Continuous Biosorption of Pb,Cu, and Cd by Phanerochaete chrysosporium in a Packed Column Reactor

The dynamic removal of lead, copper and cadmium in a single component system by Phanerochaete chrysosporium was studied in packed columns. The packed columns consisted of biomass of P. chrysosporium immobilized on polyurethane foam cubes. The performances of packed columns were described through the concept of breakthrough and the values of column parameters predicted as a function of bed depth. The column biosorption data were evaluated in terms of maximum (equilibrium) capacity of the column, the amount of metal loading and the yield of the process. The maximum capacities for lead, copper and cadmium were 70.7, 43.7 and 70.8 mg, respectively, and their yields were 39.2, 40.6 and 41%, respectively. The kinetic and mass transfer aspects of the dynamic removal of the three metals were studied using three mathematical models commonly used to describe the column performance in adsorption processes. Column studies showed good agreement between the experimental data and the simulated breakthrough curves obtained with Adams-Bohart or the Wolborska model and the Clark model. While the initial segment of the breakthrough curve was defined by the Adams-Bohart and Wolborska models, the whole breakthrough curve was well predicted by the Clark model for all the three metals studied.     

Expanded bed chromatography of proteins in small diameter columns. I. Scale down and validation

The use of large columns for expanded-bed chromatography in protein adsorption and purification can pose limitations in method scouting due to the high volumes of consumables involved in optimisation runs. Scaling down this technique would provide a practical and necessary first step for demonstrating its feasibility in very small beds. The performance of three columns of diameters 5.0, 1.0 and 0.5 cm were compared in terms of the bed expansion, hydrodynamics and breakthrough for lysozyme adsorption onto STREAMLINE-SP . This represented a scale-down factor of a 100-fold from the 5-cm column and the success was judged by the insignificant changes in performance based on the selected criteria. Bed characterisation and breakthrough runs indicated good plug flow behaviour, despite the high particle size to column diameter ratio in the smaller columns. The column efficiency was found to be sensitive to the vertical alignment, making it an important issue in scale down. The results of these investigations show that small diameter columns can be effectively used for mimicking the behaviour in scale up systems providing a useful tool for method scouting studies.     

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.     

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.     

Effect of fouling on the capacity and breakthrough characteristics of a packed bed ion exchange chromatography column

This study examined the impact of fouling with yeast homogenate on capacity and breakthrough performance of an ion exchange packed bed column. Column performance was assessed by analysis of breakthrough curves obtained with BSA as a test protein. The overall impact of fouling on breakthrough performance depended heavily on the level of clarification of the feed stream. Challenging the column with particulate-free homogenate caused no change in column performance. Loading successive small volumes of poorly clarified homogenate, interspersed with frequent column salt washes, did not alter significantly the column capacity. By contrast, when the column was challenged with an equivalent cumulative volume of poorly clarified homogenate, dynamic binding capacity decreased significantly and changes in breakthrough curves suggested increased intraparticle and external mass transfer limitations. These changes were ascribed to deposition of solid particulates in void spaces in the bed and colloidal contaminants in the bead pores.     

Ultra scale‐down approach to correct dispersive and retentive effects in small‐scale columns when predicting larger scale elution profiles

Ultra scale‐down approaches represent valuable methods for chromatography development work in the biopharmaceutical sector, but for them to be of value, scale‐down mimics must predict large‐scale process performance accurately. For example, one application of a scale‐down model involves using it to predict large‐scale elution profiles correctly with respect to the size of a product peak and its position in a chromatogram relative to contaminants. Predicting large‐scale profiles from data generated by small laboratory columns is complicated, however, by differences in dispersion and retention volumes between the two scales of operation. Correcting for these effects would improve the accuracy of the scale‐down models when predicting outputs such as eluate volumes at larger scale and thus enable the efficient design and operation of subsequent steps. This paper describes a novel ultra scale‐down approach which uses empirical correlations derived from conductivity changes during operation of laboratory and pilot columns to correct chromatographic profiles for the differences in dispersion and retention. The methodology was tested by using 1 mL column data to predict elution profiles of a chimeric monoclonal antibody obtained from Protein A chromatography columns at 3 mL laboratory‐ and 18.3 L pilot‐scale. The predictions were then verified experimentally. Results showed that the empirical corrections enabled accurate estimations of the characteristics of larger‐scale elution profiles. These data then provide the justification to adjust small‐scale conditions to achieve an eluate volume and product concentration which is consistent with that obtained at large‐scale and which can then be used for subsequent ultra scale‐down operations. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009     

