Macroporous copolymer matrix. IV. Expanded bed adsorption application

Macroporous crosslinked hydroxyethyl methacrylate-ethylene dimethacrylate copolymeric beads (HEG beads) were synthesized by suspension polymerization in the presence of a pore generating agent. These beads were coupled to alpha-cyclodextrin through a urethane spacer. These modified copolymer beads (affinity-HEG beads) so prepared were evaluated for their suitability in expanded bed chromatography. The optimum thickness of the distributor plate for stable expanded bed for use in expanded bed adsorption (EBA) was established. The affinity-HEG beads are comparable in density to Streamline diethyl amino ethane (DEAE) and exhibit better mechanical stability at higher superficial velocity under fluidization. The affinity-BEG beads were used as affinity chromatography matrices for the purification of cyclodextrin glycosyltransferase. Feeding of 5-fold diluted fermented broth to the column containing affinity-HEG beads of settled bed height 7.5 cm (I.D. 26 mm and length 42 cm) at double bed expansion resulted in a sharp breakthrough curve of alpha-cyclodextrin glycosyltransferase (CGTase). The adsorbed enzyme was eluted from the bed in 50 mM Tris-HCl buffer containing 10 mM CaCl2 at 25 degrees C in packed bed configuration.     

Analysis of aminofluorescein-fatty acid derivatives by capillary electrophoresis with laser-induced fluorescence detection at the attomole level: application to mycobacterial fatty acids

In the present work, a new method of purification for antithrombin was developed using an expanded bed chromatography technique. Milk fat was removed by centrifugation and caseins were precipitated selectively by addition of zinc chloride. Crude skim milk was then directly fed to an expanded bed column containing the ion-exchange matrix. The use of a cation-exchanger (P-11) resulted in 100% adsorption and 13% recovery whereas the use of an anion-exchanger (DE-52) resulted in 100% adsorption and 84% recovery and up to five-fold purification of antithrombin. The buffer, 25 mM Tris-HCl pH 8.0; the eluting agent, 2 M (NH4)2SO4; and 100% expansion of settled bed were determined to be the optimum conditions for the purification of antithrombin by ion-exchange expanded bed chromatography. A comparison of column diameters revealed that the elution yields increase by two-fold while the column diameter increases from 1 to 2.5 cm. However, antithrombin III was concentrated to a higher degree by using the column with an internal diameter of 1 cm.     

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.     

Preparation of monodisperse porous polymethacrylate microspheres and their application in the capillary electrochromatography of macrolide antibiotics

Monodisperse poly(glycidyl methacrylate-divinylbenzene) microspheres were prepared by a simple one-step dispersion polymerization process. Examination of the polymeric microspheres showed that they had a mean particle diameter of 3 microm and dual pore size distribution with mean pore diameters of 300 and 800 A. The microspheres were functionalized by introducing quaternary ammonium/octadecyl groups to obtain positively charged beads in a wide pH range. The functionalized beads were packed into fused-silica capillary having 50 microm inner diameter and used to separate erythromycin derivatives by capillary electrochromatography (CEC). These samples require gradient elution when separated by high-performance liquid chromatography (HPLC) or micro-HPLC, but with the new columns isocratic elution suffices for their separation by CEC. The column efficiency ranged from 40,000 to 50,000 theoretical plates.     

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.     

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     

Peak spreading in linear gradient elution chromatography with a thin monolithic disk

The peak spreading of DNAs of various sizes [12-mer, 20-mer, 50-mer and 95-mer poly(T)] in linear gradient elution (LGE) chromatography with a thin monolithic disk was investigated by using our method developed for determining HETP in LGE. Electrostatic interaction-based chromatography mode (ion-exchange chromatography, IEC) was used. Polymer-based monolithic disks of two different sizes (12 mm diameter, 3mm thickness and 0.34 mL; 5.2 mm diameter, 4.95 mm thickness and 0.105 mL) having anion-exchange groups were employed. For comparison, a 15-μm porous bead IEC column (Resource Q, 6.4mm diameter, 30 mm height and 0.97 mL) was also used. The peak width did not change with the flow velocity for the monolithic disks where as it became wider with increasing velocity. For the monolithic disks the peak width normalized with the column bed volume was well-correlated with the distribution coefficient at the peak position K(R). HETP values were constant (ca. 0.003-0.005 cm) when K(R)>5. Much higher HETP values which are flow-rate dependent were obtained for the porous bead chromatography. It is possible to obtain 50-100 plates for the 3mm monolithic disk. This results in very sharp elution peaks (standard deviation/bed volume=0.15) even for stepwise elution chromatography, where the peak width is similar to that for LGE of a very steep gradient slope.     

