Radial Flow Column Research Articles

Efficient purification of antiproliferative polysaccharides from Hypsizigus marmoreus with radial flow chromatography.

The increasing commercial significance of natural polysaccharides for use in medicinal products is stimulating the development of efficient and easy scale-up techniques for polysaccharide purification. In this research, the crude polysaccharides from submerged cultivation broth of Hypsizigus marmoreus were purified using radial flow chromatography (RFC), and the antiproliferative activity of the purified fractions was evaluated in vitro. DEAE Sepharose CL-6B was selected to be packed in the RFC column based on its good resolution, physical stability, and low cost. Compared with axial flow chromatography (AFC), an efficient chromatographic process with significantly less time and buffer consumption but yielding higher polysaccharide recovery and resolution was established in RFC, which could clearly purify the crude polysaccharides into different fractions. An acceptable linear scale-up effect of RFC from 100 to 500 mL was successfully achieved without loss of resolution and enhancement of time consumption. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays in cell cultures indicated that the purified polysaccharide fractions possess moderate antiproliferative activities in three different human cancer cell lines, but have significantly lower cytotoxicity in normal human cell lines in vitro. Among the polysaccharide fractions, the main purified acidic fraction W-I could be considered as a novel potential antitumor agent candidate for several tumors, especially for human alveolar epithelial tumors. This research confirmed for the first time that RFC would be a new fast and efficient tool for purification of polysaccharides into different fractions, both at laboratory and commercial scales.

An Efficient Separation Method of Polysaccharides: Preparation of an Antitumor Polysaccharide APS-2 from Auricularia polytricha by Radial Flow Chromatography

Auricularia polytricha is an edible mushroom, quite popular in China. Modern pharmacology research indicate that A. polytricha polysaccharides possess antitumor and immunomodulatory activities. In this paper, an antitumor Auricularia polytricha polysaccharide (APS)-2 was purified by radial flow chromatography (RFC) in a column of 500 mL bed-volume (40 cm i.d × 15 cm) packed with DEAE52-cellulose anion ion-exchange resin. The optimal separation conditions were: sample flow-velocity 15–20 mL min−1, sample volume 160–200 mL, and elution with distilled water and 0.2 M NaCl at a velocity of 35–45 mL min−1. The yield and content of APS-2 obtained under these conditions were 182 mg g−1 total polysaccharides and 98.35 %, respectively. The study provides guidelines for the separation and purification of polysaccharides using a radial flow chromatography column.

Are axial and radial flow chromatography different?

Radial flow chromatography can be a solution for scaling up a packed bed chromatographic process to larger processing volumes. In this study we compared axial and radial flow affinity chromatography both experimentally and theoretically. We used an axial flow column and a miniaturized radial flow column with a ratio of 1.8 between outer and inner surface area, both with a bed height of 5cm. The columns were packed with affinity resin to adsorb BSA. The average velocity in the columns was set equal. No difference in performance between the two columns could be observed. To gain more insight into the design of a radial flow column, the velocity profile and resin distribution in the radial flow column were calculated. Using mathematical models we found that the breakthrough performance of radial flow chromatography is very similar to axial flow when the ratio between outer and inner radius of the radial flow column is around 2. When this ratio is increased, differences become more apparent, but remain small. However, the ratio does have a significant influence on the velocity profile inside the resin bed, which directly influences the pressure drop and potentially resin compression, especially at higher values for this ratio. The choice between axial and radial flow will be based on cost price, footprint and packing characteristics. For small-scale processes, axial flow chromatography is probably the best choice, for resin volumes of at least several tens of litres, radial flow chromatography may be preferable.

COMPARISON OF RADIAL AND AXIAL FLOW CHROMATOGRAPHY FOR MONOCLONAL ANTIBODY DOWNSTREAM PROCESSING AT BENCH AND PILOT SCALES

Axial Flow Chromatography (AFC) is widely used for the purification of therapeutic Monoclonal Antibodies (MAbs). However, AFC columns can generate high pressure drops across the resin bed, preventing operation of the column at high flow rates especially at pilot or manufacturing-scales. Radial Flow Chromatography (RFC) was developed to provide lower pressure drops during chromatographic separations. In these studies, small and pilot-scale columns were used to evaluate purification of a MAb using both AFC and RFC technologies. A bench-scale, wedge RFC column (250 mL) was compared to a bench-scale AFC column at various linear velocities with resulting Residence Times (RT) using Protein A resin for the recovery of a monoclonal antibody. The bench RFC column was successfully operated at 4.5 min RT for equilibration and loading steps and 2 min RT for washing, elution and cleaning steps without compromising yield. The RFC column had approximately 50% lower pressure drop than the AFC column at similar flow rates. The process was then scaled-up to 5 L using a pilot-scale RFC column. The 5-L RFC column was operated at 4.5 min RT for equilibration and loading and 2 min. RT for washing, elution and cleaning with no loss of yield. However, pressure drop across the 5 L RFC column was higher than expected, but antibody recovery yields were similar for both column types. Subsequent investigations revealed a potential design issue with the RFC column. Overall, RFC has great potential to be used for pilot or manufacturing scale without high pressure drop concerns, which will certainly improve processing efficiency.

