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 Component levels in the withdrawn streams can be assayed using on-line analytical chromatography or samples can be collected and assayed off-line.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.     

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.     

Expanded bed adsorption: elution in expanded bed mode

Elution in expanded bed mode has been investigated in the expanded bed adsorption process. Elution was performed at different sample loads and at different liquid velocities using bovine serum albumin as a model. The effect on mixing in the liquid phase and on the volume of the eluted peak were determined. Mixing in the liquid phase was almost unaffected when elution was performed at 100 cm/h, regardless of sample load. However, mixing increased significantly when elution was carried out at high liquid velocities (300 cm/h) at high sample loads. The eluted peak volume increased with liquid velocity and increased sample load. It was approx. 80% higher in expanded bed mode than in packed bed from an adsorbent completely saturated with protein eluted at 300 cm/h.     

A new fluid distribution system for scale-flexible expanded bed adsorption

A new fluid distribution system designed for expanded bed adsorption was introduced and studied in a 150-cm diameter column. Based on fluid application through a rotating distributor, it eradicates the need for perforated plates, meshes, or local mixers. The effect of rotation rate on column performance was examined by fluidizing a 30-cm high bed of supports with tap water and introducing pulses of dye or acetone tracer. Linear bed expansion was seen as the superficial fluid velocity was raised from 170 x h(-1) to 450 cm x h(-1) (3000 L x h(-1) to 8000 L x h(-1)), and there was little change in expansion characteristics as distributor rotation rate was increased from 2.5 to 10 rpm. The distributor was observed to generate a flow pattern suitable for expanded bed adsorption when the supports were fluidized at a superficial fluid velocity of 283 cm center dot h(-1) and dye pulses introduced. At a rotation rate of 2.5 rpm, no significant dead zones were observed, and a discrete band was formed that moved up through the bed. Furthermore, the pattern of dye movement could be used to calculate interstitial linear fluid velocities of 460 cm x h(-1) and 572 cm x h(-1) at the column wall and center, respectively, indicating a parabolic flow profile. The distributor rotation rate giving the best operating conditions was found to be 2.5 rpm when the bed was fluidized at a flow velocity of 283 cm x h(-1) and the residence time distribution of acetone tracer examined. Under these conditions, the coefficient of axial dispersion was 6.1 x 10(-6) m(2) x s(-1) and 29 theoretical plates were measured. When the rotation rate was raised to 10 rpm, the coefficient of axial dispersion increased to 8.08 x 10(-6) m(2) x s(-1) and the number of theoretical plates decreased to 22.     

Determining particle density distribution of expanded bed adsorbents

This study presents an experimental approach to measure the density distribution of expanded bed adsorption (EBA) matrices. We report on the use of a series of solutions of caesium trifluoroacetate (CsTFA) of varying density spun in a laboratory centrifuge so as to separate representative matrix samples on the basis of bead density. Mass data was used to plot a decumulative density distribution for the matrix. By performing laser light scattering-based measurements on the same samples of matrix the variation in particle size with density was determined. Particle settling velocity distributions were then calculated using these data and compared with a settling velocity distribution calculated on the basis of an assumed constant bead density. The study demonstrates a reliable and simple method for the characterisation of matrix density distribution. For the case of the Streamline matrices tested the particle size distribution is constant with varying bead density. Bead densities varied from 1.5 to 2.1 g/cm3 in the CsTFA solutions. These were then adjusted using bead porosity to give a density range of 1.11-1.33 g/cm3 in aqueous buffer (assumed 1.0 g/cm3) The differences in resultant settling velocity distributions when based upon measured density distribution than when based upon an assumed mean density value were shown to be insignificant. This result confirms experimentally that an assumption of a single constant mean density for EBA particles is acceptable for hydrodynamic modelling and performance prediction purposes.     

Cell/adsorbent interactions in expanded bed adsorption of proteins

Expanded bed adsorption (EBA) is an integrated technology for the primary recovery of proteins from unclarified feedstock. A method is presented which allows a qualitative and quantitative understanding of the main mechanisms governing the interaction of biomass with fluidised resins. A pulse response technique was used to determine the adsorption of various cell types (yeast, Gram positive and Gram negative bacteria, mammalian cells and yeast homogenate) to a range of commercially available matrices for EBA. Cells and cell debris were found to interact with the ligands of agarose based resins mainly by electrostatic forces. From the adsorbents investigated the anion exchange matrix showed the most severe interactions, while cation exchange and affinity adsorbents appeared to be less affected. Within the range of biologic systems under study E. coli cells had the lowest tendency of binding to all matrices while hybridoma cells attached to all the adsorbents except the protein A affinity matrix. The method presented may be employed for screening of suitable biomass/adsorbent combinations, which yield a robust and reliable initial capture step by expanded bed adsorption from unclarified feedstock.     

