Affinity Filtration Research Articles

Modification of 3D-Printed PLA Structures Using Photo-Iniferter Polymerization: Toward On-Demand Antimicrobial Water Filters.

Water filtration is an important application to ensure the accessibility of clean drinking water. As requirements and contaminants vary on a local level, adjustable filter devices and their evaluation with contaminants are required. Within this work, modular filter devices are designed featuring an adjustable surface functionalization. For this purpose, 3D-printed structures are created consisting of bio-based poly(lactic acid) (PLA) that are manufactured by extrusion printing. The surface of PLA is activated with amino groups that are used to install xanthates as chain transfer agents. Subsequently, photo-iniferter (PI) polymerization is used to create cationic polymer brushes on the surface of PLA substrates. Multiple surface characterization techniques are employed to prove successful growth of polymer brushes on PLA. After initial optimization studies on flat surfaces, filter devices are printed, functionalized, and used to remove bacteria from contaminated water. Significant reduction of the number of microorganisms is detected after filtration (single filtration or cycling) and contaminating organism can also be removed from freshwater samples by simple incubation with a 3D-printed filter. The herein developed setup for producing functional filter devices and probing their performance in affinity filtration is a useful platform technology, enabling the rapid testing of polymer brushes for such applications.

Molecular Recognition in Confined Space Elucidated with DNA Nanopores and Single-Molecule Force Microscopy.

The binding of ligands to receptors within a nanoscale small space is relevant in biology, biosensing, and affinity filtration. Binding in confinement can be studied with biological systems but under the limitation that essential parameters cannot be easily controlled including receptor type and position within the confinement and its dimensions. Here we study molecular recognition with a synthetic confined nanopore with controllable pore dimension and molecular DNA receptors at different depth positions within the channel. Binding of a complementary DNA strand is studied at the single-molecule level with atomic force microscopy. Following the analysis, kinetic association rates are lower for receptors positioned deeper inside the pore lumen while dissociation is faster and requires less force. The phenomena are explained by the steric constraints on molecular interactions in confinement. Our study is the first to explore recognition in DNA nanostructures with atomic force microscopy and lays out new tools to further quantify the effect of nanoconfinement on molecular interactions.

Selective, user-friendly, highly porous, efficient, and rapid (SUPER) filter for isolation and analysis of rare tumor cells

Rapid, efficient, and selective separation of tumor cells from complex body fluids is urgently needed for clinical application of tumor-cell-based liquid biopsy. Herein, a size-selective affinity filtration system, named selective, user-friendly, highly porous, efficient, and rapid filter (SUPER Filter), was developed for high-performance tumor cell isolation and analysis. SUPER Filter enabled selective interaction of tumor cells with size-optimized and antibody-coated micropore walls during filtration, achieving a high efficiency of 91.0 ± 6.1% in buffer and 83.7 ± 6.4% in whole blood. Meanwhile, its larger micropore size than those of traditional filtration devices greatly reduced the nonspecific capture of background cells (55-126 cells per mL blood) with enrichment factors of 1.1 × 104-1.0 × 105 and a purity of 52.7 ± 4.2%. Moreover, its high porosity enabled ultra-fast (<5 s for 1 mL of blood or 10 mL of buffer samples) and user-friendly gravity-driven filtration. Finally, SUPER Filter demonstrated rapid, efficient, and selective separation of tumor cells from blood and large-volume pleural and ascetic fluid samples from cancer patients for morphological and molecular analysis. We expect that this size-selective affinity filtration strategy facilitates the clinical application of tumor-cell-based liquid biopsy.

Affinity filtration by a coating of pyrolyzed fish scale colloids on microfibres for removing phenol/quinone compounds from alcohols