Desaltation of the Polysaccharide of Nostoc Flagelliforme with Radial Flow Chromatography

.The process for the desaltation of the polysaccharide from the mixotrophic liquid culture of Nostoc flagelliforme with radial flow chromatography (RFC) column packed with Sephadex G-100 gel was investigated. The RFC column having 7.7 cm of bed length and 229.6 cm3 of bed volume was used in this study. The effects of some operating factors such as the sampling weight, the flow rate of eluent on the desaltation efficiency were examined. It was found that the separation efficiency increases with the decrease of sampling weight and flow rate of eluent. With 2.4 mg of sampling weight and 0.3 ml/min of flow rate of eluent, the salt content of polysaccharide was lowered from 90.42% to 2.36%. As a conclusion, this technique can be used to get rid of the eluant in the polysaccharide introduced by the ion exchange process.

High throughput purification of recombinant human growth hormone using radial flow chromatography

Recombinant human growth hormone (r-hGH) was expressed in Escherichia coli as inclusion bodies. Using fed-batch fermentation process, around 670 mg/L of r-hGH was produced at a cell OD600 of 35. Cell lysis followed by detergent washing resulted in semi-purified inclusion bodies with more than 80% purity. Purified inclusion bodies were homogenous in preparation having an average size of 0.6 μm. Inclusion bodies were solubilized at pH 12 in presence of 2 M urea and refolded by pulsatile dilution. Refolded protein was purified with DEAE-anion exchange chromatography using both radial and axial flow column (50 ml bed volume each). Higher buffer flow rate (30 ml/min) in radial flow column helped in reducing the batch processing time for purification of refolded r-hGH. Radial column based purification resulted in high throughput recovery of diluted refolded r-hGH in comparison to axial column. More than 40% of inclusion body protein could be refolded into bioactive form using the above method in a single batch. Purified r-hGH was analyzed by mass spectroscopy and found to be bioactive by Nb2 cell line proliferation assay. Inclusion body enrichment, mild solubilization, pulsatile refolding and radial flow chromatography worked co-operatively to improve the overall recovery of bioactive protein from inclusion bodies.

Dispersion effects in preparative polymethacrylate monoliths operated in radial-flow columns

Monolithic media have found widespread use as excellent tools for fast analytical separations of small molecules, proteins, pDNA and viruses. Polymethacrylate monoliths with large channels are attractive for capturing large molecules, like immunoglobulins, DNA, and viruses. For preparative purposes, these monoliths are operated in radial flow mode. Band spreading in monoliths is extremely low and mostly dominated by the contribution of extra column effects. The model used here had a single axial dispersion coefficient which lumps together extra column effects and the intrinsic band spreading of the monolithic material to characterize the adsorption of proteins and pDNA on polymethacrylate ion-exchange monoliths. Due to the fact that the performance of the monolith was unaffected by the velocity within the applied range, and due to highly favourable adsorption isotherms, a constant pattern model could be applied to predict preparative runs on radial flow units assuming axial flow for modelling.

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 μm. 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 β-galactosidase and used in the production of lactose-free milk.

Chromatography of human prothrombin from Nitschmann fraction III on Q Sepharose Fast Flow using axial and radial flow column

An axial column (Hitrap Q 5 ml, 2.5 x 1.6 cm) and a radial flow column (3.5 x 5 cm) packed with Q Sepharose Fast Flow media had been evaluated for the separation of human prothrombin. Nitschmann fraction III dissolved in buffered saline (0.10 M sodium chloride buffered with 0.06 M Tris/HCl to pH 7.5) was the starting material. Effects of sample flow rate of the two columns were screened. Under radial flow conditions using the radial column, sample flow rate up to 15 ml/min (i.e. 18 bed volumes/h) was achieved and the operating pressure was below 0.2 MPa eventhough the elution velocity was 30 ml/min. Breakthrough capacity was determined by analyzing the total protein and prothrombin activity of the target protein-containing fraction under subsaturating conditions and both columns had almost the same breakthrough capacity per ml media, indicating that the sample loading was independent of radial column geometry. It was concluded that the radial column is an attractive alternate to traditional axial packed bed column, exhibiting very good potential for use in the separation of human prothrombin.