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.     

Immobilised metal affinity chromatography of beta-galactosidase from unclarified Escherichia coli homogenates using expanded bed adsorption

The development of an expanded bed process for the direct extraction and partial purification of beta-galactosidase from unclarified Escherichia coli homogenates using its natural affinity for metal loaded STREAMLINE Chelating is described. Small packed beds were used to determine the effect of chelated metal ion (Cu2+, Ni2+, Co2+ or Zn2+), loading pH and ionic strength on the selective binding capacity, and recovery of beta-galactosidase from clarified homogenates. An elution protocol was developed using the competitive displacer, imidazole, to recover beta-galactosidase in 87% yield and 3.4-fold purification. These results were then used to develop a separation for the recovery of beta-galactosidase from unclarified homogenates in a 2.5-cm diameter expanded bed. Although Ni2+ loaded STREAMLINE Chelating had a 5% dynamic capacity for beta-galactosidase of just 118 U ml(-1) (0.39 mg ml(-1)), the low capacity was thought to be due to the large size of the target (464,000) relative to the exclusion limit of the macroporous adsorbent. Despite this low capacity, Ni2 STREAMLINE Chelating was used successfully to recover beta-galactosidase from an unclarified homogenate in 86.4% yield and at 5.95-fold purification. The degree of purification relative to a commercial standard, as assessed using the purification factor and sodium dodecyl sulphate-polyacrylamide gel electrophoresis was high suggesting that this pseudo-affinity procedure compared favourably with alternative methods.     

Application of a chromatography model with linear gradient elution experimental data to the rapid scale-up in ion-exchange process chromatography of proteins

We applied the model described in our previous paper to the rapid scale-up in the ion exchange chromatography of proteins, in which linear flow velocity, column length and gradient slope were changed. We carried out linear gradient elution experiments, and obtained data for the peak salt concentration and peak width. From these data, the plate height (HETP) was calculated as a function of the mobile phase velocity and iso-resolution curve (the separation time and elution volume relationship for the same resolution) was calculated. The scale-up chromatography conditions were determined by the iso-resolution curve. The scale-up of the linear gradient elution from 5 to 100mL and 2.5L column sizes was performed both by the separation of beta-lactoglobulin A and beta-lactoglobulin B with anion-exchange chromatography and by the purification of a recombinant protein with cation-exchange chromatography. Resolution, recovery and purity were examined in order to verify the proposed method.     

Superporous agarose anion exchangers for plasmid isolation

Superporous agarose beads have wide, connecting flow pores allowing large molecules such as plasmids to be transported into the interior of the beads by convective flow. The pore walls provide additional surface for plasmid binding thus increasing the binding capacity of the adsorbent. Novel superporous agarose anion exchangers have been prepared, differing with respect to bead diameter, superpore diameter and type of anion-exchange functional group (poly(ethyleneimine) and quaternary amine). The plasmid binding capacities were obtained from breakthrough curves and compared with the binding capacity of homogeneous agarose beads of the same particle size. Significantly, the smaller diameter superporous agarose beads were found to have four to five times higher plasmid binding capacity than the corresponding homogeneous agarose beads. The experimentally determined plasmid binding capacity was compared with the theoretically calculated surface area for each adsorbent and fair agreement was found. Confocal microscopy studies of beads with adsorbed, fluorescently labelled plasmids aided in the interpretation of the results. Superporous poly(ethyleneimine)-substituted beads with a high ion capacity (230 micromol/ml) showed a plasmid binding of 3-4 mg/ml adsorbent. Superporous quaternary amine-substituted beads had a lower ion capacity (81 micromol/ml) and showed a correspondingly lower plasmid binding capacity (1-2 mg/ml adsorbent). In spite of the lower capacity, the beads with quaternary amine ligand were preferred, due to their much better plasmid recovery (70-100% recovery). Interestingly, both capacity and recovery was improved when the plasmid adsorption step was carried out in the presence of a moderate salt concentration. The most suitable superporous bead type (45-75 microm diameter beads; 4 microm superpores; quaternary amine ligand) was chosen for the capture of plasmid DNA from a clarified alkaline lysate. Two strategies were evaluated, one with and one without enzymatic digestion of RNA. The strategy without RNase gave high plasmid recovery, quantitative removal of protein and a 70% reduction in RNA.     

