Effects of adsorbent properties on zone spreading in expanded bed chromatography

The mixing performance as well as the adsorption performance in expanded bed chromatography (EBC) was investigated by using various types of adsorption media (average particle size = 100–700 m, density = 1100–1700 kg/m³, base matrix = hydroxyapatite, styrene-divinylbenzene, cross-linked agarose). The scale down study with 0.8 cm diameter columns was also attempted. Pulse response curves were measured with vitamin B₁₂ as a tracer [Residence time distribution RTD experiments], and the HETP (height equivalent to a theoretical plate or plate height) values were calculated from the peak variance and the peak retention time. The HETP values for different types of packing media tested showed very similar values (0.5–1.0 cm), which did not depend on the flow-rate or the column diameter (0.8–2.6 cm). Dynamic binding capacity (DBC) values of lactic acid on a Dowex anion-exchange resin were determined from breakthrough curve (BTC) measurements for both EBC and fixed bed chromatography (FBC). The DBC values for EBC were similar to those for FBC. When the liquid feed contained insoluble particles (yeast cells) the degree of mixing increased. However, the contribution of the mixing to the total spreading of BTCs for EBC was usually small so that this increase in the mixing did not affect the adsorption performance or the DBC values significantly.     

Expanded bed adsorption on supermacroporous cross-linked cellulose matrix

Rigid spherical macroporous adsorbent beads (CELBEADS) prepared by cross-linking of cellulose were characterised and found eminently suitable for use as expanded bed affinity chromatography matrix. Chromatographic runs were performed on a 10 mm diameter column with three solutes tyrosine, papain and bovine serum albumin under non-retaining conditions on CELBEADS and Streamline^TM DEAE, a commercial agarose based expanded bed matrix. Performance of the runs was measured in terms of height equivalent to theoretical plate, HETP. Variation in HETP with velocity on Streamline^TM DEAE gave flat profiles in packed bed and increasing trend in expanded bed. On CELBEADS, the HETP curves in both packed and expanded bed modes followed profiles typical of macroporous adsorbents i.e. increasing and levelling with velocity. HETP values obtained for papain and bovine serum albumin on CELBEADS were lower than those obtained on Streamline^TM DEAE at all velocities. Lactate dehydrogenase was purified from porcine muscle homogenate using Cibacron blue conjugated to CELBEADS using a protocol reported for supports with surface hydroxyl groups. Elution of the enzyme was investigated both in packed mode as well as in expanded mode at a flow rate of 1 ml min^-1. The purification procedure took about 60 minutes and a purification fold of about 14 was achieved in both cases. The adsorbent could be cleaned in place with 5 M urea and used repeatedly without loss of performance.     

Submerged Macrophyte-bed Effects on Water-Column Phosphorus,Chlorophyll <Emphasis Type="Italic">a</Emphasis>, and Bacterial Production

Submerged macrophytes are a major component of freshwater ecosystems, yet their net effect on water column phosphorus (P), algae, and bacterioplankton is not well understood. A 4-month mass-balance study during the summer quantified the net effect of a large (5.5 ha) undisturbed macrophyte bed on these water-column properties. The bed is located in a slow-flowing (0.05–0.1 cm s^–1) channel between two lakes, allowing for the quantification of inputs and outputs. The P budget for the study period showed that, despite considerable short-term variation, the macrophyte bed was a negligible net sink for P (0.06 mg m^–2 day^–1, range from –0.76 to +0.79 mg m^–2 day^–1), demonstrating that loading and uptake processes in the weedbed roughly balance over the summer. Chlorophyll a was disproportionately retained relative to particulate organic carbon (POC), indicating that the algal component of the POC was preferentially trapped. However, the principal contribution of the weedbed to the open water was a consistent positive influence on bacterioplankton production over the summer. Conservative extrapolations based on measured August specific exports (m^–2 day^–1) of P and bacterial production exiting the weedbed applied to five regional lakes varying in lake morphometry and macrophyte cover suggest that even in the most macrophyte dominated of lakes (66% cover), P loading from submerged weedbeds never exceeds 1% day^–1 of standing epilimnetic P levels, whereas subsidization of bacterioplankton production can reach upward of 20% day^–1. The presence of submerged macrophytes therefore differentially modifies algae and bacteria in the water column, while modestly altering P dynamics over the summer.     

