Direct purification of recombinant hepatitis B core antigen from two different pre-conditioned unclarified Escherichia coli feedstocks via expanded bed adsorption chromatography

Direct recovery of hepatitis B core antigen (HBcAg) from unclarified Escherichia coli homogenates via expanded bed adsorption chromatography (EBA) has been explored in this study. Streamline DEAE was selected as the anion exchanger to recover HBcAg from heat-treated and non-heat-treated unclarified feedstocks. The use of anion-exchanger for direct extraction of proteins from unclarified feedstock is not preferred due to lack of specificity of its ligand. In this study, thermal treatment of the unclarified feedstock at 60 degrees C has resulted in 1.2- and 1.8-fold increases in yield and purity of HBcAg, respectively, compared with that purified from non-heat-treated feedstock. Heating the crude feedstock has resulted in denaturation and precipitation of contaminants in the feedstock, hence reducing non-specific interactions between the cell debris and adsorbent. The selectivity of the anion-exchanger has also been increased as shown in the breakthrough curve obtained. Enzyme-linked immunosorbent assay showed that the antigenicity of the HBcAg from heat-treated unclarified feedstock is still preserved.     

Use of expanded bed adsorption to purify flavonoids from Ginkgo biloba L.

Three techniques (liquid–liquid extraction, packed bed adsorption and expanded bed adsorption) have been compared for the purification of flavonoids from the leaves of Ginkgo biloba L. A crude Ginkgo extract was obtained by refluxing with ethanol for 3 h. The yield of flavonoids achieved by this crude extraction was about 19% (w/w) and the purity of flavonoids in the concentrated extract was between 1.9 and 2.3% (w/w). The crude extract was then dissolved in deionized water and centrifuged where necessary to prepare clarified feedstock for further purification. For the method using liquid–liquid extraction with ethyl acetate, the purity, concentration ratio and yield of flavonoids were 25.4–31.0%, 16–18 and >98%, respectively. For the method using packed bed adsorption, Amberlite XAD7HP was selected as the adsorbent and clarified extract was used as the feedstock. The dynamic adsorption breakthrough curves and elution profiles were measured. For a feedstock containing flavonoids at a concentration of 0.25 mg/mL, the appropriate loading volume to reach a 5% breakthrough point during the adsorption stage was estimated to be 550–600 mL for a packed bed of volume 53 mL and a flow rate of 183 cm/h. The results from the elution stage indicated that the majority of impurities were eluted by ethanol concentrations of 40% (v/v) or below and efficient separation of flavonoids from the impurities could be achieved by elution of the flavonoids with 50–80% ethanol reaching an average purity of ∼25%. The recovery yield of flavonoids using the packed bed purification method was about 60% of the flavonoids present in the clarified feedstock (corresponding to around 30% for the total flavonoids in the unclarified crude extract). For the method using expanded bed adsorption also conducted with Amberlite XAD7HP as the adsorbent, the optimal operation conditions scouted during the packed bed experiments were used but unclarified crude extract could be loaded directly into the column. For an expanded bed with a settled bed height of 30 cm, the loss of flavonoids in the column flow-through was about 30%. The two-step elution protocol again proved to be effective in separating the adsorbed impurities and flavonoids. More than 96% of the bound impurities were completely removed by 40% ethanol in the first elution stage and less than 4% remained in the final product eluted by 90% ethanol in the second elution stage. Also, ∼74% of the adsorbed flavonoids on column (corresponding to 51% of the total flavonoids in the unclarified feedstock) were recovered in the product. In addition to higher recovery yield, the average process time to obtain the same amount of product was decreased in the expanded bed adsorption (EBA) process. The results suggest that the adoption of EBA procedures can greatly simplify the process flow sheet and in addition reduce the cost and time to purify flavonoids from Ginkgo biloba. These results clearly demonstrate the potential for the use of EBA to purify pharmaceuticals from plant sources.     

