Expanded Bed Column Research Articles

Adsorption of lactoferrin and bovine serum albumin nanoparticles on pellicular two-layer agarose-nickel at reactive blue 4 in affinity chromatography

In this study, adsorption of lactoferrin (Lf) and bovine serum albumin (BSA) nanoparticles on pellicular two-layer agarose-nickel immobilized by reactive blue 4 dye-ligand (2L-AN@RB4) was investigated. 2L-AN@RB4 was prepared using the three-phase emulsion method. The dynamic light scattering was first employed to quantify size distribution of Lf and BSA nanoparticles. Then, scanning electron microscopy (SEM) and atomic force microscope (AFM), respectively, were used to characterize structures of the two types of nanoparticles and adsorbent beads. SEM and AFM images demonstrate that shapes of the nanoparticles are globular and relatively uniform, where no adhesions were observed. Finally, adsorption behaviors of both Lf and BSA nanoparticles on 2L-AN@RB4 in affinity chromatography were investigated, with focus on the adsorption kinetics. Influences of contact time, pH, and initial concentration were analyzed to investigate the adsorbent behaviors in the expanded bed column. The influence of contact time on adsorption of Lf versus BSA nanoparticles indicates that 4 h is enough to adsorb protein models equal to 45%. Results indicate that Lf nanoparticles have a higher rate of around 83% than that of BSA nanoparticles. The increases in pH have negative effect on adsorption while opposite trend was found for initial concentration. Adsorption isotherm results reveal that the Langmuir and Freundlich isotherm models fit both adsorption kinetics of Lf and BSA very well.

Fabrication of novel agarose–nickel bilayer composite for purification of protein nanoparticles in expanded bed adsorption column

In the present work, a novel agarose–nickel bilayer composite (ANBC) adsorbents modified with Cibacron Blue 3GA (CB3) dye-ligand were fabricated via the three-phase emulsion method. The surface morphology of the novel adsorbents was evaluated using an optical microscope, scanning electron microscopy, and atomic force microscopy. Also, the commercially purchased adsorbents were modified with the same ligand and compared to novel composite (ANBC-CB3). Also, Lactoferrin (Lf) nanoparticles synthesized and employed as a protein model. Residence time distribution experiments were performed to consider the effect of particle density and fluid viscosity. The results established that the ANBC-CB3 bilayer composite follows a typical size range of 60–300μm and a mean diameter of 125μm with a spherical appearance. In batch adsorption, ANBC-CB3 revealed a higher dynamic binding capacity (DBC) of Lf versus Streamline™-CB3. Furthermore, the experimental data were consistent with the Langmuir isotherm model. Finally, The DBC at 10% breakthrough and the two-fold flow rate was more than 70% for the novel adsorbent. It was proved that ANBC-CB3 as high selective adsorbents for protein nanoparticle purification is suitable for high flow rate operations in an expanded bed column.

Purification of Peroxidase from Rice Bran Using Expanded-Bed Ion-Exchange Chromatography

Peroxidase catalyzes the oxidation of various substrates at the expense of hydrogen peroxide. Among the various techniques used for the purification of enzymes, expanded-bed adsorption chromatography is particularly popular because it offers several advantages, such as greater interactions between adsorbents and target molecules, increased overall yield, potential for a scale-up and shorter process times. It relies on the interaction between charged molecules in the mobile phase (i.e. buffer and sample) and oppositely charged groups coupled to the resin in the expanded-bed form. Other chromatographic techniques are also commonly used for peroxidase purification and characterization; however, there are no reports in the literature about the use of expanded-bed adsorption chromatography for this purpose. In this paper, the purification of peroxidase from rice bran using ion-exchange chromatography (expanded-bed column) was investigated. Chromatographic assays were carried out using STREAMLINE SP cationic re...

Characterization of a Novel Agarose–Nickel Composite Matrix for Protein Nanoparticles Affinity Chromatography in Expanded Bed

