Fabrication and characterisation of a novel pellicular adsorbent customised for the effective fluidised bed adsorption of protein products

Abstract

A dense pellicular solid matrix has been fabricated by coating 4% agarose gel on to dense zirconia-silica (ZS) spheres by water-in-oil emulsification. The agarose evenly laminated the ZS bead to a depth of 30 μm, and the resulting pellicular assembly was characterised by densities up to 2.39 g/mL and a mean particle diameter of 136 μm. In comparative fluidisation tests, the pellicular solid phase exhibited a two-fold greater flow velocity than commercial benchmark adsorbents necessary to achieve common values of bed expansion. Furthermore, the pellicular particles were characterised by improved qualities of chromatographic behaviour, particularly with respect to a three-fold increase in the apparent effective diffusivity of lysozyme within a pellicular assembly modified with Cibacron Blue 3GA. The properties of rapid protein adsorption/desorption were attributed to the physical design and pellicular deployment of the reactive surfaces in the solid phase. When combined with enhanced feedstock throughput, such practical advantages recommend the pellicular assembly as a base matrix for the selective recovery of protein products from complex, particulate feedstocks (whole fermentation broths, cell disruptates and biological extracts).

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

References

  1. [1]

    Wells, C. M., K. Patel, and A. Lyddiatt (1987) Liquid fluidised bed adsorption. pp. 200–209. In: M. J. Verrall and M. J. Hudson (eds.):Separations for Biotechnology: Ellis-Horwood, Chichester, UK.

    Google Scholar 

  2. [2]

    Draeger N. M., and H. A. Chase (1990) Liquid fluidised bed adsorption in the presence of cells.Bioseparation 2: 67–80.

    Google Scholar 

  3. [3]

    Hansson M., S. Stahl, R. Hjorth, M. Uhlen, and T. Mokes (1994) Single-step recovery of a secreted recombinant protein by expanded bed adsorption.Bio/Technology 12: 285–288.

    Article  CAS  Google Scholar 

  4. [4]

    Chang Y. K., G. E. McCreath, and H. A. Chase (1995) Development of an expanded bed technique for an affinity purification of G6PDH from unclarified yeast cell homogenates.Biotechnol. Bioeng. 48: 355–366.

    Article  CAS  Google Scholar 

  5. [5]

    Thoemmes J., S. Halfar, S. Lenz, and M-R. Kula (1995) Purification of monoclonal antibodies from whole hybridoma fermentation broth by fluidized bed adsorption.Biotechnol. Bioeng. 45: 205–211.

    Article  CAS  Google Scholar 

  6. [6]

    Chang Y. K., and H. A. Chase (1996) Ion exchange purification of G6PDH from unclarified yeast cell homogenates using expanded bed adsorption.Biotechnol. Bioeng. 49: 204–216.

    Article  CAS  Google Scholar 

  7. [7]

    Hamilton G. E., P. H. Morton, T. W. Young, and A. Lyddiart (1999) Process intensification by direct product sequestration from batch fermentations: Application of a fluidized bed, multi-bed external loop contactor.Biotechnol. Bioeng. 64: 310–321.

    Article  CAS  Google Scholar 

  8. [8]

    Hamilton, G. E., F. Luechau, S. C. Burton, and A. Lyddiatt (2000) Development of a mixed mode adsorption process for the direct sequestration of an extracellular protease from microbial batch cultures.J. Biotechnol. 79: 103–115.

    Article  CAS  Google Scholar 

  9. [9]

    Bierau, H., R. Hinton, and A. Lyddiatt, A. (2001) Direct process integration of cell disruption and fluidised bed adsorption in the recovery of labile microbial enzymes.Bioseparation 10: 73–85.

    Article  CAS  Google Scholar 

  10. [10]

    Hjorth, R. (1997) Expanded bed adsorption in industrial bioprocessing: Recent developments.Trends Biotechnol. 15: 230–235.

    Article  CAS  Google Scholar 

  11. [11]

    Chase, H. A. (1994) Purification of proteins by adsorption chro-matography in expanded beds.Trends Biotechnol. 12: 296–303.

    Article  CAS  Google Scholar 

  12. [12]

    Voute, N., and E. Boschetti (1999) Highly dense beaded sorbents suitable for fluidised bed applications.Bioseparation 8: 115–120.

    Article  CAS  Google Scholar 

  13. [13]

    Gilchrist G. R., M. T. Burns, and A. Lyddiatt (1994) Solid phases for protein adsorption in fluidised beds: Comparison of commercial and custom assembled particles. pp. 186–192. In: M. J. Verrall and M. J. Hudson (eds.),Separations for Biotechnology: Ellis-Horwood, Chichester, UK.

    Google Scholar 

  14. [14]

    Finette G. M. S., Q-M. Mao, and M. T. W. Hearn (1998) Examination of protein adsorption in fluidized bed and packed bed columns at different temperature using frontal chromatographic methods.Biotechnol. Bioeng. 58: 35–46.

    Article  CAS  Google Scholar 

  15. [15]

    Thoemmes J., M. Weiher, A. Karau, and M-R. Kula (1995) Hydrodynamics and performance in fluidized bed adsorption.Biotechnol. Bioeng. 48: 367–374.

