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Bed Adsorption (bed + adsorption)
Selected AbstractsIntensified Process for the Purification of an Enzyme from Inclusion Bodies Using Integrated Expanded Bed Adsorption and RefoldingBIOTECHNOLOGY PROGRESS, Issue 4 2006Matthew H. Hutchinson This work describes the integration of expanded bed adsorption (EBA) and adsorptive protein refolding operations in an intensified process used to recover purified and biologically active proteins from inclusion bodies expressed in E. coli. ,5 -3-Ketosteroid isomerase with a C-terminal hexahistidine tag was expressed as inclusion bodies in the cytoplasm of E. coli. Chemical extraction was used to disrupt the host cells and simultaneously solubilize the inclusion bodies, after which EBA utilizing immobilized metal affinity interactions was used to purify the polyhistidine-tagged protein. Adsorptive refolding was then initiated in the column by changing the denaturant concentration in the feed stream from 8 to 0 M urea. Three strategies were tested for performing the refolding step in the EBA column: (i) the denaturant was removed using a step change in feed-buffer composition, (ii) the denaturant was gradually removed using a gradient change in feed-buffer composition, and (iii) the liquid flow direction through the column was reversed and adsorptive refolding performed in the packed bed. Buoyancy-induced mixing disrupted the operation of the expanded bed when adsorptive refolding was performed using either a step change or a rapid gradient change in feed-buffer composition. A shallow gradient reduction in denaturant concentration of the feed stream over 30 min maintained the stability of the expanded bed during adsorptive refolding. In a separate experiment, buoyancy-induced mixing was completely avoided by performing refolding in a settled bed, which achieved comparable yields to refolding in an expanded bed but required a slightly more complex process. A total of 10% of the available KSI,(His6) was recovered as biologically active and purified protein using the described purification and refolding process, and the yield was further increased to 19% by performing a second iteration of the on-column refolding operation. This process should be applicable for other polyhistidine tagged proteins and is likely to have the greatest benefit for proteins that tend to aggregate when refolded by dilution. [source] Modeling of the Wall Effect in Packed Bed AdsorptionCHEMICAL ENGINEERING & TECHNOLOGY (CET), Issue 11 2004W. Kwapinski Abstract A model developed for catalytic packed bed reactors and consistently accounting for the influence of the tube wall on porosity, flow and transport phenomena is used in order to simulate the operation of packed bed adsorbers. By comparison of simulation results with reduced versions of the model the influence of the wall on the adsorber performance is worked out and found to be major at low ratios between tube and particle diameter. The interaction between maldistribution, thermal effects and intraparticle resistances in such adsorber tubes is discussed. [source] Fabrication by three-phase emulsification of pellicular adsorbents customised for liquid fluidised bed adsorption of bioproductsJOURNAL OF CHEMICAL TECHNOLOGY & BIOTECHNOLOGY, Issue 11 2003Mohsen Jahanshahi Abstract A novel dense pellicular adsorbent, custom-designed for liquid fluidised bed adsorption of protein bioproducts, has been fabricated by coating zirconia,silica particles with agarose gel in a three-phase emulsification process. A slurry feedstock comprising solid zirconia,silica particles (120 µm average diameter) suspended in an aqueous solution of agarose was emulsified in an oil,surfactant mixture in a stirred vessel to yield composite droplets. These were subsequently stabilised by cooling to form spherical pellicular particles characterised by a porous, pellicular coat cast upon a solid core. The impact of agitation speed, surfactant concentration, oil viscosity and slurry composition upon the pellicle depth and overall particle diameter was investigated. Pellicle depth decreased with increasing impeller speed and decreased oil viscosity, whilst increased slurry viscosity enhanced that parameter. Initial increases from low concentrations of Span 80 surfactant (0.1% w/v oil) reduced the depth of the agarose pellicle, but the highest values investigated (1.5% w/v oil) promoted particle aggregation. The fluidisation behaviour of particles fabricated under various conditions was characterised by the measurement of expansion coefficients and axial dispersion coefficients for the liquid phase when operated in a standard fluidised bed contactor. Both parameters were found to be comparable or superior to those reported for conventional, composite fluidised bed adsorbents. The controlled coating of porous agarose upon a solid core to yield specific pellicular geometries is discussed in the context of the fabrication of adsorbents customised for the recovery of a variety of bioproducts (macromolecules, nanoparticulates) from complex particulate feedstocks (whole broths, cell disruptates and unclarified bio-extracts). Given the agreement between the size of the pellicular particles and the trends expected from theory, the large-scale manufacture of such particles for customised industrial use is recommended. Copyright © 2003 Society of Chemical Industry [source] Improved design and optimization models for the fixed bed adsorption of acid dye and