Expanded Bed Chromatography Research Articles

Purification of his-tagged proteins using printed monolith adsorption columns

Bio-separation is a crucial process in biotechnology and biochemical engineering for separating biological macromolecules, and the field has long relied on bead-based and expanded bed chromatography. Printed monolith adsorption (PMA) is a new alternative to which uses a 3D-printed monolithic structure containing self-supporting, ordered flow channels. PMA allows for direct purification of biological molecules from crude cell lysates and cell cultures, and like the other technologies, can functionalized to specifically target a molecule and enable affinity chromatography. Here we have combined PMA technology with an immobilized metal affinity ligand (iminodiacetic acid) to provide selectivity of binding to polyhistidine-tagged proteins during PMA chromatography. Two different PMA structures were created and tested for both static and dynamic protein-binding capacity. At comparative linear flow rates, the dynamic binding capacity of both columns was ≈3 mg/mL, while static capacity was shown to differentiate based on column voidage. We show that a polyhistidine-tagged protein can be directly purified from crude lysate with comparable results to the available commercial providers of IMAC, and with a substantially reduced purification time.

Purification of lactoferrin from sweet whey using ultrafiltration followed by expanded bed chromatography

Lactoferrin is an important protein found in sweet whey that has several biological activities, such as nutritional, antimicrobial, antiviral, anti-inflammatory, antioxidant, anticarcinogenic and immunomodulation. However, it is found in very low concentration in whey and its capture and purification is a challenge. This study proposes a simple strategy in which two operations for the separation and purification of lactoferrin are integrated. Sweet whey containing 0.12 mg/mL of lactoferrin was subjected to microfiltration for clarification, followed by ultrafiltration and diafiltration in order to reduce in 10 times the total volume with simultaneous concentration and pre-purification of lactoferrin to 1.1 mg/mL. In the second operation, the protein was purified by a preparative chromatography based on cationic exchange expanded bed chromatography (EBC). Adsorption isotherms were determined which allowed establishing the conditions for the chromatographic step. Hydrodynamic studies of the expanded bed and its dynamic binding capacity were also accomplished. The final concentration of the purified lactoferrin was 17.4 mg/mL, resulting in a purity of 92.7% and recovery of 87.0%.

Single-step purification of peroxidases from rice bran: Evaluation of the main expanded-bed chromatography parameters

Peroxidases catalyze the oxidation-reduction reactions of organic substrates and present several applications in the biotechnological, clinical, environmental, and industrial areas. Among the chromatographic strategies applied to peroxidase purification to allow future uses, expanded-bed adsorption (EBA) stands out due to the large volumes of purified enzyme and potential for scale-up. In this paper, the main EBA parameters were evaluated in the purification of rice bran peroxidases. Sodium acetate buffer (pH 4.5) and expansion degree of 2.5 were the most favorable conditions to the enzyme adsorption onto Streamline® SP resin, showing an enzymatic activity at equilibrium on the solid phase (q*) of 2.68 U/mL resin, a partition coefficient (f) of 38.35 and a dynamic binding capacity (Q10%) of 0.19 U/mL resin. Rice bran peroxidase was purified in 14.1-fold (± 1.3) with 49.8% (± 2.5) of recovery by using 25 mmol/L acetate buffer pH 5.5 in the washing step, and elution combining stepwise (0.15 mol/L NaCl) and linear gradient (0.15–1 mol/L NaCl), using acetate buffer pH 5.5 with 1 mmol/L of CaCl2. Based on the results obtained, EBA showed to be a viable single-step strategy for peroxidase purification.

