Unclarified Feedstock Research Articles

Experimental investigation and modeling of copper ion adsorption in packed and expanded bed

Expanded bed adsorption (EBA) is well demonstrated integration technology for capturing bio-products from un-clarified feedstock. Liquid mixing and axial dispersion plays a crucial role in the performance of an expanded bed. In this study, a high density ion exchange resin (INDION225H) is explored for using in expanded bed adsorption. Liquid mixing and axial dispersion in the packed and expanded bed has been examined using RTD studies. Breakthrough experiments on adsorption of copper ions were performed in a packed and an expanded bed. The study provides an insight into dispersion in the packed and expanded bed, which would be useful in commercial operations like water and waste water treatment.Adsorption breakthrough experiments were further simulated using an axially dispersed plug flow model. Simulation results are in good agreement with the experimental outcome. Plug flow behavior was observed up to a velocity of 0.00135m/s, which corresponds to a bed expansion of 18%. Results endorse that proper choice of adsorbent and superficial velocity can allow for carrying out bigger scale adsorptive separations like waste water treatment in expanded bed, with a minimum impact on the bed efficiency. A new non dimensional number has also been proposed for the performance analysis of expanded bed.

5-Aminobenzimidazole as new hydrophobic charge-induction ligand for expanded bed adsorption of bovine IgG

Expanded bed adsorption (EBA) can capture target proteins directly from unclarified feedstock without prior solid–liquid separation. Hydrophobic charge-induction chromatography (HCIC) is a promising technology for biomolecule separation with high capacity, good selectivity and relatively low cost without the pretreatment of dilution or salt addition. In this work, EBA and HCIC were combined to develop a new separation technology, hydrophobic charge-induction EBA. Two HCIC ligands, 4-mercapto-ethyl-pyridine (MEP) and 5-aminobenzimidazole (ABI), were coupled onto agarose beads containing tungsten carbide to prepare the resins for EBA, named T-MEP and T-ABI, respectively. The static adsorption and dynamic binding behaviors of bovine IgG (bIgG) were investigated. Two resins had similar saturated adsorption capacities and salt-tolerant properties, but T-ABI showed higher dynamic binding capacity than T-MEP, indicating that ABI ligand was more suitable for EBA. The performances in expanded bed were verified. With the protein mixture (2mg/ml bIgG and 10mg/ml bovine serum albumin) as the model feedstock, the effects of loading and elution pH, expansion factor and loading volume on the separation performance of bIgG were evaluated. Finally, T-ABI EBA was used to separate bIgG directly from bovine whey with optimized operation conditions. The purity and recovery of bIgG reached 90.6% and 78.2%, respectively. The purification factor was about 19.3. The results demonstrated that the combination of HCIC and EBA would be a potential platform for antibody capture with less feedstock pretreatments, high efficiency and relatively low cost.

Evaluation and characterization of axial distribution in expanded bed: II. Liquid mixing and local effective axial dispersion

Expanded bed adsorption (EBA) is a promising technology to capture proteins directly from unclarified feedstock. In order to better understand liquid mixing along the bed height in expanded beds, an in-bed sampling method was used to measure residence time distribution at different bed heights. A 2cm diameter nozzle column was tested with agarose raw beads (3% crosslinked agarose containing tungsten carbide). Two settled bed heights (11.5 and 23.1cm) with different expansion factors (1.4–2.6) were investigated and the number of theoretical plates (N), the height equivalent of theoretical plate (HETP) and the local effective axial dispersion coefficient (Dax) were calculated for each bed height-defined zone. The effects of expansion factor, settled bed height and mobile phase were evaluated. The results showed that N increased with the increase of expansion factors, but Dax was unaffected under fixed bed heights. Dax and HETP were found similar as a function of relative bed height for two settled bed heights tested. Higher mobile phase viscosity resulted in stronger axial dispersion. In addition, the local effective Dax under the expansion factor near 2.0 had a different profile which showed minimum values at 0.6–0.8 relative bed height, and the potential mechanism was discussed. These results would be useful for the characterization of axial dispersion and modeling protein adsorption in expanded beds under varying operation conditions.