Miniaturised expanded-bed column with low dispersion suitable for fast flow-ELISA analyses

Flow-ELISA measurements of the monoclonal antibody concentration in cultivation broth containing murine hybriboma cells were carried out using a small expanded-bed column (0.5×2.5 cm) charged with protein A. A new specialised pellicular agarose/stainless steel matrix designed for high flow rates with fast mass transport properties was used. Special care was taken to get an efficient flow distribution. The axial dispersion coefficient was very low (2×10^–6 m² s^–1 for latex particles at a linear velocity of 10 cm min^–1). Breakthrough curves for polyclonal IgG on the protein A-derivatised support (at 2–11 cm min^–1) further emphasised its advantageous properties. No significant change in dynamic capacity was found over the entire speed range.     

Design of expanded bed supports for the recovery of plasmid DNA by anion exchange adsorption

In this study we detail the rational design of new chromatographic adsorbents tailored for the capture of plasmid DNA. Features present on current chromatographic supports that can significantly enhance plasmid binding capacity have been identified in packed bed chromatography experiments and blueprints for improved expanded bed adsorbents have been put forward. The characterisation and testing of small (20-40 m) high density (>3.7 g cm^–3) pellicular expanded bed materials functionalised with various anion exchange structures is presented. In studies with calf thymus DNA, dynamic binding capacities of 1.2 and 3.4 mg ml^–1 were recorded for prototype diethylaminoethyl-and polyethylene imine-linked adsorbents which were respectively 25 and 70 fold higher than those of equivalently derivatised commercial expanded bed materials. The prototype polyethylene imine-coupled material exhibited severe sensitivity to inter-particle bridging by nucleic acid polymers, gave low DNA recoveries (<37%) and proved difficult to regenerate. In contrast, few operational difficulties were experienced with the diethylaminoethyl-linked prototype adsorbent and successful high capacity (>0.8 mg ml^–1) capture of plasmid DNA from crude neutralised E. coli lysate was demonstrated.     

Expanded bed adsorption for recovery of patatin from crude potato juice

An expanded bed adsorption process was used to isolate patatin possessing esterase activity, from a crude juice of potato tubers. Patatin is the major storage protein of potato tubers and is released in ample amounts in the processing effluent during starch milling. We employed mixed mode affinity resins, where the binding depends primarily on the pH, and is almost independent of the ionic strength. From a library of mixed mode chemistries involving both charged and hydrophobic functions, we screened for ligands with binding specificity for patatin. The dynamic binding capacity of two high density (1.45–1.5 g ml^-1) patatin-binding agarose-glass resins in response to change of linear velocity (85–230 cm h^-1) was tested in packed (25 ml) and expanded (250 ml) column modes. The column operation included a loading step at low expansion; H/H_o~1.2. Adsorption from crude juice at pH 7.5, retained patatins up to a breakthrough level of 50%. The eluate fraction at pH 3.5, now effectively stripped from the pigments, provided a 2.5-fold enzyme enrichment and produced 4 g protein per cycle. Column productivity was 122 kAU L^-1 h^-1. The study, using potato juice as model feedstock, demonstrated the feasibility of expanded bed-recovery of potentially valuable proteins from plant biomass.This revised version was published online in October 2005 with corrections to the Cover Date.     

Isolation of human fibrinogen by axial and radial flow chromatography from Nitschmann fraction I

An axial column (3×2.6 cm) and a radial flow column (3.5×5 cm) packed with DEAE Sepharose Fast Flow media had been evaluated for the separation of human fibrinogen. Nitschmann fraction I dissolved in buffered saline (0.015 M NaCl buffered with 0.06 M Tris/HCl to pH 7.5) was the starting material. Under radial flow conditions, sample flow up to 15 ml min^–1 (i.e., 18 bed volumes h^–1) was achieved. The operating pressures were below 0.2 MPa, even though the elution velocity was 30 ml min^–1 (i.e., 36 bed volumes h^–1).     