Factors affecting dispersion in expanded bed chromatography

Factors affecting the dispersion of solutes in expanded bed chromatography were experimentally investigated to characterize the behavior in small columns. Pulse response curves were measured with a vitamin B¹² tracer, and HETP (height equivalent to a theoretical plate) values were calculated from peak variance and retention time. Approximately 15 min were required to attain a stable steady state expanded bed height with constant HETP values. HETP values ranged from 0.8 to 1.6 cm and did not change appreciably with the degree of expansion (1.5–3.5 fold), column diameter (1.6 and 2.6 cm), column temperature (293–308 K) or settled bed height (ca. 4–11 cm). A very small column (1.6 cm diam. and 4.2 cm-settled bed height) was successfully expanded and axial mixing measured could be useful for conducting scale down experiments.     

Partial purification of nattokinase from Bacillus subtilis by expanded bed adsorption

Nattokinase was purified from unclarified Bacillus subtilisculture filtrate using an expanded bed with a purification factor of 8.2 and at a yield of 95%. The optimal pHs for adsorption and elution were 6.0 and 7.0, respectively. The expanded bed route shortened the process time by 8–10 h and increased the yield by 50% when compared with the conventional method.     

Interfacing Pichia pastoris cultivation with expanded bed adsorption

For improved interfacing of the Pichia pastoris fed-batch cultivation process with expanded bed adsorption (EBA) technique, a modified cultivation technique was developed. The modification included the reduction of the medium salt concentration, which was then kept constant by regulating the medium conductivity at low value (about 8 mS/cm) by salt feeding. Before loading, the low conductivity culture broth was diluted only to reduce viscosity, caused by high cell density. The concept was applied to a one-step recovery and purification procedure for a fusion protein composed of a cellulose-binding module (CBM) from Neocallimastix patriciarum cellulase 6A fused to lipase B from Candida antarctica (CALB). The modified cultivation technique resulted in lower cell death and consequently lower concentration of proteases and other contaminating proteins in the culture broth. Flow cytometry analysis showed 1% dead (propidium-stained) cells compared to 3.5% in the reference process. During the whole process of cultivation and recovery, no proteolysis was detected and in the end of the cultivation, the product constituted 87% of the total supernatant protein. The lipase activity in the culture supernatant increased at an almost constant rate up to a value corresponding to 2.2 g/L of CBM-CALB. In the EBA process, no cell-adsorbent interaction was detected but the cell density had to be reduced by a two-times dilution to keep a proper bed expansion. At flow velocity of 400 cm/h, the breakthrough capacity was 12.4 g/L, the product yield 98%, the concentration factor 3.6 times, the purity about 90%, and the productivity 2.1 g/L x h.     

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.     

Routine manufacture of recombinant proteins using expanded bed adsorption chromatography

Two different recombinant human proteins were purified directly from Pichia pastoris whole cell fermentation broth, containing 30–44% biomass (wet weight percent), by strong cation exchange expanded bed adsorption chromatography. Expanded bed adsorption chromatography provided clarification, product purification and product concentration in a single unit operation at large scale (2000-l nominal fermentation volume). The efficiency of expanded bed adsorption chromatography resulted in a short process time, high process yield, and limited proteolytic degradation of the target proteins. The separations were operated using a 60-cm (d) column run at 14 l/min. For one protein, expanded bed adsorption chromatography resulted in an average product recovery of 113% (relative to fermentation supernatant) and a purity of 89% (n=10). For the other protein, the average product recovery was 99% (relative to fermentation supernatant) and the purity was 62.1 (n=10). Laboratory experiments showed that biomass reduced product dynamic binding capacity for protein 2.     

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.     

