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 2 1.6 cm) and a radial flow column (3.5 2 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.     

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).     

New system for preparative electrochromatography of proteins

Electrochromatography employs an axial electric field across a chromatographic stationary phase to separate proteins and other molecules based on differences in electrophoretic mobility. Because the separation is electrically driven, the need for additional chemical reagents is reduced. Two major impediments to scale-up of electrochromatography columns, removal of heat and electrolysis gases, have historically limited the diameter of packed columns to 2.5 cm ID with volumes of approximately 55 mL. We report a novel electrochromatography column that effectively removes electrolysis gases and minimizes heating. A vital component of this system is a new electrode design that couples a platinum gauze with an ultrafiltration membrane across both ends of the column. Use of a methacrylate base stationary phase enabled axial voltage gradients of 10 to 20 V/cm. Thermocouples inserted radially in the column at four axial positions showed that the flow of a 4 degrees C mobile phase coupled with heat conduction through the column walls controlled the temperature to 28 degrees C. The new column design, with dimensions of 3.81 cm ID x 38.1 cm long and bed volume of 400 mL, was demonstrated by separating mixtures of BSA and myoglobin. The column was operated in a horizontal position with radial sample injection and withdrawal at the ends of the packed bed. These experiments are a first step in demonstrating that scale-up of electrochromatography columns can be achieved by choosing appropriate flow rates, voltage gradients, and stationary phase.     

Evaluation of radial chromatography versus axial chromatography, practical approach

Developments in packing and packing port design of radial columns in recent years have resulted in a claimed significant increase in performance of this process chromatography technology. In this first study, the main chromatographic parameters as efficiency, capacity factor, asymmetry and resolution were evaluated in a unique one-to-one comparison between a 120 ml bed-volume and 6 cm bed length radial chromatography mini-process column against a 50 mm diameter, 6 cm bed height and 120 ml bed-volume axial chromatography column. Radial chromatography showed an increase in efficiency by 31% in the number of plates per meter while the equilibration could be reduced by 0.4-0.5 column volumes. The asymmetry factor for bovine serum albumin in radial chromatography showed a reduction of 20% while the reduction of the asymmetry factor of the smaller protein ovotransferrin decreased even by 46% in comparison to the performance of the comparative axial chromatography column. Therefore in radial chromatography resolution improved up to 20%. The retention volume was similar in both cases. For radial chromatography, the decrease in "width at half height" at Height Equivalent of Theoretical Plates (HETP) measurements was 40% while the decrease of the over-all width of the peak was 27%. For adsorbed/desorbed proteins, the elution peak showed similar results: "width at half height" decreased to 45% while the over-all width of the peak decreased by 28%. The concentration of the non-retained protein in the flow-through (lysozyme), increased by 35% while the concentration of the eluted fraction (serum albumin bovine), increased with 40% in the radial chromatography columns. The better results obtained with the radial column were probably the consequence of the geometrical design of this device (larger inlet surface area and small outlet surface area which concentrate the eluted fraction).     

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.     

Considerations on the flow configuration of membrane chromatography devices for the purification of human basic fibroblast growth factor from crude lysates

Membrane chromatography possesses numerous advantages such as operation at high flow rates, low back pressure, ease of handling and scale up, which make the membrane adsorber process a viable alternative to conventional packed column chromatography. A purification process for the isolation of human recombinant basic fibroblast growth factor (FGF‐2) based on membrane chromatography was investigated using devices with different flow configurations. In the first process, the FGF‐2 capture step was performed with an axial flow device, while the alternative method achieved direct capture of FGF‐2 from unclarified cell lysate with a tangential flow device. In both processes, FGF‐2 purities exceeded 82% and the purified cytokine displayed high biological activity. Binding capacity (BC) from fermentation broth of the axial flow device was 28 mg/mL. This was 50% higher than the BC obtained with the tangential flow device under particle‐free supernatant conditions (18 mg/mL) and 150% higher compared to the BC achieved with unclarified cell lysate (11 mg/mL). While membrane chromatography in tangential flow mode omits clarification and thus reduces the number of stages in the downstream process, it displays lower peak resolution and leads to a lower overall process yield.     

