Expanded bed adsorption is a new downstream processing technique forcapture of proteins directly from unclarified feedstocks. Expanded bed adsorption reduces the number of operations in purification processes by combining clarification, concentration, and capture into one operation. It is based on stable fluidization and uses adsorbent particles with well-defined size and density distributions, together with columns designed to giveeven liquid flow distribution. The bed expands as the adsorbent particles are lifted by an upward liquid flow through the column. The behavior of the expanded bed is similar to a packed chromatography bed due to very little back-mixing of the adsorbent particles. The major benefit of using anexpanded bed is that adsorption can be carried out with unclarified feedstocks; there is no need for centrifugation or filtration to remove cells and debris. When the feedstock is applied, the target protein is captured by the adsorbent while cells and debris pass through the column unhindered. Washing is performed with the bed in an expanded mode, followed by elution of bound protein in a sedimented mode with downward flowDescribed in this article is the use of expanded bed adsorption for pilot scale recovery of recombinant human placental annexin V from an Escherichia coli ho mogenate. The description includes the whole procedure, from small-scale method optimization to pilot scale. The recovery of annexin V was approximately 95% at both lab scale and pilot scale. During the trials, it was discovered that the expanded bed was affected by the biomass content and viscosity of the homogenate. The upper limits for these parameters were therefore investigated further. For the E. coli used in the application described here, homogenates with biomass dry weightup to 5% and viscosities up to 10 mPa s (at a shear rate of 1 s(-1)) worked best. It was, however, feasible to use homogenates with dry weight up to 7-8% and viscosities up to 50 mPa s (1 s(-1)). (c) 1994 John Wiley & Sons, Inc.

Expanded bed adsorption is a technique for recovery of biomolecules directly from unclarified feedstocks. The work described here demonstrates that expanded bed adsorption is a scaleable technique. The methods used to test scaleability were ''determination of degree of bed expansion'', ''determination of axial dispersion'' and ''determination of protein breakthrough capacity''. The performance of a production scale expanded bed column with 600 mm diameter was tested using these methods and the results were found to be consistent with the results obtained from lab scale and pilot scale expanded bed columns.The scaleability and function of the expanded bed technique was also tested by performing a ''process example'': a purification mimicking a real process using a yeast culture spiked with bovine serum albumin as feedstock. The results show that the 600 mm diameter production scale column was as efficient as a 25 mm diameter lab scale column in recovering bovine serum albumin from the unclarified yeast culture. The production scale runs were fully automated using a software controlled system containing an adaptor position sensor and an adsorbent sensor.A cleaning study was performed which showed that after use of a proper cleaning protocol, no surviving microorganisms could be detected in the column or in the adsorbent.