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.     

Development of a polishing step using a hydrophobic interaction membrane adsorber with a PER.C6®‐derived recombinant antibody

Membrane chromatography has already proven to be a powerful alternative to polishing columns in flow‐through mode for contaminant removal. As flow‐through utilization has expanded, membrane chromatography applications have included the capturing of large molecules, including proteins such as IgGs. Such bind‐and‐elute applications imply the demand for high binding capacity and larger membrane surface areas as compared to flow‐through applications. Given these considerations, a new Sartobind Phenyl? membrane adsorber was developed for large‐scale purification of biomolecules based on hydrophobic interaction chromatography (HIC) principles. The new hydrophobic membrane adsorber combines the advantages of membrane chromatography—virtually no diffusion limitation and shorter processing time—with high binding capacity for proteins comparable to that of conventional HIC resins as well as excellent resolution. Results from these studies confirmed the capability of HIC membrane adsorber to purify therapeutic proteins with high dynamic binding capacities in the range of 20 mg‐MAb/cm³‐membrane and excellent impurity reduction. In addition the HIC phenyl membrane adsorber can operate at five‐ to ten‐fold lower residence time when compared to column chromatography. A bind/elute purification step using the HIC membrane adsorber was developed for a recombinant monoclonal antibody produced using the PER.C6® cell line. Loading and elution conditions were optimized using statistical design of experiments. Scale‐up is further discussed, and the performance of the membrane adsorber is compared to a traditional HIC resin used in column chromatography. Biotechnol. Bioeng. 2010; 105: 296–305. © 2009 Wiley Periodicals, Inc.     

Transport and binding characterization of a novel hybrid particle impregnated membrane material for bioseparations

The transport and binding properties of a novel hybrid particle-nonwoven membrane medium are described. In this construct, a polymeric chromatographic resin is entrapped between two layers of a nonwoven polypropylene membrane. The membrane-supported resin medium offers the advantage of increased interstitial pore diameter to allow passage of cells and other debris in the feed, while providing sufficiently high surface area for product capture within the resin particles. Columns packed with PIM displayed excellent flow distribution and had interstitial porosities of 0.48 ± 0.01, 25-60% larger than those typical of a packed bed. These columns were able to pass over 95% of E. coli cells and human red blood cell concentrate in 30 column volumes while maintaining a pressure drop significantly lower than that of a packed bed with a similar amount of resin. The dynamic binding capacity of bovine serum albumin (BSA) to the chromatographic resin entrapped in the PIM packed column was essentially the same as that observed with the same volume of resin in a packed bed. The General Rate (GR) model of chromatography was used to analyze experiments indicating the breakthrough behavior of the PIM columns is predictable, and very similar to those of a normal packed bed. These results suggest that PIM constructs can be designed to process viscous mobile phases containing particulates while retaining the desirable binding characteristics of the embedded chromatographic resin and could find uses in adsorption separation processes from complex feed streams such as whole blood, cell culture, and food processing.     

