Use of dimensionless residence time to study variations in breakthrough behaviour in expanded beds formed from varied particle size distributions

This work demonstrates an experimental method for studying breakthrough behaviour in expanded beds. The behaviour of beds made with differently sized particles were studied at varying flowrates. The use of a dimensionless residence time measurement allowed a more valid comparison of breakthrough characteristics in expanded bed operation by compensating for the changes in bed volume that occur during expansion. We demonstrate that bed breakthrough behaviour can be compared directly even when the beds contain different-sized particles and hence have different expanded volumes. By utilising this concept we demonstrate that, in the case of the Alcohol Dehydrogenase (ADH) / STREAMLINE Phenyl system used here, there was little or no variation in ADH breakthrough behaviour between beds of differently sized particles operating at flowrates above 100 cm/h. This suggests that the higher specific surface area and hence binding capacity of smaller particles is negated in this case due to mass transfer limitations and the increase in system void volume even at normal operating flowrates of 200-300 cm/h.     

Polyelectrolyte-coated ion exchangers for cell-resistant expanded bed adsorption

Adsorption chromatography in expanded beds is a widely used technology for direct capture of target proteins from fermentation broths. However, in many cases this method cannot be applied as a result of the strong tendency of cells or cell debris to interact with the adsorbent beads. To prevent contamination of the expanded bed with the biomass, STREAMLINE DEAE, anion exchanger designed for expanded bed adsorption, was modified with a layer of poly(acrylic acid) (PAA). The shielding layer of polyelectrolyte was attached to the surface of the matrix beads via electrostatic interactions. PAA with a high degree of polymerization was chosen to prevent diffusion of large polymer molecules into the pores of adsorbent. Thus, the shielding layer of PAA was adsorbed only at the mouth of the pores of STREAMLINE DEAE beads and only marginally decreased the binding capacity of the ion exchanger for bovine serum albumin, the model protein in this study. PAA-coated STREAMLINE DEAE practically did not interact with yeast cells, which otherwise bound strongly to the native adsorbent at neutral conditions. Cell-resistant PAA-coated anion exchanger was successfully used for isolation of BSA from the model protein mixture containing BSA, lysozyme (positively charged at applied conditions), and yeast cells. The layer of PAA was stable under mild elution conditions, and the modified adsorbent could be used in the repeated purification cycles.     

Development of an expanded bed technique for an affinity purification of G6PDH from unclarified yeast cell homogenates

The use of an expanded bed of STREAMLINE Red H-7B for the purification of the intracellular glycolytic enzyme glucose 6-phosphate dehydrogenase (G6PDH) directly from untreated preparations of disrupted yeast cells has been investigated. Small-scale experiments, carried out in packed beds, have shown that the optimal pH for adsorption is 6.0 and have enabled optimization of elution conditions using a series of eluents. The dynamic capacity of the adsorbent for G6PDH was determined in a small expanded bed to be 28 units/mL. These results were used to develop a preparative scale separation of G6PDH in a STREAMLINE 50 expanded bed column. G6PDH was purified directly from an unclarified yeast homogenate in 99% yield with an average purification factor in the eluted fraction of 103. Cleaning-in-place (CIP) procedures using 0.5 M NaOH and 4M urea in 60% (v/v) ethanol have demonstrated that the adsorbent can be regenerated with no loss of adsorption capacity of alteration of bed expansion characteristics after many cycles of operation. (c) 1995 John Wiley & Sons, Inc.     

Capture of proteins from mammalian cells in pilot scale using different STREAMLINE adsorbents

This presentation compares three different expanded bed matrices. STREAMLINE rProtein A, STREAMLINE SP-XL and STREAMLINE Chelating were monitored in respect to their ability to clarify the broth, to concentrate and to purify the distinct target protein. The capture of a mouse IgG₁ and a recombinant prothrombin (PT) was carried out in pilot scale using a 100-l bioreactor and STREAMLINE 100 and 200 columns, respectively. The robustness of the process was also estimated monitoring the expansion behaviour and the cell and debris concentrations during the load and in the eluat. In all cases the capture of the target proteins was comparable to conventional chromatographic systems. The purification success was mainly dependent on the selectivity of the ligand used. The affinity process resulted in a highly purified product. The ion exchanger and chelating material mainly concentrated the product. In all three cases 100 l of cell broth were successfully processed in one run. The robustness of the ion exchanger process was poor, because of strong cell matrix interaction. However, for the chelating and especially for the affinity matrix a highly reproducible process was 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.     