    Article  Google Scholar 

  16. [16]

    McCreath G. E., H. A. Chase, R. O. Owen, and C. R. Lowe (1995) Expanded bed affinity chromatography of dehydrogenase from baker's yeast using dye-ligand perfluoropolymer supports.Biotechnol. Bioeng. 48: 341–354.

    Article  CAS  Google Scholar 

  17. [17]

    Zhu J., A. Lyddiatt, A. W. Pacek, and A. W. Nienow (1997) Fabrication and characterisation of agar/zircon sand composite adsorbents for protein recovery in liquid fluidised beds. pp 103–114. In: Proceedings Fourth International Conference on Bioreactor and Bioprocess Fluid Dynamics. BHR Group/Inst. Mechanical Engineering Publications, London, UK.

    Google Scholar 

  18. [18]

    Gibson, N. B. (1991).Liquid Fluidised Bed Adsorption in Protein Recovery: Assembly and Characterisation of Dedicated Solid Phases. PhD thesis, University of Birmingham, UK.

    Google Scholar 

  19. [19]

    Gibson, N. B., and A. Lyddiatt (1993) Cellulose composites in liquid fluidised bed adsorption and recovery of proteins. pp. 55–62. In: J. F. Kennedy, G. O. Phillips and P. A. Williams (eds.).Cellulosics Materials for Selective Separations and Other Technologies, Ellis-Horwood, Chichester, UK

    Google Scholar 

  20. [20]

    Lyddiatt A., and D. A. O'Sullivan DA (1998) Biochemical recovery and purification of gene therapy vectors.Curr. Opin. Biotechnol. 9: 177–185.

    Article  CAS  Google Scholar 

  21. [21]

    He, L-Z., Y-R. Gan, and Y. Sun (1997) Adsorption-desorption of BSA to highly-substituted dye-ligand adsorbents: quantitative study of the effect of ionic strength.Bioprocess Eng. 17: 301–305.

    Article  CAS  Google Scholar 

  22. [22]

    Wright, P. R., F. J. Muzzio, and B. J. Glasser (1993) Batch uptake of lysozyme: Effect of solution viscosity and mass transfer on adsorption.Biotechnol. Prog. 14: 913–921.

    Article  Google Scholar 

  23. [23]

    Levenspiel, O. (1999)Chemical Reaction Engineering, 3rd ed, John Wiley & Sons, New York, USA.

    Google Scholar 

  24. [24]

    Barnfield Frej, A-K., H. J. Johansson, S. Johansson, S, and P. Leijon (1997) Expanded bed adsorption at production scale: Scale up verification, process example and sanitisation of column and adsorbent.Bioprocess Eng. 15: 57–61.

    Article  Google Scholar 

  25. [25]

    Sexena, V. P., and D. B. Wetlaufer (1970) Formation of three dimensional structure in proteins: I, rapid nonenzymic reactivation of reduced lysozyme.Biochemistry 9: 5015–5022.

    Article  Google Scholar 

  26. [26]

    Bird, B., W. E. Stewart, and I. Lighfoot (1960)Transport Phenomena, Wiley and Sons, NY, USA.

    Google Scholar 

  27. [27]

    Geankopolis C. J. (1983)Transport Processes and Unit Operations. 2nd ed. Allyn and Bacon, NY, USA.

    Google Scholar 

  28. [28]

    Tyn M. T. and T. W. Guesk (1990) Prediction of diffusion coefficients of proteins.Biotechnol. Bioeng. 35: 326–338.

    Article  Google Scholar 

  29. [29]

    Clemmitt, R. H., L. J. Bruce, and H. A. Chase (1999) Online monitoring of the purification of GST-(His)6 from an unclarifiedEscherichia coli homogenate within an immobilised metal affinity expanded bed.Bioseparation 8: 53–67.

    Article  CAS  Google Scholar 

  30. [30]

    Richardson, J. F. and W. N. Zaki (1954) Sedimentation and fluidization: Part I.Trans. Inst. Chem. Eng. 32: 35–53.

    CAS  Google Scholar 

  31. [31]

    Chang, Y. K., and H. A. Chase (1996) Development of operating conditions for protein purification using expanded bed techniques: the effect of the degree of bed expansion on adsorption performance.Biotechnol. Bioeng. 49: 512–526.

    Article  CAS  Google Scholar 

  32. [32]

    Bierau, H., Z. Zhang, and A. Lyddiatt (1999) Direct process integration of cell disruption and fluidised bed adsorption for the recovery of intracellular proteins.J. Chem. Technol. Biotechnol. 74: 208–212.

    Article  CAS  Google Scholar 

Download references

Author information

Affiliations

Authors

Corresponding author

Correspondence to Andrew Lyddiatt.

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Sun, Y., Pacek, A.W., Nienow, A.W. et al. Fabrication and characterisation of a novel pellicular adsorbent customised for the effective fluidised bed adsorption of protein products. Biotechnol. Bioprocess Eng. 6, 419–425 (2001). https://doi.org/10.1007/BF02932323

Download citation

Keywords

  • pellicular adsorbent
  • adsorption equilibrium
  • adsorption kinetics
  • fluidised bed hydrodynamics