Separation Of Bovine Serum Albumin Research Articles

Thin alumina nanoporous membranes for similar size biomolecule separation

Anodic aluminum oxide (AAO) membranes are chemically and thermally stable and inert, and can be produced with wide variety of pore size distribution. We report thin alumina nanoporous membranes (0.7–1 μm thickness) with narrow pore size distribution (20–30 nm diameter) obtained by anodizing aluminum films that were deposited on silicon substrates for a biomolecule separation platform. We demonstrate the electrostatic sieving effect for separation of proteins with similar molecular weights with the thin nanoporous anodic aluminum oxide membranes. Without separate modification of the membrane surface, we have achieved high throughput (>10 −8 M cm −2 s −1) and high selectivity (>42) for separation of bovine serum albumin (BSA) and bovine hemoglobin (BHb) at pH = 4.7, the isoelectric point of BSA.

Protein adsorption on needle-shaped hydroxyapatite prepared by hydrothermal treatment of mixture composed of CaHPO4 2H2O and .BETA.-Ca3(PO4)2

The behavior of protein adsorption on needle-shaped hydroxyapatite (HAp) crystals was investigated and compared with that on irregularly shaped HAp crystals in phosphate buffer solutions. The proteins used were bovine serum albumin (BSA) and lysozyme chloride (LSZ). The effect of phosphate ions in the buffered solutions was evaluated at concentrations in the range 2-200 mol·m-3. In 50 mol·m-3 or lower buffer concentrations, the saturated amount of BSA adsorbed on needle-shaped HAp was greater than that on irregularly shaped HAp. The saturated amount of BSA adsorbed on needle-shaped HAp decreased with increasing phosphate buffer concentration. This resulted in little difference in BSA adsorption between the needle-shaped HAp and the irregularly shaped HAp at phosphate concentrations of 100 mol·m-3 or higher. In contrast, the saturated amount of adsorbed LSZ increased with increasing phosphate buffer concentrations up to 10 mol·m-3 and subsequently decreased with increasing phosphate concentrations between 10 and 200 mol·m-3. There were no differences in the protein adsorption behaviors between the needle-shaped HAp and irregularly shaped HAp at a phosphate concentration of 200 mol·m-3. A specific adsorption, useful for the separation of BSA and LSZ proteins was observed for the needle-shaped HAp at a low phosphate buffer concentration. This separation was caused by the large a-face surface area of the needle-shaped HAp by comparison with that of the irregularly shaped HAp.

Global Model for Optimizing Crossflow Microfiltration and Ultrafiltration Processes: A New Predictive and Design Tool

A global model and algorithm that predicts the performance of crossflow MF and UF process individually or in combination in the laminar flow regime is presented and successfully tested. The model accounts for solute polydispersity, ionic environment, electrostatics, membrane properties and operating conditions. Computer programs were written in Fortran 77 for different versions of the model algorithm that can optimize MF/UF processes rapidly in terms of yield, purity, selectivity, or processing time. The model is validated successfully with three test cases: separation of bovine serum albumin (BSA) from hemoglobin (Hb), capture of immunoglobulin (IgG) from transgenic goat milk by MF, and separation of BSA from IgG by UF. These comparisons demonstrate the capability of the global model to conduct realistic in silico simulations of MF and UF processes. This model and algorithm should prove to be an invaluable technique to rapidly design new or optimize existing MF and UF processes separately or in combination in both pressure-dependent and pressure-independent regimes.

Adsorption of trypsin onto magnetic ion‐exchange beads of poly(glycidylmethacrylate‐co‐ethyleneglycoldimethacrylate)

AbstractMagnetic beads were prepared from glycidyl methacrylate (GMA), and ethyleneglycol dimethylmethacrylate (EGDMA) in the presence of Fe3O4 nano‐powder via suspension polymerization. The magnetic beads were characterized by surface area measurement, electron spin resonance (ESR), and scanning electron microscopy (SEM). ESR data revealed that the beads were highly super‐paramagnetic. The effects of contact time, pH, ionic strength, and temperature on the adsorption process have been studied. Adsorption equilibrium was established in about 120 min. The maximum adsorption of trypsin on the magnetic beads was obtained as 84.96 mg g−1 at around pH 7.0. At increased ionic strength, the contribution of the electrostatic component to the overall binding decreased, and so the adsorption capacity. The experimental equilibrium data obtained trypsin adsorption onto magnetic beads fitted well to the Langmuir isotherm model. The result of kinetic analyzed for trypsin adsorption onto magnetic ion‐exchange beads showed that the second order rate equation was favorable. It was observed that after six adsorption–elution cycle, magnetic beads can be used without significant loss in trypsin adsorption capacity. Finally, the magnetic beads were used for separation of bovine serum albumin (BSA) and trypsin from binary solution in a batch system. © 2007 Wiley Periodicals, Inc. J Appl Polym Sci, 2008

