2088 CrossRefPubMedGoogle Scholar
  24. 24.
    Hsu CC, Nguyen HM, Yeung DA et al (1995) Surface denaturation at solid-void interface—a possible pathway by which opalescent particulates from during the storage of lypholized tissue-type plasminogen activator at high temperatures. Pharm Res 12:69–77.  https://doi.org/10.1023/A:1016270103863 CrossRefPubMedGoogle Scholar
  25. 25.
    Webb SD, Cleland JL, Carpenter JF, Randolph TW (2003) Effects of annealing lyophilized and spray-lyophilized formulations of recombinant human interferon-gamma. J Pharm Sci 92:715–729.  https://doi.org/10.1002/jps.10334 CrossRefPubMedGoogle Scholar
  26. 26.
    Fransen GJ, Salemink PJM, Crommelin DJA (1986) Critical parameters in freezing of liposomes. Int J Pharm 33:27–35.  https://doi.org/10.1016/0378-5173(86)90035-9 CrossRefGoogle Scholar
  27. 27.
    Kolhe P, Amend E, Singh SK (2010) Impact of freezing on pH of buffered solutions and consequences for monoclonal antibody aggregation. Biotechnol Prog 26:727–733.  https://doi.org/10.1002/btpr.377 CrossRefPubMedGoogle Scholar
  28. 28.
    Hernández-Jiménez J, Martínez-Ortega A, Salmerón-García A et al (2018) Study of aggregation in therapeutic monoclonal antibodies subjected to stress and long-term stability tests by analyzing size exclusion liquid chromatographic profiles. Elsevier B.V, AmsterdamCrossRefGoogle Scholar
  29. 29.
    Rašković B, Popović M, Ostojić S et al (2015) Fourier transform infrared spectroscopy provides an evidence of papain denaturation and aggregation during cold storage. Spectrochim Acta Part A Mol Biomol Spectrosc 150:238–246.  https://doi.org/10.1016/j.saa.2015.05.061 CrossRefGoogle Scholar
  30. 30.
    Radmanovic N, Serno T, Joerg S, Germershaus O (2013) Understanding the freezing of biopharmaceuticals: first-principle modeling of the process and evaluation of its effect on product quality. J Pharm Sci 102:2495–2507.  https://doi.org/10.1002/jps CrossRefPubMedGoogle Scholar
  31. 31.
    Wöll AK, Schütz J, Zabel J, Hubbuch J (2019) Analysis of phase behavior and morphology during freeze−thaw applications of lysozyme. Int J Pharm 555:153–164.  https://doi.org/10.1016/j.ijpharm.2018.11.047 CrossRefPubMedGoogle Scholar
  32. 32.
    Kröner F, Hubbuch J (2013) Systematic generation of buffer systems for pH gradient ion exchange chromatography and their application. J Chromatogr A 1285:78–87.  https://doi.org/10.1016/j.chroma.2013.02.017 CrossRefPubMedGoogle Scholar
  33. 33.
    Column P-10 D (2007) PD-10 desalting columnGoogle Scholar
  34. 34.
    Baumgartner K, Galm L, Nötzold J et al (2015) Determination of protein phase diagrams by microbatch experiments: exploring the influence of precipitants and pH. Int J Pharm 479:28–40.  https://doi.org/10.1016/j.ijpharm.2014.12.027 CrossRefPubMedGoogle Scholar
  35. 35.
    Galm L, Morgenstern J, Hubbuch J (2015) Manipulation of lysozyme phase behavior by additives as function of conformational stability. Int J Pharm 494:370–380.  https://doi.org/10.1016/j.ijpharm.2015.08.045 CrossRefPubMedGoogle Scholar
  36. 36.
    Vitkup D, Ringe D, Petsko GA, Karplus M (2000) Solvent mobility and the protein “glass” transition. Nat Struct Biol 7:34–38.  https://doi.org/10.1038/71231 CrossRefPubMedGoogle Scholar
  37. 37.
    Kumar P, Yan Z, Xu L et al (2006) Glass transition in biomolecules and the liquid-liquid critical point of water. Phys Rev Lett 97:1–4.  https://doi.org/10.1103/PhysRevLett.97.177802 CrossRefGoogle Scholar
  38. 38.
    Muschol M, Rosenberger F (1997) Liquid–liquid phase separation in supersaturated lysozyme solutions and associated precipitate formation/crystallization. J Chem Phys.  https://doi.org/10.1063/1.474547 CrossRefGoogle Scholar
  39. 39.
    Singh SK, Nema S (2010) Freezing and thawing of protein solutions. In: Formulation and process development strategies for manufacturing biopharmaceuticals, pp 625–675.  https://doi.org/10.1002/9780470595886.ch26 CrossRefGoogle Scholar
  40. 40.
    Cocks FH, Brower WE (1974) Phase diagram relationships in cryobiology. Cryobiology 11:340–358.  https://doi.org/10.1016/0011-2240(74)90011-X CrossRefPubMedGoogle Scholar
  41. 41.