Expanded-bed chromatography in primary protein purification

Chromatography in stable expanded beds enables proteins to be recovered directly from cultivations of microorganisms or cells and preparations of disrupted cells, without the need for prior removal of suspended solids. The general performance of an expanded bed is comparable to a packed bed owing to reduced mixing of the adsorbent particles in the column. However, optimal operating conditions are more restricted than in a packed bed due to the dependence of bed expansion on the size and density of the adsorbent particles as well as the viscosity and density of the feedstock. The feedstock composition may become the most limiting restriction owing to interactions of adsorbent particles with cell surfaces, DNA and other substances, leading to their aggregation and consequently to bed instabilities and channeling. Despite these difficulties, expanded-bed chromatography has found widespread applications in the large scale purification of proteins from mammalian cell and microbial feedstocks in industrial bioprocessing. The basics and implementation of expanded-bed chromatography, its advantages as well as problems encountered in the use of this technique for the direct extraction of proteins from unclarified feedstocks are addressed.     

Affinity purification of proteins using expanded beds.

The use of expanded beds of affinity adsorbents for the purification of proteins from feedstocks containing whole or broken cells is described. It is demonstrated that such feedstocks can be applied to the bed without prior removal of particulate material by centrifugation or filtration thus showing considerable potential for this approach in simplifying downstream processing flow-sheets. A stable, expanded bed can be obtained using simple equipment adapted from that used for conventional packed bed adsorption and chromatography processes. Circulation and mixing of the adsorbent particles is minimal and liquid flow through the expanded bed shows characteristics similar to those of plug flow. Frontal analysis performed with the highly selective affinity system involving the adsorption of human polyclonal immunoglobulin G onto Protein A Sepharose Fast Flow indicate that the adsorption performance of the expanded bed is similar to that achieved when the same amount of adsorbent is used in a packed configuration at the same volumetric flow-rate. The adsorption performance of the expanded bed was not diminished when adsorption was carried out in the presence of intact yeast cells. Batch adsorption experiments also indicated that the adsorption characteristics of the affinity system were not greatly altered in the presence of cells in contrast to results from a less selective ion-exchange system. An expanded bed of Cibacron Blue Sepharose Fast Flow was used to purify phosphofructokinase from feedstock of disrupted yeast prepared by high pressure homogenisation without the need for prior removal of particulate material. The potential for the use of expanded beds in large scale purification systems is discussed.     

DNA binding during expanded bed adsorption and factors affecting adsorbent aggregation

DNA-induced aggregation and contraction of expanded bed adsorption chromatography beds have been examined using strong anion exchanger Q HyperZ and calf thymus DNA in buffers containing added NaCl. Two batches of adsorbent with different ionic capacities were used allowing the effects of different ligand densities to be examined. Very high dynamic binding capacities at 10% breakthrough were found in the absence of added salt. However, the highest binding capacities ( approximately 10 and approximately 19mgDNAml(-1) gel) were found in buffers containing added salt at concentrations of either 0.25 or 0.35M, for the low and high ligand density adsorbents, respectively. Bed contraction was observed, but did not correlate with dynamic binding capacity or with the amount of DNA loaded. No differences in bed contraction were seen by varying the concentration of DNA loaded in the range of 20-80mugml(-1) even though the dynamic binding capacity was reduced as DNA concentration was increased. The extent of bed contraction during DNA loading was found to be a function of added salt concentration and ligand density of the adsorbent. The results imply that ligand density significantly affects the salt tolerance of anion exchangers when binding DNA. However, more importantly, with the adsorbents examined here, attempts to reduce bed aggregation by feedstock conditioning with added salt may increase DNA binding leading to a reduction in expanded bed adsorption performance compromising protein capture in real feedstocks.     

