Expanded bed adsorption (EBA) is a special chromatography technique with perfect classification of adsorbent particles in the column, thus the performance of protein adsorption in expanded beds is particular, obviously nonuniform and complex along the column. Detailed description of the complex adsorption kinetics of proteins in expanded bed is essential for better analyzing of adsorptive mechanisms, the design of chromatographic processes and the optimization of operation parameters of EBA processes. In this work, a theoretical model for the prediction of protein adsorption kinetics in expanded beds was developed by taking into account the classified distribution of adsorbent particles along the bed height, the nonuniform behaviors of axial liquid dispersion, the axial variation of local bed voidage as well as the axial changes of target component mass transfer. The model was solved using the implicit finite difference scheme combining with the orthogonal collocation method, and then applied to predict the breakthrough behaviors of bovine serum albumin (BSA) on StreamlineDEAEand lysozyme on Streamline SP along the bed height in expanded beds under various conditions. In addition, the experiments of front adsorption of BSA on Streamline DEAE at different axial column positions were carried out to reveal the adsorption kinetics of BSA along the bed height in a 20mm I.D. expanded bed, and the influences of liquid velocity and feed concentration on the breakthrough behaviors were also analyzed. The breakthrough behaviors predicted by the present model were compared with the experimental data obtained in this work and in the literature published. The agreement between the prediction and the experimental breakthrough curves is satisfied.

Expanded bed adsorption (EBA) is an integrated technology for the primary recovery of proteins from crude feedstock. Interactions between solid matter in the feed suspension and fluidised adsorbent particles influence bed stability and therefore have a significant impact on protein adsorption in expanded beds. In order to design efficient and reliable EBA processes a strategy is needed, which allows to find operating conditions, where these adverse events do not take place. In this paper a methodological approach is presented, which allows systematic characterisation and minimisation of cell/adsorbent interactions with as little experimental effort as possible. Adsorption of BSA to the anion exchanger Streamline Q XL from a suspension containing S. cerevisiae cells was chosen as a model system with a strong affinity of the biomass towards the stationary phase. Finite bath biomass adsorption experiments were developed as an initial screening method to e stimate a potential interference. The adhesiveness of S. cerevisiae to the anion exchanger could be reduced significantly by increasing the conductivity of the feedstock. A biomass pulse response method was used to find optimal operation conditions showing no cell/adsorbent interactions. A good correlation was found between the finite bath test and the pulse experiment for a variety of suspensions (intact yeast cells, E. coli homogenate and hybridoma cells) and adsorbents (Streamline Q XL, DEAE and SP) , which allows to predict cell/adsorbent interactions in expanded beds just from finite bath adsorption tests. Under the optimised operating conditions obtained using the prior methods, the stability of the expanded bed was investigated during fluidisation in biomass containing feedstock (up to 15% yeast on wet weight basis) employing residence time distribution analysis and evaluation by an advanced model. Based on these studies threshold values were defined for the individual experiments, which have to be achieved in order to obtain an efficient EBA process. Breakthrough experiments were conducted to characterise the efficiency of BSA adsorption from S. cerevisiae suspensions in EBA mode under varying operating conditions. This allowed to correlate the stability of the expanded bed with its sorption efficiency and therefore could be used to verify the threshold values defined. The approach presented in this work provides a fast and simple way to minimise cell/adsorbent interactions and to define a window of operation for protein purification using EBA.

Expanded bed adsorption is an integrated technology that allows the introduction of particle-containing feedstock without the risk of blocking the bed. The biomass particles contained in the feedstock have to be treated as an integral part of the process and potential interactions between suspended biomass and the adsorbent must be excluded during process design. Because the electrostatic forces dominate the interactions between the biomass and adsorbent, the zeta potential has been studied as a tool to characterize biomass/adsorbent electrostatic interactions. The zeta potentials of four types of biomass (yeast intact cells, yeast homogenate, Escherichia coli intact cells, and E. coli homogenate) and two types of ion exchanger were measured systematically at varying process conditions. Using the cell transmission index from biomass pulse-response experiments as a parameter, the relations between zeta potential and the biomass/adsorbent interaction were evaluated. Combining the influences from zeta potential of adsorbent (ζa), zeta potential of biomass (ζb), and biomass size (d), parameter (-ζaζbd) was found to be an appropriate indicator of the biomass/adsorbent interactions in expanded beds under various liquid-phase conditions for different types of biomass. The threshold value of parameter (-ζaζbd) can be defined as 120mV2 μm for cell transmission of >90%, which means that systems with (-ζaζbd)<120 may have a considerable probability of forming stable expanded beds in a biomass suspension under the particular experimental conditions.