This study utilizes the elementary grains of fish scale to construct a filtration membrane, which manifests organic solvent nanofiltration (OSN) efficacy relying on a microflow-promoted adsorption percolation mechanism. Specifically, a colloidal suspension of Barramundi fish scales (BFS) in concentrated sulfuric acid was blended with polyvinyl alcohol (PVA) in aqueous medium. The resulting suspension was thereafter coated on two pieces of cellulose filtration paper (CFP), followed by sandwiching the two coating layers together. The resulting stacked sheet was then subjected to partial pyrolysis under slight compression to complete the membrane preparation. The resulting membrane possesses a fibrous matrix in which individual cellulose fiber is wrapped by a selective layer comprising of cubic-shaped grains that aggregate together and leave nano-crevices. The tortuous pores amongst the fibers act as permeation channels where the coating layer chafes with the permeate stream. The membrane offers high retentions (> 90%) for Sudan IV and Rose Bengal in solvent solutions of ethanol and isopropanol respectively. The assessments suggest that the membrane separation proceeds via a hybrid mechanism, by which convective feed flowing through the permeation channels disperses into the nano-crevices of the selective layer, where pore diffusion facilitates adsorption due to the increase in liquid-solid contact. In addition, the fact that liquid streams chafe with the rough selective layer amidst the permeation channels increases residence time. The friction, pore diffusion, adsorption and buildup mutually sustain the solute retention rates.

Biophysical Insights on the Enrichment of Cancer Cells from Whole Blood by (Affinity) Filtration

Circulating tumor cells (CTCs) play a key role during the metastatic process of human cancers and their reliable detection and characterization could enable new and effective ways of cancer diagnosis, monitoring and treatment. However, due to their ultralow concentration in patient blood, the CTCs must first be enriched before such analysis can be performed. Classical microfiltration is an important and widely used method for the mechanical enrichment of CTCs. This method exploits that CTCs are generally larger than the accompanying blood cells, however, does not differentiate the cells in other ways. In an affinity filtration, selectivity is added by functionalizing the membrane with specific antibodies against a CTC-characteristic surface protein such as the epithelial cell adhesion molecule (EpCAM). A common shortcoming of both filtration approaches is that there is still a poor understanding of the enrichment process and the systems developed so far are frequently operated under non-optimized conditions. To address this, systematic filtration experiments are performed in this work using the EpCAM+ cell line MCF-7 as CTC-model and standard track-etched membranes modified with or without antibodies against EpCAM. The influences of the key filtration parameters time and applied pressure are studied and it is found that in all cases the extent of cell recovery is limited by a lysis process which occurs on the membrane surface. Counterintuitively, it is found that filtration at rather high pressures is advantageous to ensure high recovery rates. To describe the pressure-induced lysis process a biophysical model is developed. This model allows the determination of optimum filtration conditions to achieve both high cancer cell recovery and large blood sample throughput. It is demonstrated that this way practically 100% of spiked cancer cells can be recovered from milliliters of undiluted whole blood within seconds.

Cyclodextrin-Functionalized Fiber Yarns Spun from Deep Eutectic Cellulose Solutions for Nonspecific Hormone Capture in Aqueous Matrices.

A wood based yarn platform for capturing pharmaceutical molecules from water was developed. Cellulose fiber yarns were modified with cyclodextrins, and the capture of 17α-ethinyl estradiol (EE2), a synthetic estrogen hormone used as contraceptive, from water was tested. The yarns were prepared by spinning a deep eutectic solution (DES) of cellulose in choline chloride-urea. Despite their high porosity and water sorption capacity (5 g/g), the spun fiber yarns displayed high wet strength, up to 60% of that recorded in dry condition (128 MPa with 17% strain at break). Cyclodextrin irreversible attachment on the yarns was achieved with adsorbed chitosan and the conjugation reactions and capture of EE2 by the cyclodextrin-modified cellulose were confirmed via online detection with Surface Plasmon Resonance (SPR). The facile synthesis of the bioactive yarns and EE2 binding capacity from aqueous matrices (as high as 2.5 mg/g) indicate excellent prospects for inexpensive platforms in disposable affinity filtration. The study presents a strategy to produce a wood fiber based yarn to be used as a platform for human and veterinary pharmaceutical hormone capture.

High-efficiency expression and purification of the soluble glycoprotein extracellular domain of Rabies virus strain CTN

Objective To express and purify the glycoprotein extracellular domain (Ex-GP) of Rabies virus strain CTN in soluble form with high efficiency. Methods A recombinant expression plasmid containing the gene encoding the Ex-GP was constructed. Various expression conditions were screened to obtain an optimum prokaryotic expression system for Ex-GP in soluble form. The expressed target protein was purified using affinity chromatography and gel filtration chromatography. Results The target protein Ex-GP with high antigenicity was efficiently expressed in soluble form by using the recombinant PBCX expression system and effectively purified by using affinity and gel filtration chromatography. Conclusion The soluble form of Ex-GP is successfully expressed and purified in a simple and convenient way. This study paves the way for further researches on the biological functions of rabies virus glycoprotein, the pathogenic mechanism of rabies and the development of diagnostic reagent and vaccines for rabies virus. Key words: Rabies virus; Glycoprotein extracellular domain; Soluble protein expression

An experimental model of affinity filtration for the isolation of egg white Lysozyme using Cibacron Blue immobilized to yeast cells.

