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Affinity purification of proteins using expanded beds.

H A Chase1, N M Draeger

  • 1Department of Chemical Engineering, University of Cambridge, UK.

Journal of Chromatography
|April 24, 1992
PubMed
Summary
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Expanded beds of affinity adsorbents allow direct protein purification from cell-containing feedstocks, simplifying downstream processing. This method maintains high adsorption performance even with intact cells, offering efficient bioprocessing solutions.

Area of Science:

  • Biochemical Engineering
  • Protein Purification
  • Chromatography

Background:

  • Traditional protein purification often requires extensive pre-treatment steps like centrifugation or filtration to remove particulate matter.
  • This pre-treatment adds complexity and cost to downstream processing flow-sheets.
  • Developing methods that can directly process crude feedstocks is crucial for efficient bioseparation.

Purpose of the Study:

  • To describe and evaluate the use of expanded beds of affinity adsorbents for direct protein purification.
  • To demonstrate the feasibility of applying feedstocks containing whole or broken cells without prior particulate removal.
  • To assess the performance of expanded beds compared to conventional packed beds in protein purification.

Main Methods:

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  • Utilized expanded beds packed with affinity adsorbents (Protein A Sepharose Fast Flow, Cibacron Blue Sepharose Fast Flow).
  • Applied feedstocks containing whole or broken cells directly to the expanded beds.
  • Performed frontal analysis and batch adsorption experiments to evaluate performance.
  • Compared adsorption performance with conventional packed bed configurations.

    • Main Results:

    • Stable expanded beds were achieved with minimal adsorbent particle circulation and plug-flow-like liquid dynamics.
    • Expanded beds demonstrated similar adsorption performance to packed beds for human polyclonal immunoglobulin G purification.
    • Adsorption performance was not diminished in the presence of intact yeast cells.
    • Successfully purified phosphofructokinase from disrupted yeast feedstock without pre-treatment.

    Conclusions:

    • Expanded beds of affinity adsorbents offer a simplified approach to protein purification by eliminating the need for pre-filtration.
    • This technology maintains high purification efficiency and selectivity, even with complex, cell-containing feedstocks.
    • Expanded bed technology holds significant potential for large-scale bioprocessing and downstream purification applications.