Skip to main content

Advertisement

Springer Nature Link
Log in

Apparent protein cloud point temperature determination using a low volume high-throughput cryogenic device in combination with automated imaging

  • Research Paper
  • Published:
Bioprocess and Biosystems Engineering Aims and scope Submit manuscript

Abstract

Short-term parameters correlating to long-term protein stability, such as the protein cloud point temperature (Tcloud), are of interest to improve efficiency during protein product development. Such efficiency is reached if short-term parameters are obtained in a low volume and high-throughput (HT) manner. This study presents a low volume HT detection method for (sub-zero) Tcloud determination of lysozyme, as such an experimental method is not available yet. The setup consists of a cryogenic device with an automated imaging system. Measurement reproducibility (median absolute deviation of 0.2 °C) and literature-based parameter validation (Pearson correlation coefficient of 0.996) were shown by a robustness and validation study. The subsequent case study demonstrated a partial correlation between the obtained apparent Tcloud parameter and long-term protein stability as a function of lysozyme concentration, ion type, ionic strength, and freeze/thaw stress. The presented experimental setup demonstrates its ability to advance short-term strategies for efficient protein formulation development.

Graphical Abstract

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

Access this article

Log in via an institution

Subscribe and save

Springer+
from $39.99 /Month
  • Starting from 10 chapters or articles per month
  • Access and download chapters and articles from more than 300k books and 2,500 journals
  • Cancel anytime
View plans

Buy Now

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4

Similar content being viewed by others

Explore related subjects

Discover the latest articles and news from researchers in related subjects, suggested using machine learning.

Notes

  1. 87 g/L lysozyme at pH 7.0 in a 20 mM Tris buffer with 0.5 M NaCl [26, 27].

References

  1. Cromwell MEM, Hilario E, Jacobson F (2006) Protein aggregation and bioprocessing. AAPS J 8:E572–E579. https://doi.org/10.1208/aapsj080366

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  2. Mahler H-C, Friess W, Grauschopf U, Kiese S (2009) Protein aggregation: pathways, induction factors and analysis. J Pharm Sci 98:2909–2934. https://doi.org/10.1002/jps.21566

    Article  PubMed  CAS  Google Scholar 

  3. Rosenberg AS (2006) Effects of protein aggregates: an immunologic perspective. AAPS J 8:E501–E507. https://doi.org/10.1208/aapsj080359

    Article  PubMed  PubMed Central  Google Scholar 

  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

    Article  PubMed  CAS  Google Scholar 

  5. Philo JS, Arakawa T (2009) Mechanisms of protein aggregation. Curr Pharm Biotechnol 10:348–351. https://doi.org/10.2174/138920109788488932

    Article  PubMed  CAS  Google Scholar 

  6. 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

    Article  PubMed  CAS  Google Scholar 

  7. 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

    Article  PubMed  CAS  Google Scholar 

  8. International Council for Harmonization (1995) Stability testing of biotechnological/biological products Q5C. ICH Harmon Tripart Guidel, pp 1–8

  9. Asherie N (2004) Protein crystallization and phase diagrams. Methods 34:266–272. https://doi.org/10.1016/j.ymeth.2004.03.028

    Article  PubMed  CAS  Google Scholar 

  10. 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

    Article  PubMed  CAS  Google Scholar 

  11. 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

    Article  CAS  Google Scholar 

  12. Goldberg DS, Bischop SM, Shah AU, Sathish HA (2011) Formulation development of therapeutic monoclonal antibodies using high-throughput fluorescence and static light scattering techniques: role of conformational and colloidal stability. J Pharm Sci 100:1306–1315. https://doi.org/10.1002/jps

    Article  PubMed  CAS  Google Scholar 

  13. George A, Wilson WW (1994) Predicting protein crystallization from a dilute solution property. Acta Crystallogr Sect D Biol Crystallogr 50:361–365. https://doi.org/10.1107/s0907444994001216

    Article  CAS  Google Scholar 

  14. Bauer KC, Göbel M, Schwab M-L et al (2016) Concentration-dependent changes in apparent diffusion coefficients as indicator for colloidal stability of protein solutions. Int J Pharm 511:276–287. https://doi.org/10.1016/j.ijpharm.2016.07.007

    Article  PubMed  CAS  Google Scholar 

  15. 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

    Article  PubMed  CAS  Google Scholar 

  16. Maddux NR, Iyer V, Cheng W et al (2014) High throughput prediction of the long-term stability of pharmaceutical macromolecules from short-term multi-instrument spectroscopic data. J Pharm Sci 103:828–839. https://doi.org/10.1002/jps.23849