Evaluation of new high-density ion exchange adsorbents for expanded bed adsorption chromatography

New adsorbents Q HyperZ and CM HyperZ composed of hydrogel-filled porous zirconium oxide particles were evaluated for expanded bed adsorption applications in the present work. The HyperZ adsorbents have wet density of 3.16 g ml(-1), particle size of 44.5-100.8 microm and average sphere diameter of 67 microm. The bed expansion as the function of flow velocity and fluid viscosity was measured and correlated with Richardson-Zaki equation. The suitable expansion factor was considered less than 2.5, while the corresponding flow velocity was about 450 cmh(-1). Liquid mixing in the bed was determined to evaluate the stability of expanded bed. The Bodenstein numbers tested were higher than 40 and the axial mixing coefficients (D(ax)) were between 0.5 and 9.7x10(-6)m(2)s(-1), which demonstrated that a stable expanded bed could be formed under suitable operation conditions. Bovine serum albumin (BSA) and lysozyme were used as model proteins to estimate the adsorption capacities of Q and CM HyperZ, respectively. The maximum equilibrium adsorption of Q and CM HyperZ could reach 45.7 and 27.2 mg g(-1) drained adsorbents, respectively. It was found that yeast cells had little influence on the adsorption capacities of the two adsorbents tested. The dynamic adsorption capacity of BSA at 10% breakthrough with Q HyperZ was 35.9 mg g(-1) drained adsorbent at flow velocity of 100 cm h(-1) for packed bed adsorption. The values for expanded bed adsorption were 34.4 mg g(-1) drained adsorbent at flow velocity of 200 cm h(-1), 33.6 mg g(-1) drained adsorbent at 300 cm h(-1) and 31.7 mg g(-1) drained adsorbent 400 cm h(-1). The results demonstrated that Q HyperZ and CM HyperZ are suitable for expanded bed adsorption of biomolecules.     

Preparation and evaluation of polymer-coated adsorbents for the expanded bed recovery of protein products from particulate feedstocks

A novel prototype polymer-coated adsorbent (PCA) has been developed for the effective expanded bed recovery of protein products from particulate feedstocks. The adsorbents were manufactured using the three-phase emulsification process by which the selected core phases (anion- and cation-exchangers and a custom-assembled pseudo-affinity adsorbent) were coated by an agarose gel. This new non-stick exterior coating acts as a sieve reducing the non-specific binding of cell and cell debris without diminution of selective capture of target protein from complex feedstocks such as whole microbial broths and cell disruptates. The new coated adsorbents were subjected to physical and hydrodynamical comparison with the performance of their uncoated adsorbents. Hydrodynamic characteristics (e.g. axial dispersion coefficient (D(axl)) and Bodenstein number (B(o))) of PCA demonstrated a marked robustness in the face of biomass loading disrupted yeast cells. In addition, each adsorbent was compared with its uncoated native form during the expanded bed adsorption of one of two intracellular proteins (i.e. glyceraldehyde 3-phosphate dehydrogenase and cytochrome c) from a 20% (ww/v) yeast disruptate. The performance parameters of efficiency of washing, purification factor, turbidity of the eluted product and protein recovery in all analysed cases were favourable to the coated materials. In particular, exploiting PCA reduced significantly undesirable adsorption of cells without significant loss of binding capacity for the target product. The generic application of such adsorbents and their potential for the recovery of target products from complex feedstock is discussed, whilst other application such as the subtractive purification of nanoparticles were detailed in our previous publication.     

Direct recovery of recombinant nucleocapsid protein of Nipah virus from unclarified Escherichia coli homogenate using hydrophobic interaction expanded bed adsorption chromatography

A direct recovery of recombinant nucleocapsid protein of Nipah virus (NCp-NiV) from crude Escherichia coli (E. coli) homogenate was developed successfully using a hydrophobic interaction expanded bed adsorption chromatography (HI-EBAC). The nucleic acids co-released with the recombinant protein have increased the viscosity of the E. coli homogenate, thus affected the axial mixing in the EBAC column. Hence, DNase was added to reduce the viscosity of feedstock prior to its loading into the EBAC column packed with the hydrophobic interaction chromatography (HIC) adsorbent. The addition of glycerol to the washing buffer has reduced the volume of washing buffer applied, and thus reduced the loss of the NCp-NiV during the washing stage. The influences of flow velocity, degree of bed expansion and viscosity of mobile phase on the adsorption efficiency of HI-EBAC were studied. The dynamic binding capacity at 10% breakthrough of 3.2 mg/g adsorbent was achieved at a linear flow velocity of 178 cm/h, bed expansion of two and feedstock viscosity of 3.4 mPa s. The adsorbed NCp-NiV was eluted with the buffer containing a step gradient of salt concentration. The purification of hydrophobic NCp-NiV using the HI-EBAC column has recovered 80% of NCp-NiV from unclarified E. coli homogenate with a purification factor of 12.5.     