The novel agarose–nickel (Ag–Ni) expanded bed matrix was investigated with regard to suitability for practical recovery of nano-bioproducts (NBPs) such as protein nanoparticles as drug delivery carriers. The matrix was immobilized by Reactive Green 19 (RG19) dye–ligand and was subjected to biochemical evaluation through batch adsorption studies (isotherm and kinetic studies) and column chromatography of bovine serum albumin nanoparticles (BSA NPs) with average size of 85–95 nm as a model system. Based on adsorption isotherm investigations, the adsorption phenomenon appeared to follow the Langmuir isotherm model with maximum binding capacity of 24.9 mg/ml adsorbent. Subsequently adsorption data were modeled using the pseudo-first-order and pseudo-second-order kinetics equation. The results demonstrated that the adsorption process kinetics followed the pseudo-first-order kinetic model. The dynamic binding capacity (DBC) for BSA NP adsorption was calculated at various flow velocities which showed favorable column efficiency at relatively high flow rates. BSA NPs recovery was studied in the expanded bed column which resulted in 74 % recovery. The results indicated that the novel resin is a promising chromatographic medium for protein nanoparticle separation with high adsorption capacity and column efficiency at reasonably high flow rates. The generic application of such dye–ligand immobilized composite matrix for the adsorption and purification of BSA NPs as a nanoparticulate bioproduct was discussed.

Chromium(III) biosorption onto spent grains residual from brewing industry: equilibrium, kinetics and column studies

The use of industrial wastes for wastewater treatment as a strategy to their re-use and valorisation may provide important advances toward sustainability. The present work gives new insights into heavy metal biosorption onto low-cost biosorbents, studying chromium(III) biosorption onto spent grains residual from a Portuguese brewing industry both in batch and expanded bed column systems. Experimental studies involved unmodified spent grains and spent grains treated with NaOH. Metal uptake followed a rapid initial step, well described by the pseudo-second-order kinetic model up to 2–7 h, indicating chemisorption to be the rate-limiting step. Beyond this period intraparticle diffusion assumed an important role in the uptake global kinetics. The best fit for equilibrium data was obtained using the Langmuir model, with unmodified spent grains having the higher maximum uptake capacity (q max = 16.7 mg g−1). In open system studies, using expanded bed columns, the best performance was also achieved with unmodified spent grains: Breakthrough time (C/C i = 0.25) and total saturation time (C/C i = 0.99) occurred after 58 and 199 h of operation, corresponding to the accumulation of 390 mg of chromium(III), 43.3 % of the total amount entering the column. These results suggest that alkali treatment does not improve spent grains uptake performance. Changes in biomass composition determined by Fourier transform infrared spectroscopy suggested hydroxyl groups and proteins to have an important role in chromium(III) biosorption. This study points out that unmodified spent grains can be successfully used as low-cost biosorbent for trivalent chromium.

Modeling of ion exchange expanded-bed chromatography for the purification of C-phycocyanin

This work is focused on the experimental evaluation and mathematical modeling of ion exchange expanded-bed chromatography for the purification of C-phycocyanin from crude fermentative broth containing Spirulina platensis cells. Experiments were carried out in different expansion degree to evaluate the process performance. The experimental breakthrough curves were used to estimate the mass transfer and kinetics parameters of the proposed model, using the Particle Swarm Optimization algorithm (PSO). The proposed model satisfactorily fitted the experimental data. The results from the model application pointed out that the increase in the initial bed height does not influence the process efficiency, however enables the operation of expanded-bed column at high volumetric flow rates, improving the productivity. It was also shown that the use of mathematical modeling was a good and promising tool for the optimization of chromatographic processes.

Investigation of hydrodynamic parameters in a novel expanded bed configuration: local axial dispersion characterization and an empirical correlation study

Study of liquid behavior in an expanded bed adsorption (EBA) system is important for understanding, modeling and predicting nanobioproduct/biomolecule adsorption performance in such processes. In this work, in order to analyze the local axial dispersion parameters, simple custom NBG (Nano Biotechnology Group) expanded bed columns with 10 and 26 mm inner diameter were modified by insertion of sampling holes. Based on this configuration, the particles and liquid can be withdrawn directly from various axial positions of the columns. Streamline DEAE particles were used as solid phase in this work. The effects of factors such as liquid velocity, viscosity, settled bed height and column diameter on the hydrodynamic parameters were investigated. Local bed voidages in different axial bed positions were measured by a direct procedure within the column with 26 mm diameter. Increasing trend of voidage with velocity at a certain position of the bed and with bed height at a certain degree of expansion was observed. Residence time distribution (RTD) analysis at various bed points showed approximately uniform hydrodynamic behavior in the column with 10 mm diameter while a decreasing trend of mixing/dispersion along the bed height at a certain degree of expansion was seen in the column with 26 mm diameter. Also lower mixing/dispersion occured in the smaller diameter column. Finally, a combination of two empirical correlations proposed by Richardson-Zaki and Tong-Sun was successfully employed for identification of the bed voidage at various bed heights (RSSE=99.9%). Among the empirical correlations presented in the literatures for variation of the axial dispersion coefficient, the Yun correlation gave good agreement with our experimental data (RSSE=87%) in this column.