    Article  PubMed  CAS  Google Scholar 

  17. Hirano A, Hamada H, Okubo T et al (2007) Correlation between thermal aggregation and stability of lysozyme with salts described by molar surface tension increment: an exceptional propensity of ammonium salts as aggregation suppressor. Protein J 26:423–433. https://doi.org/10.1007/s10930-007-9082-3

    Article  PubMed  CAS  Google Scholar 

  18. Galm L, Amrhein S, Hubbuch J (2017) Predictive approach for protein aggregation: correlation of protein surface characteristics and conformational flexibility to protein aggregation propensity. Biotechnol Bioeng 114:1170–1183. https://doi.org/10.1002/bit.25949

    Article  PubMed  CAS  Google Scholar 

  19. Schermeyer MT, Wöll AK, Kokke B et al (2017) Characterization of highly concentrated antibody solution—a toolbox for the description of protein long-term solution stability. MAbs 9:1169–1185. https://doi.org/10.1080/19420862.2017.1338222

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  20. Klijn ME, Hubbuch J (2019) Correlating multidimensional short-term empirical protein properties to long-term protein physical stability data via empirical phase diagrams. Int J Pharm 560:166–174. https://doi.org/10.1016/j.ijpharm.2019.02.006

    Article  PubMed  CAS  Google Scholar 

  21. Kumar V, Dixit N, Zhou L, Fraunhofer W (2011) Impact of short range hydrophobic interactions and long range electrostatic forces on the aggregation kinetics of a monoclonal antibody and a dual-variable domain immunoglobulin at low and high concentrations. Int J Pharm 421:82–93. https://doi.org/10.1016/j.ijpharm.2011.09.017

    Article  PubMed  CAS  Google Scholar 

  22. Thiagarajan G, Semple A, James JK et al (2016) A comparison of biophysical characterization techniques in predicting monoclonal antibody stability. MAbs 8:1088–1097. https://doi.org/10.1080/19420862.2016.1189048

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  23. Broide ML, Tominc TM, Saxowsky MD (1996) Using phase transitions to investigate the effect of salts on protein interactions. Phys Rev E 53:6325–6335. https://doi.org/10.1103/PhysRevE.53.6325

    Article  CAS  Google Scholar 

  24. Heijna MCR, Van Enckevort WJP, Vlieg E (2007) Crystal growth in a three-phase system: diffusion and liquid-liquid phase separation in lysozyme crystal growth. Phys Rev 76:1–7. https://doi.org/10.1103/PhysRevE.76.011604

    Article  CAS  Google Scholar 

  25. Liu C, Asherie N, Lomakin A et al (1996) Phase separation in aqueous solutions of lens gamma-crystallins: special role of gamma s. Proc Natl Acad Sci USA 93:377–382. https://doi.org/10.1073/pnas.93.1.377

    Article  PubMed  CAS  Google Scholar 

  26. Grigsby JJ, Blanch HW, Prausnitz JM (2001) Cloud-point temperatures for lysozyme in electrolyte solutions: effect of salt type, salt concentration and pH. Biophys Chem 91:231–243. https://doi.org/10.1016/S0301-4622(01)00173-9

    Article  PubMed  CAS  Google Scholar 

  27. Park EJ, Bae YC (2004) Cloud-point temperatures of lysozyme in electrolyte solutions by thermooptical analysis technique. Biophys Chem 109:169–188. https://doi.org/10.1016/j.bpc.2003.11.001

    Article  PubMed  CAS  Google Scholar 

  28. Raut AS, Kalonia DS (2016) Effect of excipients on liquid-liquid phase separation and aggregation in dual variable domain immunoglobulin protein solutions. Mol Pharm 13:774–783. https://doi.org/10.1021/acs.molpharmaceut.5b00668

    Article  PubMed  CAS  Google Scholar 

  29. Taratuta VG, Holschbach A, Thurston GM et al (1990) Liquid-liquid phase separation of aqueous lysozyme solutions: effects of pH and salt identity. J Phys Chem 94:2140–2144. https://doi.org/10.1021/j100368a074

    Article  CAS  Google Scholar 

  30. Curtis RA, Prausnitz JM, Blanch HW (1998) Protein-protein and protein-salt interactions in aqueous protein solutions containing concentrated electrolytes. Biotechnol Bioeng 57:11–21. https://doi.org/10.1002/(SICI)1097-0290(19980105)57:1%3c11:AID-BIT2%3e3.0.CO;2-Y

    Article  PubMed  CAS  Google Scholar 

  31. Boire A, Menut P, Morel MH, Sanchez C (2013) Phase behaviour of a wheat protein isolate. Soft Matter 9:11417–11426. https://doi.org/10.1039/c3sm51489g

    Article  CAS  Google Scholar 

  32. Muschol M, Rosenberger F (1997) Liquid-liquid phase separation in supersaturated lysozyme solutions and associated precipitate formation/crystallization. J Chem Phys 107:1953–1962. https://doi.org/10.1063/1.474547