Monitoring of α-ketoglutarate in a fermentation process using expanded bed enzyme reactors

A bienzyme flow injection system is presented for the monitoring of α-ketoglutarate produced in a fermentation process, using glutamate dehydrogenase (GDH) and glutamate oxidase (GlOx) immobilised in two serially connected expanded bed reactors. The use of expanded bed resulted in unhindered passage of the bacterial cells through the columns, and thereby the need of a separate filtering step (e.g. microdialysis) was avoided. In the first reactor, α-ketoglutarate was converted to -glutamate by GDH in the presence of ammonia and NADH. In the following reactor, -glutamate was converted by GlOx to α-ketoglutarate, ammonia and hydrogen peroxide, which was detected in an electrochemical flow-through cell at +650 mV vs. Pt/(0.1 M KCl). The detection limit of α-ketoglutarate in the coupled packed bed reactors was 1 μM (defined as 3 S/N), the linear range 0–100 μM, and the sensitivity 0.80 nA/μM (R² 0.99). In the coupled expanded bed reactors, the detection limit of α-ketoglutarate was 7 μM (defined as 3 S/N), the linear range and the sensitivity being 0–500 μM and 0.11 nA/μM (R² 1.00), respectively. The response time (defined as the time between peak rise and return to baseline) was 5 min for coupled packed beds (injection of supernatant), and 12 min for coupled expanded beds (injection of sample containing cellular and particulate matter). Several other parameters, such as reactor stability, flow rate dependency, bed expansion, glutamate interference, etc. were investigated and characterised. When analysing real samples from a fermentation broth, the same results were obtained independent of the nature of the reactor system (packed or expanded bed). The hereby described system can easily be automatised and controlled from a personal computer.     

High gradient magnetic separation versus expanded bed adsorption: a first principle comparison

A robust new adsorptive separation technique specifically designed for direct product capture from crude bioprocess feedstreams is introduced and compared with the current bench mark technique, expanded bed adsorption. The method employs product adsorption onto sub-micron sized non-porous superparamagnetic supports followed by rapid separation of the loaded adsorbents from the feedstock using high gradient magnetic separation technology. For the recovery of Savinase® from a cell-free Bacillus clausii fermentation liquor using bacitracin-linked adsorbents, the integrated magnetic separation system exhibited substantially enhanced productivity over expanded bed adsorption when operated at processing velocities greater than 48 m h^–1. Use of the bacitracin-linked magnetic supports for a single cycle of batch adsorption and subsequent capture by high gradient magnetic separation at a processing rate of 12 m h^–1 resulted in a 2.2-fold higher productivity relative to expanded bed adsorption, while an increase in adsorbent collection rate to 72 m h^–1 raised the productivity to 10.7 times that of expanded bed adsorption. When the number of batch adsorption cycles was then increased to three, significant drops in both magnetic adsorbent consumption (3.6 fold) and filter volume required (1.3 fold) could be achieved at the expense of a reduction in productivity from 10.7 to 4.4 times that of expanded bed adsorption.     

HETP values of large-scale columns for liquid chromatography

Two columns of 100 mm and 200 mm in diameter for bench-scale liquid chromatography were designed to obtain a small dead volume and an appropriately uniform distribution of liquid, and the values of the height equivalent to a theoretical plate were measured for three kinds of packing beads, Sepharose 4B, Cellulofine GC-700 m and Silica. A suitable pressure drop through a distribution plate and/or a packed bed helps to distribute liquid flow uniformly over the column diameter, and reasonable HETP values were obtained.     