In-situ extraction and separation of salvianolic acid B from Salvia miltiorrhiza Bunge by integrated expanded bed adsorption

This article describes the development of an integrated expanded bed extraction and separation method for in-situ recovery of salvianolic acid B from Salvia miltiorrhiza Bunge. Twenty-five grams of the herb was extracted in a dynamic extraction column for 3 h, while the target was simultaneously absorbed onto an expanded bed column without any further processing. Then, the expanded bed column was eluted with 1 bed volume (BV) of water and 5 BVs of 50% (v/v) ethanol aqueous solution with a flow rate of 2.4 BV/h. From this, 1438.8 mg of salvianolic acids, containing 55.7% of pure salvianolic acid B, was harvested with an 88.0% recovery. Six evaluation parameters – purity, recovery, process throughput, process efficiency, process environmental impact and general process evaluation – were used as indices for evaluation of different methods. The results indicate that the in-situ method we have developed is a promising process for the recovery of salvianolic acid B from the herb of S. miltiorrhiza Bunge.

Adsorption Strategy of Plasmid DNA Nanoparticulate: Preparative Purification by a Simple Custom Expanded Bed Column

This paper summarizes the critical examination of the hydrodynamic performance of the NBG expanded bed contactor operated with streamline-DEAE adsorbent under various operating conditions for expanded bed adsorption of plasmid DNA nanoparticles from alkaline lysate. The purification process is not RNase-free. In this study, a rapid and efficient scaleable purification protocol obtaining, plasmid DNA nanoparticles (average size of 40 nm) with a high purity level for use as therapeutic agent in customized NBG expanded bed columns was developed. This technique allows efficient levels of binding to the column media and vector purification without centrifugation or filtration steps. Residence time distribution (RTD) studies were exploited to achieve the optimal condition of plasmid DNA nanoparticle (pDNA) recovery upon anion exchange adsorbent in this contactor. In addition, the purification experiments were carried out in the expanded bed columns with settle bed height of 6.0 ± 0.2 cm. NaCl gradient elution enabled the isolation of supercoiled plasmid from low-Mr RNA, cDNA and plasmid variants. Subsequently dynamic binding capacity of the adsorbent was calculated while these values decreased with increase in flow velocity. Moreover, the effect of pH upon the performance of this recovery process and the feedstock volume upon the expanded bed anion exchange purification was investigated. The results demonstrated that separation of low-Mr RNA from plasmid DNA isoforms in the range of pH between 5.5 and 7.5 is achievable in this column. The yield of recovery of pDNA in optimal condition was higher than 88.51% which was a superior result in one-pass frontal chromatography. The generic application of simple customized NBG expanded bed column and its potential for the purification and recovery of plasmid DNA as a nanoparticulate bioproduct is strongly discussed.

Expanded Bed Chromatography as a Tool for Nanoparticulate Separation: Kinetic Study and Adsorption of Protein Nanoparticles

Expanded bed adsorption was investigated together with its suitability for the practical recovery of nanoparticulate mimics of products such as plasmid DNA and viruses as putative gene therapy vectors. The study assessed the binding of protein nanoparticles fabricated from bovine serum albumin (BSA) with average size of 80 nm as a model system and viral size/charge mimic to the streamline DEAE adsorbent in the expanded bed column chromatography. The adsorption kinetics and adsorption mechanism for the BSA nanoparticles on the adsorbent were studied. In batch adsorption studies, the factors nanoparticle concentration, contact time and adsorbent amount, affecting adsorption isotherms were investigated. Subsequently the data were regressed against the Lagergren equation, which represents a first-order kinetics equation and also against a pseudo-second-order kinetics equation. The results demonstrated that the adsorption process followed a Langmuir isotherm equation. The kinetics of the adsorption process followed a pseudo-second-order kinetics model with a rate constant value of 0.025 g mg−1 min−1. The dynamic binding capacity of the BSA nanoparticles on an expanded bed was calculated. The recovery of the nanoparticles was more than 85%.

Mathematical modeling and simulation of inulinase adsorption in expanded bed column

A mathematical model for an expanded bed column was developed to predict breakthrough curves for inulinase adsorption on Streamline SP ion-exchange adsorbent, using a crude fermentative broth with cells as the feedstock. The kinetics and mass transfer parameters were estimated using the PSO (particle swarm optimization) heuristic algorithm. The parameters were estimated for each expansion degree (ED) using three breakthrough curves at initial inulinase concentrations of 65.6 U mL −1. In sequence, the model parameters for an ED of 2.5 were validated using the breakthrough curve at an initial concentration of 114.4 U mL −1. The applicability of the validated model in process optimization was investigated, using the model as a process simulator and experimental design methodology to optimize the column and process efficiencies. The results demonstrated the usefulness of this methodology for expanded bed adsorption processes.