Affinity purification of immunoglobulins from chicken egg yolk using a new synthetic ligand

Due to the peculiar composition of the egg yolk and the lack of specific affinity ligands, Y immunoglobulins are normally purified using complex and time consuming procedures involving a combination of precipitation and chromatographic steps first to extract and capture and then to polish IgY. In this study, we have examined the applicability for IgY affinity purification of TG19318, a synthetic ligand for immunoglobulin, obtained from the screening of combinatorial libraries, and already characterized for its capability to purify immunoglobulins of class G, M, E and A. Soluble proteins were separated from the lipidic fraction of egg yolk by the water dilution method and loaded on to TG19318 affinity columns prepared by immobilizing the ligand on the commercially available support Emphaze™. In a single chromatographic step TG19318 affinity columns led to an efficient capture of IgY directly from crude samples, and with a purity degree higher than 90%, as determined by densitometric scanning of SDS–PAGE analysis of bound fractions, and with full recovery of antibody activity, as determined by ELISA assay. Higher recovery and purity of IgY was obtained by using loading buffers at pH close to 6.5. Column capacity, determined by applying 4× excess IgY to 1 ml bed volume column, and eluting the retained immunoglobulins, was close to 65 mg of IgY per ml of resin. Chemical and chromatographic stability of TG19318/Emphaze was tested before and after various treatments. The derivatized matrix was found to be very stable, in terms of ligand leakage and maintenance of IgY binding capacity, under conditions of normal column usage, cleaning and storage.     

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.     

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.     

Pilot scale recovery of monoclonal antibodies by expanded bed ion exchange adsorption

The aim of the investigations was to estimate the scale up properties of an efficient chromatographic first capture step for the recovery of murine IgG₁ from undiluted and unclarified hybridoma cell culture broth using an ion exchange matrix in expanded bed mode. The tested new sulfopropyl-based ion exchange matrix (Streamline^TM SP XL, Amersham Pharmacia Biotech) stands out due to its enhanced capacity compared to its precursor (Streamline^TM SP). Defining the working pH in preliminary electrophoretic analyses (titration curve, SDS-PAGE) and small-scaled chromatographic binding studies showed, that the optimal value for the IgG purification was pH 4.6, where a co-chromatography of the medium supplement albumin (500 mg l^-1, pI = 4.8) could not be avoided. Further scouting experiments dealt with the dynamic capacity of the matrix, which was evaluated by frontal adsorption analysis. In packed bed mode no break-through of the target protein was achieved even after 6.5 mg IgG per ml matrix were applied. These results could not be reproduced in expanded bed mode with cell-free supernatant, where the dynamic capacity was found to be only 1.5 mg IgG/ml SP XL. Processing cell-containing broth resulted in an additional decrease of the value down to 0.5 mg ml^-1, presumably caused by the remarkable biomass adsorption to the matrix. The search for the reasons led to the examination of the hydrodynamic conditions. Buffer experiments with a tracer substance (acetone) pointed out, that the flow in expanded bed was significantly more influenced by back-mixing effects and channel formations than in packed bed. These effects could be compensated with an enhanced viscosity of the liquid phase, which was achieved by the addition of glucose. As a result of the improved hydrodynamic conditions in the expanded bed, the dynamic capacity could be increased from 0.5 to more than 4.5 mg IgG/ml matrix for the processing of cell culture broth with 400 mM glucose. Finally, the scale up from a Streamline^TM 25 to a Streamline^TM 200 column was performed under conditions, which proved to be optimal: 100 L of unclarified hybridoma broth were concentrated with a binding rate of 95% in less than 3.5 hours. Loading the column no break-through of the target protein was achieved. However, the eluate still contained debris and cells, which points out the major disadvantage of the method: the biomass attachment to the matrix.     

Development and scale up of a capture step (expanded bed chromatography) for a fusion protein expressed intracellularly in Escherichia coli

A capture step was developed using the expanded bed adsorption technology to separate a protein of interest on a cation exchanger from a crude Escherichia coli homogenate. This method was developed in bench-top scale using a STREAMLINE 25 column (Amersham Pharmacia Biotech, Sweden) and STREAMLINE SP. The development was based on earlier experiments performed in a packed bed column (SP-Sepharose FF) to investigate the conditions for sample application, wash and elution. The packed bed method was transformed into an expanded bed method by slightly modifying the wash procedure and cleaning in place (CIP). This method was then scaled-up to pilot scale and used for production of the fusion protein according to cGMP.The yield over the step in pilot scale was 70-85% compared with only 30-50% in small scale. Pressure build-up, attachment of biomass to the adsorbent and collapses of the expanded bed were phenomena seen in small scale but not in pilot scale. The scale-up of the step significantly improved the performance of the step.     