Effect of phase inversion and separation on hepatitis B core antigen extraction from unclarified bacterial feedstock using aqueous two-phase system

Conventional purification methods for hepatitis B core antigen (HBcAg) such as sucrose-gradient ultracentrifugation and size-exclusion chromatography are tedious and time-consuming. Therefore, a new approach using aqueous two-phase system (ATPS) was employed in this study to purify HBcAg from unclarified Escherichiacoli feedstock. Phase inversion and separation are important factors in protein partitioning, particularly in ATPS. Therefore, the objectives of this study were to first generate phase diagrams for various MW PEGs and salts, followed by determination of phase inversion and separation effects on the partitioning of HBcAg in the selected systems. Based on the phase diagrams generated in this study, polyethylene-glycol (PEG) 6000–sodium sulphate system required the lowest concentration of phase components for biphasic formation compared to other systems. When unclarified feedstock was introduced into the systems, it was observed that most of the HBcAg were partitioned to the top PEG rich phase. Moreover, it was shown that phase inversion and separation had significant effects on partitioning of HBcAg. Vigorous inversion method using a vortex mixer was favoured over gentle inversion method using a rotary shaker used in this study due to its higher HBcAg recovery. Furthermore, centrifugation was an ideal method to use for phase separation as this method shortened the overall phase separation time with better recovery and purification factor compared to systems that were left on the bench to achieve phase separation. The percent recovery and purification factor obtained were 68% and 1.93 respectively when the systems were vortexed for 1min and centrifuged at 1000g for 3min at 4°C. When these results were compared with those obtained using sucrose gradient ultracentrifugation, the percent recovery obtained using the current method was more than 20 times higher. However, the latter purification methods produced higher purity of HBcAg of more than 90% compared to that obtained in the present study (∼71%). When the purified HBcAg was analysed with enzyme-linked immunosorbent assay (ELISA), it was observed that the antigenicity was still preserved and comparable to those purified with sucrose gradient ultracentrifugation.

Direct recovery of polyhydroxyalkanoates synthase from recombinant Escherichia coli feedstock by using aqueous two-phase systems

Polyhydroxyalkanoates (PHAs) are intracellular bacterial polyesters which play a role as carbon and energy storage materials. The types of polymers produced depend on the available carbon sources, the flexibility of the organism's intermediary metabolism, and the substrate specificity of the PHA biosynthetic enzymes. The recombinant Escherichia coli harboring phaC gene obtained from Cupriavidus necator H16 was constructed and conducted in this work. The overexpressed proteins in the cell broth were directly recovered in the aqueous two-phase systems (ATPS). The optimized concentration of ATPS for separation of PHA synthase from unclarified feedstock was found to compose polyethylene glycol 6000 (30%, w/w) and potassium phosphate (8%, w/w) in the absence of NaCl at pH 8.7 and 4°C. The results indicated that the partition coefficient of activity and protein content are 8.30 and 0.23, respectively. The specific activity, selectivity, purification factor and recovery of phaCRe can achieve 1.82Umg−1, 35.30, 16.62 and 96.42%, respectively. The increment of concentration of loading biomass and temperature demonstrated a decreasing trend in terms of selectivity and purification factor. The present results demonstrated that ATPS can be applied to directly recover PHA synthase from crude feedstocks and preparation of large quantity of PHA synthase on synthesis of P(3HB) in vitro.

Evaluation and characterization of axial distribution in expanded bed. I. Bead size, bead density and local bed voidage