Surfactant-aided size-exclusion chromatography for the purification of immunoglobulin G

In the production of monoclonal antibodies, separate chains of the antibody are often present in the product mixture as well as other contaminating proteins. These fragments should be removed from the whole antibodies. This paper shows the purification of monoclonal immunoglobulin G (IgG) from its heavy chain contaminant. The heavy chain fragment is simulated experimentally using bovine serum albumin (BSA), which has approximately the same molecular weight. The purification is performed using traditional size-exclusion chromatography (SEC) and using surfactant-aided SEC (SASEC), testing two different surfactants (C 12E 23 and Tween20) and two different gels (Sephacryl S200HR and Sephacryl S300 HR). Pulse experiments show that with SASEC both BSA and IgG are more distributed towards the solid phase than compared to using SEC. This effect is larger on IgG, the largest component than on BSA. As a consequence, azeotropes will be formed at a specific surfactant concentration. Above this concentration the selectivity is reversed and increased to values higher than obtained with conventional SEC. At 7.5% (w/w) of C 12E 23, BSA actually elutes before IgG. These experiments further show that when using SASEC larger productivity, higher yields and lower solvent consumption can be achieved without loss of purity of IgG when compared to conventional SEC. Mathematical simulation of the separation of BSA and IgG using simulated moving bed (SMB) chromatography indicates a large increase in productivity when applying a surfactant gradient in SASEC SMB compared to conventional isocratic SEC-SMB. Furthermore, solvent consumption reductions with a factor 15 prove possible as well as concentrating the IgG by a factor 2.

Effects of Ionic Environments on Bovine Serum Albumin Fouling in a Cross‐Flow Ultrafiltration System

AbstractThe influence of electrostatic interactions on membrane fouling during the separation of bovine serum albumin (BSA) from solution was studied in a cross‐flow ultrafiltration system. Experiments were carried out at different pH values between 3.78 and 7.46; and for different ionic strengths between 0.001 M and 0.1 M. The changes in permeate flux, cake layer resistance, zeta potentials of BSA and polyether sulfone (PES) membranes, and electrostatic interaction energies, were evaluated. At all of the ionic conditions studied, PES membranes are negatively charged. However, BSA molecules are either negatively or positively charged depending on the ionic environment. Whereas the cake layer resistance decreased with increasing pH and ionic strength, the permeate fluxes increased. The calculated electrostatic energy was a minimum at the isoelectric point (IEP) of BSA. However, at this point, the cake resistances corresponding to fouling at each ionic strength, were not minimized. Below the IEP of BSA, the electrostatic forces were attractive, while above the IEP, repulsive electrostatic forces were dominant.

Modification with DEAE-dextran, an alternative way to prepare anion-exchange monolithic column with lower pressure drop

Poly(methacrylate-co-ethylene dimethacrylate) monolithic column (100 mm × 4.6 mm i.d.) subsequently modified with DEAE-dextran had been prepared for the separation of proteins with anion-exchange mode. The column, which possessed the specific surface area of 5.3 m 2/g and immobilized amine group of 9.93 μmol/g media, showed even better permeability of 607 psi at flow rate of 1445 cm/h in comparison to diethylamine modification procedure. The dynamic binding capacity of bovine serum albumin (BSA) was 23.8 mg/g media. Fast separation of lysozyme, hemoglobin and BSA on the column was achieved within 3 min at flow rate of 1445 cm/h.

Preparation, characterization and effect of annealing on performance of cellulose acetate/sulfonated polysulfone and cellulose acetate/epoxy resin blend ultrafiltration membranes

Polymeric membranes based on cellulose acetate (CA)––sulfonated polysulfone blends at three different polymer compositions were prepared by solution blending and phase inversion technique, characterized and subjected to annealing at 70, 80 and 90 °C. The permeate water flux, separation of bovine serum albumin and its flux by the blend membranes before and after thermal treatment, have been compared and discussed. Similarly, CA and epoxy resin (diglycidyl ether of bisphenol-A) were blended in various compositions, in the presence and in the absence of polyethyleneglycol 600 as non-solvent additive, using N,N′-dimethylformamide as solvent, and used for preparing ultraflltration membranes by phase inversion technique. The polymer blend composition, additive concentration, casting and gelation conditions were optimized. Blend membranes were characterized in terms of compaction, pure water flux, water content and membrane resistance. The effects of polymer blend composition and additive concentration on the above parameters were determined and the results are discussed.