一种球形纤维素/钛白粉扩张床吸附剂的研究--组成对性能的影响

采用反相悬浮再生法 ,以超细钛白粉颗粒作增重剂 ,包埋于纤维素骨架之中 ,经环氧氯丙烷活化后与二乙胺连接 ,制得一种球形扩张床吸附剂 .研究表明 ,吸附剂的密度、机械强度和孔结构可以随钛白粉用量的变化而改变 ;钛白粉颗粒的掺入有利于基质的活化 ,活化后环氧基含量可达 2 2 0 μmol mL .吸附剂具有良好的扩张床性能 ,扩张床中的蛋白质吸附行为与填充床中相似 ,吸附容量为 4 8 9mg牛血清白蛋白 mL吸附剂     

Modeling of the whole expanded-bed protein adsorption process with yeast cell suspensions as feedstock

Expanded bed adsorption of bovine serum albumin (BSA) directly from a feedstock containing whole yeast cells has been investigated with an anion-exchanger DEAE Spherodex M. In the presence of 6% (w/w) yeast cells, the axial liquid-phase dispersion coefficient was found in the order of 10(-6) m2/s, which felled into the common range of 1.0 x 10(-6)-1.0 x 10(-5) m2/s observed previously without the use of cell suspensions as mobile phase. We found that the static and dynamic binding capacity of BSA decreased with increasing the yeast cell concentration due to the competitive adsorption of cells onto the outer surface of the anion-exchanger. However, because of the small size of the adsorbent, the large pore diffusivity of protein and the favorable column efficiency (low axial dispersion coefficient), the dynamic binding capacity of BSA in the presence of 6% (w/w) cells in the expanded bed reached 86% that of the equilibrium adsorption density. Then, the whole expanded bed adsorption process of BSA in the presence of cells, including feedstock loading, washing and elution steps, was predicted using a mathematical model with parameters all determined independently. In the elution stage, the steric mass-action adsorption isotherm with salt concentration as one of the model parameters was used to predict the step-gradient elution process with salt concentration increases. Computer simulations showed that the model was in good agreement with the experimental results for the whole operation process.     

二次嫁接法制备氨基修饰的硅基二氧化碳吸附剂

采用二次嫁接法制备了一系列氨基修饰的SBA-15二氧化碳(CO₂)吸附剂,利用XRD、BET、热重、元素分析、电镜、红外等表征手段对吸附剂进行了表征,并通过固定床测量穿透曲线的方法研究了其CO₂吸附性能,分别考察了不同制备途径和不同有机胺类型等因素对吸附剂结构以及其CO₂吸附性能的影响,同时,还通过多次吸脱附实验考察了吸附剂循环吸脱附的稳定性。结果表明,超声法所制备的聚乙烯亚胺(PEI)嫁接的吸附剂具有最优的CO₂吸附性能,在CO₂分压为10 kPa,反应温度为25 ℃时,吸附量达到1.72 mmol/g;进行多次循环吸脱附实验后,吸附量未见明显降低,表明吸附剂具有稳定的循环吸附性能。     

Hydrophobic interaction chromatography of proteins. IV. Protein adsorption capacity and transport in preparative mode