Superporous pellicular κ-Carrageenan–Nickel composite beads; morphological, physical and hydrodynamics evaluation for expanded bed adsorption application

Expanded bed adsorption (EBA) is an authentic separation method for the purification of bioproducts. Novel pellicular κ-Carrageenan–Nickel (KC–Ni) and superporous κ-Carrageenan–Nickel (SP-KC–Ni) adsorbent beads have been fabricated and evaluated using water in oil emulsification and granule leaching methods. The morphology, structure and porosity of the fabricated composite matrices were investigated. A proper spherical shape and porous structure with nano sized pores in the range of about 50–190nm has been detected for the KC–Ni beads and upper than 50% of the SP-KC–Ni superpores was at the range of 600–5400nm. The results from the image processing indicated that the KC–Ni particles had normal size distribution with the range of 60–320μm. As a result of physical properties measurements, a proper wet density at the range of 1.43–2.42g/mL was resulted for the adsorbent beads. The porosity and effective porosity of egg albumin (EA) for SP-KC–Ni3 was measured about 98.89% and 73.87% respectively. Residence time distribution (RTD) measurements were applied to investigate hydrodynamic characteristics of KC–Ni and SP-KC–Ni matrices in expanded bed. The results showed that proper physical and hydrodynamic characteristics of KC–Ni and SP-KC–Ni matrices confirm the potential of use in high speed expanded bed chromatography.

Modeling and simulation of Bacillus cereus chitosanase activity during purification using expanded bed chromatography

A phenomenological model was used to describe sequentially the three steps (flowthrough, washing and elution) of expanded bed adsorption chromatography for recovery of chitosanases from Bacillus cereus. Additionally, a hybrid strategy for model parameter estimation was carried out using particle swarm optimization and Gauss-Newton algorithms. The model was validated with independent experimental data and the statistical criteria (χ 2 and mean squared error tests) showed that the hybrid strategy was more promising than just the heuristic method. With the calibrated model, surface response methodology was applied to obtain the optimal operational conditions, and experiments were performed to confirm these results. Overall, a value of 41.08% for yield was obtained using 700 mM NaCl during elution. In summary, all approach employed in this work was relevant for maximizing the yield of the chromatographic process.

Single-step purification of chitosanases from Bacillus cereus using expanded bed chromatography

A chitosanase-producing strain was isolated and identified as Bacillus cereus C-01. The purification and characterization of two chitosanases were studied. The purification assay was accomplished by ion exchange expanded-bed chromatography. Experiments were carried out in the presence and in the absence of cells through different expansion degree to evaluate the process performance. The adsorption experiments demonstrated that the biomass does not affect substantially the adsorption capacity of the matrix. The enzyme bound to the resin with the same extent using clarified and unclarified broth (0.32 and 0.30U/g adsorbent, respectively). The fraction recovered exhibited 31% of the yield with a 1.26-fold increase on the specific activity concerned to the initial broth. Two chitosanases from different elution steps were recovery. Chit A and Chit B were stable at 30–60°C, pH 5.5–8.0 and 5.5–7.5, respectively. The highest activity was found at 55°C, pH 5.5 to Chit A and 50°C, pH 6.5 to Chit B. The ions Cu2+, Fe2+ and Zn2+ indicated inhibitory effect on chitosanases activities that were significantly activated by Mn2+. The methodology applied in this study enables the partial purification of a stable chitosanase using a feedstock without any pre-treatment using a single-step purification.

Ion exchange expanded bed chromatography for the purification of an extracelular chitosanase from Bacillus cereus

Ion exchange expanded bed chromatography for the purification of an extracelular chitosanase from Bacillus cereus

Cost-effective nanoporous Agar–Agar polymer/Nickel powder composite particle for effective bio-products adsorption by expanded bed chromatography