Continuous biosorption of cadmium by Moringa olefera in a packed column

The biosorption of Cd(II) by Moringa oleifera using a batch system and a continuous up flow mode in a fixed bed column was studied. Batch adsorption experiments were performed as a function of pH, biosorbent dose, contact time, volume of the solution, and initial metal concentration. The adsorption isotherms obtained fitted well into the Freundlich and Langmuir isotherms. The dynamic removal of cadmium by powdered seed of the Moringa oleifera was studied in a packed column. The effect of bed height (4 and 8 cm) and flow rate (2 and 5mL/min) on biosorption process was investigated and the experimental breakthrough curves were obtained. Results showed that by increasing the bed height and decreasing the flow rate, the breakthrough and exhaustion times increased. The break-through time was considered as a measure of the column performance. The maximum break-through time of 320 min was achieved at the operating condition of 2 mL/min influent flow rate and bed height of 8 cm.     

Optimization and scale up of the adsorption fractionation of cod pyloric caeca deoxyribonuclease using axial and radial flow columns.

The key step in the purification of a deoxyribonuclease (DNase) from extracts of cod (Gadus morhua L.) pyloric caeca, is the selective retention of the enzyme by anion exchange chromatography. The cod DNase purification on Q-Sepharose Fast Flow (Pharmacia) was optimized, using a 60 ml fixed-bed column. In combination with titration curve analysis, we have screened the effect of buffer pHs, feed conductivity and protein loading, on the product recovery and purity. We have developed elution conditions which allow effective separation of the cod DNase from bounded impurities, such as proteinases and nucleic acids. Low levels of these impurities were regarded as essential for the desired product quality. The optimum resolution and maximum purification (ca. 20-fold increase in specific activity) of DNase, was, however, achieved at low protein loading (2.6 mg ml-1 gel), corresponding to less than 4% of the dynamic bed capacity. Scale-up to a 2.5 l pilot scale column (axial flow) and a 0.25 l radial flow column showed that the separation and yield obtained at laboratory scale was retained, and was independent of column geometry and bed height. The implications for a production scale scenario of 100 g of fractionated protein, are also discussed, as well as process hygiene. The optimization described herein adds further knowledge to the treatment of fish waste and the downstream processing of valuable biochemicals from marine raw material.     

Expanded bed protein A affinity chromatography of a recombinant humanized monoclonal antibody: process development, operation, and comparison with a packed bed method.

We show that expanded bed protein A affinity chromatography using Streamline rProtein A media is an efficient method for purifying a recombinant humanized monoclonal antibody from unclarified Chinese hamster ovary cell culture fluid and that it provides purification performance comparable to using a packed bed. We determined that the dynamic capacity of the expanded bed media is related to flow rate (measured in column volumes per hour) by a power function, which allows a high capacity at a low flow rate. At 250 cm h-1 with a 25 cm bed height (10 column volumes h-1), the dynamic capacity is 30 g l-1. The yield and purity (measured by the amount of host cell proteins, DNA, SDS-PAGE, and turbidity) of the antibody purified by expanded bed is comparable to the yield and purity obtained on a standard packed bed method using Prosep A media.     

Partial purification properties of human epidermal growth factor from recombinant Escherichia coli by expanded bed adsorption

Human epidermal growth factor is a polypeptide hormone having many diverse biological functions. This paper first presents the recovery results of human epidermal growth factor (hEGF) immediately from the fermentation broth of recombinant Escherichia coli by using an expanded bed system (a couple of STREAMLINE25 and ÄKTA explorer 100). The influences of operational conditions such as linear flow rate, gradient length of NaCl concentration, pH and sample concentration on the purification performances of hEGF in expanded and packed bed modes with STREAMLINE DEAE resin were systematically evaluated. After optimization, the practical recovery procedure in the expanded bed mode was carried out on a scaled-up system under the conditions of linear flow rates of 183 cm/h (upward) and 37 cm/h (downward), sample volume of 300 ml and column bed height of 13.8 cm which yielded a primary product of hEGF from the cell-free supernatant containing hEGF after centrifugation at 4000 rev/min for 15 min. As a result, the hEGF concentration in the product was higher than 20% (w/v), the concentration factor was greater than 4.3 and the total yield was higher than 80%, respectively. At the same time, the results of hEGF recovery by using expanded bed adsorption (EBA), packed bed chromatography (PBC) and salting out were compared. The results show that the procedure of hEGF recovery in expanded bed adsorption has some advantages over the other two procedures, because of its higher concentration factor, recovery yield, productivity, hEGF concentration in the primary product and shorter duration of purification run.     

