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Expanded-bed adsorption immobilized-metal affinity chromatography

Nature Protocols volume 1, pages 1213–1222 (2006)Cite this article

Abstract

The protocol describes a method for capture of secreted hexahistidine-tagged proteins using expanded-bed adsorption immobilized-metal affinity chromatography. The starting material for the procedure is any crude feedstock that contains a histidine (His)-tagged target protein. The protocol is exemplified using unclarified broth from Pichia pastoris fermentation as feedstock. The protocol can be used for laboratory studies or as part of a process for production of recombinant biotherapeutics to standards of good manufacturing practice. It takes approximately 5 h to purify proteins from 10 liters of feedstock and a further 5–6 h to sterilize and regenerate the column.

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Figure 1: Expanded-bed systems.
Figure 2: Addition of the biomass to the column.
Figure 3: Anticipated sample trace and pictures of key stages during expanded-bed chromatography.
Figure 4: Anticipated product purification during EBA, illustrated with sm3e, a humanized scFv (ref. 25).

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Acknowledgements

The authors would like to thank J. Bhatia, T. Hillyer and K. Vigor for contributions to this manuscript. Work is supported by Cancer Research UK, The Royal Free Cancer Research Trust, The Copley May Foundation and NTRAC.

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Authors and Affiliations

  1. Department of Oncology, Royal Free and University College Medical School, University College London, Rowland Hill Street, London, NW3 2PF, UK

    Berend Tolner, Lisa Smith, Richard H J Begent & Kerry A Chester

Authors
  1. Berend Tolner
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  2. Lisa Smith
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  3. Richard H J Begent
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  4. Kerry A Chester
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Correspondence to Berend Tolner.

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Supplementary information

Supplementary Methods 1

Aseptic handlings, pichia spillage and disposal of waste. (PDF 74 kb)

Supplementary Methods 2

Guidelines for expanded bed adsorption - IMAC process development. (PDF 80 kb)

Supplementary Note

Expanded Bed Adsorption - IMAC Process Log Sheet (PDF 110 kb)

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Tolner, B., Smith, L., Begent, R. et al. Expanded-bed adsorption immobilized-metal affinity chromatography. Nat Protoc 1, 1213–1222 (2006). https://doi.org/10.1038/nprot.2006.127

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