    Kerwin BA, Heller MC, Levin SH, Randolph TW (1998) Effects of Tween 80 and sucrose on acute short-term stability and long-term storage at −20 ˚C of a recombinant hemoglobin. J Pharm Sci 87:1062–1068.  https://doi.org/10.1021/js980140v CrossRefPubMedGoogle Scholar
  42. 42.
    Jameel F, Pikal MJ (2010) Design of a formulation for freeze drying. In: Part III: development of a formulation for lyophilized dosage form, Chapt 18. pp 459–492Google Scholar
  43. 43.
    Desai KG, Pruett WA, Martin PJ et al (2017) Impact of manufacturing scale freeze-thaw conditions on a mAb solution. BioPharm Int 30:30–36Google Scholar
  44. 44.
    Chi EY, Krishnan S, Randolph TW, Carpenter JF (2003) Physical stability of proteins in aqueous solution: mechanism and driving forces in nonnative protein aggregation. Pharm Res 20:1325–1336.  https://doi.org/10.1023/A:1025771421906 CrossRefPubMedGoogle Scholar
  45. 45.
    Asherie N (2004) Protein crystallization and phase diagrams. Methods 34:266–272.  https://doi.org/10.1016/j.ymeth.2004.03.028 CrossRefPubMedGoogle Scholar
  46. 46.
    Saridakis E, Chayen NE (2008) Towards a ‘ universal ’ nucleant for protein crystallization. Cell Press.  https://doi.org/10.1016/j.tibtech.2008.10.008 CrossRefGoogle Scholar
  47. 47.
    Ataka M, Tanaka S (1986) The growth of large single crystals. Biopolymers 67:337–350.  https://doi.org/10.1021/ed067p410 CrossRefGoogle Scholar
  48. 48.
    Rodrigues MA, Miller MA, Glass MA et al (2011) Effect of freezing rate and dendritic ice formation on concentration profiles of proteins frozen in cylindrical vessels. J Pharm Sci.  https://doi.org/10.1002/jps CrossRefPubMedGoogle Scholar
  49. 49.
    Ataka M (1993) Protein crystal growth: an approach based on phase diagram determination. Phase Transitions A Multinatl J 45:205–219.  https://doi.org/10.1080/01411599308223724 CrossRefGoogle Scholar
  50. 50.
    Garcıa-Ruiz JM (2003) Nucleation of protein crystals. J Struct Biol 142:22–31.  https://doi.org/10.1016/S1047-8477(03)00035-2 CrossRefGoogle Scholar
  51. 51.
    Chayen NE (2004) Turning protein crystallisation from an art into a science. Curr Option Struct Biol 14:577–583.  https://doi.org/10.1016/j.sbi.2004.08.002 CrossRefGoogle Scholar
  52. 52.
    McPherson A (2004) Introduction to protein crystallization. Methods 34:254–265.  https://doi.org/10.1016/j.ymeth.2004.03.019 CrossRefPubMedGoogle Scholar
  53. 53.
    Chernov AA (2003) Protein crystals and their growth. J Struct Biol 142:3–21.  https://doi.org/10.1016/S1047-8477(03)00034-0 CrossRefPubMedGoogle Scholar
  54. 54.
    Lin C, Zhang Y, Liu JJ, Wang XZ (2017) Study on nucleation kinetics of lysozyme crystallization. J Cryst Growth 469:59–64.  https://doi.org/10.1016/j.jcrysgro.2016.10.028 CrossRefGoogle Scholar
  55. 55.
    Körber C (1988) Phenomena at the advancing ice—liquid interface: solutes particles and biological cells. Q Rev Biophys 21:229–298.  https://doi.org/10.1017/S0033583500004303 CrossRefPubMedGoogle Scholar
  56. 56.
    Butler MF (2001) Instability formation and directional dendritic growth of ice studied by optical interferometry. Cryst Growth Des 1:213–223.  https://doi.org/10.1021/cg005534q CrossRefGoogle Scholar
  57. 57.
    Brody HD (1966) Solute redistribution in dendritic solidification. Trans Metall Soc AIME 236:615–624Google Scholar
  58. 58.
    Butler MF (2002) Freeze concentration of solutes at the ice/solution interface studied by optical interferometry. Cryst Growth Des 2:541–548CrossRefGoogle Scholar
  59. 59.
    Weiss WF IV, Young TM, Roberts CJ (2009) Principles, approaches, and challenges for predicting protein aggregation rates and shelf life. Int J Drug Dev Res 98:1246–1277.  https://doi.org/10.1002/jps CrossRefGoogle Scholar
  60. 60.
    Zeelen JP (2007) Interpretation of crystallization drop results. Molecular Dimension Monograph Series© Google Scholar

Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2019

Authors and Affiliations

  • Anna Katharina Wöll
    • 1
  • Monika Desombre
    • 1
  • Lena Enghauser
    • 1
  • Jürgen Hubbuch
    • 1
    Email author
  1. 1.Institute of Engineering in Life Sciences, Section IV: Biomolecular Separation EngineeringKarlsruhe Institute of Technology (KIT)KarlsruheGermany

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