Separation of proteins by high-performance anion-exchange chromatography

The chromatographic separation of four proteins, cytochrome c, alpha 1-acid glycoprotein, ovalbumin, and beta-lactoglobulin, was achieved on a 4.6 X 250-mm wide-pore polyethyleneimine (PEI)-silica gel column (5-micron particles, 330-A pore size) with essentially baseline resolution using a 20-min linear gradient from 0.025 M potassium phosphate, pH 6.80, to 0.50 M potassium phosphate, pH 6.80. The back pressure of this anion-exchange column was 1000 psi at a flow rate of 1.0 ml/min. Protein recoveries averaged over 95% and protein capacity exceeded 33 mg for a single protein. Isocratic elution (0.040 M potassium phosphate, pH 6.8; flow rate, 0.50 ml/min) of ovalbumin gave a column efficiency of 15,700 plates/m with a peak asymmetry factor of 1.27. Resolution of these same four proteins on a 4.6 X 50-mm PEI-silica gel column occurred within 2 min. Nucleoside monophosphates were separated on the short PEI-silica column within 1 min with 0.01 M potassium phosphate, pH 2.58, at a flow rate of 6 ml/min which generated a column back pressure of 2000 psi.     

Isocratic separation of DNA bases and deoxyribonucleosides by high-performance liquid chromatography on anex OSTION.

A high-performance liquid chromatographic method has been developed for the resoiusion of DNA bases and deoxyribonucleosides in mixture. The isocratic separation based on ion exelusion was achieved in column of 250-mm length and 2 mm i.d. packed with the spherical, totally porous anex OSTION LGAT 0800 of particle size 10–12 μm. The mobile phase consists of 5 mm ammonium formate pH 4.5 and complete resolution of an eight-component system is possible at a flow rate of 2 ml/h in 100 min. Rapid separation of the components, with exception of Cyt-dCyd, is reached at flow rate of 20 ml/h in 10 min.     

Continuous adsorption and recovery of Cr(VI) in different types of reactors

This study reports the results of experiments on continuous adsorption and desorption of Cr(VI) ions by a chemically modified and polysulfone-immobilized biomass of the fungus Rhizopus nigricans. A fixed quantity of polymer-entrapped biomass beads corresponding to 2 g of dry biomass powder was employed in packed bed, fluidized bed, and stirred tank reactor for monitoring the continuous removal and recovery of Cr(VI) ions from aqueous solution and synthetic chrome plating effluent. Parameters such as flow rate (5, 10 and 15 mL/min), inlet concentration of Cr(VI) ions (50, 100, 150 and 250 mg/L) and the depth of biosorbent packing (22.8, 11.2 and 4.9 cm) were evaluated for the packed bed reactor. The breakthrough time and the adsorption rates in the packed bed column were found to decrease with increasing flow rate and higher Cr inlet concentrations and to increase with higher depths of sorbent packing. To have a comparative analysis of Cr adsorption efficiency in different types of reactors, the fluidized bed reactor and stirred tank reactor were operated using the same quantities of biosorbent material. For the fluidized bed reactor, Cr(VI) solution of 100 mg/L was pumped at 5 mL/min and fluidized by compressed air at a flow rate of 0.5 kg/cm.(2) The stirred tank reactor had a working volume of 200 mL capacity and the inlet/outlet flow rate was 5 mL/min. The maximum removal efficiency (mg Cr/g biomass) was obtained for the stirred tank reactor (159.26), followed by the fluidized reactor (153.04) and packed bed reactor (123.33). In comparison to the adsorption rate from pure chromate solution, approximately 16% reduction was monitored for synthetic chrome plating effluent in the packed bed. Continuous desorption of bound Cr ions from the reactors was effective with 0.01 N Na(2)CO(3) and nearly 80-94% recoveries have been obtained for all the reactors.     