Catalytic Synthesis of Glycerol Monoacetate Using a Continuous Expanded Bed Column Reactor Packed with Cation-Exchange Resin

The selective and continuous production of monoacetin using an expanded-bed column reactor, in which the reactive fluid flows vertically upward and the catalyst is hydrodynamically floated, was studied. Acetic acid and glycerol were used as the substrates and the proton-type cation-exchange resin Amberlyst 16 was used as the catalyst. Batch reaction experiments under various conditions were first conducted to investigate the details of the heterogeneous catalytic reaction mechanism. The esterification of acetic acid with glycerol to produce the target product monoacetin was proved to proceed predominantly over the undesired consecutive esterification reactions to diacetin and triacetin. The intraparticle and extraparticle mass-transfer resistances for the catalytic resin were negligible. The Eley−Rideal-type catalytic reaction model was successfully applied to the experimental data for the various conditions. Next, the hydrodynamic correlation between the flow rate of the reactive fluid and the corresponding expanded-bed height on the continuous reactor was determined experimentally. Furthermore, the reaction and transport model for a continuous expanded-bed column reactor was constructed based on the kinetic model for the batch system. The numerical calculation simulated the experimental data well, and the optimum operating conditions were successfully determined by the theoretical analysis.

Hydrodynamic behaviour and biochemical characterization of a simple custom expanded bed column for protein purification

Hydrodynamic behaviour and biochemical characterization of a simple custom expanded bed column made by Nanobiotechnology Group (NBG) was compared with a commercial expanded bed contactor. Hydrodynamic characteristics of the columns (Daxl and Bo) with various settled bed height (SBH = 5 - 10 cm, increment of 1 cm) along with variety of column diameters (1.0, 1.6, 2.0 and 2.5 cm) were investigated. All parameters were found to be comparable or superior to those reported for conventional, expanded bed contactors. However, the experimental values of Richardson-Zaki coefficient determined here were close to the value of 4.8, commonly used in the laminar flow regime. The expansion coefficient and terminal velocity of the adsorbent (Streamline ™ DEAE) were theoretically calculated based on correlation stated in the literature and also experimentally determined. The adsorbent was also used in batch binding experiments, as well as in commercial and custom assembled expanded bed contactors with various feedstocks comprising bovine serum albumin and egg albumin. This evaluation exhibited good capacities and adsorption/desorption performance of NBG column in compare with commercial expanded bed column. The hydrodynamic behaviour of expanded bed adsorption and the generic application of simple NBG column and its potential for the purification and recovery of protein products are thoroughly discussed.

Single-step purification of recombinant green fluorescent protein on expanded beds of immobilized metal affinity chromatography media

Abstract Immobilized metal ion affinity chromatography (IMAC) in expanded bed mode is used for purifying recombinant green fluorescent protein (GFP) overexpressed in Escherichia coli. The purification is carried out on two different matrices, i.e. Ni2+ Streamline™ and Ni2+ cross-linked alginate beads. The binding isotherms to both IMAC media followed the Langmuir model. The maximum binding capacity (qmax) of Ni2+ Streamline™ and Ni2+ cross-linked alginate for the GFP was 1,42,860 FU ml−1 and 18,000 FU ml−1, respectively. The expanded bed column chromatography using Ni2+ Streamline™ gave 2.7-fold purification with 89% of GFP recovery, while Ni2+ alginate gave 3.1-fold purification with 91% of GFP recovery. SDS-PAGE of purified GFP in both cases showed single band. The results obtained in the expanded bed chromatography are compared with those obtained in packed bed chromatography.

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.

Large-scale recovery of C-phycocyanin from Spirulina platensis using expanded bed adsorption chromatography

C-phycocyanin was purified on a large scale by a combination of expanded bed adsorption, anion-exchange chromatography and hydroxyapatite chromatography from inferior Spirulina platensis that cannot be used for human consumption. First, phycobiliproteins were extracted by a simple, scaleable method and then were recovered by Phenyl-Sepharose chromatography in an expanded bed column. The purity (the A 620/ A 280 ratio) of C-phycocyanin isolated with STREAMLINE™ column was up to 2.87, and the yield was as high as 31 mg/g of dried S. platensis. After the first step, we used conventional anion-exchange chromatography for the purification steps, with a yield of 7.7 mg/g of dried S. platensis at a purity greater than 3.2 and with an A 620/ A 650 index higher than 5.0. The fractions from anion-exchange chromatography with a level of purity that did not conform to the above standard were subjected to hydroxyapatite chromatography, with a C-PC yield of 4.45 mg/g of dried S. platensis with a purity greater than 3.2. The protein from both purification methods showed one absolute absorption peak at 620 nm and a fluorescence maximum at 650 nm, which is consistent with the typical spectrum of C-phycocyanin. SDS-PAGE gave two bands corresponding to 21 and 18 kDa. In-gel digestion and LC-ESI-MS showed that the protein is C-phycocyanin.