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.     

Immobilised peptide displaying phages as affinity ligands. Purification of lactoferrin from defatted milk

An affinity purification procedure for the direct purification of lactoferrin from defatted (skimmed) milk has been developed. The procedure is based on using selected phage clones expressing a peptide with high binding affinity for lactoferrin which were covalently coupled to macroporous poly(dimethylacrylamide) monolithic column. Large pore size (10-100 microm) of macroporous poly(dimethylacrylamide) makes it possible to couple long (1 microm) phage particles as ligands without any risk of blocking the monolithic column. Bound lactoferrin was eluted using 1M NaCl with a purity of >95%. The technique presents a good alternative to conventional immunoaffinity chromatography for purification of a protein of interest from complex samples due to (i) the robustness of the system in terms of recovery and ligand leakage and (ii) economical aspect in terms of low ligand cost.     

Preparation of hydrophobic interaction chromatographic packings based on monodisperse poly(glycidylmethacrylate-co-ethylenedimethacrylate) beads and their application

The monodisperse, poly(glycidylmethacrylate-co-ethylenedimethacrylate) beads with macroporous in the range of 8.0-12.0 microm were prepared by a single-step swelling and polymerization method. The seed particles prepared by dispersion polymerization exhibited good absorption of the monomer phase. The pore size distribution of the beads was evaluated by gel permeation chromatography and mercury instrusion method. Based on this media, a hydrophobic interaction chromatographic (HIC) stationary phase for HPLC was synthesized by a new chemically modified method. The prepared resin has advantages for biopolymer separation, high column efficiency, low column backpressure, high protein mass recovery and good resolution for proteins. The dynamic protein loading capacity of the synthesized HIC packings was 40.0 mg/ml. Six proteins were fast separated in less than 8.0 min using the synthesized HIC stationary phase. The HIC resin was firstly used for the purification and simultaneous renaturation of recombinant human interferon-gamma (rhIFN-gamma) in the extract solution containing 7.0 mol/l guanidine hydrochloride with only one step. The purity and specific bioactivity of the purified of rhIFN-gamma was found more than 95% and 1.3 x 10(8) IU/mg, respectively.     

High-capacity composite adsorbents for nucleic acids

Cytopore™ is a bead-shaped, macroporous and easily compressible cellulose-based anion-exchange material intended for cultivation of anchor-dependent animal cells. Reticulated vitreous carbon (RVC) is a strong, non-compressible, high voidage (97%) matrix material that can be cut to desired geometrical shapes. Cytopore and RVC were combined to cylindrical composites (25 mm × 10 mm) fitted inside chromatography columns. The composite combined the advantageous properties of both its constituents, making it suitable for column chromatography. The composite could withstand very high flow rates without compaction of the bed (>25 column volumes/min; 4000 cm h⁻¹). Chromatography runs with tracers showed a low HETP value (0.3 mm), suggesting that pore flow was in operation. The dynamic binding capacities (10% breakthrough) per gram of dry weight Cytopore were determined for several compounds including DNA and RNA and were found to be 240–370 mg/g. The composite was used to isolate pUC 18-type plasmids from a cleared alkaline lysate in a good yield. Confocal microscopy studies showed that plasmids were bound not only to the surface of the Cytopore material but also within the matrix walls, thus offering an explanation to the very high binding capacities observed. The concept of using a composite prepared from a mechanically weak, high-binding material and a strong scaffold material may be applied to other systems as well.     