The adsorption isotherms of four model proteins (lysozyme, α-lactalbumin, ovalbumin, and BSA) on eight commercial phenyl hydrophobic interaction chromatography media were measured. The isotherms were softer than those usually seen in ion-exchange chromatography of proteins, and the static capacities of the media were lower, ranging from 30 to 110 mg/mL, depending on the ammonium sulfate concentration and the protein and adsorbent types. The protein-accessible surface area appears to be the main factor determining the binding capacity, and little correlation was seen with the protein affinities of the adsorbents. Breakthrough experiments showed that the dynamic capacities of the adsorbents at 10% breakthrough were 20-80% of the static capacities, depending on adsorbent type. Protein diffusivities in the adsorbents were estimated from batch uptake experiments using the pore diffusion and homogeneous diffusion models. Protein transport was affected by the adsorbent pore structures. Apparent diffusivities were higher at lower salt concentrations and column loadings, suggesting that adsorbed proteins may retard intraparticle protein transport. The diffusivities estimated from the batch uptake experiments were used to predict column breakthrough behavior. Analytical solutions developed for ion-exchange systems were able to provide accurate predictions for lysozyme breakthrough but not for ovalbumin. Impurities in the ovalbumin solutions used for the breakthrough experiments may have affected the ovalbumin uptake and led to the discrepancies between the predictions and the experimental results.     

Nd-Fe-B alloy-densified agarose gel for expanded bed adsorption of proteins

Novel dense composite adsorbents for expanded bed adsorption of protein have been fabricated by coating 4% agarose gel onto Nd-Fe-B alloy powder by a water-in-oil emulsification method. Two composite matrices, namely Nd-Fe-B alloy-densified agarose (NFBA) gels with different size distributions and densities, NFBA-S (50-165 microm, 1.88 g/ml) and NFBA-L (140-300 microm, 2.04 g/ml), were produced. Lysozyme was used as a model protein to test the adsorption capacity and kinetics for the NFBA gels modified by Cibacron blue 3GA (CB-NFBA gels). Liquid-phase dispersion behavior in the expanded beds was examined by measurements of residence time distributions, and compared with that of Streamline SP (Amersham-Pharmacia Biotech, Sweden). The dependence of axial mixing in the expanded beds on flow velocity, bed expansion degree. settled bed height, and viscosity of liquid phase was investigated. Breakthrough curves of lysozyme in the expanded beds of the CB-NFBA gels were also examined. The dynamic binding capacity at 5% breakthrough was 23.3 mg/ml matrix for the CB-NFBA-S gels, and 16.7 mg/ml matrix for the CB-NFBA-L, at a flow velocity of 220 cm/h. The results indicate that the NFBA gels are promising for expanded bed adsorption of proteins.     

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.     

Preparation and characterization of poly glycidyl methacrylete–zirconium dioxide–β-cyclodextrin composite matrix for separation of isoflavones through expanded bed adsorption

The specially prepared adsorbent is most important in realizing the expanded bed adsorption (EBA) process. In the present work, a novel poly glycidyl methacrylete–zirconium dioxide–β-cyclodextrin (PGMA–ZrO₂–β-CD) composite matrix for EBA has been first prepared. Wet density, water content and pore properties of the composite beads have been investigated, which shows good expansion and stability in EBA. The application of custom-made adsorbent has been investigated to recover isoflavones from soy molasses. The recovery is up to 90% and the purity of isoflavones obtained is 75.4%. Compared with the traditional purification processes, EBA has the advantage of high efficiency and integrality, which leads to large reduction in operation time and cost.     

Adsorption of sodium dodecylsulfate on modified carbon adsorbents

The adsorption of anionic surfactants on carbon adsorbents modified with water-soluble derivatives of natural polymers, cellulose and chitin, is considered with sodium dodecylsulfate taken as an example. It is shown that such modification leads to changes in the adsorption structural characteristics and the particle size distribution of carbon-water suspensions of the original adsorbent, and to the emergence of new functional groups on its surface that are able to interact selectively with adsorbate molecules. It is assumed that adsorption of anionic surfactant on carbon adsorbents under equilibrium conditions proceeds via stepwise filling of the carbon??s porous structure: we first observe volume filling of micropores according to their sizes, and then the formation of a surfactant??s monolayer in mesopores and on the outer surface of the adsorbate. It is established by thermal analysis that the thermal stability of carbon adsorbents is enhanced through the preferential localization of anionic surfactants in micropores. The filling of mesopores and the outer carbon surface by surfactant molecules leads to a regular decrease in thermal stability and an increase in the adsorbent surface??s hydrophilicity.     