Expanded bed adsorption (EBA) is an innovative chromatography technology that allows the adsorption of target proteins directly from unclarified feedstock, and the most important property of an expanded bed is the perfectly classified fluidization of resin beads in the column. Due to the variation of both size and density of bulk resin beads, the axial distributions of bead size, bead density and bed voidage are the inherent characteristics of an expanded bed. However, the understanding on these properties is quite limited. In this study, raw beads (3% crosslinked agarose containing tungsten carbide) and 2cm-diameter nozzle column were used as the model system and mean bead size, bead density and local bed voidage along the bed height were measured systematically with the in-bed sampling method for two settled bed heights (11.5 and 23.1cm) and different expansion factors (1.4–2.6). With the increase of bed height, mean bead size and wet density of the beads decreased from 140 to 90μm and from 4 to 2g/ml, respectively. The local bed voidage increased from 0.6 to 0.9 with the increasing bed height. The relative bed height and relative bed voidage were introduced to describe the general rule of axial distribution. Some empirical equations were used to correlate the mean bead size, bead density and local bed voidage along the bed height with the standard deviations of 10.6%, 6.1% and 5.5, respectively. In addition, a general equation was proposed to predict the axial distributions of bead size, bead density and local bed voidage in the column with standard deviations less than 10% for most of the experimental data, which would be useful for the characterization of resin beads distribution in an expanded bed under varying operation conditions.

A Comparative Study of Agarose-Nickel prototype Composite Adsorbent and Commercial Streamline DEAE Adsorbent: Physical and Hydrodynamical Assessments

Expanded bed adsorption (EBA) is an integration technology for capturing target bioproducts directly from unclarified feedstock, designing and applying a good adsorbent affects its performance. In present work, a novel matrix has been developed for expanded bed adsorption process. The prepared matrix was manufactured using water-in-oil emulsification method. Desirable adsorbents tend to be of small size, high density as well as high porosity. Here, the prepared matrices had regular spherical shape, wet density of 1.95, followed logarithmic normal size distribution within the range of 50-200 µm, mean diameter of 126.4 µm and 91.93% prosily. The Richardson-Zaki equation expressed the bed expansion and operation flow rate for the prepared matrix and Streamline DEAE. In addition, the theoretical prediction of correlation parameters (U and n) was also measured. t To characterize the hydrodynamic properties in the expanded bed, the residence time distribution method was used. The physical and hydrodynamic properties of the fabricated matrix were studied and compared with those of the commercial Streamline DEAE matrix. The results indicated that the prepared matrices showed good expansion and stability in the bed and are thus suitable for expanded bed applications.

Integrated fluidized bed affinity recovery of an anti-MUC1 mucin recombinant diabody from Escherichia coli lysates

The fluidized bed affinity recovery of C595 diabody fragment (dbFv) from Escherichia coli lysates in an integrated system mode has been investigated. An amino acid epitope sequence, RPAP was immobilized onto Upfront adsorbent ( ρ = 1.35 g cm −3). The recovery performance of various feedstocks (clarified, unclarified and concentrated unclarified feedstocks) were compared and evaluated. It was demonstrated that the values of operational productivity for clarified and unclarified feedstock were 198.18 and 129.11 mg L −1, respectively. A relatively high elution yield (87–93%) was achieved for all types of feedstock evaluated.

Preparation of cellulose–tungsten carbide composite beads with ionic liquid for expanded bed application

AbstractExpanded bed adsorption (EBA) is an integrated technology for capturing target biomolecules directly from unclarified feedstock. The adsorbents should be designed specially to ensure the perfect performance of the expanded bed. In this study, an ionic liquid (IL), 1‐butyl‐3‐methylimidazolium chloride, was used to dissolve cellulose directly to prepare cellulose–tungsten carbide composite beads with water‐in‐oil suspension and cooling/ethanol regeneration. The preparation conditions were optimized, especially for four key factors, the cellulose concentration, ratio of the oil phase to the cellulose solution, stirring speed, and addition of tungsten carbide powder. The results showed that the tungsten carbide powder emerged well in the cellulose matrix as an inert densifier. The composite beads had a spherical appearance, a suitable size and size distribution, an appropriate wet density of 1.32–1.68 g/mL, a porosity of 90–96%, a pore radius of 45–65 nm, and a specific surface area of 30–42 m2/mL. These results show the potential application for this EBA process. Compared to the traditional technology with cellulose xanthate viscose, the new method with the IL as the nonderivatizing dissolution solvent is technically feasible, has concise processing, and is environmentally friendly. © 2010 Wiley Periodicals, Inc. J Appl Polym Sci, 2011