Partial purification properties of human epidermal growth factor from recombinant Escherichia coli by expanded bed adsorption

Human epidermal growth factor is a polypeptide hormone having many diverse biological functions. This paper first presents the recovery results of human epidermal growth factor (hEGF) immediately from the fermentation broth of recombinant Escherichia coli by using an expanded bed system (a couple of STREAMLINE25 and ÄKTA explorer 100). The influences of operational conditions such as linear flow rate, gradient length of NaCl concentration, pH and sample concentration on the purification performances of hEGF in expanded and packed bed modes with STREAMLINE DEAE resin were systematically evaluated. After optimization, the practical recovery procedure in the expanded bed mode was carried out on a scaled-up system under the conditions of linear flow rates of 183 cm/h (upward) and 37 cm/h (downward), sample volume of 300 ml and column bed height of 13.8 cm which yielded a primary product of hEGF from the cell-free supernatant containing hEGF after centrifugation at 4000 rev/min for 15 min. As a result, the hEGF concentration in the product was higher than 20% (w/v), the concentration factor was greater than 4.3 and the total yield was higher than 80%, respectively. At the same time, the results of hEGF recovery by using expanded bed adsorption (EBA), packed bed chromatography (PBC) and salting out were compared. The results show that the procedure of hEGF recovery in expanded bed adsorption has some advantages over the other two procedures, because of its higher concentration factor, recovery yield, productivity, hEGF concentration in the primary product and shorter duration of purification run.     

Anion exchange purification of plasmid DNA using expanded bed adsorption

Recent developments in gene therapy with non-viral vectors and DNA vaccination have increased the demand for large amounts of pharmaceutical-grade plasmid DNA. The high viscosity of process streams is of major concern in the purification of plasmids, since it can cause high back pressures in column operations, thus limiting the throughput. In order to avoid these high back pressures, expanded bed anion exchange chromatography was evaluated as an alternative to fixed bed chromatography. A Streamline 25 column filled with 100 ml of Streamline QXL media, was equilibrated with 0.5 M NaCl in TE (10 mM Tris, 1 mM EDTA, pH=8.0) buffer at an upward flow of 300 cmh^-1, E. coli lysates (obtained from up to 3 liters of fermentation broth) were injected in the column. After washing out the unbound material, the media was allowed to sediment and the plasmid was eluted with 1 M NaCl in TE buffer at a downward flow of 120 cmh^-1. Purification factors of 36±1 fold, 26±0.4 plasmid purity, and close to 100% yields were obtained when less than one settled column volume of plasmid feed was injected. However, both recovery yield and purity abruptly decreased when larger amounts were processed–values of 35±2 and 5±0.7 were obtained for the recovery yield and purity, respectively, when 250 ml of feedstock were processed. In these cases, gel clogging and expansion collapse were observed. The processing of larger volumes, thus larger plasmid quantities, was only possible by performing an isopropanol precipitation step prior to the chromatographic step. This step led to an enhancement of the purification step.     

Evaluation of the effect of in-bed sampling on expanded bed adsorption

An expanded bed adsorption (EBA) column (5 cm diameter) has been modified to allow the abstraction of liquid samples from various positions along the height of an expanded bed. As the adsorbent particles were fluidized, in-bed monitoring of key component concentrations during feedstock application, washing and elution was achieved by the withdrawal of liquid samples from the voids within the expanded bed through ports along the wall of the column. Component levels in the withdrawn streams can be assayed using on-line analytical chromatography or samples can be collected and assayed off-line. On-line monitoring can be used to control the duration of the loading stage and as a tool to provide information about the hydrodynamic and adsorption/desorption processes that occur during expanded bed adsorption. Studies of residence time distributions indicated that the modifications to the column do not significantly affect liquid dispersion. Using the adsorption of glucose-6-phosphate dehydrogenase from yeast homogenate on Streamline DEAE as a model system, comparison of breakthrough curves for runs when in-bed monitoring was and was not performed also suggested that separation efficiency is not appreciably affected by in-bed sampling.     

