Expanded Bed Absorption Chromatography

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The expanded bed absorption chromatography (short EBA chromatography ) is a preparative chromatographic reduced method that enables the analysis of unpurified biological samples, or the number of steps required for this purpose. The advantages of EBA chromatography are the reduction in processing time and cost. It was first mentioned in the 1990s in connection with the analysis of samples containing cells . It was used to separate the samples with the same level of accuracy as was previously only possible with classical chromatography methods. The EBA chromatography differs from fluidized bed methods used in the industry are used.

functionality

Methods such as filtration or centrifugation are normally used to remove cells or cell components . EBA chromatography combines these methods, but reduces the disadvantages (long processing times, high costs, etc.) that would otherwise arise when the two methods are used in combination . The principles for binding proteins are the same as in classical chromatography. The usual ligands for ion exchange and affinity chromatography can therefore also be used.

First the column is filled and the filled granules are expanded by applying an upward current strong enough to fluidize the granules. The samples are then introduced directly into the column and the target proteins or target substances bind to the column, while the cell debris can pass through the column.

Process of a typical EBA.

After completion of this adsorption step , the fluidized bed is washed out with a buffer in order to bind all unbound particles. Usually, the adsorbed proteins or substances are eluted with a flow in the opposite direction . In most cases, a fluidized bed is not used, but a "packed" bed is used to collect the dissolved substances in a smaller volume. Recently (as of 2018), a new generation of EBA was developed that maintains the extended status in order to obtain higher purity and a higher yield while using even smaller volumes of eluents. The column is then cleaned and brought back to equilibrium using reagents such as sodium hydroxide .

While classic chromatography columns use a solid stationary phase , EBA uses particles that are in a liquid phase. In the best case scenario, these are expanded by a factor of 2. EBA differs from other forms of chromatography with fluidized stationary phase on the one hand by the varying size and density of the particles in the stationary phase, which results in a gradient when the expansion takes place, on the other hand by the formation of flows behind of expansion.

Individual evidence

  1. ↑ E. Lenkiewicz, A. Ferencowa, E. Szewczykowa: [Subconjunctival autohemotherapy of eye burns in our cases] . In: Klinika Oczna . tape 94 , no. 4 , 1992, ISSN  0023-2157 , pp. 113-114 , PMID 1405409 (Polish).
  2. ^ Howard A. Chase, Nicholas M. Draeger: Affinity purification of proteins using expanded beds . In: Journal of Chromatography A . tape 597 , no. 1-2 , April 1992, pp. 129-145 , doi : 10.1016 / 0021-9673 (92) 80103-2 .
  3. ↑ RR Vennapusa, p Binner, R. Cabrera, M. Fernandez-Lahore: Surface Energetics to Assess Microbial Adhesion Onto Fluidized Chromatography Adsorbents . In: Engineering in Life Sciences . tape 8 , no. 5 , October 2008, ISSN  1618-0240 , p. 530-539 , doi : 10.1002 / elsc.200800027 .
  4. ^ Robert M. Kennedy: Expanded-Bed Adsorption Chromatography . In: Current Protocols in Protein Science . John Wiley & Sons, Inc., Hoboken, NJ, USA 2005, ISBN 0-471-14086-4 , pp. 8.8.1–8.8.25 , doi : 10.1002 / 0471140864.ps0808s40 .
  5. ^ Howard A. Chase, Nicholas M. Draeger: Affinity purification of proteins using expanded beds . In: Journal of Chromatography A . tape 597 , no. 1-2 , 1992, pp. 129-145 , doi : 10.1016 / 0021-9673 (92) 80103-2 .
  6. ↑ Expanded Bed Absorption. (PDF) Hebrew University of Jerusalem , accessed June 11, 2018 .
  7. ^ R. Hjorth: Expanded-bed adsorption in industrial bioprocessing: recent developments . In: Trends in Biotechnology . tape 15 , no. 6 , June 1997, p. 230-235 , doi : 10.1016 / S0167-7799 (97) 01045-7 , PMID 9183866 .