Effect of chemical additives on biomass deposition onto beaded adsorbents

Common limitations encountered during the direct recovery of bioproducts from an unclarified feedstock are related to the presence of biomass in such processing systems. Biomass-related effects can be described as biomass-to-support deposition and cell-to-cell aggregation. In this work, a number of chemical additives were screened for their ability to inhibit either biomass deposition, cell aggregation, or a combination of both effects. Several interacting pairs were screened. These were composed of (i) a commercial chromatographic matrix harbouring a variety of ligand types and (ii) intact yeast cells - as a model biomass type. Studies were performed based on partitioning tests, colloid deposition experiments, and sorption performance studies in expanded beds. Results indicated that the coating of anion-exchanger beads with the synthetic polymer PVP 360 alleviated biomass deposition and consequently restored EBA process performance. This behaviour correlated well with calculations performed according to the XDLVO approach: the secondary (interaction) free energy pockets decreased from -230 kT to -100 kT in the absence and in the presence of PVP 360, respectively. Experiments performed in parallel demonstrated that total binding capacity for the model protein (BSA) - under dynamic conditions - remained almost constant (≈ 55.7 kg m(-3)). Other combinations of additives and adsorbents were tested. However, no solution chemistry was able to inhibit biomass deposition onto strong (composite) ion exchangers. Moreover, yeast cell deposition was only marginally decreased when hydrophobic interaction and pseudo-affinity supports were explored. The utilization of non-toxic polymers could help to avoid detrimental biomass deposition during expanded bed adsorption of bioproducts and other direct contact sequestration methods.

Use of expanded bed adsorption to purify flavonoids from Ginkgo biloba L.

Three techniques (liquid–liquid extraction, packed bed adsorption and expanded bed adsorption) have been compared for the purification of flavonoids from the leaves of Ginkgo biloba L. A crude Ginkgo extract was obtained by refluxing with ethanol for 3 h. The yield of flavonoids achieved by this crude extraction was about 19% (w/w) and the purity of flavonoids in the concentrated extract was between 1.9 and 2.3% (w/w). The crude extract was then dissolved in deionized water and centrifuged where necessary to prepare clarified feedstock for further purification. For the method using liquid–liquid extraction with ethyl acetate, the purity, concentration ratio and yield of flavonoids were 25.4–31.0%, 16–18 and >98%, respectively. For the method using packed bed adsorption, Amberlite XAD7HP was selected as the adsorbent and clarified extract was used as the feedstock. The dynamic adsorption breakthrough curves and elution profiles were measured. For a feedstock containing flavonoids at a concentration of 0.25 mg/mL, the appropriate loading volume to reach a 5% breakthrough point during the adsorption stage was estimated to be 550–600 mL for a packed bed of volume 53 mL and a flow rate of 183 cm/h. The results from the elution stage indicated that the majority of impurities were eluted by ethanol concentrations of 40% (v/v) or below and efficient separation of flavonoids from the impurities could be achieved by elution of the flavonoids with 50–80% ethanol reaching an average purity of ∼25%. The recovery yield of flavonoids using the packed bed purification method was about 60% of the flavonoids present in the clarified feedstock (corresponding to around 30% for the total flavonoids in the unclarified crude extract). For the method using expanded bed adsorption also conducted with Amberlite XAD7HP as the adsorbent, the optimal operation conditions scouted during the packed bed experiments were used but unclarified crude extract could be loaded directly into the column. For an expanded bed with a settled bed height of 30 cm, the loss of flavonoids in the column flow-through was about 30%. The two-step elution protocol again proved to be effective in separating the adsorbed impurities and flavonoids. More than 96% of the bound impurities were completely removed by 40% ethanol in the first elution stage and less than 4% remained in the final product eluted by 90% ethanol in the second elution stage. Also, ∼74% of the adsorbed flavonoids on column (corresponding to 51% of the total flavonoids in the unclarified feedstock) were recovered in the product. In addition to higher recovery yield, the average process time to obtain the same amount of product was decreased in the expanded bed adsorption (EBA) process. The results suggest that the adoption of EBA procedures can greatly simplify the process flow sheet and in addition reduce the cost and time to purify flavonoids from Ginkgo biloba. These results clearly demonstrate the potential for the use of EBA to purify pharmaceuticals from plant sources.