Ion exchange purification of G6PDH from unclarified yeast cell homogenates using expanded bed adsorption

The use of expanded beds of STREAMLINE ion exchange adsorbents for the direct extraction of an intracellular enzyme glucose-6-phosphate dehydrogenase (G6PDH) from unclarified yeast cell homogenates has been investigated. It has been demonstrated that such crude feedstocks can be applied to the bed without prior clarification steps. The purification of G6PDH from an unclarified yeast homogenate was chosen as a model system containing the typical features of a direct extraction technique. Optimal conditions for the purification were determined in small scale, packed bed experiments conducted with clarified homogenates. Results from these experiments were used to develop a preparative scale separation of G6PDH in a STREAMLINE 50 EBA apparatus. The use of an on-line rotameter for measuring and controlling the height of the expanded bed when operated in highly turbid feedstocks was demonstrated. STREAMLINE DEAE has been shown to be successful in achieving isolation of G6PDH from an unclarified homogenate with a purification factor of 12 and yield of 98% in a single step process. This ion exchange adsorbent is readily cleaned using simple cleaning-in-place procedures without affecting either adsorption or the bed expansion properties of the adsorbent after many cycles of operation. The ability of combining clarification, capture, and purification in a single step will greatly simplify downstream processing flowsheets and reduce the costs of protein purification. (c) 1996 John Wiley & Sons, Inc.     

Cell separation by expanded bed adsorption: use of ion exchange chromatography for the separation of <Emphasis Type="Italic">E. coli</Emphasis> and <Emphasis Type="Italic">S. cerevisiae</Emphasis>

In a wide variety of biotechnological and medical applications it is necessary to separate different cell populations from one another. A promising approach to cell separations is demonstrated to be the adoption of chromatographic techniques conducted in expanded beds. The high voidage between the adsorbent beads in an expanded bed allows for the efficient capture of particulate entities such as cells together with washing and subsequent elution without entrapment and loss. In addition, the combination of a gentle hydrodynamic environment, a high surface area and low mixing within the expanded bed make this technique highly favourable. A model system for the separation of two types of microbial cells using STREAMLINE DEAE adsorbent in expanded bed procedures has been investigated. The use of a less selective ligand such as an ion exchange group, which is often characterised by gentle elution procedures, has been investigated as an alternative to affinity ligands whose strong binding characteristics can result in harsh elution procedures with consequent loss of yield and cell viability. Expanded bed experiments have demonstrated selective and high capacity capture of cells from feedstocks containing either a single type of cell or as a mixture of cells of Saccharomyces cerevisiae and Eschericia coli. The capture, washing and elution phases of the separation have been studied with respect to capacity, selectivity and yield of released cells. In these procedures, separation of cell types is achieved by the presence of multiple equilibrium stages within the expanded bed. The results show the potential for carrying out cell separations in expanded beds as an alternative to immunomagnetic cell separations. The combination of these recently developed technologies promises to be a powerful, but economic technique for cell separations involving simple equipment that can readily be scaled up.     

Physical and biochemical characterization of a simple intermediate between fluidized and expanded bed contactors

Physical and biochemical comparison has been made of the performance of a simple fluidised bed contactor and a commercial expanded bed contactor, characterised by identical dimensions, and operated at various settled bed heights with two anion exchange adsorbents. The contactors were tested with various feedstocks comprising bovine albumin in the absence and presence of 20 g dry cell weight biomass litre^-1. Earlier classification of the simple contactor as a single-stage, well mixed fluidised bed is reviewed. The relative merits of STREAMLINE DEAE and DEAE Spherodex LS as fluidisable, anion exchange adsorbents are discussed.     

Studies on protein adsorption in a vortex flow reactor

The performance of a vortex flow reactor (VFR) with suspended particles for protein adsorption was studied under varying operating conditions, and resin volume fractions. The VFR behaved as an expanded bed in the regimen of laminar vortices flow. Streamline DEAE was used for bovine serum albumin (BSA) adsorption. Expanded bed VFR experiments were performed with varying geometric aspect ratios (14.6, 28.6 and 40.0) and axial superficial velocity (100–300 cm h⁻¹) to investigate their influence on productivity and dynamic capacity. The results are compared with literature data on an expanded bed column (EBC). Adsorption breakthrough curves were fitting to a simple two-parameter model.     

On-line monitoring of glucose and/or lactate in a fermentation process using an expanded micro-bed flow injection analyser

A novel flow injection biosensor system for monitoring fermentation processes has been developed using an expanded micro bed as the enzyme reactor. An expanded bed reactor is capable of handling a mobile phase containing suspended matter like cells and cell debris. Thus, while the analyte is free to interact with the adsorbent, the suspended particulate matter passes through unhindered. With the use of a scaled down expanded bed in the flow injection analysis (FIA) system, it was possible to analyse samples directly from a fermentor without the pretreatment otherwise required to extract the analyte or remove the suspended cells. This technique, therefore, provides a means to determine the true concentrations of the metabolites in a fermentor, with more ease than possible with other techniques.Glucose oxidase immobilised on STREAMLINE was used to measure glucose concentration in a suspension of dead yeast cells. There was no interference from the cell particles even at high cell densities such as 15 gm dry weight per litre. The assay time was about 6 min. Accuracy and reproducibility of the system was found to be good. In another scheme, lactate oxidase was covalently coupled to STREAMLINE for expanded bed operation. With the on-line expanded micro bed FIA it was possible to follow the fermentation with Lactobacillus casei.     