In the present work a novel kind of dense nanoporous composite matrix for expanded bed application has been successfully first prepared with Nickel powder as a densifier and was covered with Agar–Agar layer as a skeleton, through the method of water-in-oil emulsification. Agar–Agar is a porous and inexpensive polymer. In order to fabricate cost-effective adsorbent with favorable qualities Agar–Agar polymer was used. Thereafter, the customized composite particle was modified by pseudo-affinity dye–ligand, Reactive Blue 4 (RB4), aimed at preparing a pseudo-affinity adsorbent (RB4-Agar-Ni) for bioprodut adsorption from aqueous solution. Bovine Serum Albumin (BSA) was selected as a model protein to investigate the adsorption behavior in batchwise and expanded bed chromatography, and the obtained results were evaluated with that of Streamline™ (Amersham–Pharmacia Biotech, Sweden). Spherical appearance and porous structure of composite particles were observed by the optical microscope (OM) and scanning electronic microscope (SEM). The results suggested that the matrices followed the logarithmic normal size distribution with the range of 65–300μm and average diameter of 126.81–151.47μm, proper wet density of 1.64–2.78g/ml, water content of 62.74–34%, porosity of 98–90% and pore size of about 38–130nm. For better comprehension of the impact of solid phase properties on the performance of the expanded bed, the expansion and hydrodynamic properties of a composite matrix with a series of densities was evaluated and estimated by the retention time distribution method (RTD) in an expanded bed and was compared with that of other matrices. According to obtained results the expansion factors under the same fluid velocity decreased by increasing the matrix density. Moreover, the axial dispersion coefficient (Dax) is the most appropriate parameter for evaluating the stability of expanded bed, on various operating conditions, such as different flow velocity, bed expansion degree, viscosity of the liquid phase and the density of adsorbent. It was observed that the application of matrix with high density was proper for high operation, fluid velocity, since the addition of densifier improves the rigidity of the matrix. Three momentous factors, pH, ionic strength and initial concentration of BSA were analyzed. The best results showed that the adsorption equilibrium isotherms seems to follow a typical Langmuir isotherm and also the maximum adsorption capacity (qm) of BSA on RB4-Agar-Ni (64.01mg/ml adsorbent) was higher than that on RB4-Streamline commercial adsorbent (about 54mg.ml adsorbent). Additionally kinetic adsorption processes were characterized by the pseudo-first-order and pseudo-second-order kinetics equations. The experimental data followed the pseudo-first-order kinetic equation. Also the breakthrough curves were investigated. It was found that dynamic binding capacity (DBC) decreased with increasing the flow rate and the values of DBC decreased from 21.08 to 11.15mg/ml adsorbent when the density of composite beads increased from 1.64 to 2.78g/ml. All results indicate that the prepared composite is promising for efficient bioproduct adsorption with good hydrodynamic characteristics, high stability and it is suitable for expanded bed usage as a cost-effective adsorbent.

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.

エクスパンデットベッドカラムクロマトグラフィーのマイナーアクチノイド分離プロセスへの適応性

軽水炉及び高速増殖炉（FBR）から発生する使用済核燃料において，高レベル放射性廃棄物（High-Level radioactive Waste; HLW）の処理・処分法の確立は極めて大きな開発課題である。特に放射性廃棄物を地層処分するためには，環境負荷軽減や処分場の占有面積低減等の観点から，HLW に含まれる長半減期核種であるマイナーアクチノイド（MA）の分離が重要となる。現在，HLW からの MA の分離回収には抽出クロマトグラフィーが検討されているが，放射性分解ガスや溶解スラッジによるカラムの通液阻害等が懸念されている。そこで，本研究ではカラムの下方から通液し，充填層を粗に保ちながらクロマトグラフィーの操作を行うエクスパンデットベッドカラムクロマトグラフィー（Expanded bed Chromatography: EBC）に着目した。本研究では，MA 分離用に開発された含浸吸着材を用いて，EBC の動的吸着・溶離特性を検討した。本研究の成果から，EBC によっても良好なクロマトグラムを得られることができ，ガス・スラッジの影響を抑制可能であることが示された。

Expanded Bed Adsorption: An Option For Energy Savings In Multiple Proteins Purification