Separation of proteins on polymeric stationary phases grafted with various amine groups

Six polymeric stationary phases with various amine groups were prepared by surface grafting of glycidyl methacrylate on silica gel surface and its subsequent amination. The six kinds of amines, namely, triethylamine, diethylamine, ethylenediamine (EDA), hexaethylenediamine (HEDA), diethylenetriamine (DETA) and triethylenetetraamine (TETA) were used in this study. The separation of bovine serum albumin (BSA) and chicken egg albumin (CEA) on the polymeric stationary phase with various amines was investigated. The affinity degree of BSA was higher than CEA for the EDA, HEDA and DETA columns, whereas the affinity degree of CEA was higher than BSA for the TETA column.

Optimization of azeotropic protein separations in gradient and isocratic ion-exchange simulated moving bed chromatography

The separation of dilute binary mixtures of proteins by salt aided ion-exchange simulated moving bed (SMB) chromatography is optimized with respect to throughput, desorbent consumption and salt consumption. The optimal flow-rate ratios are analytically determined via an adopted “triangle theory”. Azeotropic phenomena are included in this procedure. The salt concentrations in the feed and recycled liquid are subsequently determined by numerical optimization. The azeotropic separation of bovine serum albumin and a yeast protein is used to illustrate the procedure. Gradient operation of the SMB is generally preferred over isocratic operation. A feed of azeotropic salt concentration can only be separated in a gradient SMB. Desorbent and salt consumption are always lower in gradient than in isocratic SMB chromatography.

Free-flow zone electrophoresis: a novel approach and scale-up for preparative protein separation.

Different continuously working free-flow zone electrophoresis (FFZE) chambers have already been developed [1, 2]. All of them deal with the problem of distinctive Joule heating. The resulting temperature gradients cause an unstable density field which leads to thermal convection and thus to an intermixing of the different fractions within the chamber. The most promising and simple approach to stabilize the flow is to build chambers with one very small dimension (e.g., h = 0.5 mm) to assure efficient heat withdrawal. This in turn presents substantial disadvantages, namely limited throughput and restricted scale-up potential. The novel approach combines a simplified design and assembly with the possibility of straightforward scale-up. It still operates with one small dimension (d = 1-2 mm) to handle the Joule heating. Here, however, not the dimension perpendicular to the electric field but the dimension parallel to the electric field (separation distance) is chosen as the smallest dimension. The efficiency of the new device is shown by the separation of bovine serum albumin (BSA) and cytochrome c with an overall protein throughput of up to 1.1 g/h, using a cell with a separation volume of less than 20 mL.

Separation of bovine serum albumin and its monoclonal antibody from their immunocomplexes by sodium dodecyl sulfate–capillary gel electrophoresis and its application in capillary electrophoresis-based immunoassay

A non-competitive immunoassay was performed by sodium dodecyl sulfate–capillary gel electrophoresis with UV detection using bovine serum albumin (BSA) and monoclonal anti-BSA. BSA, anti-BSA and their immunocomplexes were well resolved under non-denaturing conditions. A linear calibration curve was obtained and can be used for the quantification of anti-BSA. The limit of detection of anti-BSA was 0.1 μ M under the present conditions. Compared with capillary zone electrophoresis, we believed that this method has the potential to be used as a more general format for performing capillary electrophoresis-based immunoassay of medium- and large-sized analytes.

HPCPC Separation of Proteins using Polyethylene Glycol-Potassium Phosphate Aqueous Two-Phase

ABSTRACT High Performance Centrifugal Partition Chromatography (HPCPC) is a practical and suitable method, particularly on the preparative scale, for the separation of biomolecules such as proteins, enzymes, etc. Aqueous two-phase system is also very attractive for the isolation of biomolecules. Aqueous polymer phase system composed of polyethylene glycol 6000-potassium phosphate has been used for the countercurrent chromatographic separation of bovine serum albumin (BSA) and lysozyme using HPCPC. The separation of BSA and lysozyme under various conditions such as various flow rates, rotational speeds, pH of the solvent, and the retention of stationary phase has been studied in the present investigation. The baseline separation of BSA and lysozyme has been also observed. The results of this study demonstrate that HPCPC is useful for separation of proteins with aqueous two-phase systems.

Studies on Box-Behnken design experiments: cellulose acetate-polyurethane ultrafiltration membranes for BSA separation

Ultrafiltration membranes were prepared from mixtures of cellulose acetate-polyurethane blend membranes. During the last 1 or 2 decades, the concentration purification and separation of Albumin by ultrafiltration through semipermeable membranes have been put into practice and hence membrane separation is considered as the unit operation. The blend solution was prepared from cellulose acetate and polyurethane in polar solvent in presence of polyvinylpyrrolidone as additive. The performance of modified blend membranes applied for Bovine Serum Albumin (BSA) separation by ultrafiltration technique using Box-Behnken design with three variables: additive, time and pressure. Three different levels was studied to identify a significant correlation between the effect of these variables on the amount of separation of BSA. The methodology identifies the principal experimental variables, which have the greatest effect on the separation process. The experimental values are in good agreement with predicted values, the correlation coefficient was found to be 0.9871.