    Article  CAS  Google Scholar 

  33. Bloustine J, Virmani T, Thurston GM, Fraden S (2006) Light scattering and phase behavior of lysozyme-poly (ethylene glycol) mixtures. Phys Rev Lett 96:1–4. https://doi.org/10.1103/PhysRevLett.96.087803

    Article  CAS  Google Scholar 

  34. Galkin O, Vekilov PG (2000) Control of protein crystal nucleation around the metastable liquid-liquid phase boundary. Proc Natl Acad Sci 97:6277–6281. https://doi.org/10.1073/pnas.110000497

    Article  PubMed  CAS  Google Scholar 

  35. Pincemaille J, Banc A, Chauveau E et al (2018) Methods for screening cloud point temperatures. Food Biophys 13:422–431. https://doi.org/10.1007/s11483-018-9548-1

    Article  Google Scholar 

  36. Williamson AP, Kiefer J (2014) Automatic low-cost method to determine the solubility of liquid-liquid mixtures by continuous-flow cloud point titration. Chem Eng Technol 37:1736–1740. https://doi.org/10.1002/ceat.201400091

    Article  CAS  Google Scholar 

  37. 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

    Article  PubMed  CAS  Google Scholar 

  38. Wöll AK, Desombre M, Enghauser L, Hubbuch J (2019) A phase diagram based toolbox to assess the impact of freeze/thaw ramps on the phase behavior of proteins. Bioprocess Biosyst Eng. 1:1. https://doi.org/10.1007/s00449-019-02215-5

    Article  CAS  Google Scholar 

  39. 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

    Article  PubMed  CAS  Google Scholar 

  40. GE Healthcare (2007) PD-10 desalting column

  41. Klijn ME, Hubbuch J (2018) Application of empirical phase diagrams for multidimensional data visualization of high-throughput microbatch crystallization experiments. J Pharm Sci 107:2063–2069. https://doi.org/10.1016/j.xphs.2018.04.018

    Article  PubMed  CAS  Google Scholar 

  42. Rodgers JL, Nicewander WA (1988) Thirteen ways to look at the correlation coefficient. Am Stat 42:59–66. https://doi.org/10.2307/2685263

    Article  Google Scholar 

  43. Leskovec J, Rajaraman A, Ullman JD (2014) Mining of massive datasets. Cambridge University Press

  44. Lewis GN, Randall M (1921) The activity coefficient of strong electrolytes. J Am Chem Soc 43:1112–1154. https://doi.org/10.1021/ja01438a014

    Article  CAS  Google Scholar 

  45. Hofmeister F (1888) Zur Lehre von der Wirkung der Salze. Arch für Exp Pathol und Pharmakologie 25:1–30. https://doi.org/10.1007/BF01838161

    Article  Google Scholar 

  46. Schwierz N, Horinek D, Sivan U, Netz RR (2016) Reversed Hofmeister series—the rule rather than the exception. Curr Opin Colloid Interface Sci 23:10–18. https://doi.org/10.1016/j.cocis.2016.04.003

    Article  CAS  Google Scholar 

  47. Boström M, Tavares FW, Finet S et al (2005) Why forces between proteins follow different Hofmeister series for pH above and below pI. Biophys Chem 117:217–224. https://doi.org/10.1016/j.bpc.2005.05.010

    Article  PubMed  CAS  Google Scholar 

  48. Zhang Y, Cremer PS (2009) The inverse and direct Hofmeister series for lysozyme. Proc Natl Acad Sci 106:15249–15253. https://doi.org/10.1073/pnas.0907616106

    Article  PubMed  CAS  Google Scholar 

  49. Wetter LR, Deutsch HF (1951) Immunological studies on egg white proteins IV. Immunochemical and physical studies of lysozyme. J Biol Chem 192:237–242

    PubMed  CAS  Google Scholar 

  50. Ries-Kautt MM, Ducruix AF (1989) Relative effectiveness of various ions on the solubility and crystal growth of lysozyme. J Biol Chem 264:745–748

    PubMed  CAS  Google Scholar 

  51. Retailleau P, Riès-Kautt M, Ducruix A (1997) No salting-in of lysozyme chloride observed at low ionic strength over a large range of pH. Biophys J 73:2156–2163. https://doi.org/10.1016/S0006-3495(97)78246-8

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  52. Collins KD (2004) Ions from the Hofmeister series and osmolytes: effects on proteins in solution and in the crystallization process. Methods 34:300–311. https://doi.org/10.1016/j.ymeth.2004.03.021

    Article  PubMed  CAS  Google Scholar 

  53. Ataka M, Tanaka S (1986) The growth of large single crystals of lysozyme. Biopolymers 25:337–350. https://doi.org/10.1002/bip.360250213