An experimental model of affinity filtration process was designed using a macroligand composed by Cibacron Blue F3GA immobilized to yeast cells. Its performance was evaluated, at bench scale, through the recovery of egg white Lysozyme. The selective and reversible binding between the Cibacron ligand molecule and the enzyme is described. The separation of Lysozyme from the protein mixture included the application of stages such as affinity adsorption, concentration, diafiltration and elution. A tangential microfiltration system with an inorganic membrane was designed. The main finding was the development of the diafiltration operation, key stage in the enzyme isolation. The macroligand particle kept its integrity along the whole process and the degree of purity of the isolated Lysozyme was significant.

Improved Refolding Efficacy of Recombinant Human Interferon α-2b via pH Modulation

Purpose: To increase the refolding yield of Recombinant Human Interferon α-2b in order to achieve a highly potent product. Methods: Interferon α-2b inclusion body was dissolved in tris-HCl buffer containing 6 M guanidine-HCl and CuSO4. Different refolding buffers were employed for refolding the target protein. The refolded proteins were then purified by affinity and gel filtration chromatography. The purified proteins were subjected to circular dichroism (CD) spectropolarimetry and assayed for biological activity in vitro. Results: Increment of pH to 8.5 improved refolding efficacies from 42.28 % to 71.22 %. However, the relative potency significantly increased up to pH 8.0 (from 19353546 to 28633902, p < 0.05) and then decreased to 21081305.00 at pH 8.5. The CD spectra demonstrated that by increasing pH to 8.5, the secondary structure of the protein was altered, probably due to increase in alpha-helix from 23.7 % at pH 7.0 to 28.1 %. Conclusion: Employing a low-cost and simple method, such as alteration of refolding buffer pH, results in higher refolding yield in downstream processing of rhIFN α-2b.

Towards High Performance Detection of Circulating Tumor Cells in Whole Blood

A novel dead-end affinity filtration system is presented for the enrichment of circulating tumor cells (CTCs) from whole blood with high efficiency, selectivity and throughput. At the heart of the system lies an affinity membrane, which was prepared by chemical surface modification of a commercial micro-filter to exclusively allow for the specific adhesion of CTCs. First filtration experiments showed that the prepared membranes are highly permeable for blood cells and allow the efficient capture of cancer cells at high filtration pressure. This promising combination is expected to enable high quality enrichments, which are required for subsequent CTC identification and characterization.

Affinity filtration coupled with capillary-based affinity purification for the isolation of protein complexes

The isolation of complex macromolecular assemblies at the concentrations required for structural analysis represents a major experimental challenge. Here we present a method that combines the genetic power of site-specific recombination in order to selectively “tag” one or more components of a protein complex with affinity-based rapid filtration and a final step of capillary-based enrichment. This modified form of tandem affinity purification produces highly purified protein complexes at high concentrations in a highly efficient manner. The application of the method is demonstrated for the yeast Arp2/3 heptameric protein complex involved in mediating reorganization of the actin cytoskeleton.

IgG PURIFICATION USING AFFINITY FILTRATION WITH SULFAMETHAZINE-AFFINITY CARRIERS

Immunoglobulin G (IgG) antibodies are used extensively for analytical, diagnostic, and therapeutic applications. However, there are some disadvantages to purify IgG antibodies by protein A and G affinity chromatography. Therefore, it is necessary to find an effective alternative and nonchromatographic method to purify IgG. Dextran microparticles were activated and coupled with sulfamethazine to form sulfamethazine-affinity carriers. Then the carriers were used to purify IgG by affinity filtration. Quantitative and qualitative determination proved that sulfamethazine would successfully bond to the surface of dextran microparticles with a density of 85.5 μmol/g (wet). Affinity carriers were proved to withstand high shear force and reveal rare sulfamethazine leakage under filtration conditions between pH 3 to 11. The maximum IgG-binding capacity of affinity carriers was 8.03 mg IgG/g (wet). The affinity filtration process obtained a recovery yield above 80% and purity above 90%. Thus, this work involved in both the advantages of membrane filtration and affinity purification. The results, for the first time, proved that it is possible to use the small ligand sulfamethazine for affinity filtration of IgG. It is an attractive alternative to conventional protein A or G affinity chromatography.