Continuous flow isoelectric focusing for purification of proteins.

To estimate the feasibility of isoelectric focusing in a continuous mode, the conditions for obtaining stable and linear pH gradients were first investigated, using a mixture of carrier ampholytes. Then the operating parameters allowing the isoelectric focusing of bovine serum albumin as model protein were determined. A theoretical approach was used to find conditions minimizing the electrohydrodynamic effect, mainly responsible for protein dispersion, for the separation of bovine serum albumin and alpha-lactalbumin from bovine milk.

A Newly Designed Adsorbent Prepared from Hydroxyapatite Originating from Cattle-Bones for Chromatographic Separation of Albumin and Lysozyme

Abstract Functional spherical particles 5—30 μm in size were prepared by a spray-drying method with ultrasonic dispersion from hydroxyapatite (r-HAp) which was derived from bones of cattle. The liquid chromatographic characteristics for the separation of bovine serum albumin (BSA) and lysozyme from egg white (LSZ) on the r-HAp particles were investigated in comparison with those of stoichiometric hydroxyapatite (s-HAp) derived from reagents. The specific surface area (SAD), the crystallite size (CS), and the microstructure of the HAp’s particles were strongly influenced by the heat-treatment at 273—1073 K (designated by HAp(273—1073)) in a stream of water vapor. In liquid chromatography (LC) analysis with columns packed with the HAp powders, for r-HAp, mixed solutions of 0.5—2.0 mg cm−3 BSA and LSZ were sufficiently eluted using a linear molarity gradient of sodium phosphate buffers at 313 K. The resolutions (RS) of the proteins and the widths for each half of the chromatographic curves (WHB for BSA or WHL for LSZ) were sensitively varied in connection with SAD and CS. The optimal values of the LC operating parameters for all the HAp’s were evaluated as RS of r-HAp(673) and WHB and WHL of r-HAp(873).

Continuous separation of proteins by poly(vinyl alcohol) shielded Multichannel Flow Electrophoresis

A hydrophilic polysulphone membrane spaced multicompartment electrolyzer for Multichannel Flow Electrophoresis (MFE) was developed. Application of poly(vinyl alcohol) (PVA) in MFE resulted in a 35% increase of protein transmembrane flux. Continuous separation of bovine serum albumin (BSA) and haemoglobin bovine blood (HBB) model mixture by PVA shielded MFE yielded 56mg BSA and 48mg HBB per hour. The average recovery was 65%.

Influence of additives on resolution and focusing efficiency in free-flow isoelectric focusing

In theory, the resolution of isoelectric focusing (IEF) depends directly on the electric field strength. However, at higher electric field strengths with free-flow IEF, focusing of proteins is impaired by electrodispersive effects. These effects cause a widening of protein peaks sharply focused at lower voltage. This was dependent on the electric field strength and thus reduced the focusing efficiency and resolution. Using 4-nitroaniline as a visible marker substance, the influence of concentration and molecular mass of various additives on the electrodispersive effects as a function of electric field strength was investigated. The efficiency to reduce electrodispersive effects was determined by various properties of the additives. However, the molecular mass of the additives only marginally influenced electrodispersive effects. In the most favourable case, the resolution R for the separation of bovine serum albumin and haemoglobin applied as a standard mixture increased from R = 0.4 without additive to R = 3.4 with 2% saccharose and 0.3% Servalyte 3–10 as ampholyte. Improvements in the resolution for the separation for the standard mixture were independent of the ampholyte used (Bio-Lyte, Ampholine, Servalyte).

Enhanced separation of albumin-poly(ethylene glycol) by combination of ultrafiltration and electrophoresis

Separation of bovine serum albumin (BSA) from poly(ethylene glycol) (PEG) through ultrafiltration membranes has been investigated. Concentration polarization by BSA represents a strong limitation to the transmission of PEG through a porous membrane. The application of an electric field normal to the membrane can help to reduce BSA concentration polarization and to enhance PEG transmission, both in ultrafiltration and in diafiltration mode. A range of solution compositions of BSA and PEG has been investigated. The contribution of electrokinetic phenomena is measured by specific experiments.

Selective separation of proteins by microfiltration with formed-in-place membranes

The separation of bovine serum albumin and bovine gamma globulin from their mixtures in solution by microfiltration with porous stainless steel supported titanium dioxide formed-in-place membranes was investigated. Strong dependencies of the separation factors on protein concentration, pH and ionic strength were observed. High separation factors were obtained for dilute solutions at pH near 8.3 and without added salt.