Simplified and more robust EBA processes by elution in expanded bed mode

This paper illustrates the feasibility of eluting EBA columns in the expanded bed mode as an alternative to the generally used method of packed bed elution. It is shown that at linear flow rates of 1 – 3 cm/min the difference in total elution volume between expanded bed elution and packed bed elution is less than 20%. It is suggested that expanded bed elution offers a range of significant advantages, while the drawbacks will be insignificant in most applications. The key to the success of this method seems to be the use of EBA matrices with a relatively low degree of expansion (i.e. a high density) at the linear flow rates employed for elution of bound product.     

Ethanol production by immobilized whole cells ofZymomonas mobilis in a continuous flow expanded bed bioreactor and a continuous flow stirred tank bioreactor

Continuous ethanol fermentation by immobilized whole cells ofZymomonas mobilis was investigated in an expanded bed bioreactor and in a continuous stirred tank reactor at glucose concentrations of 100, 150 and 200 g L^–1. The effect of different dilution rates on ethanol production by immobilized whole cells ofZymomonas mobilis was studied in both reactors. The maximum ethanol productivity attained was 21 g L^–1 h^–1 at a dilution rate of 0.36 h^–1 with 150 g glucose L^–1 in the continuous expanded bed bioreactor. The conversion of glucose to ethanol was independent of the glucose concentration in both reactors.     

Expanded bed protein A affinity chromatography of a recombinant humanized monoclonal antibody: process development, operation, and comparison with a packed bed method.

We show that expanded bed protein A affinity chromatography using Streamline rProtein A media is an efficient method for purifying a recombinant humanized monoclonal antibody from unclarified Chinese hamster ovary cell culture fluid and that it provides purification performance comparable to using a packed bed. We determined that the dynamic capacity of the expanded bed media is related to flow rate (measured in column volumes per hour) by a power function, which allows a high capacity at a low flow rate. At 250 cm h-1 with a 25 cm bed height (10 column volumes h-1), the dynamic capacity is 30 g l-1. The yield and purity (measured by the amount of host cell proteins, DNA, SDS-PAGE, and turbidity) of the antibody purified by expanded bed is comparable to the yield and purity obtained on a standard packed bed method using Prosep A media.     

On-line monitoring of the purification of GST-(His)6 from an unclarified Escherichia coli homogenate within an immobilised metal affinity expanded bed

The use of a rapid chromatographic assay to monitor the level of a specific protein during its downstream processing by expanded bed adsorption is described. An expanded bed column (5 cm diameter) has been modified to allow the abstraction of liquid samples at various heights along the bed, in an automated, semi-continuous manner throughout the separation. The withdrawn samples were filtered in-line and the level of the target protein assayed by a rapid on-line chromatographic method. Using this technique it was possible to monitor the development of adsorbate profiles during the loading, washing and elution phases of the application of an unclarified feedstock. The potential of the technique is demonstrated using the separation of histidine tagged glutathione s-transferase (GST-(His)₆) from an unclarified Escherichia coli homogenate using an expanded bed of Ni²⁺ loaded STREAMLINE Chelating^TM. The level of GST-(His)₆ in the abstracted homogenate samples was measured using Zn²⁺ loaded NTA-silica as an affinity chromatographic sensor. The approach described demonstrates potential for the on-line monitoring and control of expanded bed separations and for providing a greater understanding of adsorption/desorption and hydrodynamic processes occurring within the bed.     

The effect of column verticality on separation efficiency in expanded bed adsorption

The effect of column verticality on liquid dispersion and separation efficiency in expanded bed adsorption columns was investigated using 1 and 5 cm diameter columns. Column misalignment of only 0.15° resulted in the reduction of the Bodenstein number from 140 to 50 for the 1 cm dia. column and from 75 to 45 for the 5 cm dia. column. This degree of misalignment was not detectable by visual assessment of adsorbent particle movement within the column. Depending on the relative importance of transport limitations, kinetic limitations and dispersion to any specific separation, this increase in dispersion with column alignment can significantly affect separation efficiency. Pure protein breakthrough profiles resulting from the application of bovine serum albumin onto STREAMLINE Q XL demonstrated that, at 10% breakthrough, 7.8% more protein could be applied to a vertical 1 cm dia. column compared to the same column misaligned by 0.15°. When an unclarified yeast homogenate was applied to a 1 cm dia. vertical column packed with STREAMLINE DEAE, 10% breakthrough of glucose-6-phosphate dehydrogenase (G6PDH) corresponded to a load 55% greater compared to the same column aligned 0.185° off-vertical. The G6PDH breakthrough curves for vertical and 0.15° off-vertical runs performed using a 5 cm column were essentially indistinguishable.     