Combination of Crossflow Ultrafiltration, Monolithic Affinity Filtration, and Quantitative Reverse Transcriptase PCR for Rapid Concentration and Quantification of Model Viruses in Water

We present a rapid and effective adsorption-elution method based on monolithic affinity filtration (MAF) for the concentration and purification of waterborne viruses. The MAF column consists of a hydrolyzed macroporous epoxy-based polymer. High recoveries were achieved by columns for the bacterial virus (bacteriophage) MS2 110 (±19)%, as model organism, as well as for human adenoviruses 42.4 (±3.4)% and murine noroviruses 42.6 (±1.9)%. This new concentration and purification method was combined with crossflow ultrafiltration (CUF). Because of the adsorption of the examined viruses to the macroporous surface of the MAF column at pH 3, concentrated matrix components by CUF can be removed. Bacteriophages MS2 were spiked in tap water and concentrated with the new CUF-MAF concentration method by a volumetric factor of 10(4) within 33 min. Furthermore, the detection limit for quantification of bacteriophage MS2 by quantitative reverse transcriptase PCR (qRT-PCR) could be improved from 79.47 to 0.0056 GU mL(-1) by a factor of 1.4 × 10(4). In a first study, we have shown that this method could also be applied for river water containing naturally MS2 and MS2-like phages.

Affinity and size capture of circulating tumor cells: A platform for increased sensitivity

Abstract Background: The potential clinical value of evaluating circulating tumor cells (CTCs) from blood is clear. CTC analysis can aid in cancer diagnosis, prognosis, and treatment prediction. However, since CTCs are rare, current analysis is limited by low efficiencies. Variability of expression of cell surface antigens by CTCs make capture mechanisms based on antibody affinity less than ideal. We report here data from a CTC platform that combines affinity capture with size filtration capture. Methods: A spiked cell model was used, involving whole EDTA blood spiked with SKBR-3 human breast cancer cells. Two microfluidic chips were analyzed, a standard chip (T7) with constant gap size between posts, and a gradient chip (MA3) with a gap-size gradient between posts starting at 40 μ and decreasing to 12 μ. Chips were coated with either an anti-EpCAM monoclonal antibody or with no Ab. An anti-cytokeratin (CK) Ab in combination with a nuclear specific fluorescent stain on an automated cell imaging platform was used to identify CTCs. Results: 45% of cells were captured by size alone (MA3 with no Ab), 16% of cells were captured by affinity alone (T7 with MAb), and 65% of cells were captured by a combination of antibody affinity and size gradient (MA3 with MAb). This increased capture efficiency has been confirmed in clinical samples from patients with metastatic breast cancer, with a greater than 40% increase in capture efficiency utilizing both mechanisms. Whole blood from healthy donors run through either chip type yielded virtually no CTCs. FACS analysis showed that 75% of cultured SKBR-3 cells express high levels of EpCAM. This expression level will vary, as will, presumably, the relative contributions of the antibody capture mechanism and the size capture mechanism. A “heat map” analysis can demonstrate visually the relative contributions of each and these will be presented. Conclusions: The MA3 microfluidic chip captures CTCs using a combination of affinity and size filtration. This allows for the capture of CTCs with varying expression levels of EpCAM by exploiting the size differential between these CTCs and other blood elements. This dual-capture mechanism may allow for more efficient capture than affinity alone. Further work evaluating this platform in late-stage breast cancer patients is ongoing.

