Advertisement

Chromatographic Techniques in the Downstream Processing of Proteins in Biotechnology

  • Ruth Freitag
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1104)

Abstract

The purification of the product, the so-called downstream process (DSP), tends to be one of the most costly aspects of modern bioprocessing, especially in the case of proteins. In such cases, chromatography is still the major tool on all levels of the DSP from the first capture to the final polishing step. In this chapter, we will first outline the commonly used methods and their setup, in particular ion exchange chromatography (IEX), hydrophobic interaction chromatography (HIC), affinity chromatography (AC), and gel filtration (GPC, SEC), but also some less-known alternatives such as hydroxyapatite chromatography (HAC). Then the rational design of a downstream process, which usually comprises three orthogonal chromatographic steps, is discussed. Finally, process variants deviating from the usual batch-column/gradient elution approach will be presented, including expanded bed, displacement, and continuous chromatography, but also affinity precipitation. A most recent trend observable in the biotechnical DSP is the drive towards disposable elements (single-use technologies). Some options for this will be discussed as well.

Key words

Affinity Capture Chromatography DSP GPC HIC IEX Isolation Protein Purification SEC 

References

  1. 1.
    Wurm FM (2004) Production of recombinant protein therapeutics in cultivated mammalian cells. Nat Biotechnol 22:1393–1398CrossRefGoogle Scholar
  2. 2.
    Hagel L, Jagschies G, Sofer GK (2007) Handbook of process chromatography: development, manufacturing, validation and economics. Academic, Burlington, MAGoogle Scholar
  3. 3.
    Carta G, Jungbauer A (2010) Protein chromatography: process development and scale up. Wiley-VCH Verlag GmbH & Co. KGaA, WeinheimGoogle Scholar
  4. 4.
    Antony J (2003) Design of experiments for engineers and scientists. Butterworth-Heinemann, OxfordGoogle Scholar
  5. 5.
    Molnar I, Rieger H-J, Monks KE (2010) Aspects of the “design space” in high pressure liquid chromatography method development. J Chromatogr A 1217:3193–3200CrossRefGoogle Scholar
  6. 6.
    Turkova J (1993) Bioaffinity chromatography (J. Chromatogr. Library, Vol. 55), 2nd edn. Elsevier, AmsterdamGoogle Scholar
  7. 7.
    Becker K, Val AJ, Bülow L (2008) Multipurpose peptide tags for protein isolation. J Chromatogr A 1202:40–46CrossRefGoogle Scholar
  8. 8.
    Terpe K (2003) Overview of tag protein fusions: from molecular and biochemical fundamentals to commercial systems. Appl Microbiol Biotechnol 60:523–533Google Scholar
  9. 9.
    Freitag R, Hilbrig F (2012) Isolation and purification of recombinant proteins, antibodies and plasmid DNA with hydroxyapatite chromatography. Biotechnol J 7:90–102CrossRefGoogle Scholar
  10. 10.
    Gagnon P (2009) Monoclonal antibody purification with hydroxyapatite. New Biotechnol 25:287–293CrossRefGoogle Scholar
  11. 11.
    Gagnon P, Ng P, Zhen J et al (2006) A ceramic hydroxyapatite-based purification platform. Simultaneous removal of leached Protein A, aggregates, DNA, and endotoxins from MABs. Proc Int 4:50–60Google Scholar
  12. 12.
    Kennedy RM (2005) Expanded-bed adsorption chromatography. Curr Protoc Protein Sci. Chapter 8:Unit 8.8Google Scholar
  13. 13.
    Freitag R (1998) Displacement chromatography: application to downstream processing in biotechnology. In: Subramanian G (ed) Bioseparation and bioprocessing, biochromatography, vol 1. VCH Verlagsgruppe Weinheim, New York, pp 89–112CrossRefGoogle Scholar
  14. 14.
    Guiochon G, Felinger A, Shirazi DGG et al (2006) Fundamentals of preparative and non-linear Chromatography, 2nd edn. Academic, AmsterdamGoogle Scholar
  15. 15.
    Freitag R, Wandrey C (2003) Synthetic displacers for preparative biochromatography. In: Freitag R (ed) Synthetic polymers in biotechnology and medicine, Biotechnology Intelligence Unit 4. Landes Biosciences, Eurekah-Com, Georgetown, Chapter 4Google Scholar