A method for the analysis of acetate turnover in a coastal marine sediment

The concentrations of volatile fatty acids were measured in the pore water of sediment from the Limfjorden, Denmark. The pore water was freeze-dried and the acids, which were redissolved in formic acid, were analyzed by gas chromatography on a Carbopack column. The limit of detection was 0.1 mol l^–1 pore water. The concentration ranges (mol l^–1 pore water) were as follows: 0.1 to 6.0 for acetate; <0.1 to 0.6 for propionate, and <0.1 to 0.5 for butyrate. The rate constants for the disappearance of injected tracer concentrations of U-¹⁴C-acetate were measured at 2 cm depth intervals in sediment strata (0 to 10 cm). The rate constant for acetate turnover at 4 to 6 cm depth did not vary greatly with season, 2.1 h^–1, SD 0.6 for 7 values. In spring, the rate constants were highest in the 0 to 2 cm stratum and decreased with sediment depth. The calculated rates for acetate turnover of 7.2 mmol m^–2 day^–1 for early spring (2°C) and of 19.6 mmol m^–2 day^–1 for late autumn (7°C) were higher than would be expected from published values for carbon oxidation by sulfate in these sediments.     

Vegetative growth response of young olive trees (Olea europaea L., cv. Arbequina) to soil salinity and waterlogging

High-density olive orchards are increasing around the world, many of which may be potentially affected by salinity and waterlogging (hypoxia), two important stresses common in irrigated fields in arid and semi-arid climates. However, the response of olive to these stresses under field conditions is not well established. Therefore, our objective was to evaluate the vegetative growth response of young olive trees (Olea europaea L., cv. Arbequina) grown in a spatially variable waterlogged, saline-sodic field. We monitored the growth in trunk diameter of 341, 3-year-old olives between September 1999 and September 2000. Field contour maps were developed delineating soil salinity (ECa), relative ground elevation (RGE) and water table depth (WTD). Soil samples were also collected and analyzed for ECe and SARe in order to characterize the salinity and sodicity profiles and develop the ECa-ECe calibration equation. The infiltration rate (IR) of the crusted and uncrusted soil and the penetration resistance (PR) were also measured. The field was characterized by spatially variable ECe (2–15 dS m^–1), SARe (3–40), RGE (–4 to +4 cm) and WTD (0.5–1.9 m, with corresponding ground water EC values between 12 and 6 dS m^–1). Steady-state IR of crusted soil was only 7% of the uncrusted soil. Since the field was heavily irrigated by flooding, waterlogging conditions were related to low RGE values. Soil salinity was negatively correlated (R ² = 0.83, P<0.001) with RGE (ponded water) and WTD (upward flux), due to the evapo-concentration of water and salts at the soil surface. Thus, inverted salinity profiles developed in high salinity areas. Fifty-five percent of the olives were dead 3.5 years after planted, and most of them were located in areas of high ECe (> 10 dS m^–1), low RGE (< – 1.5 cm) and low WTD (< 1.2 m). The surviving trees had vegetative salinity tolerance values of ECe threshold = 4 dS m^–1 and slope = –12% (i.e., percent decline per unit increase in ECe above the treshold), indicating that the Arbequina olive is moderately tolerant to salinity. The RGE and WTD thresholds for olive's survival were > 0.1 cm and > 1.6 m, respectively. Thus, very small changes in ground elevation had a significant effect on olive's survival or death. The coupled effects of salinity and waterlogging (hypoxia) stresses were most detrimental for olive's growth.