Scale‐up of controlled‐shear affinity filtration using computational fluid dynamics

Controlled shear affinity filtration (CSAF) is an integrated bioprocess that positions a contoured rotor above a membrane affinity chromatography column to permit the capture and purification of a secreted protein product directly from cell culture. Here, computational fluid dynamics (CFD) simulations previously used on a laboratory-scale unit (Francis et al., Biotechnol. Bioeng. 2005, 95, 1207-1217) are extended to study the fluid hydrodynamics and expected filter performance of the CSAF device for rotor sizes up to 140 cm in radius. We show that the fluid hydrodynamics within the rotor chamber of larger-scale CSAF units are complex and include turbulent boundary layers; thus, CFD likely provides the only reliable route to CSAF scale-up. We then model design improvements that will be required for CSAF scale-up to permit processing of industrial feedstock. The result is the in silico design of a preparative CSAF device with an optimized rotor 140 cm in radius. The scaled up device has an effective filtration area of 5.93 m(2), which should allow for complete processing in ca. 2 h of 1000 L of culture harvested from either a perfusion, fed-batch or batch bioreactor. Finally, a novel method for the parallelization of CSAF units is presented for use in bioprocessing operations larger than 1000 L.

In this issue: Biotechnology Journal 5/2009

AbstractIn silico filtration scale‐upFrancis and Haynes, Biotechnol. J. 2009, 5, 674–683Controlled shear affinity filtration (CSAF) is an integrated bioprocess that positions a contoured rotor above a membrane affinity chromatography column to purify a secreted protein product directly from cell culture, e.g. monoclonal antibodies. However, for industrial application an effective method for scale‐up is needed. To achieve this Francis and Haynes from Vancouver (Canada) used computational fluid dynamics (CFD) simulations to determine fluid hydrodynamics and expected filter performance for CSAF units with increasing size. This resulted in an in silico design of a preparative CSAF device, which allows processing of 1000 L cell culture in two hours. In addition, the authors report a method for the parallelization of CSAF units that can be applied to volumes exceeding 1000 L.Substrate absorption in gas/solid biocatalysisDimoulal, et al., Biotechnol. J. 2009, 5, 712–721Gas/solid biocatalysis has various potential applications, including fine chemical synthesis and toxic pollutant degradation. To gain a deeper insight into the enzyme microenvironment of a gas/solid system, researchers from Aachen and Jülich (Germany) studied the adsorption of water and substrates to dry deposited alcohol dehydrogenase from Lactobacillus brevis. To monitor the system the well‐characterized conversion of acetophenone catalyzed by alcohol dehydrogenase was chosen. While acetophenone is adsorbed to the deposited enzyme preparation at a significant level, comparable to the amount of adsorbed water, 2‐propanol was not adsorbed at a detectable level. Reaction kinetics will to be studied in the future.Inclusion bodies resolvedDürauer, et al., Biotechnol. J. 2009, 5, 722–729Recombinant proteins produced in Escherichia coli are often deposited in so‐called inclusion bodies (IBs). The purification of proteins in IBs requires several steps, including solubilization and refolding of non‐native proteins. Researchers from Vienna (Austria) report a new method for high‐throughput screening of solubilization conditions and evaluation of dissolution kinetics carried out in conventional 96‐well plates. This involves simple measurements of absorbance at 600 nm and 280 nm to determine turbidity and protein concentration. This new method will contribute to a faster development in the early stage of recombinant protein recovery from E. coli.

Optimizing the rotor design for controlled-shear affinity filtration using computational fluid dynamics

Controlled shear affinity filtration (CSAF) is a novel integrated processing technology that positions a rotor directly above an affinity membrane chromatography column to permit protein capture and purification directly from cell culture. The conical rotor is intended to provide a uniform and tunable shear stress at the membrane surface that inhibits membrane fouling and cell cake formation by providing a hydrodynamic force away from and a drag force parallel to the membrane surface. Computational fluid dynamics (CFD) simulations are used to show that the rotor in the original CSAF device (Vogel et al., 2002) does not provide uniform shear stress at the membrane surface. This results in the need to operate the system at unnecessarily high rotor speeds to reach a required shear stress of at least 0.17 Pa at every radial position of the membrane surface, compromising the scale-up of the technology. Results from CFD simulations are compared with particle image velocimetry (PIV) experiments and a numerical solution for low Reynolds number conditions to confirm that our CFD model accurately describes the hydrodynamics in the rotor chamber of the CSAF device over a range of rotor velocities, filtrate fluxes, and (both laminar and turbulent) retentate flows. CFD simulations were then carried out in combination with a root-finding method to optimize the shape of the CSAF rotor. The optimized rotor geometry produces a nearly constant shear stress of 0.17 Pa at a rotational velocity of 250 rpm, 60% lower than the original CSAF design. This permits the optimized CSAF device to be scaled up to a maximum rotor diameter 2.5 times larger than is permissible in the original device, thereby providing more than a sixfold increase in volumetric throughput.