Direct purification of recombinant hepatitis B core antigen from two different pre-conditioned unclarified Escherichia coli feedstocks via expanded bed adsorption chromatography

Direct recovery of hepatitis B core antigen (HBcAg) from unclarified Escherichia coli homogenates via expanded bed adsorption chromatography (EBA) has been explored in this study. Streamline DEAE was selected as the anion exchanger to recover HBcAg from heat-treated and non-heat-treated unclarified feedstocks. The use of anion-exchanger for direct extraction of proteins from unclarified feedstock is not preferred due to lack of specificity of its ligand. In this study, thermal treatment of the unclarified feedstock at 60 °C has resulted in 1.2- and 1.8-fold increases in yield and purity of HBcAg, respectively, compared with that purified from non-heat-treated feedstock. Heating the crude feedstock has resulted in denaturation and precipitation of contaminants in the feedstock, hence reducing non-specific interactions between the cell debris and adsorbent. The selectivity of the anion-exchanger has also been increased as shown in the breakthrough curve obtained. Enzyme-linked immunosorbent assay showed that the antigenicity of the HBcAg from heat-treated unclarified feedstock is still preserved.

A study of the influence of yeast cell debris on protein and α‐glucosidase adsorption at various zones within the expanded bed using In‐Bed sampling

Expanded bed adsorption chromatography is used to capture products directly from unclarified feedstocks, thus combining solid-liquid separation, product concentration and preliminary purification into a single step. However, when non-specific ion-exchangers are used as the adsorbent in the expanded bed, there is the possibility that electrostatic interactions of cells or cell debris with the adsorbent may interfere with the adsorption of soluble products. These interactions depend on the particle size of the cell debris and its surface charge, which in turn depend on the extent of disruption used to release the intracellular products. The interactions occurring during expanded bed adsorption between the anionic ion-exchanger STREAMLINE DEAE and particulate yeast homogenates obtained by high pressure homogenisation at different intensities of disruption achieved by operating at different pressures were studied, while maintaining all other parameters constant. In-bed sampling from the expanded bed using ports fitted up the height of expanded bed was used to study the retention of yeast cells and cell debris within the bed and its influence on the adsorption of total soluble protein and alpha-glucosidase within various zones of the expanded bed. The retention of the biomass present in the homogenate obtained at a lower intensity of disruption was found to be high at the lower end of the column (17% from 13.8 MPa sample compared to 1% from 41.4 MPa sample). This interaction of the particulate material with the adsorbent was found to reduce the dynamic binding capacity of the adsorbent for total soluble protein from 3.6 mg/mL adsorbent for 41.4 MPa sample to 3.0 mg/mL adsorbent for 13.8 MPa sample. The adsorption of alpha-glucosidase was found to increase with an increase in the concentration of the enzyme in the feed, which increased with the intensity of disruption. Selective adsorption of 6,732 U alpha-glucosidase per mg of total protein bound, was noticed for the feedstock prepared at a higher disruption intensity at 41.4 MPa compared to adsorption of 1,262 U/mg of total protein bound for that prepared at 13.8 MPa. The selective adsorption of alpha-glucosidase due to its high concentration together with simultaneous high specific activity of the enzyme in the feed indicated the significance of selective release of enzymes during microbial cell disruption for efficient expanded bed adsorption processes.

Direct purification and immobilization of recombinant hyaluronan lyase from unclarified feedstock using immobilized metal affinity magnetite for oligo-hyaluronan preparation

Submicron-size magnetite with metal chelating capability was prepared to directly and specifically recover recombinant hyaluronan (HA) lyase from unclarified E. coli cells lysate. The unclarified contaminants did not have significant effect on reducing the yield and purity of the recovered 6xHis tagged HA lyase. The recombinant HA lyase recovered on this immobilized metal affinity magnetite (IMAM) (12.0 mg/g magnetite) was used as an immobilized enzyme to digest high molecular weight HA for the preparation of oligo-HA. Once immobilized, HA lyase activity decreased significantly to about 10% of its free activity. After an abrupt activity decrease in the first two batch reaction, 40% of the initial activity of IMAM immobilized HA lyase was maintained at the following three repeated batch reaction. The average size of the oligo-HA prepared was estimated to be about 1660 Da by using the unsaturated oligo-HA concentration measured at OD 232. However, the average molar mass of oligo-HA determined by MALDI-TOF-MS was around 1400.