Fluid Dynamics of Radial-Flow Ion Exchange in Partially Filled Columns

An experimental and numerical study demonstrated that completely filling the resin bed is essential if a radial-flow ion-exchange column is to work properly. The first phase of the study developed and verified a finite-element model of both an infinitely long column and finite-height column that was completely filled with resin. The porosity and tortuosity of the support columns of the ion-exchange column were calculated using a least-squares analysis of experimental pressure drop versus flow rate data in an empty column in conjunction with a theoretical analysis based on a layered-cylinder model. Numerical analysis of a partially filled column (i.e., one in which the top 2% of the resin bed was empty headspace) demonstrated that approximately 9% of the fluid would flow through the headspace. This fluid would never come in contact with the ion-exchange resin and would thus leave the column untreated. Modifying the column so that the top 10% of the support membranes was impermeable changed the flow lines such that the headspace was filled with a stagnant fluid and that, after this space was filled, all subsequent fluid entering the column flowed through the ion-exchange bed. This provides a method in which one could ensure that all the fluid would flow through the ion-exchange resin and be treated even if the column could not be or was not completely filled.

Chromatography of human prothrombin from Nitschmann fraction III on DEAE Sepharose Fast Flow using axial and radial flow column.

An axial column (3 x 2.6 cm) and a radial flow column (3.5 x 5 cm) packed with DEAE Sepharose Fast Flow media was evaluated for the separation of human prothrombin from Nitschmann fraction III. Under radial flow conditions, a sample flow rate up to 14 mL/min (approximately 18 bed vols/h) was achieved. Breakthrough capacity was determined and both columns had almost the same breakthrough capacity per mL media, indicating that the sample loading was independent of radial column geometry.

Isolation of human fibrinogen by axial and radial flow chromatography from Nitschmann fraction I

An axial column (3×2.6 cm) and a radial flow column (3.5×5 cm) packed with DEAE Sepharose Fast Flow media had been evaluated for the separation of human fibrinogen. Nitschmann fraction I dissolved in buffered saline (0.015 M NaCl buffered with 0.06 M Tris/HCl to pH 7.5) was the starting material. Under radial flow conditions, sample flow up to 15 ml min−1 (i.e., 18 bed volumes h−1) was achieved. The operating pressures were below 0.2 MPa, even though the elution velocity was 30 ml min−1 (i.e., 36 bed volumes h−1).

Concomitant purification and characterization of malate dehydrogenase, aspartate transaminase, nucleoside diphosphate kinase and enolase from rabbit liver cytosol.

A procedure was developed for purifying the cytosolic isoforms of malate dehydrogenase, aspartate transaminase, enolase and nucleoside diphosphate kinase from a single preparation of rabbit liver. The procedure includes chromatography on reactive-dye, radial-flow columns, and elution of enzymes from columns by substrates, to obtain high yields in a minimal amount of time. The scheme avoids steps used in previous methods that are either difficult to execute in large-scale preparations, or alter the native forms of the enzymes. Examination of the purified enzymes by SDS-PAGE indicated that nearly homogeneous preparations had been obtained. The native molecular weight, subunit molecular weight, and isoelectric point(s) were determined for each enzyme. Although preparations of nucleoside diphosphate kinase purified from cytosol showed only a single band on SDS-PAGE, isoelectric focusing revealed the presence of multiple isoforms.

Ligand efficiency in axial and radial flow immunoaffinity chromatography of factor IX

Radial flow (RF) columns are attractive for process chromatography primarily because larger throughputs and lower pressure drops are achievable in such columns. Large scale immunoaffinity processes using soft resins can benefit most from this configuration. In this study, we compared immobilized ligand efficiency in axial flow (AF) and RF columns using monoclonal antibody against factor IX as the immobilized ligand and a coagulation factor IX complex as the source material. We examined the effects of flow-rate, total protein loading, feed antigen concentration and direction of flow (centrifugal or centripetal for RF, and downward or upward for AF) on immobilized antibody capacity (measured as mg antigen bound per mg antibody). Our results corroborate earlier work, and suggest that none of the factors, in the ranges examined, significantly altered the efficiency of the monoclonal antibody (MAb) in binding factor IX. We also investigated the efficiency of the immobilized antibody upon reuse and found that, over twenty cycles, there was no significant decrease in antibody efficiency. Our results demonstrated that efficiencies obtainable in AF columns can be achieved in RF columns with the same bed thickness, suggesting that radial dispersion, mass transfer and intraparticular diffusion may not have a significant influence on immunoaffinity chromatography efficiency in RF and AF columns.