Experimental and modeling studies on the sorption breakthrough behaviors of butanol from aqueous solution in a fixed-bed of KA-I resin

Removal of biobutanol from acetone-butanolethanol (ABE) fermentation broth can be achieved by fixed-bed sorption by means of KA-I resin, and the relevant breakthrough curves would provide much valuable information to help design a continuous fixed-bed sorption process in field application. In the present study, the effects of several important design parameters, i.e., initial butanol concentration (C _f: 3.0 ～ 30.0 g/L), inlet flow rate (Q _f: 0.5 ～ 5.5 mL/min) and adsorbent bed height (Z: 4.2 ～ 18.0 cm), on the adsorption breakthrough curves of KA-I resin in a fixed-bed column were investigated. It was found that the amount of adsorbed butanol at breakthrough point was increased with an increase in the value of C _f and Z; and with decrease in the value of Q _f. However, the maximum sorption capacities of butanol at saturated point were basically unchanged. Three well-established fixed-bed adsorption models, namely Thomas, Yoon-Nelson and Adams-Bohart, were applied to predict the breakthrough curves and to determine the characteristic parameters of fixed-bed column, which are the basis for the process design at a real scale. Good agreement between the theoretical breakthrough curves and the experimental result were observed using Thomas and Yoon-Nelson models.     

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.     

Continuous biosorption of cadmium by Moringa olefera in a packed column

The biosorption of Cd(II) by Moringa oleifera using a batch system and a continuous up flow mode in a fixed bed column was studied. Batch adsorption experiments were performed as a function of pH, biosorbent dose, contact time, volume of the solution, and initial metal concentration. The adsorption isotherms obtained fitted well into the Freundlich and Langmuir isotherms. The dynamic removal of cadmium by powdered seed of the Moringa oleifera was studied in a packed column. The effect of bed height (4 and 8 cm) and flow rate (2 and 5mL/min) on biosorption process was investigated and the experimental breakthrough curves were obtained. Results showed that by increasing the bed height and decreasing the flow rate, the breakthrough and exhaustion times increased. The break-through time was considered as a measure of the column performance. The maximum break-through time of 320 min was achieved at the operating condition of 2 mL/min influent flow rate and bed height of 8 cm.     

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.     

Pressure-flow relationships for packed beds of compressible chromatography media at laboratory and production scale

Pressure drop across chromatography beds employing soft or semirigid media can be a significant problem in the operation of large-scale preparative chromatography columns. The shape or aspect ratio (length/diameter) of a packed bed has a significant effect on column pressure drop due to wall effects, which can result in unexpectedly high pressures in manufacturing. Two types of agarose-based media were packed in chromatography columns at various column aspect ratios, during which pressure drop, bed height, and flow rate were carefully monitored. Compression of the packed beds with increasing flow velocities was observed. An empirical model was developed to correlate pressure drop with the aspect ratio of the packed beds and the superficial velocity. Modeling employed the Blake-Kozeny equation in which empirical relationships were used to predict bed porosity as a function of aspect ratio and flow velocity. Model predictions were in good agreement with observed pressure drops of industrial scale chromatography columns. A protocol was developed to predict compression in industrial chromatography applications by a few laboratory experiments. The protocol is shown to be useful in the development of chromatographic methods and sizing of preparative columns.     

Fixed-Bed Studies on Removal of Arsenic from Simulated Aqueous Solutions Using Chitosan Nanoparticles

Arsenic is a toxic element and may be found in natural as well as in industrial water; therefore, before using water for drinking purpose, its proper treatment is required. Thus, the aim of this work was to evaluate the potential of chitosan nanoparticles, in a continuous-flow method, for the removal of arsenic (III) and (V) from aqueous solutions. All experiments were conducted in fixed-bed columns. Experiments were carried out as a function of varying liquid flow rate (0.3–1.0 ml/min), initial metal concentration (0.5–1.5 mg/L), and bed height (3–9 cm) of adsorbent. The total adsorbed quantity, equilibrium uptake, and total percentage removal of arsenic ions were determined by evaluating the breakthrough curves obtained at different flow rates, initial concentrations, and bed heights. The results showed that the column performed well at the lowest flow rate. Also, column bed capacity and exhaustion time were found to increase with increasing bed height. When initial metal ion concentration was increased from 0.5 to 1.5 mg/L, the corresponding adsorption bed capacity decreased from 0.076 to 0.028 mg/g. The bed depth service time model (BDST) model was used to analyze the experimental data and the model parameters were evaluated. The calculated values of N _o and K _a were found to be 19.28 × 10^?2 mg/L and 0.662 L/mg·min, respectively. Good agreement was found between the experimental breakthrough curves and the model predictions.     