An improved method for purification of recombinant truncated heme oxygenase-1 by expanded bed adsorption and gel filtration

Recombinant truncated human heme oxygenase-1 (hHO-1) expressed in Escherichia coli was efficiently separated and purified from feedstock by DEAE-ion exchange expanded bed adsorption. Protocol optimization of hHO-1 on DEAE adsorbent resulted in adsorption in 0 M NaCl and elution in 150 mM NaCl at a pH of 8.5. The active enzyme fractions separated from the expanded bed column were further purified by a Superdex 75 gel filtration step. The specific hHO-1 activity increased from 0.82 +/- 0.05 to 24.8 +/- 1.8 U/mg during the whole purification steps. The recovery and purification factor of truncated hHO-1 of the whole purification were 72.7 +/- 4.7 and 30.2 +/- 2.3%, respectively. This purification process can decrease the demand on the preparation of feedstock and simplify the purification process.

Dye–ligand expanded bed adsorption of G6PDH from highly dense unclarified yeast extract

The application of dye–ligand expanded bed chromatography adsorption (EBA) of glucose-6-phosphate dehydrogenase (G6PDH) from unclarified yeast extract was undertaken by using a commercially available expanded bed column (20 mm i.d.) and UpFront adsorbent ( ρ = 1.5 g/mL) from UpFront Chromatography. The influence of biomass concentration on the adsorption capacity was explored by employing yeast extracts containing various biomass concentrations (5–30%, w/v). It was demonstrated that the biomass concentration had little effect on G6PDH adsorption performance. Feedstock containing 15% (w/v) biomass gave a relatively high recovery yield (>90%) of G6PDH compared to feedstock containing 30% (w/v) biomass, which gave a recovery of 75% G6PDH. Nevertheless, the enzyme specific activity of 7 U mg −1 with a purification factor of 6 was achieved in the feedstock containing biomass concentration of 30% (w/v). The generic applicability of dye–ligand as an affinity tool in expanded bed chromatography is discussed.

Screen-less expanded bed column: new approach for the recovery and purification of a malaria transmission blocking vaccine candidate from Pichia pastoris

An experimental malaria transmission blocking vaccine antigen, Pfs25H, expressed and secreted from Pichia pastoris was recovered and purified using a screenless expanded bed column equipped with a rotating fluid distribution system. This column was able to accommodate feed stock, containing 30% biomass, at a flow rate of 300-400 cm/h without affecting column stability. This capability is three times higher than the capability of the expanded bed column currently in use, which is equipped with a perforated plate fluid distribution system; this design could accommodate biomass concentrations of only up to 10%. The screen-less design did not affect the binding capacity, purification level or process yield and, therefore, shorten the process. Purified Pfs25H of 6.4 g were recovered from 37 l of Pichia pastoris culture in one step.

Expanded Bed Adsorption Chromatography for Recovery of Phycocyanins from the Microalga Spirulina Platensis

We carried out the purification of C-phycocyanin and allophycocyanin from Spirulina platensis taking advantage of the adsorption properties of the expanded beds. Initially, phycobiliproteins were released from the microalga cells by osmotic shock. Next, phycocyanins were recovered by applying the centrifuged cell suspension directly to the anion exchanger Streamline-DEAE using expanded bed columns, equilibrated with 50 mM sodium phosphate buffer, pH 7.0. After adsorption, washing was carried out in the expanded-bed mode. Having removed unbound proteins and cellular debris, the bed was allowed to sediment and phycocyanins rich solution was eluted with a downward flow of 500 mM sodium phosphate buffer, pH 7.0. Finally, we utilized conventional gel filtration and ion exchange chromatography methods for separation and purification of C-phycocyanin and allophycocyanin. The purification steps were monitored using sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and the purity of recovered phycocyanins was confirmed by absorption and emission spectroscopy. The main advantage of this new method is the high yield achieved in the steps of product extraction and adsorption by expanded bed adsorption, so reducing both processing times and costs.