Protein Band Dispersion in Axial and Radial Flow Chromatography

Abstract Radial flow (RF) column configurations have been developed for larger scale production liquid chromatography, with the primary purpose of increasing throughput rates and decreasing trans-bed pressure drops in comparison to conventional axial flow (AF) columns. The RF columns have been quite successful in attaining these two objectives. In this work, we investigated the nature of protein band dispersion in both axial and radial flow chromatography column configurations, utilizing S-200 Sephacryl gel filtration media with bovine serum albumin as the applied protein. The effects of input feed flow rate as well as input feed albumin concentration on the nature of protein band dispersion were studied. Protein band dispersion was quantified by examining the shape of the eluting protein peak and measuring peak height-to-width (HTW) ratio's for the various flow rates and feed concentrations used in the study. Our results indicate that protein dispersion was larger in RF columns as indicated by smaller peak HTW ratio's than those obtained for AF columns, operated at the same flow rates and feed protein concentrations. We also found that moderately increasing the flow rate increased peak HTW ratio's and resulted in sharper peaks for both AF and RF columns.

Rapid and simple isolation of multigram goat IgG from serum using avid aL™ and radial flow column

Multigram quantities (2.5-10 g) of highly purified IgG were obtained within 4 h from serum by using Avid AL packed in a radial-flow column. Avid AL is an affinity gel containing a synthetic, low-mol-wt ligand capable of selectively binding IgG from serum of all animal species tested. By packing the gel in a radial-flow column up to 500 mL, a high flow rate of 50 mL/min can be achieved without adversely affecting the performance of the gel and the purity of the isolated antibody.

A theoretical study of multicomponent radial flow chromatography

A theoretical study of radial flow chromatography was carried out based on a general nonlinear multicomponent rate model which considers radial dispersion, external mass transfer, intraparticle diffusion, and nonliner multicomponent isotherms. Radial dispersion and mass transfer coefficients were treated as variables which are dependent on the radial coordinate of the radial flow column. The model was solved numerically by using the finite element method and the orthogonal collocation method for the discretization of partial differential equations and Gear's stiff method for the solution of the resulting ordinary differential equation system. Computer simulations were carried out for various multicomponent chromatographic operations. Dispersion and mass transfer effects were investigated. The question of whether radial dispersion and mass transfer coefficients should be treated as variables was discussed. The difference between inward flow and outward flow in radial flow chromatography and the comparison between conventional axial flow chromatography and radial flow chromatography were studied. Langmuir isotherms were used in this work.

Scale-up of “myxalin” purification by a pseudoaffinity method using a radial flow column

Myxalin is a newly defined bacterial anticoagulant. It has been previously purified on Histidyl-Sepharose, on a small scale. We describe a scale-up of this procedure using a radial flow column (Sepragen®). This design permits the use of the same soft gel matrix with 15-fold increase in flow rate compared to the linear system and a simple extrapolation of the operating conditions when optimized on a smaller scale gave no decrease in resolution. There are moreover, better activity yields and very interesting economic advantages.

Semi-preparative purification of recombinant human renin and prorenin

Chinese hamster ovary (CHO) cells, transfected with a vector containing cDNA coding for preprorenin, have been shown to secrete authentic prorenin into the culture supernatant. Purification of the expressed prorenin and purification of active renin, generated by solid-phase trypsin treatment of the conditioned media, have been achieved by conventional chromatographic methods. Scale-up of the initial steps of these procedures is described, including the use of radial-flow columns and automation with fast protein liquid chromatography valves and pumps. This semi-preparative scheme has allowed hundreds of milligrams of both proteins to be isolated.

An Application of the Water Bell Liquid Flow Phenomenon in Visual Art

The authors point out their interest in the artistic aspects ofpatterns andforms caused by flowing viscous liquids. In particular, they describe the design, construction and operation of a flow system that produces a central 'water bell' sculpturalform. The various factors that influence the form of the sheet of liquidfalling from a radial-flow column or stack are presented, as well as the results obtained by adding colorants to the liquid. Photographs of especially interesting examples of the types offlow obtained illustrate their text. A study of artistic form has been produced that is both an example offruitful collaboration of artist and scientist and offaculty and students in separate academic institutions.