Expanded bed adsorption processing of mammalian cell culture fluid: comparison with packed bed affinity chromatography

A comparison between expanded bed adsorption and conventional packed bed Protein A Fast Flow to purify the anti-rHBsAg mAbs from feedstock is presented in this work. Direct capture by STREAMLINE expanded bed adsorption chromatography resulted in 92% product recovery and sevenfold more concentrated product with similar purity levels compared to that obtained by the standard packed method. The process time and buffer consumption were reduced in the expanded bed adsorption method not only with the binding-elution conditions but also with the use of NaOH during the cleaning-in-place step. The latter is the most widely accepted agent in downstream processing, being a cost effective technique that provides not only efficient cleaning but also sanitizes complete column systems and destroys pirogens.     

On-column refolding of recombinant human interferon-gamma inclusion bodies by expanded bed adsorption chromatography

A refolding strategy was described for on-column refolding of recombinant human interferon-gamma (rhIFN-gamma) inclusion bodies by expanded bed adsorption (EBA) chromatography. After the denatured rhIFN-gamma protein bound onto the cation exchanger of STREAMLINE SP, the refolding process was performed in expanded bed by gradually decreasing the concentration of urea in the buffer and the refolded rhIFN-gamma protein was recovered by the elution in packed bed mode. It was demonstrated that the denatured rhIFN-gamma protein could be efficiently refolded by this method with high yield. Under appropriate experimental conditions, the protein yield and specific activity of rhIFN-gamma was up to 52.7% and 8.18 x 10(6) IU/mg, respectively.     

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

Expanded bed adsorption is a technique for recovery of biomolecules directly from unclarified feedstocks. The work described here demonstrates that expanded bed adsorption is a scaleable technique. The methods used to test scaleability were “determination of degree of bed expansion”, “determination of axial dispersion” and “determination of protein breakthrough capacity”. The performance of a production scale expanded bed column with 600?mm diameter was tested using these methods and the results were found to be consistent with the results obtained from lab scale and pilot scale expanded bed columns. The scaleability and function of the expanded bed technique was also tested by performing a “process example”: a purification mimicking a real process using a yeast culture spiked with bovine serum albumin as feedstock. The results show that the 600?mm diameter production scale column was as efficient as a 25?mm diameter lab scale column in recovering bovine serum albumin from the unclarified yeast culture. The production scale runs were fully automated using a software controlled system containing an adaptor position sensor and an adsorbent sensor. A cleaning study was performed which showed that after use of a proper cleaning protocol, no surviving microorganisms could be detected in the column or in the adsorbent.     

Purification of biomolecules by the method of the expanded bed adsorption chromatography. III. Method optimization. Aplications

This review introduces the principles of the expanded bed adsorption (EBA) and serves as a practical guide to the use STREAMLINE adsorbent and columns available on the market. Critical operational parameters will be discussed as well as the principles for the method design and optimization that will ensure maximum operation of this unique unit. The review is illustrated with the examples of different types of biological molecules which have been purified when using the expanded bed adsorption.     

Bioprocess considerations for expanded-bed chromatography of crude canola extract: sample preparation and adsorbent reuse

Compared to the conventional microbial and mammalian systems, transgenic plants produce proteins in a different matrix. This provides opportunities and challenges for downstream processing. In the context of the plant host Brassica napus (canola), this work addresses the bioprocessing challenges of solid fractionation, resin fouling by native plant components (e.g., oil, phenolics, etc.), hydrodynamic stability, and resin reuse for expanded bed adsorption for product capture. Plant tissue processing and subsequent protein extraction typically result in an extract with a high content of solids containing a wide particle-size distribution. Without removal of larger particles, the column inlet distributor plugged. The larger particles (> 50 microm) were easily removed through centrifugal settling comparable to that attainable with a scroll decanter. The remaining solids did not affect the column performance. Less than 4% of the lipids and phenolics in the fed extract bound to STREAMLINE trade mark DEAE resin, and this small proportion could be satisfactorily removed using recommended clean-in-place (CIP) procedures. Hydrodynamic expansion and adsorption kinetics of the STREAMLINE trade mark DEAE resin were maintained throughout 10 cycles of reuse, as was the structural integrity of the resin beads. No significant accumulation of N-rich (e.g., proteins) and C/O-rich components (e.g., oil and phenolics) occurred over the same period.