Expanded bed adsorption is an integrated technology that allows the introduction of a particle containing feedstock without the risk of blocking the bed. Provided a perfectly classified fluidized bed (termed expanded bed) is formed in the crude feed, a sorption performance comparable to packed beds is found. During the application of biomass containing samples to stable expanded beds an increase in bed expansion due to the higher density and viscosity of the feed is encountered. In this article it is investigated whether the expanded bed condition is also fulfilled during the transition in bed expansion from lower to higher density (i.e., from an equilibration buffer to a biomass containing feedstock). Residence time distribution analyses were performed by using model systems and a yeast suspension during this transition phase. It is shown that in systems in which the biomass does not interact with the fluidized stationary phase, the perfectly classified fluidization is maintained also during this transition phase regardless of the type of feedstock. Additional bed expansion takes place in an "ordered" manner without compromising bed stability. In case of biomass/adsorbent interactions, a deterioration in bed stability is found directly when the crude feed is loaded.

Expanded bed adsorption is an innovative chromatographic technology that allows the introduction of particle-containing feedstock without the risk of blocking the bed. Provided a perfectly classified fluidized bed (termed expanded bed) is formed in the crude feedstock and the biomass is not influencing protein transport towards the adsorbent surface, a sorption performance comparable to packed beds is found. The influence of biomass on the hydrodynamic stability of expanded beds is essential and was investigated systematically in this article. Residencetime distribution analyses were performed using model systems and a yeast suspension under various fluid-phase conditions. It is demonstrated that three factors (biomass/adsorbent interactions, biomass concentration, and flow rate) play an interdependent role disturbing the classified fluidization of an expanded bed. Aclear correlation between the degree of aggregative fluidization-obtained by PDE modeling of RTD data-and the expansion behavior of the fluidized bed has been found. Thus, combining three analytical methods, namely cell transmission index analysis, expansion analysis, and RTD analysis provides a solid base for understanding and control of the fluidization behavior and thus further process design during the initial phase of process development. B 2004 Wiley Periodicals, Inc. Keywords

Expanded bed adsorption is an integrative technology in downstream processing allowing the direct capture of target proteins from biomass (cells or cell debris) containing feedstocks. Potential adhesion of biomass on the surface of adsorbent, however, may hamper the application of this technique. Since the electrostatic forces dominate the interactions between biomass and adsorbent, the concept of zeta potential was introduced to characterize the biomass/adsorbent electrostatic interactions during expanded bed application. The criterion of zeta potential evaluation proposed in the previous paper (Biotechnol Bioeng, 83 (2): 149-157, 2003) was verified further with the experimental validation. The zeta potential of intact cells and homogenates of four microorganisms (Escherichia coli, Bacillus subtilis, Pichia pastoris, and S. cerevisiae) were measured under varying pH and salt concentration, and two ion-exchange adsorbents (Streamline DEAE and Streamline QXL) were investigated. The biomass transmission index (BTI) from the biomass pulse response experiments was used as the indicator of biomass adhesion in expanded bed. Combining the influences from zeta potential of adsorbent (ζa), zeta potential of biomass (ζb) and biomass size (d), a good relationship was established between the zeta potential parameter (-ζaζbd) and BTI for all experimental conditions. The threshold value of parameter (-ζaζbd) can be defined as 120mV2 mmfor BTI above 0.9. This means that the systems with (-ζaζbd)<120 show neglectable electrostatic bio-adhesion, and would have a considerable probability of forming stable expanded beds in a biomass suspension under the particular experimental conditions.

For better understanding the influences of solid phase properties on the performance of the expanded bed, the expansion and hydrodynamic properties of cellulose-stainless steel powder composite matrix with a series of densities was investigated and analyzed in an expanded bed. Two kinds of matrix particle diameter fractions, the small one (60-125m) and the large (125-300m), were used in the present work. In general, the expansion factors decreased obviously with the increase of matrix ensity. A linear relation between the mean density of matrix and superficial velocity at expansion factor of 2.5 was found for same series of atrices. The Richardson-Zaki equation could correlate the bed expansion and operation fluid velocity for all matrices tested. The theoretical prediction of correlation parameters (the terminal settling velocity Ut and expansion index n) was improved with the modification of equations in the literature. The residence time distributions were investigated to haracterize the hydrodynamic property in expanded bed. Compared with three evaluation factors (the height equivalent of theoretical plate, Bo number and axial distribution coefficient Dax), the results indicated that Dax is the best parameter to analyze the bed stability of expanded bed under various operation conditions and matrix properties. In addition, it was found that fluid velocity is the most essential factor to influence the hydrodynamic properties in the bed. A linear relation between the Dax and superficial fluid velocity for all matrices tested was established.