Purification of monoclonal antibodies from whole hybridoma fermentation broth by fluidized bed adsorption

To achive the coarse purification of a monoclonal antibody from whole hybridoma fermentation broth a fluidized bed cation exchange process was used. The procedure consisted of application of the crude sample and washing of the bed in a fluidized mode and elution in a fixed bed mode. A completely clarified eluate was obtained with purification factors between 4 and 8 and a concentration of the desired product (monoclonal antibody) by a factor of more than 3 was achived. Thus, a combination of the three early steps of the downstream process clarification, concentration and coarse purification was possible. Two different materials were tested: a commercially available agarose-based matrix (Stream-line-SP), and a self-derivatized material based on controlled-pore glass (Bioran). Initial experiments were performed to describe the fluidization of the glass material. Comparison with the agarose material showed several differences, the agarose matrix allowing liquid flow closer to plug flow than the glass material. Increased backmixing in the liquid phase was detected when fluidizing the glass adsorbent compared with the agarose-based matrix. Despite this fact, comparison of the two materials with respect to antibody binding and elution demonstrated a similar performance. (c) 1995 John Wiley & Sons, Inc.     

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.     

Single-step recovery of ephedrine hydrochloride from raw materials using expanded bed adsorption

Expanded bed adsorption, using a cation resin 001 x 7 Styrene-DVB, was used to recover and purify ephedrine hydrochloride from a powdered herb. The axial liquid-phase dispersion coefficient was about 10(-5) m(2) s(-1) and the recovery yield and purification reached 86% and 22, respectively. Compared with using conventional extraction with dimethylbenzene, this method is safer and also more efficient.     

Buoyancy-induced mixing during wash and elution steps in expanded bed adsorption

Buoyancy-induced mixing occurs during expanded bed adsorption processes when the feed stream entering the bottom of the system has a lower density than that of the fluid above it. In the absence of a headspace, mixing in the expanded bed can be modeled as a single, well-mixed vessel, with first-order dynamics. In the presence of a headspace, the system exhibits second-order dynamics for the densities typically encountered in protein chromatography, and can be modeled as two well-mixed vessels (the expanded bed and the headspace) arranged in series. In this paper, the mixing dynamics of the expanded bed are described and a mathematical model of the system is presented. Experimental measurements of density changes during the dilution of sucrose and salt solutions in a STREAMLINE 25 column are presented. These show excellent agreement with predictions using the model. A number of strategies for wash and elution in expanded mode, both in the presence and absence of headspace, are discussed.     

Continuous protein separations in a magnetically stabilized fluidized bed using nonmagnetic supports

Continuous protein separations were performed using a magnetically stabilized fluidized bed (MSFB) and a commercially available affinity adsorption resin that contained no magnetically susceptible material. These nonmagnetic materials can be stabilized at relatively low fields (<75 G requiring <30 W) if sufficient magnetically susceptible particles are also present in the stabilized bed. The minimum amount of magnetic particles necessary to stabilize the bed is as low as 20% by volume and is a function of various parameters including the size and density of both particles, the magnetic field strength, and the fluidization velocity. Advantages of these beds for performing separations include true continuous, countercurrent liquid-solids contact, mass-transfer efficiencies nearly equal to that of packed beds, and the ability of handle suspended cells or cell debris. A variety of commercially available affinity, ion-exchange, and adsorptive supports can be used in the bed for continuous separations; results are presented for the adsorption and recovery of lysozyme from an aqueous mixture of lysozyme and myoglobin using an affinity resin.     

Minimising biomass/adsorbent interactions in expanded bed adsorption processes: a methodological design approach

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 estimate 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.     

Isolation of bacteriocins through expanded bed adsorption using a hydrophobic interaction medium

Two lactic acid bacterium bacteriocins were isolated from fermentation medium through expanded bed adsorption using a hydrophobic interaction gel. First, amylovorin L471, produced by Lactobacillus amylovorus DCE 471, was selected for the optimisation of the loading and eluting conditions. Secondly, the results of the optimisation were applied for the isolation of enterocin RZS C5, a bacteriocin produced by Enterococcus faecium RZS C5. Optimal adsorption was obtained for a medium with concentration of 1.0 M ammonium sulphate and adjusted to pH 4.0 (94.9% for amylovorin L471 and 75.0% for enterocin RZS C5). Elution with 50% ethanol, buffered at pH 6.0, resulted in an optimal total recovery of the bacteriocin activity of 47.6 and 57.6%, respectively. The highest fold purification expressed as the increase in specific activity (AU/mg) corresponded to the highest recovery, being 140- and 1677-fold, respectively. Nevertheless, a total recovery of only 25.6% with an increase of the specific activity of 121 times was obtained after conventional isolation by ammonium sulphate precipitation.