The performance of anion exchange expanded bed adsorption chromatography on the recovery of G6PDH from unclarified feedstock with high biomass concentration

The bed stability of Streamline DEAE (p=1.2 g/mL) in a 20 mm (i.d.) glass expanded bed contactor, and its performance on the recovery of glucose 6-phosphate dehydrogenase (G6PDH) from unclarified yeast homogenate were investigated. A residence time distribution study showed that a stable expanded bed was achieved. The theoretical plate and Bodenstein numbers determined were 25 and 53, respectively. A recovery yield of 87% and purification factor of 4.1 were achieved in the operation using 5% (w/v) biomass concentration feedstock. The performance of the anion exchange EBAC was still considerable good at a biomass concentration as high as 15% (w/v).

Heat treatment of unclarified Escherichia coli homogenate improved the recovery efficiency of recombinant hepatitis B core antigen

Heat precipitation procedure has been regularly incorporated as a selective purification step in various thermostable proteins expressed in different hosts. This method is efficient in precipitation of most of the host proteins and also deactivates various host proteases that can be harmful to the desired gene products. In this study, introduction of heat treatment procedure in the purification of hepatitis B core antigen (HBcAg) produced in Escherichia coli has been investigated. Thermal treatment of the cell homogenate at 60 °C for 30 min prior to subsequent clarification steps has resulted in 1.4 times and 18% higher in purity and recovery yield, respectively, compared to the non-heat-treated cell homogenate. In direct capture of HBcAg by using anion-exchangers from unclarified feedstock, pre-conditioning the feedstock by heat treatment at 60 °C for 45 min has increased the recovery yield of HBcAg by 2.9-fold and 42% in purity compared to that treated for 10 min. Enzyme-linked immunosorbent assay (ELISA) analysis showed that the antigenicity of the core particles was not affected by the heat treatment process.

Preparation and Characterization of Cellulose−Stainless Steel Powder Composite Particles Customized for Expanded Bed Application

Expanded bed adsorption (EBA) is an integrated technology for capturing target proteins directly from unclarified feedstock. The specially designed adsorbent is essential for the formation of a stable expanded bed. Using the method of water-in-oil suspension thermal regeneration, a novel type of composite porous particles for expanded bed application was prepared with stainless steel powder (SSP) as the densifier and cellulose as the skeleton. The preparation conditions were explored, and two size fractions with different SSP contents were obtained with mean particle sizes of 100 and 170 μm. The composite particles showed a spherical appearance, suitable size and size distribution, appropriate wet density of 1.2−1.8 g/mL, water content of 50−80%, porosity of 80−90%, pore radius of 30−40 nm, and specific surface area of about 50 m2/mL of wet particles, and the stable expanded bed with an expansion factor of 2−3 could be achieved under varying flow rates. In addition, the influences of SSP addition on the physical properties were analyzed, which provided some useful information on the design of EBA adsorbents. A linear relationship between the particle wet density and the SSP/cellulose viscose ratio was found. The addition of SSP into the cellulose skeleton showed little effect on the porosity, specific surface area, and pore radius in the particle, which verified the prepared composite particles are potentially suitable for expanded bed application of biomolecule separation.