zinc ions from effluentsJOURNAL OF CHEMICAL TECHNOLOGY & BIOTECHNOLOGY, Issue 12 2002Danny C Abstract The bed depth service time (BDST) design model, which accounts for the change of bed adsorption capacity with service time, has been modified to expand its application and overcome the limiting assumptions of the original BDST analysis. Column experiments were undertaken to test the new model for two adsorption systems, namely zinc ion,bone char and Acid Blue 80 dye-activated carbon. It was found that the percentage of saturation capacity could be correlated using a square-root dependence on the service time and this correlation was incorporated into the original BDST analysis to replace the total sorption capacity term, giving the model a much wider application to real systems. The empty bed residence time optimization approach was modified using the same time-dependent capacity expression and was successfully applied to the metal ion,bone char and the dye-activated carbon system with the use of equilibrium saturated bed capacity. These modifications to the BDST design model and the EBRT optimization model will give more accurate scale-up data for the design of large-scale column adsorption systems. © 2002 Society of Chemical Industry [source] Micro biochemical engineering to accelerate the design of industrial-scale downstream processes for biopharmaceutical proteinsBIOTECHNOLOGY & BIOENGINEERING, Issue 3 2008N.J. Titchener-Hooker Abstract The article examines how a small set of easily implemented micro biochemical engineering procedures combined with regime analysis and bioprocess models can be used to predict industrial scale performance of biopharmaceutical protein downstream processing. This approach has been worked on in many of our studies of individual operations over the last 10 years and allows preliminary evaluation to be conducted much earlier in the development pathway because of lower costs. It then permits the later large scale trials to be more highly focused. This means that the risk of delays during bioprocess development and of product launch are reduced. Here we draw the outcomes of this research together and illustrate its use in a set of typical operations; cell rupture, centrifugation, filtration, precipitation, expanded bed adsorption, chromatography and for common sources, E. coli, two yeasts and mammalian cells (GS-NSO). The general approach to establishing this method for other operations is summarized and new developments outlined. The technique is placed against the background of the scale-down methods that preceded it and complementary ones that are being examined in parallel. The article concludes with a discussion of the advantages and limitations of the micro biochemical engineering approach versus other methods. Biotechnol. Bioeng. 2008;100: 473,487. © 2008 Wiley Periodicals, Inc. [source] The influence of homogenisation conditions on biomass-adsorbent interactions during ion-exchange expanded bed adsorptionBIOTECHNOLOGY & BIOENGINEERING, Issue 3 2006Jürgen J. Hubbuch Expanded bed adsorption (EBA) is an integrative step in downstream processing allowing the direct capture of target proteins from cell-containing feedstocks. Extensive co-adsorption of biomass, however, may hamper the application of this technique. The latter is especially observed at anion exchange processes as cells or cell debris are negatively charged under common anion exchange conditions. The restrictions observed under these conditions are, however, directly related to processing steps prior to fluidised bed application. In this study, it could be shown that the effective surface charge of cell debris obtained during homogenisation is closely related to the debris size and thus to the homogenisation method and conditions. The amount and thus effect of cells binding to the adsorbent could be significantly decreased when optimising the homogenisation step not only towards optimal product release but towards a reduction of debris size and charge. The lower size and charge of the debris results not only in a reduced retention probability but also, in a lower collision probability between debris and adsorbent. The applicability was shown in an example where the homogenisation conditions of E. coli were optimised towards EBA applications. In a previous report (Reichert et al., 2001) studying the suitability of EBA for the capture of formate dehydrogenate from E. coli homogenate the pseudo affinity resin Streamline Red was identified as the only suitable adsorbent. The new approach, however, led to a system where anion exchange as capture step became possible, however, to the cost of binding capacity. © 2006 Wiley Periodicals, Inc. [source] Removal of poly-histidine fusion tags from recombinant proteins purified by expanded bed adsorptionBIOTECHNOLOGY & BIOENGINEERING, Issue 4 2005N. Abdullah Abstract Enzymatic methods have been used to cleave the C- or N-terminus polyhistidine tags from histidine tagged proteins following expanded bed purification using immobilized metal affinity chromatography (IMAC). This study assesses the use of Factor Xa and a genetically engineered exopeptidase dipeptidyl aminopeptidase-1 (DAPase-1) for the removal of C-terminus and N-terminus polyhistidine tags, respectively. Model proteins consisting of maltose binding protein (MBP) having a C- or N-terminal polyhistidine tag were used. Digestion of the hexahistidine tag of MBP-His6 by Factor Xa and HT15-MBP by DAPase-1 was successful. The time taken