Novel adsorbent hollow fibres for oxygen concentration

The research examined the development of adsorbent hollow fibres as a low pressure drop structure for the production of oxygen-enriched air. The potential benefits of using a low pressure drop flexible adsorbent structure with molecular sieving properties over a bed packed with pellets include a low attrition resistance which could extend the life of the adsorbent structure. Highly macroporous, highly adsorbent loaded (up to 90 wt%) fibres were produced. By increasing adsorbent density, the separative performance and nitrogen loading were improved. The separative performance of the adsorbent fibre was found to be slightly inferior to that of the bed of smaller 0.4–0.8 mm beads, as the diffusion path length was longer in the fibres and caused increased mass transfer resistances within the macroporous structure. The pressure drop through the fibre was found to be 40 to 70 times lower than that through an equivalent packed bed of 0.4–0.8 mm beads. This experimental feasibility study has demonstrated that the novel zeolite fibre configuration shows good potential for the production of oxygen-enriched air in a low energy, short cycle time, pressure swing process. The challenges of improving the performance of the adsorbent fibres and their operating parameters are described.     

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.     

Rapid purification of antisteroid antibodies by high-performance liquid affinity chromatography

An adsorbent for the high-performance affinity chromatography of antisteroid antibodies was prepared, based on a commercial pre-packed column. The column contained activated microparticulate silica beads bearing epoxide functions, on which the steroid dexamethasone was covalently linked. The column was used successfully for the rapid and complete isolation of several hundred microgram amounts of specific antidexamethasone antibodies from rabbit antisera. The practical aspects of the purification procedure, especially the optimization of the washing and of the elution steps, are detailed. Despite non-biospecific elution with 20% acetonitrile in an acidic buffer, the purification yield was very satisfactory and the biological activity of the purified immunoglobulins appeared excellent.     

Continuous superporous agarose beds in radial flow columns

Continuous superporous agarose beds constitute a new support material for chromatography, biocatalysis and electrophoresis. The bed consists of a single piece of agarose gel, homogeneously transected by flow-carrying pores, which easily can be varied in the range of 10-100 microm. In this work, large diameter beds (60 mm) were prepared and used in specially designed radial flow columns. The basic chromatographic properties of the beds were investigated by size-exclusion chromatography experiments. In an affinity chromatography application one bed was derivatized with Cibacron Blue 3GA and used for the purification of lactate dehydrogenase from a crude bovine heart extract. In a biotransformation application one bed was provided with immobilized beta-galactosidase and used in the production of lactose-free milk.     

Expansion and hydrodynamic properties of cellulose-stainless steel powder composite matrix for expanded bed adsorption

For better understanding the influences of solid phase properties on the performance of the expanded bed, the expansion and hydrodynamic properties of cellulose-stainless steel powder composite matrix with a series of densities was investigated and analyzed in an expanded bed. Two kinds of matrix particle diameter fractions, the small one (60-125 microm) and the large (125-300 microm), were used in the present work. In general, the expansion factors decreased obviously with the increase of matrix density. A linear relation between the mean density of matrix and superficial velocity at expansion factor of 2.5 was found for same series of matrices. The Richardson-Zaki equation could correlate the bed expansion and operation fluid velocity for all matrices tested. The theoretical prediction of correlation parameters (the terminal settling velocity U(t) and expansion index n) was improved with the modification of equations in the literature. The residence time distributions were investigated to characterize the hydrodynamic property in expanded bed. Compared with three evaluation factors (the height equivalent of theoretical plate, Bo number and axial distribution coefficient D(ax)), the results indicated that D(ax) is the best parameter to analyze the bed stability of expanded bed under various operation conditions and matrix properties. In addition, it was found that fluid velocity is the most essential factor to influence the hydrodynamic properties in the bed. A linear relation between the D(ax) and superficial fluid velocity for all matrices tested was established.