Purification of recombinant nucleocapsid protein of Newcastle disease virus from unclarified feedstock using expanded bed adsorption chromatography

In the present work, a single-step purification of recombinant nucleocapsid protein (NP) of the Newcastle disease virus (NDV) directly from unclarified feedstock using an expanded bed adsorption chromatography (EBAC) was developed. Streamline 25 column (ID = 25 mm) was used as a contactor and Streamline chelating adsorbent immobilized with Ni 2+ ion was used as affinity adsorbent. The dynamic binding capacity of Ni 2+-loaded Streamline chelating adsorbent for the NP protein in unclarified feedstock was found to be 2.94 mg ml −1 adsorbent at a superficial velocity of 200 cm h −1. The direct purification of NP protein from unclarified feedstock using expanded bed adsorption has resulted in a 31% adsorption and 9.6% recovery of NP protein. The purity of the NP protein recovered was about 70% and the volume of processing fluid was reduced by a factor of 10. The results of the present study show that the IMA-EBAC developed could be used to combine the clarification, concentration and initial purification steps into a single-step operation.

Development of operating conditions for protein purification using expanded bed techniques: The effect of the degree of bed expansion on adsorption performance

A strategy for the optimization of an expanded bed adsorption process has been developed by studying a model system involving the adsorption of lysozyme onto the adsorbent STREAMLINE SP. The hydrodynamic and adsorption properties of this ion exchange adsorbent in a variety of viscosities of feedstocks have been compared by analyzing bed expansion characteristics, liquid phase dispersion characteristics, equilibrium adsorption isotherms, and mass transfer characteristics. Additionally, the influences of the degree of bed expansion on adsorption performance have been investigated by frontal analysis. In these experiments, viscous feedstocks were simulated by the inclusion of glycerol in the adsorption buffers. Breakthrough curves for lysozyme were characterized and compared in terms of overall purification processing time and productivity. On the basis of these results, the relative productivities of different operating modes with the same process liquid were found to be almost the same. However, the processing time for each purification cycle decreased with increasing velocity of process liquid. It is demonstrated that an adsorption process carried out at a constant degree of bed expansion (twice its settled bed height, corresponding to bed voidage of 0.7) is more efficient, when characterized by the apparent dynamic binding capacity, than operation at a constant liquid velocity of 300 cm/h. These results have significant implications on the design and operation of the expanded bed adsorption procedures. The advantages and problems encountered in the use of expanded bed techniques for the direct extraction of proteins from unclarified feedstocks are also discussed.

The influence of cell adsorbent interactions on protein adsorption in expanded beds

Expanded bed adsorption (EBA) is a primary recovery operation allowing the adsorption of proteins directly from unclarified feedstock, e.g. culture suspensions, homogenates or crude extracts. Thus solid–liquid separation is combined with adsorptive purification in a single step. The concept of integration requires that the solid components of the feed solution are regarded as a part of the process, which influences stability, reproducibility, and overall performance. This aspect is investigated here at the example of the influence of presence and concentration of intact yeast cells ( S. cerevisiae) on the adsorption of model proteins (hen egg white lysozyme and bovine serum albumin) to various stationary phases (cation and anion-exchange, hydrophobic interaction, immobilised metal affinity). The interaction of the cells with the adsorbents is determined qualitatively and quantitatively by a pulse response method as well as by a finite bath technique under different operating conditions. The consequence of these interactions for the stability of expanded beds in suspensions of varying cell concentration is measured by residence time distributions (RTDs) after tracer pulse injection (NaBr, LiCl). Analysis of the measured RTD by the PDE model allows the calculation of the fraction of perfectly fluidised bed ( ϕ), a parameter which may be regarded as a critical quantity for the estimation of the quality of fluidisation of adsorbents in cell containing suspensions. The correlation between bed stability and performance is made by analysing the breakthrough of model proteins during adsorption from unclarified yeast culture broth. A clear relationship is found between the degree of cell/adsorbent interaction, bed stability in terms of the ϕ parameter, and the sorption efficiency. Only beds characterised by a ϕ value larger than 0.8 in the presence of cells will show a conserved performance compared to adsorption from cell free solutions. A drop in ϕ, which is due to interactions of the fluidised adsorbent particles with cells from the feed, will directly result in a reduced breakthrough efficiency. The data presented highlight the importance of including the potential interaction of solid feedstock components and the expanded adsorbents into the design of EBA processes, as the interrelation found here is a key factor for the overall performance of EBA as a truly integrated operation.