    Article  PubMed  CAS  Google Scholar 

  54. Riès-Kautt M, Ducruix A (1997) [3] Inferences drawn from physicochemical studies of crystallogenesis and precrystalline state. Methods Enzymol 276:23–59. https://doi.org/10.1016/S0076-6879(97)76049-X

    Article  PubMed  Google Scholar 

  55. Burke MW, Leardi R, Judge RA, Pusey ML (2001) Quantifying main trends in lysozyme nucleation: the effect of precipitant concentration, supersaturation, and impurities. Cryst Growth Des 1:333–337. https://doi.org/10.1021/cg0155088

    Article  CAS  Google Scholar 

  56. Chernov AA (2003) Protein crystals and their growth. J Struct Biol 142:3–21. https://doi.org/10.1016/S1047-8477(03)00034-0

    Article  PubMed  CAS  Google Scholar 

  57. Garcıa-Ruiz JM (2003) Nucleation of protein crystals. J Struct Biol 142:22–31. https://doi.org/10.1016/S1047-8477(03)00035-2

    Article  CAS  Google Scholar 

  58. Singh SK, Nema S (2010) Freezing and thawing of protein solutions. In: Formulation and process development strategies for manufacturing biopharmaceuticals. Wiley, pp 625–675

  59. Mullin JW (1992) Crystallization, 3rd edn. Butterworth Heinemann, Oxford

    Google Scholar 

  60. Kestin J, Sokolov M, Wakeham WA (1978) Viscosity of liquid water in the range −8 °C to 150 °C. J Phys Chem Ref Data 7:941–948. https://doi.org/10.1063/1.555581

    Article  CAS  Google Scholar 

  61. Aleksandrov AA, Dzhuraeva EV, Utenkov VF (2012) Viscosity of aqueous solutions of sodium chloride. High Temp 50:354–358. https://doi.org/10.1134/S0018151X12030029

    Article  CAS  Google Scholar 

  62. Goldsack DE, Franchetto RC (1978) The viscosity of concentrated electrolyte solutions. II. Temperature dependence. Can J Chem 56:1442–1450. https://doi.org/10.1139/v78-236

    Article  CAS  Google Scholar 

  63. Pusey ML (1992) Continuing adventures in lysozyme crystal growth. J Cryst Growth 122:1–7. https://doi.org/10.1016/0022-0248(92)90219-9

    Article  CAS  Google Scholar 

  64. Vekilov PG (2010) Nucleation. Cryst Growth Des 10:5007–5019. https://doi.org/10.1021/cg1011633

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  65. Lu J, Carpenter K, Li RJ et al (2004) Cloud-point temperature and liquid-liquid phase separation of supersaturated lysozyme solution. Biophys Chem 109:105–112. https://doi.org/10.1016/j.bpc.2003.10.021

    Article  PubMed  CAS  Google Scholar 

  66. Wang W (2000) Lyophilization and development of solid protein pharmaceuticals. Int J Pharm 203:1–60. https://doi.org/10.1016/S0378-5173(00)00423-3

    Article  PubMed  CAS  Google Scholar 

Download references

Acknowledgements

This work was financially supported by the BE-Basic Foundation (https://www.be-basic.org), under project FS2.003. The authors want to thank academic and industrial partners for scientific discussions during the development of this work. Additionally, the authors would like to thank Lena Enghauser for contributing to the experimental work.

Author information

Author notes

  1. Marieke E. Klijn and Anna K. Wöll contributed equally to this work.

Authors and Affiliations

  1. Institute of Engineering in Life Sciences, Section IV: Biomolecular Separation Engineering, Karlsruhe Institute of Technology (KIT), Fritz-Haber-Weg 2, 76131, Karlsruhe, Germany

    Marieke E. Klijn, Anna K. Wöll & Jürgen Hubbuch

Authors
  1. Marieke E. Klijn
    View author publications

    Search author on:PubMed Google Scholar

  2. Anna K. Wöll
    View author publications

    Search author on:PubMed Google Scholar

  3. Jürgen Hubbuch
    View author publications

    Search author on:PubMed Google Scholar

Corresponding author

Correspondence to Jürgen Hubbuch.

Ethics declarations

Conflict of interest

The authors report no conflict of interest.

Additional information

Publisher's Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Electronic supplementary material

Below is the link to the electronic supplementary material.

Supplementary material 1 (PDF 1262 kb)

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Klijn, M.E., Wöll, A.K. & Hubbuch, J. Apparent protein cloud point temperature determination using a low volume high-throughput cryogenic device in combination with automated imaging. Bioprocess Biosyst Eng 43, 439–456 (2020). https://doi.org/10.1007/s00449-019-02239-x

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00449-019-02239-x

Keywords