Expanded Bed Adsorption enables the protein recovery directly from cultivations of microorganisms or cells and preparations of disrupted cells, without the need for prior removal of suspended solids. The performance ofan expanded bed is comparable to a packed bed owing to reduced mixing of the adsorbent particles, clogging/plugging of solid particles in the column, while fluidized bed requires more velocity which may hinder the adsorption equilibrium. However,optimal operating conditions are more restricted than in a packed bed/fluidized bed due to the dependence of bed expansion on the sizeand density of the adsorbent particles as well as the viscosity and density of the feedstock. These difficulties can beovercomes in expanded-bed chromatography. In this work the steady state hydrodynamic behaviour of expanded beds studied experimentally.The present study focused on hydrodynamics of multiple particles separation and subsequently can be applied to the multiple proteins separation in a single expanded bed.

EMERGING TRENDS IN DESIGNING SHORT AND EFFICIENT PROTEIN PURIFICATION PROTOCOLS

Protein purification is carried out in both academi c and industrial sectors. Both non-chromatographic approaches (precipitation, crystallization, partiti oning) and chromatographic methods are used. When recombinant proteins are over expressed in foreign hosts, inclusion bodies are obtained. Many purification methods can be used to refold proteins . Many strategies allow one to avoid formation of inclusion bodies. The concept of affinity based sep aration extends to non-chromatographic methods as well. Affinity precipitation and Macro-(affinity li gand) Facilitated Three Phase Partitioning (MLFTPP) are two examples of this. Produced as fusion protei ns, most of the proteins are purified via interacti on of their affinity tag coupled to a solid matrix. Na notechnology has made magnetic based methods even more powerful. Expanded bed chromatography, aqueous two phase systems and MLFTPP allow one to deal directly with feed containing particulate matt er. Membrane based separations are another powerful option. Finally crystallization at the ind ustrial level has further evolved to reoccupy an important place in protein purification. Overall, t he main trends are: integrate upstream and downstream phases and reduce the number of steps required for purifying a protein. This has become possible by adopting variety of non-chromatographic methods. More important, bringing affinity based steps earlier in the purification have helped achie ving this.

Surface modification of chromatography adsorbents by low temperature low pressure plasma

In this study we show how low temperature glow discharge plasma can be used to prepare bi-layered chromatography adsorbents with non-adsorptive exteriors. The commercial strong anion exchange expanded bed chromatography matrix, Q HyperZ, was treated with plasmas in one of two general ways. Using a purpose-designed rotating reactor, plasmas were employed to either: (i) remove anion exchange ligands at or close to the exterior surface of Q HyperZ, and replace them with polar oxygen containing functions (‘plasma etching and oxidation’); or (ii) bury the same surface exposed ligands beneath thin polymer coatings (‘plasma polymerization coating’) using appropriate monomers (vinyl acetate, vinyl pyrrolidone, safrole) and argon as the carrier gas. X-ray photoelectron spectroscopy analysis (first ∼10 nm depth) of Q HyperZ before and after the various plasma treatments confirmed that substantial changes to the elemental composition of Q HyperZ's exterior had been inflicted in all cases. The atomic percent changes in carbon, nitrogen, oxygen, yttrium and zirconium observed after being exposed to air plasma etching were entirely consistent with: the removal of pendant Q (trimethylammonium) functions; increased exposure of the underlying yttrium-stabilised zirconia shell; and introduction of hydroxyl and carbonyl functions. Following plasma polymerization treatments (with all three monomers tested), the increased atomic percent levels of carbon and parallel drops in nitrogen, yttrium and zirconium provided clear evidence that thin polymer coats had been created at the exteriors of Q HyperZ adsorbent particles. No changes in adsorbent size and surface morphology, nor any evidence of plasma-induced damage could be discerned from scanning electron micrographs, light micrographs and measurements of particle size distributions following 3 h exposure to air (220 V; 35.8 W L −1) or ‘vinyl acetate/argon’ (170 V; 16.5 W L −1) plasmas. Losses in bulk chloride exchange capacity before and after exposure to plasmas enabled effective modification depths within hydrated Q HyperZ adsorbent particles to be calculated as 0.2–1.2 μm, depending on the conditions applied. The depth of plasma induced alteration was strongly influenced by the power input and size of the treated batch, i.e. dropping the power or increasing the batch size resulted in reduced plasma penetration and therefore shallower modification. The selectivity of ‘surface vs. core’ modification imparted to Q HyperZ by the various plasma treatments was evaluated in static and dynamic binding studies employing appropriate probes, i.e. plasmid DNA, sonicated calf thymus DNA and bovine serum albumin. In static binding studies performed with adsorbents that had been exposed to plasmas at the 5 g scale (25 g L −1 of plasma reactor), the highest ‘surface/core’ modification selectivity was observed for Q HyperZ that had been subjected to 3 h of air plasma etching at 220 V (35.8 W L −1). This treatment removed ∼53% of ‘surface’ DNA binding at the expense of a 9.3% loss in ‘core’ protein binding. Even more impressive results were obtained in dynamic expanded bed adsorption studies conducted with Q HyperZ adsorbents that had been treated with air (220 V, 3 h) and ‘vinyl acetate/argon’ (170 V, 3 h) plasmas at 10.5 g scale (52.5 g L −1 of plasma reactor). Following both plasma treatments: the 10% breakthrough capacities of the modified Q HyperZ adsorbents towards ‘surface’ binding DNA probes dropped very significantly (30–85%); the DNA induced inter-particle cross-linking and contraction of expanded beds observed during application of sonicated DNA on native Q HyperZ was completely eradicated; but the ‘core’ protein binding performance remained unchanged cf. that of the native Q HyperZ starting material.

