Expanded‐Bed Adsorption Chromatography

Robert M. Kennedy1

1 GE Healthcare, Piscataway, New Jersey
Publication Name:  Current Protocols in Protein Science
Unit Number:  Unit 8.8
DOI:  10.1002/0471140864.ps0808s40
Online Posting Date:  June, 2005
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Abstract

Expanded‐bed adsorption (EBA) chromatography is a convenient and effective technique for the capture of proteins directly from unclarified crude sample. In EBA chromatography, the settled bed is first expanded by upward flow of equilibration buffer. The crude feed, a mixture of soluble proteins, contaminants, cells, and cell debris, is then passed upward through the expanded bed. Target proteins are captured on the adsorbent, while particulates and contaminants pass through. A change to elution buffer while maintaining upward flow results in desorption of the target protein in expanded‐bed mode. Alternatively, if the flow is reversed, the adsorbed particles will quickly settle and the proteins can be desorbed by an elution buffer. The mode used for elution (expanded‐bed versus settled‐bed) depends on the characteristics of the feed. After elution, the adsorbent is cleaned with a predefined cleaning‐in‐place (CIP) solution, with cleaning followed by either column regeneration (for further use) or storage.

     
 
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Table of Contents

  • Strategic Planning
  • Basic Protocol 1: Expanded‐Bed Adsorption Chromatography
  • Support Protocol 1: Estimation of the Equilibrium Ratio of Target Protein to Resin
  • Support Protocol 2: Development of a Cleaning‐In‐Place Process for Expanded‐Bed Adsorption Chromatography
  • Alternate Protocol 1: Ion‐Exchange Expanded‐Bed Adsorption Chromatography
  • Alternate Protocol 2: Hydrophobic‐Interaction Expanded‐Bed Adsorption Chromatography
  • Alternate Protocol 3: Affinity Expanded‐Bed Adsorption Chromatography
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Expanded‐Bed Adsorption Chromatography

  Materials
  • Chromatography resin (see )
  • Start buffer
  • Equilibration buffer
  • 0.25% (v/v) acetone in equilibration buffer
  • Feedstock under constant stirring (e.g., by a magnetic stir plate or overhead mixer)
  • Wash buffer
  • Elution buffer
  • Cleaning‐in‐place (CIP) solution
  • 20% (v/v) ethanol (optional)
  • Sintered glass funnel (optional)
  • Water aspirator (optional)
  • Chromatography column (see ) with or without hydraulic pump
  • Chromatography apparatus (see Fig. , Fig. , Fig. , Fig. , and Fig. for various schematics) consisting of the following:
    • Peristaltic pump(s) (e.g., Cole‐Parmer Masterflex tubing pump; do not use peristaltic pumps designed for laboratory chromatography, such as P1, Microperpex, or Rabbit pumps, as these pumps do not produce sufficiently high flow rates) attached to Masterflex size 16 silicon pump tubing
    • Two‐channel, four‐port valve for flow reversal
    • Single‐channel, three‐ or four‐port valves
    • Pressure gauge
    • Flowthrough UV monitor with S2‐type or 6‐mm‐i.d. flow cell (do not use flow cells from analytical instruments, since cell debris may clog them)
    • Chart recorder
    • Flowthrough conductivity meter (optional)
    • Flowthrough pH meter (optional)
  • Wash bottle
  • Spirit level
  • Label tape
  • Additional reagents and equipment for CIP (see protocol 3)
NOTE: See Expanded Bed Adsorption: Principles and Methods (Amersham Biosciences; see ) for additional details regarding setup of the chromatography apparatus.

Support Protocol 1: Estimation of the Equilibrium Ratio of Target Protein to Resin

  Materials
  • Unclarified feedstock containing 0.5 mg/ml target protein
  • Chromatography resin (see )
  • End‐over‐end test tube mixer
  • Additional reagents and equipment for protein assays (unit 3.4)

Support Protocol 2: Development of a Cleaning‐In‐Place Process for Expanded‐Bed Adsorption Chromatography

  • CIP solution: 1.0 M NaOH, 6 M urea, or 6 M guanidine⋅HCl
  • Additional components for inclusion in CIP solution (if necessary; e.g., isopropyl alcohol, acetic acid, benzonase)
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Figures

Videos

Literature Cited

   Batt, B.V., Yabannavar, V.M., and Singh, S. 1995. Expanded bed adsorption process for protein recovery from whole mammalian cell culture broth. Bioseparation 5:42‐53.
   Chase, H.A. 1994. Purification of proteins by adsorption chromatography in expanded beds. Trends Biotechnol. 12:296‐303.
   Hjorth, R. 1997. Expanded bed adsorption in industrial bioprocessing: Recent developments. Trends Biotechnol. 15:230‐235.
   Kula, M.R., Lin, D.Q., Knieps, E., Reichert, U., and Thommes, J. 2001. Process design in expanded bed adsorption: Integration of biomass influence into optimizing operation conditions. Presented at Recovery of Biological Products X, Cancun, Mexico.
   Shiloach, J. and Kennedy, R.M. 2000. Expanded bed adsorption process for protein capture. In Handbook of Bioseparations (S. Ahuja, ed.) pp. 431‐451. Academic Press, New York.
   Sofer, G. and Hagel, L. 1997. Handbook of Process Chromatography: A Guide to Optimization, Scale‐Up and Validation. Academic Press, New York.
   Thommes, J. 1997. Fluidized bed adsorption as a primary recovery step in protein purification. Adv. Biochem. Eng. Biotechnol. 58:185‐230.
Internet Resources
   http://www.bo‐conf.com
  Contains program information from the biennial international congress on EBA. Manuscripts from the second congress (1998) have been collected and published in Volume 8 of the journal Bioseparation (pp. 1‐267). Manuscripts from the third congress (2000) have been collected and published in Volume 10 of Bioseparation (pp. 1‐132).
   http://www.chromatography.amershambiosciences.com
  Collection of various chromatography resources published by Amersham Biosciences.
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