Advertisement

A Compartmented Microfluidic Reactor for Protein Modification Via Solid-phase Reactions - Semi-automated Examination of Two PEGylation Routes

  • Regina Fraas
  • Jonas Ferdinand Hübner
  • Juliane Diehm
  • Ramona Faas
  • Rudolf Hausmann
  • Matthias FranzrebEmail author
Research Paper
  • 12 Downloads

Abstract

Microfluidics has emerged as a relatively new scientific field enabling fast reaction times and low demands for reactants. Pursuing these advantages, a compartmented, microfluidic reactor was developed in our group which is suitable for the semi-automated processing of complex reaction cascades including solid phases. As one of the first application examples, we analyzed the influence of different reaction paths on the modification of a model protein in a solid-phase reaction. Extensive characterization experiments were performed: Amongst others, an organic phase was identified which is immiscible with water and compatible with the designated PEGylation reactions. Such organic solvents function as separation plugs for different water based reaction plugs within the microfluidic system. Mixing within the microfluidic system was investigated, in order to ensure an efficient solid-phase reaction. Subsequently, solid-phase PEGylation of the target protein was performed within the microfluidic system via two different reaction cascades. The longest reaction cascades comprised all reactions from particle activation, via protein immobilization and PEGylation to elution and consisted of seven steps. PEGylation in the reactor took place with comparable yields and results as in the control reaction outside the reactor. Due to the modularity, the presented reactor proves to be a versatile instrument for semi-automated reactions and parameter screening, being compatible with biological systems. It combines the advantages of closed channel systems like lab on a chip microfluidics with the flexibility and preparative scale sample volume of larger liquid handling stations.

Keywords

protein modification solid-phase reaction microfluidic reactor reaction cascade reaction compartments automation 

Preview

Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.

Supplementary material

12257_2017_322_MOESM1_ESM.pdf (107 kb)
A Compartmented Microfluidic Reactor for Protein Modification Via Solid-phase Reactions - Semi-automated Examination of Two PEGylation Routes