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%.

Application of dye-ligands affinity adsorbent in capturing of rabbit immunoglobulin G

The applicability of dye-ligands attached to an expanded bed chromatography quartz base matrix (Streamline™) for the affinity bioseparation of rabbit immunoglobulin G (IgG) was investigated. Reactive Green 5 (RG-5) immobilized onto adsorbent was selected for capturing of rabbit-IgG due to its higher binding capacity compared to other dye-ligands possessing similar ligand density. Adsorption parameters such as pH, temperature, ionic strength and initial rabbit-IgG concentration were optimized for the adsorption of rabbit-IgG on the RG-5-immobilized adsorbent. The highest rabbit-IgG adsorption was recorded in pH 7.0, while the maximum binding capacity for BSA was achieved at pH 4.0. The adsorption of rabbit-IgG on RG-5-immobilized adsorbent was declined as the increase of ionic strength. There is no significant influence of temperature against adsorption efficiency of RG-5-immobilized adsorbent for rabbit-IgG. The adsorption phenomenon of rabbit-IgG on RG-5-immobilized adsorbent appeared to follow the Langmuir–Freundlich adsorption isotherm model. The theoretically maximum binding capacity ( q m) of RG-5-immobilized adsorbent estimated from this isotherm was 49.3 mg ml −1, which is very close to that obtained experimentally (49.0 mg ml −1). About 50% of bound BSA on RG-5-immobilized adsorbent in binary adsorption system was removed with washing buffer containing 1 M NaCl.

BIOCHEMISTRY CHARACTERIZATION OF α‐ AND β‐AMYLASES FROM ZEA MAYS MALT AND STATISTICAL ANALYSIS APPROACH OF THE DEGRADATION OF MANIOC STARCH