Elasticity of adsorbent beds

The practical application of adsorbents with the desired separation properties depends not only upon the adsorption characteristics of the material but also upon the mechanical properties of the packed bed. The packed bed, the vessel surrounding the bed and any internal structure that supports the bed are subjected to both static and cyclic loads during an adsorption process. In order to properly design the vessel and its internal structure, the bulk mechanical properties (most particularly the elastic properties) of the adsorbent bed must be known. The primary focus of this study was to determine the elastic properties of adsorbent beds packed with activated alumina, synthetic molecular sieve 13X or natural zeolite clinoptilolite. The bulk modulus of elasticity was found to be a linear function of applied stress for each of these materials in a range of stresses lower than the bulk crush strength. The Poisson’s ratio for the packed bed was also deduced from these results.     

Fixed bed adsorption of benzothiophene over zeolites with faujasite structure

This work addresses the adsorption of benzothiophene (BT), as a model heterocyclic and aromatic sulphur compound present in road fuels, over agglomerated zeolites with faujasite structure. Several adsorbents based on zeolites with FAU structure have been prepared with different Si/Al molar ratios and exchange cations and then agglomerated. The influence of the zeolite basicity has been studied, both in equilibrium and dynamic liquid phase adsorption experiments. Basicity of the adsorbent increased as the Si/Al molar ratio and the electronegativity of the exchange cation decreased. In equilibrium experiments, the affinity towards the adsorbent increased as the Si/Al molar ratio decreased, showing the highest affinity for exchanged low silica X zeolites with medium basicity (A-KLSX-02). Dynamic experiments showed that the less zeolite basicity, the higher fractional bed utilization and adsorption capacity at breakthrough time. Besides, zeolites with high basicity did not reach the equilibrium capacity due to the low diffusivity of BT into the micropores. Thermogravimetric analyses of the spent adsorbents showed a stronger BT adsorption onto the more basic zeolites. As main conclusion, adsorbents with medium basicity could present the best performance in fuel desulphurization due to their high affinity with sulphur compounds, although diffusion problems should be taken into account.     

Preparative chromatography of xylanase using expanded bed adsorption

Expanded bed adsorption was used to purify a marketable xylanase often used in the kraft pulp bleaching process. Experiments in packed and expanded beds were carried out mainly to study the adsorption of xylanase on to a cationic adsorbent (Streamline SP) in the presence of cells. In order to study the presence of cells, a Bacillus pumilus mass (5% wet mass) was mixed with the enzyme extract and submitted to an expanded bed adsorption system. One xylanase was purified to homogeneity in the packed bed. However, the 5% cell content hampered purification.     

Recovery of actinorhodin from fermentation broth

In the present work, a new method of purification for actinorhodin was developed using an expanded bed chromatography technique in which antibiotic capture, feedstock clarification, centrifugation, dialysis and concentration are done in one step. The cation-exchanger (P-11) resulted in 26% adsorption and 2% recovery whereas the anion-exchanger (DE-52) resulted in 99% adsorption and 56% recovery of adsorbed antibiotic using methanol buffer and 2 M NH4Cl as eluting agent. Streamline DEAE anion-exchanger, which is especially designed for EBA applications, yields 82% adsorption and 50% elution of actinorhodin fed into the chromatography column directly from the fermentation broth. Isocratic elution resulted in extremely efficient yield compared to linear gradient elution, i.e. 13.5-fold more recovery in the column with an aspect ratio (L:D) of 4. Expansion by 150% of settled bed resulted in the best recovery of actinorhodin among 100 and 200% expansions. A comparison of breakthrough profiles in packed and expanded bed adsorption showed that the performance of the expanded bed is better (by 33%) at allowing more volume of the fermentation broth to pass through the chromatography column.