PILOT-SCALE RECOVERY OF RECOMBINANT ANNEXIN-V FROM UNCLARIFIED ESCHERICHIA-COLI HOMOGENATE USING EXPANDED BED ADSORPTION

Expanded bed adsorption is a new downstream processing technique for c apture 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 operat ion. It is based on stable fluidization and uses adsorbent particles w ith well-defined size and density distributions, together with columns designed to give even liquid flow distribution. The bed expands as th e adsorbent particles are lifted by an upward liquid flow through the column. The behavior of the expanded bed is similar to a packed chroma tography bed due to very little back-mixing of the adsorbent particles . The major benefit of using an expanded bed is that adsorption can be carried out with unclarified feedstocks; there is no need for centrif ugation or filtration to remove cells and debris. When the feedstock i s applied, the target protein is captured by the adsorbent while cells and debris pass through the column unhindered. Washing is performed w ith the bed in an expanded mode, followed by elution of bound protein in a sedimented mode with downward flow. Described in this article is the use of expanded bed adsorption for pilot scale recovery of recombi nant human placental annexin V from an Escherichia coli homogenate. Th e description includes the whole procedure, from small-scale method op timization to pilot scale. The recovery of annexin V was approximately 95% at both lab scale and pilot scale. During the trials, it was disc overed that the expanded bed was affected by the biomass content and v iscosity of the homogenate. The upper limits for these parameters were therefore investigated further. For the E. coli used in the applicati on described here, homogenates with biomass dry weight up to 5% and vi scosities 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% a nd viscosities up to 50 mPa s (1 s(-1)). (C) 1994 John Wiley and Sons, Inc.