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Pilot Scale Production and Purification of a Soluble E-Selectin-IgG Chimeric Protein

  • Andreas Werner
  • Dirk Lütkemeyeri
  • I. Poggendorf
  • H. Haßelmann
  • Johannes Müthing
  • Dietmar Vestweber
  • Jürgen Lehmann
Chapter
Part of the ESACT Proceedings book series (ESACT, volume 1)

Abstract

E-selectin is a cell surface protein expressed on activated vascular endothelial cells and involved in the initial process of attachment of circulating leukocytes to endothelial cells in case of inflammation (Vestweber and Blanks, 1999). A recombinant CHO cell line secreting a soluble E-selectin-IgG chimera (Hahne et al., 1993) was cultivated under serum free conditions in DMEM/Ham’s F12 (1∶1) supplemented with transferrin, insulin, and albumin. Data of large scale cultivation of E-selectin-IgG chimera producing CHO cells in a 100 L stirred tank bioreactor and the purification steps of the soluble chimeric protein are presented. Using a repeated batch mode a final cell density of 1.25*E6 cells/ml and a maximum product concentration of 0.65 mg/l were achieved. After removal of the cells by continuous centrifugation and a depth filter clearance step, the supernatant was concentrated via ultra filtration. Purification was performed by affinity chromatography with rProtein A resulting in an overall yield of 72 mg E-selectin-IgG chimera. A following gel filtration step was used to obtain highly purified E-selectin-IgG.

Keywords

Serum Free Condition Large Scale Cultivation Stir Tank Bioreactor Elution Condition Final Cell Density 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

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References

  1. 1.
    Hahne, M., Jäger, U., Isenmann, S., Hallmann, R., and Vestweber, D. (1993). Five tumor necrosis factor-inducible cell adhesion mechanisms on the surface of mouse endothelioma cells mediate the binding of leukocytes. J. Cell Biol, 121, 655–664.PubMedCrossRefGoogle Scholar
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    Varki, A. (1994). Selectin ligands. Proc. Natl. Acad. Sci. USA, 91, 7390–7397.PubMedCrossRefGoogle Scholar
  3. 3.
    Vestweber, D. and Blanks, J.E. (1999). Mechanisms that regulate the function of the selectins and their ligands. Physiol. Rev., 79, 181–202.PubMedGoogle Scholar

Copyright information

© Springer Science+Business Media Dordrecht 2001

Authors and Affiliations

  • Andreas Werner
    • 1
  • Dirk Lütkemeyeri
    • 1
  • I. Poggendorf
    • 1
  • H. Haßelmann
    • 1
  • Johannes Müthing
    • 1
  • Dietmar Vestweber
    • 2
  • Jürgen Lehmann
    • 1
  1. 1.Institute of Cell Culture TechnologyUniversity of BielefeldGermany
  2. 2.Institute of Cell BiologyZMBEMünsterGermany

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