Pilot scale recovery of recombinant annexin V from unclarified escherichia coli homogenate using expanded bed adsorption

Abstract

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.

The influence of biomass on the hydrodynamic behavior and stability of expanded beds

“…This is based on the fact that under typical fluidphase conditions used in anion exchange processes most of the cells and cell debris carries an overall negative charge and thus predominantly interacts with a positively charged anion exchange resin. This detrimental effect has been reported by several authors (Barnfield Frej et al, 1994;Chang and Chase 1996;Chase and Draegger, 1992;Draegger and Chase, 1991;Hjorth et al, 1995). A closer understanding of the different processes influencing the reduced process performance can only be based on a combined evaluation of the hydrodynamic behavior of the bed, the influence of the feedstock onto the expansion characteristic of the bed, and the respective capacity for a given target.…”

Compatibility of column inlet and adsorbent designs for processing of corn endosperm extract by expanded bed adsorption

“…Nayak et al (2001) found that for a crude fermentation broth of Elebsiella pneumoniae pneumonia, severe channeling and inefficient adsorption (characterized by early breakthrough) were observed when the dry weight of feed was greater than c 14%. Similarly, the efficiency of EBA performance was compromised for whole Saccharomyces cerevisiae cells and S. cerevisiae homogenate at 12% wet weight (FernandezLahore et al, 1999), an Escherichia coli homogentate at loads of more than 8% dry weight (Barnfield-Frej et al, 1994), and a Pichia pastoris feed at between 10 and 12.5% wet weight . Second, interaction of the biomass with the adsorbent base matrix or ligands may interfere with product adsorption and expanded bed hydrodynamic stability.…”

Expanded bed adsorption at production scale: Scale-up verification, process example and sanitization of column and adsorbent

“…This feature generates an adsorption technique which has adsorption characteristics similar to a packed chromatography bed and yet can handle unclarified feedstocks [4,5,7]. It has already been demonstrated that the technique gives good results in lab scale [2,3,6,10,11] and pilot scale experiments [1]. The work described here shows that the expanded bed technique is scaleable and performs as well in production scale as it does in smaller scales.…”