Analysis of venous flow transients for estimation of vascular resistance, compliance, and blood flow distribution

We analysed venous flow transients using a long venous circuit and right heart bypass in 17 dogs after a rapid decrease in atrial pressure. A biphase curve was obtained which we decomposed into a two-compartment model, one with a fast time constant for venous return (0.069 min) and 52% of total circulating flow (Q), and one with a slower time constant (0.456 min) and 48% of Q. Subsequently, separate drainage from splanchnic and peripheral beds (with the renal venous return in the peripheral bed drainage) allowed comparison of time constants and venous outflow in these beds. The sum of the venous outflow volumes over time during separate drainage was indistinguishable from the single biphasic venous outflow volume curve over time observed with a long circuit and single reservoir. The fast time constant of the biphasic curve was not different from that determined by separate drainage from the peripheral circulation. The slow time constant of the single biphasic curve of 0.456 min was hybrid of two time constants, 0.216 min in the splanchnic bed and 0.862 min in the peripheral bed. Separate drainage from peripheral and splanchnic vascular beds demonstrated that the peripheral bed constituted 70% of venous outflow in the fast time constant compartment using Caldini's technique, whereas the splanchnic bed constituted 63% of venous outflow in the slow time constant compartment. It is concluded that, although Caldini's technique demonstrates biphasic venous flow transients, neither the fast nor the slow time constant compartments resolved from this analysis represent a particular anatomical region or vascular bed.     

Purification of histidine-tagged nucleocapsid protein of Nipah virus using immobilized metal affinity chromatography

Nucleocapsid (N) protein of Nipah virus (NiV) is a potential serological marker used in the diagnosis of NiV infections. In this study, a rapid and efficient purification system, HisTrap? 6 Fast Flow packed bed column was applied to purify recombinant histidine-tagged N protein of NiV from clarified feedstock. The optimizations of binding and elution conditions of N protein of NiV onto and from Nickel Sepharose? 6 Fast Flow were investigated. The optimal binding was achieved at pH 7.5, superficial velocity of 1.25 cm/min. The bound N protein was successfully recovered by a stepwise elution with different concentration of imidazole (50, 150, 300 and 500 mM). The N protein of NiV was captured and eluted from an inlet N protein concentration of 0.4 mg/ml in a scale-up immobilized metal affinity chromatography (IMAC) packed bed column of Nickel Sepharose? 6 Fast Flow with the optimized condition obtained from the method scouting. The purification of histidine-tagged N protein using IMAC packed bed column has resulted a 68.3% yield and a purification factor of 7.94.     

Experimental measurement of particle size distribution and voidage in an expanded bed adsorption system

This paper presents an experimental analysis of matrix bead size distribution and voidage variations with axial height in an expanded bed adsorption system. Use of a specially constructed expanded bed with side ports has enabled sampling from within the expanded bed along the vertical axis. Particles removed from within the bed were measured for their size distributions. Residence time distribution studies were used to estimate bed voidage. Measurements of axial and radial particle size distributions and axial voidage distribution have been made at different flow rates. Particle size was found to be radially constant, indicating constant stratification in the column. The particle size was found to decrease with increasing axial height. Voidage increased with axial height from a settled bed value of 0.39 to approaching unity for high liquid velocities and increased at a constant axial position with increased flowrate. This information provides key insight into bed stability and data for the improved modeling of this important unit operation.     

A reverse phase HPLC method for the separation of two stereo isomers of 2-[4-(methylsulfonyl)phenyl]-3-(3(R)-oxocyclopentyl)propanoic acid

This study describes successful method development and separation of two stereo isomers of 2-[4-(methylsulfonyl)phenyl]-3-(3(R)-oxocyclopentyl)propanoic acid by reverse phase high-performance liquid chromatography. Baseline resolution was achieved on a J'sphere-ODS-H80 (150 mm × 4.6 mm, 4 μm) column using mobile phase consisting of 0.05% triflouroacetic acid in water-acetonitrile (85:15, v/v) at a flow rate of 1.0 ml/min. The detection was carried out at 228 nm. The title compound, in turn, can be obtained by C-alkylation of methyl 2-[4-(methylthio)phenyl]acetate with 2(S)-iodomethyl-8,8-dimethyl-6,10-dioxaspiro[4.5]decane followed by consecutive hydrolysis and oxidation. The partially validated analytical method (system suitability, peak homogeneity, linearity, precision, robustness, and solution stability) has limit of detection and limit of quantification, 0.15 and 0.50 μg/ml respectively. Alternatively, the new method is being routinely utilized to monitor epimerization of α-carbon of the propanoic acid in the title compound by crystallization-induced dynamic resolution.     