  16. 16.
    Jacobson J, Frenz J, Horvath C (1984) Measurement of adsorption isotherms by liquid chromatography. J Chromatogr 316:53–68CrossRefGoogle Scholar
  17. 17.
    Imamoglu S (2002) Simulated moving bed chromatography (SMB) for application in bioseparation. In: Freitag R (ed) Advances in biochemical engineering/biotechnology, modern advances in chromatography, vol 76. Springer Verlag, Berlin, pp 211–231Google Scholar
  18. 18.
    Hilbrig F, Freitag R (2003) Continuous annular chromatography. J Chromatogr B 790: 1–15CrossRefGoogle Scholar
  19. 19.
    Aumann L, Morbidelli M (2007) A continuous multicolumn countercurrent solvent gradient (MCSGP) process. Biotechnol Bioeng 98:1043–1055CrossRefGoogle Scholar
  20. 20.
    Rajendran A, Paredes G, Mazzotti M (2009) Simulated moving bed chromatography for the separation of enantiomers (review). J Chromatogr A 2016:709–738CrossRefGoogle Scholar
  21. 21.
    Hilbrig F, Freitag R (2003) Affinity precipitation for protein purification. J Chromatogr B 790:79–90CrossRefGoogle Scholar
  22. 22.
    Freitag R, Hilbrig F (2008) Use of the avidin (imino) biotin system as a general approach to affinity precipitation. In: McMahon RJ (ed) Methods in molecular biology. Humana press Inc, Totowa, pp 35–50, chapter 4Google Scholar
  23. 23.
    Graumann K, Ebenbichler AA (2005) Development and scale up of preparative HIC for the purification of a recombinant therapeutic protein. Chem Eng Technol 28:1398–1407CrossRefGoogle Scholar
  24. 24.
    Schubert S, Freitag R (2007) Comparison of ceramic hydroxy- and fluoroapatite versus Protein A/G-based resins in the isolation of a recombinant human antibody from cell culture supernatant. J Chromatogr A 2007(1142):106–113CrossRefGoogle Scholar
  25. 25.
    Shukla AA, Barnthouse KA, Bae SS et al (1998) Structural characteristics of low molecular mass displacers for cation-exchange displacement chromatography. J Chromatogr 814:83–95CrossRefGoogle Scholar
  26. 26.
    Schmidt B, Wandrey C, Freitag R (2002) Mass influence in the performance of oligomeric poly(diallyldimethylammonium chloride) as displacer for cation-exchange displacement chromatography of proteins. J Chromatogr A 944(1–2):149–159CrossRefGoogle Scholar
  27. 27.
    Kasper C, Vogt S, Breier J et al (1996) Protein displacement chromatography in hydroxy- and fluoroapatite columns. Bioseparation 6:247–262Google Scholar
  28. 28.
    Freitag R, Garret-Flaudy F (2002) Salt effects on the thermoprecipitation of poly-(N-isopropylacrylamide) from aqueous solution. Langmuir 18:3434–3440CrossRefGoogle Scholar
  29. 29.
    Melander WR, Corradine D, Horvath CG (1984) Salt-mediated retention of proteins in hydrophobic-interaction chromatography. Application of solvophobic theory. J Chromatogr 317:67–85CrossRefGoogle Scholar
  30. 30.
    Hermanson GT, Mallia AK, Smith PK (1992) Immobilised affinity ligand techniques. Academic, San DiegoGoogle Scholar
  31. 31.
    Ng P, Cohen A, Gagnon P (2006) Monoclonal antibody purification with CHT. Gen Eng News 26:14Google Scholar
  32. 32.
    DeVauldt D (1943) The theory of chromatography. J Am Chem Soc 65:532–540CrossRefGoogle Scholar
  33. 33.
    Brooks CA, Cramer SM (1992) Steric mass action ion exchange: displacement profiles and induced salt gradients. AIChE J 38: 1969–1978CrossRefGoogle Scholar
  34. 34.
    Freitag R (1999) Displacement chromatography of biomolecules. In: Aboul-Enein HY (ed) Analytical and preparative separation methods for biomolecules. Marcel Dekker, New York, pp 203–252Google Scholar
  35. 35.
    Vogt S, Freitag R (1998) Displacement chromatography using the UNO™ continuous bed column as stationary phase. Biotechnol Progr 14(5):742–748CrossRefGoogle Scholar
  36. 36.
    Pörtner R (2007) Animal cell biotechnology: methods and protocols, 2nd edn. Springer, New YorkCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2014

Authors and Affiliations

  • Ruth Freitag
    • 1
  1. 1.Process BiotechnologyUniversity of BayreuthBayreuthGermany

Personalised recommendations