ABSTRACT This work examined the degradation of manioc starch by amylases from maize malt and the biochemistry characterization of enzymes. Enzyme characterization showed that the maize malt amylases have optimal pH between 4.5 and 6.5, with optimal temperature between 50 and 90C; Michaelis‐Menten constant (Km) and maximum velocity of starch hydrolysis (Vmax) values for starch hydrolysis by maize malt were 7.69 × 10−2 g/L and 7.69 × 102 g/L/min, respectively. Sodium dodecyl sulfate‐polyacrylamide gel electrophoresis analysis of the eluted sample from ion‐exchange expanded bed chromatography showed that molecular weights of α‐ and β‐amylases from maize malt were of 67.4 and 47.5 kDa, respectively. The hydrolysis times had been found between 20 and 30 h, and the higher hydrolysis yield values were about 31%; it was higher than 27% (cited by literature) and was obtained at 65C and at 9 and 11% (w/v) of any starch concentration studied.PRACTICAL APPLICATIONSThe results of this work have practical uses in the hydrolysis process of starch by food industries (beers, beverages, bakeries, syrups and others). This work shows optimal conditions of using the amylases from maize malt in starch hydrolysis. Recently, a search for new energetic sources has been unleashed after the pronounced governing of various countries on a possible energetic crisis. At present, Brazil is in search of methods that will increase the ethanol offer by production of combustible alcohol. Thus, this study shows the optimal conditions to produce fermentable syrup from manioc starch and as a potential substitute to sugarcane in alcohol production.

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.

Development of a generic transient transfection process at 100 L scale.

We have developed a generic transient transfection process at 100 L scale, using HEK293-EBNA cells and PEI as the transfection reagent for the production of recombinant IgG. The process, including large-scale plasmid preparation, expression at bioreactor scale, capture, purification and, if necessary, endotoxin removal allows reproducible production of more than 0.5 g IgG for in vitro and in vivo studies. We compared the performance of two HEK cell lines, investigated the effect of conditioned medium, optimized the DNA:PEI ratio and implemented a feed strategy to prolong the culture time to increase product yield. The transient transfection protocol developed enables a closed process from seeding culture to protein capture. The challenge of performing a medium exchange before transfection at large scale is solved by applying a continuous centrifugation step between the seeding bioreactor and the production bioreactor. After 7-8 days the harvest and capture is performed in a one-step operation using a Streamline expanded bed chromatography system. Following a polishing step the purified antibody is transferred to the final formulation buffer. The method has shown to be reproducible at 10, 50, and 100 L scale expressing between 5 and 8 mg L(-1) IgG.

Dual fluorescence confocal imaging of the accessibility and binding of F(ab′) 2 to an EBA resin with various immobilised antigen densities

Dual fluorescence confocal laser scanning microscopy has been used to visualise the binding of a fluorescently labelled polyclonal ovine anti-fluorescein F(ab′) 2 antibody to immobilised fluorescein. The fluorescent ligand was immobilised on a Streamline quartz base agarose matrix; a resin used industrially for expanded bed chromatography, using two different fluorescein initial concentrations in order to obtain two batches of immunogen-affinity adsorbent with different immobilised ligand densities. The fluorescein specific F(ab′) 2 were purified from anti-fluorescein serum pepsin digest by adsorption on immobilised antigen chromatographic resin, followed by conjugation to the fluorescent probe Alexa Fluor 660. The dual fluorescence signals from the immobilised antigen and the immuno-specific F(ab′) 2 were used to map the progressive depth of the bound F(ab′) 2 layer within individual adsorbent beads. In addition, the labelled anti-fluorescein F(ab′) 2 was diluted to identical antigen binding activity concentrations in crude serum digest and in blank buffer and the resulting fluorescence intensity profiles were comparatively assessed for any detectable differences in binding patterns that might be caused by processing the more complex mixture of crude serum digests. It was observed that the relative immobilised ligand utilisation was higher when using the immuno-adsorbent with lower immobilised antigen density. Furthermore, the progression of the adsorbed F(ab′) 2 front inside the immuno-adsorbent beads displayed closer agreement with the postulates of the shrinking core mechanism (SCM) when the immuno-adsorbent with lower immobilised antigen was used. The confocal images did not reveal any differences between the depth of the adsorption fronts of crude serum digest and pre-purified F(ab′) 2 samples.

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.