Adsorption of pure recombinant MBP-fusion proteins on amylose affinity membranes

The specific interaction between MBP-fusion proteins and amylose based affinity membranes has been presented recently. In this work, the attention is focused on the influence of ionic strength on the equilibrium isotherm and on the kinetics of adsorption and desorption. Three different MBP-fusion proteins have been used: MBP-rubredoxin (MW 51 kDa), MBP-intein-CBD (MW 97 kDa) and MBP-β galactosidase (MW 160 kDa) characterized by different dimensions. The equilibrium data follow the Langmuir isotherm, whose parameters q m and K d show a dependence on salt concentration in the feed solution. By increasing the ionic strength, the binding capacity at saturation q m decreases while the equilibrium constant K d increases, indicating that a higher concentration of NaCl reduces the affinity interaction between protein and ligand. A simple kinetic model has been investigated for the interpolation of adsorption and elution curves. No mass transfer effects have been considered within the liquid phase and inside the pores of the stationary phase. The high processing speed of membrane affinity filtration has been exploited. The kinetics of adsorption and elution have been described and the values of the kinetic constants have been obtained both for adsorption and elution conditions for all proteins. The influence of the action of a competing substrate like maltose on the removal of the immobilized product has been also determined.

Effects of Some Antibiotics on Human Erythrocyte 6-Phosphogluconate Dehydrogenase: An in vitro and in vivo Study

The in vitro and in vivo effects of some antibiotics on human erythrocyte 6-phosphogluconate dehydrogenase were investigated. Human erythrocyte 6-phosphogluconate dehydrogenase was purified with ammonium sulphate precipitation, 2′,5′ ADP-Sepharose 4B affinity and gel filtration chromatography. Some antibiotics (netilmicin sulphate, cefepime, amikacin, isepamycin, chloramphenicol, ceftazidim, teicoplanin, ampicillin, ofloxacin, levofloxacin, cefotaxime, penicillin G, gentamicin sulphate, ciprofloxacin) inhibited enzyme activity in vitro but others (cefozin, decefin, streptomycin, combisid, and meronem) were devoid of inhibitory effects. For the drugs having low IC50 values (netilmicin sulphate and cefepime), in vivo studies were performed in rats. Netilmicin sulphate at 15-mg/kg inhibited enzyme activity significantly (p < 0.001) 1h, 2h, and 3h after dosing and cefepime at 200-mg/kg very significantly (p < 0.001) inhibited the enzyme 1 h and 2 h after dosing. Netilmicin sulphate and cefepime inhibited rat erythrocyte 6-phosphogluconate dehydrogenase both in vivo and in-vitro.

Xenin plasma concentrations during modified sham feeding and during meals of different composition demonstrated by radioimmunoassay and chromatography

Xenin is a 25 amino acid peptide produced by specific endocrine cells of the duodenal mucosa. Xenin has multiple biological actions in the gastrointestinal tract. It modulates intestinal motility, affects exocrine pancreatic secretion, and gastric secretion of acid. In the present investigation, we studied plasma concentration of xenin in volunteers after modified sham feeding and after meals of different composition. Plasma xenin concentrations were determined by radioimmunoassay in unextracted plasmas and after acidic extraction using C-18 Sep-Pak chromatography and after neutral extraction using affinity filtration. Both extraction methods were followed by C 18 r.p. HPLC chromatography. Xenin plasma concentrations in unextracted and in extracted plasma rose significantly after modified sham feeding when the food was brought to the volunteers from another room immediately before sham feeding started. When the volunteers had the opportunity to observe the preparation of the meal, xenin plasma concentrations during fasting were high and no further rise was observed after sham feeding. Isocaloric feeding resulted in elevated xenin concentrations in unextracted plasma and after high-pressure liquid chromatography. The methods of extraction, acidic or neutral, did not affect the results. Conclusion: Cephalic factors, investigated by modified sham feeding, stimulate release of xenin into the circulation. Xenin may participate in the central nervous regulation of gastrointestinal function.