Application of equilibrium and mass transfer models to dynamic removal of Cr(VI) ions by Chitin in packed column reactor

The dynamic removal of hexavalent chromium by chitin flakes was studied in a packed column reactor. The values of column parameters were predicted as a function of flow rate, bed depth, particle size and inlet metal ion concentration. On evaluating the breakthrough curves, sorption isotherms were obtained and modelled according to the Langmuir, the Redlich–Peterson and the Freundlich models. Kinetic and mass transfer aspects of the dynamic removal of Cr(VI) ions by chitin were investigated using several mathematical models. Column studies showed a good correlation between the experimental data and the calculated breakthrough curves obtained by the Adams–Bohart or the Wolborska models and the Clark model. The simulation of the whole breakthrough curve was effective with the Clark model, but the breakthrough was best predicted by Adams–Bohart, or related derived models.     

Protein A immunoaffinity hollow fiber membranes for immunoglobulin G purification: experimental characterization

Immunoaffinity adsorption is increasingly used for protein purification and medical applications. Synthetic membranes have advantages as support matrices in comparison to conventional bead supports because they are not compressible and they eliminate internal diffusion limitations. The goal of this study was to explore in detail the performance of microporous hollow fibers composed of modified polysulfone to which protein A was immobilized for adsorption of human IgG. The internal matrix was characterized by scanning electron microscopy. The binding equilibrium constant was measured using both static and dynamic methods. Break-through curves up to ligand saturation were measured and used to study the effects of IgG concentration, presence of contaminant albumin, flow direction, flow mode, and especially filtrate flow rate and maximum IgG binding capacity. The highest binding capacities studied were comparable with that attainable with bead matrices. All of the breakthrough curves could be represented on a single figure when plotted versus the dimensionless relative throughput (the mass of IgG loaded on the membrane divided by the mass that would be bound when the entire fiber is in equilibrium with the feed concentration), and the effect of operating variables on the position and shape of the individual breakthrough curves could be understood in terms of a dimensional performance parameter (the product of membrane volume and maximum binding capacity divided by the filtrate flow rate). The best breakthrough curves were obtained with the highest values of the performance parameter. Based on the results, membranes as solid supports for immunoadsorption can be a useful alternative to the use of traditional columns for protein separations. (c) 1995 John Wiley & Sons, Inc.     

Demonstration of the use of windows of operation to visualize the effects of fouling on the performance of a chromatographic step

The high resolution afforded by packed bed chromatography makes it an indispensable operation in the downstream processing of therapeutic molecules. Packed bed performance is however inherently susceptible to changes in feed stream characteristics and fouling processes. The impact of fouling is seldom considered during the early stages of bioprocess development which is concerned with the selection of purification conditions. Instead these are performed with rigorously clarified feeds. Under such conditions, chromatography is effectively treated as an isolated step, independent from its preceding unit operations. In this study, we demonstrate how windows of operation could be used to visualize the impact of changes in the preceding clarification step on the fouling response of a subsequent cation exchange capture step. Laboratory columns (2,5 and 12 cm height) were subjected to varying fouling challenges of Escherichia coli lysate containing different amounts of solids carried over from the previous step. Changes in trans‐column pressure drop and breakthrough of the target protein (Fab′) were monitored. The limits of operability of the resin were determined with respect to the process material's properties. This information was used to extract the parameters for the adsorption kinetics used in the general rate (GR) model to create windows of operation for manufacturing scale operation. © 2011 American Institute of Chemical Engineers Biotechnol. Prog., 2011