Separation of ionic liquid [Mmim][DMP] and glucose from enzymatic hydrolysis mixture of cellulose using alumina column chromatography

Pretreatment of cellulose with ionic liquids (ILs) can improve the efficiency of the hydrolysis by increasing the surface area of the substrates accessible to solvents and cellulases. However, the IL methods are facing challenges to separate the hydrolyzed sugar products as well as the renewable ILs from the complex hydrolysis mixtures. In this study, an alumina column chromatography (ACC) method was developed for the separation of hydrophilic IL N-methyl-N-methylimidazolium dimethyl phosphate ([Mmim][DMP]) and glucose, which was the main ingredient of the monosaccharide hydrolyzate. The processing parameters involved in ACC separation were investigated in detail. Our results showed that the recovery yields of [Mmim][DMP] and glucose can reach up to 93.38% and 90.14%, respectively, under the optimized parameters: the sampling ratio of 1:20 between the applied sample volume and the bed volume of the column; a gradient elution using methanol (100%, 150 ml) and then water (170 ml) as eluents with 1 ml/min flow rate. The recovered [Mmim][DMP] showed qualified property and was effective in a new hydrolysis reaction. In addition, scale-up ACC separations were successfully done with satisfied separation performance. The results indicated that the ACC is one of the available methods for the separation of ILs and monosaccharides from the hydrolysis mixtures.     

A new fluid distribution system for scale-flexible expanded bed adsorption

A new fluid distribution system designed for expanded bed adsorption was introduced and studied in a 150-cm diameter column. Based on fluid application through a rotating distributor, it eradicates the need for perforated plates, meshes, or local mixers. The effect of rotation rate on column performance was examined by fluidizing a 30-cm high bed of supports with tap water and introducing pulses of dye or acetone tracer. Linear bed expansion was seen as the superficial fluid velocity was raised from 170 x h(-1) to 450 cm x h(-1) (3000 L x h(-1) to 8000 L x h(-1)), and there was little change in expansion characteristics as distributor rotation rate was increased from 2.5 to 10 rpm. The distributor was observed to generate a flow pattern suitable for expanded bed adsorption when the supports were fluidized at a superficial fluid velocity of 283 cm center dot h(-1) and dye pulses introduced. At a rotation rate of 2.5 rpm, no significant dead zones were observed, and a discrete band was formed that moved up through the bed. Furthermore, the pattern of dye movement could be used to calculate interstitial linear fluid velocities of 460 cm x h(-1) and 572 cm x h(-1) at the column wall and center, respectively, indicating a parabolic flow profile. The distributor rotation rate giving the best operating conditions was found to be 2.5 rpm when the bed was fluidized at a flow velocity of 283 cm x h(-1) and the residence time distribution of acetone tracer examined. Under these conditions, the coefficient of axial dispersion was 6.1 x 10(-6) m(2) x s(-1) and 29 theoretical plates were measured. When the rotation rate was raised to 10 rpm, the coefficient of axial dispersion increased to 8.08 x 10(-6) m(2) x s(-1) and the number of theoretical plates decreased to 22.     

Preparative high speed gel permeation chromatography of proteins on Toyopearl HW55F

A method is described for the rapid separation of protein mixtures by high speed gel permeation chromatography using Toyopearl HW55F, a new semi-rigid hydrophilic polymer. Good resolution of protein mixtures according to molecular size can be achieved on this material with high flow rates and low column pressures. Molecular weight estimations in the range between 10⁴ and 10⁶ daltons can be performed within minutes. Large-scale enzyme purification (up to 1.6 gm of starting material with a 3.2 × 105 cm column) was achieved with 86–110% recovery of enzymatic activity. Data are presented on the optimum column length, flow rate, loading capacity and eluant ionic strength.