Expanded bed adsorption (EBA) is an integrative unit operation for the primary recovery of bioproducts from crude feedstock. Biomass electrostatic adhesion often leads to bad bed stability and low adsorption capacity. The results indicate that effective cell disruption is a potential approach to reduce the biomass adhesion during anion-exchange EBA. Two common cell disruption methods (sonication treatment and high-pressure disruption with a French press) were investigated in the present work. The mean size of cell debris reduced dramatically during the cell disruption process, and the absolute value of the ζ potential of cell debris also decreased significantly as the mean size reduced. The biomass transmission index (BTI) obtained through the biomass pulse response experiment was used to quantitatively evaluate the biomass-adsorbent interaction. Combining the influences of ú potential of adsorbent (ζA), ζ potential of biomass (ζB), and biomass mean size (ζB), the parameter of (-ζA·ζB·dB) was explored as a reasonable indicator of biomass adhesion in expanded beds. A good linear correlation was confirmed between BTI and (-ζA·ζB·dB) for all biomass and cell disruption conditions tested, which was independent of the cell disruption methods. A target parameter (-ζA·ζB·dB) of 120 mV2μm was derived for BTI above 0.9, which meant a very slight influence of biomass on the stability of the expanded bed. This criterion could be used as a rational control target for cell disruption processes in EBA applications.

Ligand density is an important factor in determining the binding capacity and separation efficiency for affinity chromatography. A molecular analysis method based on the three-dimensional structure of protein and protein-ligand interactions was introduced to optimize the dye-ligand density for target protein separation. Expanded-bed adsorption (EBA) of L-lactate dehydrogenase (LDH) from rabbit muscle crude extract with Procion Red HE-3B as the dye-ligand was used as the model. After the analysis of LDH three-dimensional molecular structure and dye-protein interaction modes, the rational dye-ligand distance was predicted at about 20 Å for efficiently binding LDH. A series of dye-ligand adsorbents with different ligand densities were prepared, and the isotherm adsorption equilibria of LDH were measured. High adsorption capacity of LDH was achieved at about 1600u/mL adsorbent. Packed-bed chromatography was performed, and the elution effects were investigated. Finally, an EBA process was achieved to capture the LDH directly from rabbit muscle crude extract. The method established in the present work could be expanded to guide the screening of ligand density for other affinity chromatographic processes.

Macroporous cellulose-tungsten carbide composite beads was designed and prepared as an anionexchanger for expanded bed adsorption (EBA). The wet density of composite beads was adjusted at the range of 1.2-2.4g/ml with the control of tungsten carbide addition, and optimized for EBA at high fluid velocity. The results indicated that the wet density of composite beads could increase linearly with the increase of tungsten carbide addition, meanwhile other physical properties, such as size, porosity, specific surface area, mean pore diameter, etc., were hardly or slightly influenced. The composite beads were coupled with diethylaminoethyl (DEAE) as an anion-exchanger for EBA. The expansion characteristics in expanded bed were investigated and sensitively changed as the wet density of composite beads, corresponding to tungsten carbide addition in the preparation. The relation among the operation fluid velocity, the ratio of tungsten carbide to cellulose viscose in the preparation and the expansion factor was found, which could be used to predict the operation velocity of composite beads with varying tungsten carbide addition. The liquid mixing in expanded bed was also tested and showed good bed stability for EBA processes. With the adsorption equilibrium experiments, the saturated adsorption capacity of bovine serum albumin could reach 68.7mg/g adsorbents (equal to 97.1mg/ml adsorbents). The ratio ofQ10% (the dynamic adsorption at 10% breakthrough) in expanded bed to packed bed could reach more than 90% for the fluid velocity of 500cm/h, even 77.1% for the fluid velocity as high as 900cm/h. The chromatographic results demonstrated that the composite beads prepared are suitable for EBA applications at high fluid velocity.