References

  1. 1.
    Hübner, J., R. Brakowski, J. Wohlgemuth, G. Brenner-Weiß, and M. Franzreb (2015) Compartmented microfluidic bioreactor system using magnetic enzyme immobilisates for fast small-scale biotransformation studies. Eng. Life Sci. 15.Google Scholar
  2. 2.
    Babich, L., A. F. Hartog, L. J. C. van Hemert, F. P. J. T. Rutjes, and R. Wever (2012) Synthesis of carbohydrates in a continuous flow reactor by immobilized phosphatase and aldolase. ChemSusChem 5: 2348–2353.CrossRefGoogle Scholar
  3. 3.
    Fornera, S., P. Kuhn, D. Lombardi, A. D. Schlüter, P. S. Dittrich, and P. Walde (2012) Sequential immobilization of enzymes in microfluidic channels for cascade reactions. Chempluschem 77: 98–101.CrossRefGoogle Scholar
  4. 4.
    Grant, J., J. A. Modica, J. Roll, P. Perkovich, and M. Mrksich (2018) An immobilized enzyme reactor for spatiotemporal control over reaction products. Small 14: 1800923.CrossRefGoogle Scholar
  5. 5.
    Huang, M. C., H. Ye, Y. K. Kuan, M.-H. Li, and J. Y. Ying (2009) Integrated two-step gene synthesis in a microfluidic device. Lab. Chip. 9: 276–285.CrossRefGoogle Scholar
  6. 6.
    Lee, C.-C., T. M. Snyder, and S. R. Quake (2010) A microfluidic oligonucleotide synthesizer. Nucleic Acids Res. 38: 2514–2521.CrossRefGoogle Scholar
  7. 7.
    Pham, Q. N., K. T. L. Trinh, S. W. Jung, and N. Y. Lee (2018) Microdevice-based solid-phase polymerase chain reaction for rapid detection of pathogenic microorganisms. Biotechnol. Bioeng. 115: 2194–2204.Google Scholar
  8. 8.
    Wang, W., Y. Huang, J. Liu, Y. Xie, R. Zhao, S. Xiong, G. Liu, Y. Chen, and H. Ma (2011) Integrated SPPS on continuous-flow radial microfluidic chip. 11.Google Scholar
  9. 9.
    Li, J., R. P. Carney, R. Liu, J. Fan, S. Zhao, Y. Chen, K. S. Lam, and T. Pan (2018) Microfluidic print-to-synthesis platform for efficient preparation and screening of combinatorial peptide microarrays. Anal. Chem. 90: 5833–5840.CrossRefGoogle Scholar
  10. 10.
    Ottow, K. E., T. Lund-Olesen, T. L. Maury, M. F. Hansen, and T. J. Hobley (2011) A magnetic adsorbent-based process for semicontinuous PEGylation of proteins. Biotechnol. J. 6: 396–409.CrossRefGoogle Scholar
  11. 11.
    Veronese, F. M., A. Mero, and G. Pasut (2009) Protein PEGylation, Basic Science and Biological Applications BT - PEGylated Protein Drugs: Basic Science and Clinical Applications. (Veronese, F. M., ed), pp. 11–31, Birkhäuser BaselGoogle Scholar
  12. 12.
    Rajan, R. S., T. Li, M. Aras, C. Sloey, W. Sutherland, H. Arai, R. Briddell, O. Kinstler, A. M. K. Lueras, Y. Zhang, H. Yeghnazar, M. Treuheit, and D. N. Brems (2009) Modulation of protein aggregation by polyethylene glycol conjugation: GCSF as a case study. Protein Sci. 15: 1063–1075.CrossRefGoogle Scholar
  13. 13.
    Song, L., Y. Zhu, H. Wang, A. A. Belov, J. Niu, L. Shi, Y. Xie, C. Ye, X. Li, and Z. Huang (2014) A solid-phase PEGylation strategy for protein therapeutics using a potent FGF21 analog. Biomaterials 35: 5206–5215.CrossRefGoogle Scholar
  14. 14.
    Niu, J., Y. Zhu, Y. Xie, L. Song, L. Shi, J. Lan, B. Liu, X. Li, and Z. Huang (2014) Solid-phase polyethylene glycol conjugation using hydrophobic interaction chromatography. J. Chromatogr. A 1327: 66–72.CrossRefGoogle Scholar
  15. 15.
    Wilms, B., A. Hauck, M. Reuss, C. Syldatk, R. Mattes, M. Siemann and J. Altenbuchner (2001) High-cell-density fermentation for production of L-N-carbamoylase using an expression system based on the Escherichia coli rhaBAD promoter. Biotechnol. Bioeng. 73: 95–103.CrossRefGoogle Scholar
  16. 16.
    Sauter, A. and F. Sauter (1944) Die Erzeugung von möglichst homogenen Magnetfeldern durch Stromsysteme. Zeitschrift für Phys. 122: 120–136.CrossRefGoogle Scholar
  17. 17.
    Strohalm, M., D. Kavan, P. Novák, M. Volný, and V. Havlíček (2010) mMass 3: A cross-platform software environment for precise analysis of mass spectrometric data. Anal. Chem. 82: 4648–4651.CrossRefGoogle Scholar
  18. 18.
    Laemmli, U. K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227: 680.CrossRefGoogle Scholar
  19. 19.
    Maiser, B., K. Baumgartner, F. Dismer, and J. Hubbuch (2015) Effect of lysozyme solid-phase PEGylation on reaction kinetics and isoform distribution. J. Chromatogr. B 1002: 313–318.CrossRefGoogle Scholar
  20. 20.
    Izumi, M., S. Okumura, H. Yuasa, and H. Hashimoto (2003) Mannose-BSA Conjugates: Comparison Between Commercially Available Linkers in Reactivity and Bioactivity. 22.Google Scholar
  21. 21.
    Paulus, A., N. Till, and M. Franzreb (2015) Controlling the partitioning behavior of magnetic micro-particles via hydrophobization with alkylamines: Tailored adsorbents for continuous bioseparation. Appl. Surf. Sci. 332.Google Scholar

Copyright information

© The Korean Society for Biotechnology and Bioengineering and Springer-Verlag GmbH Germany, part of Springer Nature 2018

Authors and Affiliations

  • Regina Fraas
    • 1
    • 2
  • Jonas Ferdinand Hübner
    • 1
  • Juliane Diehm
    • 1
  • Ramona Faas
    • 1
  • Rudolf Hausmann
    • 2
  • Matthias Franzreb
    • 1
    Email author
  1. 1.Institute of Functional InterfacesKarlsruhe Institute of TechnologyEggenstein-LeopoldshafenGermany
  2. 2.Institute of Food Science and BiotechnologyUniversity of HohenheimStuttgartGermany

Personalised recommendations