to complete the conversion of MBP-His6 to MBP was 16 h, as judged by SDS,PAGE and Western blots against anti-His antibody. When the detagged protein was purified using subtractive IMAC, the yield was moderate at 71% although the overall recovery was high at 95%. Likewise, a yield of 79% and a recovery of 97% was obtained when digestion was performed with using "on-column" tag digestion. On-column tag digestion involves cleavage of histidine tag from polyhistidine tagged proteins that are still bound to the IMAC column. Digestion of an N-terminal polyhistidine tag from HT15-MBP (1 mg/mL) by the DAPase-I system was superior to the results obtained with Factor Xa with a higher yield and recovery of 99% and 95%, respectively. The digestion by DAPase-I system was faster and was complete at 5 h as opposed to 16 h for Factor Xa. The detagged MBP proteins were isolated from the digestion mixtures using a simple subtractive IMAC column procedure with the detagged protein appearing in the flowthrough and washing fractions while residual dipeptides and DAPase-I (which was engineered to exhibit a poly-His tail) were adsorbed to the column. FPLC analysis using a MonoS cation exchanger was performed to understand and monitor the progress and time course of DAPase-I digestion of HT15-MBP to MBP. Optimization of process variables such as temperature, protein concentration, and enzyme activity was developed for the DAPase-I digesting system on HT15-MBP to MBP. In short, this study proved that the use of either Factor Xa or DAPase-I for the digestion of polyhistidine tags is simple and efficient and can be carried out under mild reaction conditions. © 2005 Wiley Periodicals, Inc. [source] Protein recovery from enzyme-assisted aqueous extraction of soybeanBIOTECHNOLOGY PROGRESS, Issue 2 2010Kerry A. Campbell Abstract Enzyme-assisted aqueous oil extraction from soybean is a "green" alternative to hexane extraction that must realize potential revenues from a value-added protein co-product. Three technologies were investigated to recover protein from the skim fraction of an aqueous extraction process. Ultrafiltration achieved overall protein yields between 60% and 64%, with solids protein content of 70%, and was effective in reducing stachyose content, with fluxes between 4 and 10 L/m2 hr. Protein content was limited because of high retention of lipids and the loss of polypeptides below 13.6 kDa. Isoelectric precipitation was effective in recovering the minimally hydrolyzed proteins of skim, with a protein content of 70%, again limited by lipid content. However, protein recovery was only 30% because of the greater solubility of the hydrolyzed proteins. Recovery by the alternative of protein capture on dextran-grafted agarose quaternary-amine expanded bed adsorption resins decreased with decreasing polypeptide molecular weight. Proteins with molecular mass greater than 30 kDa exhibited slow adsorption rates. Expanded bed adsorption was most effective for recovery of proteins with molecular weight between 30 and 12 kDa. Overall, adsorption protein yields were between 14% and 17%. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2010 [source] Intensified Process for the Purification of an Enzyme from Inclusion Bodies Using Integrated Expanded Bed Adsorption and RefoldingBIOTECHNOLOGY PROGRESS, Issue 4 2006Matthew H. Hutchinson This work describes the integration of expanded bed adsorption (EBA) and adsorptive protein refolding operations in an intensified process used to recover purified and biologically active proteins from inclusion bodies expressed in E. coli. ,5 -3-Ketosteroid isomerase with a C-terminal hexahistidine tag was expressed as inclusion bodies in the cytoplasm of E. coli. Chemical extraction was used to disrupt the host cells and simultaneously solubilize the inclusion bodies, after which EBA utilizing immobilized metal affinity interactions was used to purify the polyhistidine-tagged protein. Adsorptive refolding was then initiated in the column by changing the denaturant concentration in the feed stream from 8 to 0 M urea. Three strategies were tested for performing the refolding step in the EBA column: (i) the denaturant was removed using a step change in feed-buffer composition, (ii) the denaturant was gradually removed using a gradient change in feed-buffer composition, and (iii) the liquid flow direction through the column was reversed and adsorptive refolding performed in the packed bed. Buoyancy-induced mixing disrupted the operation of the expanded bed when adsorptive refolding was performed using either a step change or a rapid gradient change in feed-buffer composition. A shallow gradient reduction in denaturant concentration of the feed stream over 30 min maintained the stability of the expanded bed during adsorptive refolding. In a separate experiment, buoyancy-induced mixing was completely avoided by performing refolding in a settled bed, which achieved comparable yields to refolding in an expanded bed but required a slightly more complex process. A total of 10% of the available KSI,(His6) was recovered as biologically active and purified protein using the described purification and refolding process, and the yield was further increased to 19% by performing a second iteration of the on-column refolding operation. This process should be applicable for other polyhistidine tagged proteins and is likely to have the greatest benefit for proteins that tend to aggregate when refolded by dilution. [source] | |||||||||||||