Analysis of diffusion models for protein adsorption to porous anion-exchange adsorbent

The ion-exchange adsorption kinetics of bovine serum albumin (BSA) and gamma-globulin to an anion exchanger, DEAE Spherodex M, has been studied by batch adsorption experiments. Various diffusion models, that is, pore diffusion, surface diffusion, homogeneous diffusion and parallel diffusion models, are analyzed for their suitabilities to depict the adsorption kinetics. Protein diffusivities are estimated by matching the models with the experimental data. The dependence of the diffusivities on initial protein concentration is observed and discussed. The adsorption isotherm of BSA is nearly rectangular, so there is little surface diffusion. As a result, the surface and homogeneous diffusion models do not fit to the kinetic data of BSA adsorption. The adsorption isotherm of gamma-globulin is less favorable, and the surface diffusion contributes greatly to the mass transport. Consequently, both the surface and homogeneous diffusion models fit to the kinetic data of gamma-globulin well. The adsorption kinetics of BSA and gamma-globulin can be very well fitted by parallel diffusion model, because the model reflects correctly the intraparticle mass transfer mechanism. In addition, for both the favorably bound proteins, the pore diffusion model fits the adsorption kinetics reasonably well. The results here indicate that the pore diffusion model can be used as a good approximate to depict protein adsorption kinetics for protein adsorption systems from rectangular to linear isotherms.     

Mechanism and kinetics of protein transport in chromatographic media studied by confocal laser scanning microscopy. Part II. Impact on chromatographic separations

The impact of different transport mechanism on chromatographic performance was studied by confocal laser scanning microscopy (CLSM) for solutions containing bovine serum albumin (BSA) and monoclonal IgG 2a under different solid- and fluid-phase conditions. During this investigation, a clear influence of the uptake mechanism on the affinity of the respective proteins for the different adsorbents and thus separation performance of the chromatographic process could be observed. For the system SP Sepharose Fast Flow at pH 4.5 pore diffusion could be ascribed to be the dominant transport mechanism for both proteins and the adsorption profiles resembled a pattern similar to that described by the 'shrinking core' model. Under these conditions a significantly higher affinity towards the adsorbent was found for BSA when compared to IgG 2a. With changing fluid- and solid-phase conditions, however, a change of the transport mode for IgG 2a could be detected. While the exact mechanism is still unresolved it could be concluded that both occurrence and magnitude of the now governing transport mechanism depended on protein properties and interaction with the adsorbent surface. For the system SP Sepharose XL at pH 5.0 both parameters leading to the change in IgG 2a uptake were combined resulting in a clear change of the system affinity towards the IgG 2a molecule, while BSA adsorption was restricted to the most outer shell of the sorbent.     

Confocal microscopy study of uptake kinetics of α‐lactalbumin and β‐lactoglobulin onto the cation‐exchanger SP Sepharose FF

Confocal laser scanning microscopy (CLSM) was used to study single‐ and two‐component protein uptake for α‐lactalbumin (ALA) and β‐lactoglobulin (BLG), as models for whey proteins, to SP Sepharose FF at pH 3.7 during batch experiments in a finite bath. By coupling a fluorescent dye with the protein molecule, the penetration into individual adsorbent particles at different times during batch uptake was visualised. In a single‐component system, BLG penetrated fast into the adsorbent beads and gradually filled them in a shell‐wise fashion, while adsorption of ALA was mostly confined to the outer shells of the adsorbent. For the two‐component studies, the results showed that ALA was able to displace BLG despite its lower affinity to the adsorbent under the employed conditions. CLSM results were then compared both qualitatively and quantitatively to their counterparts obtained in traditional experiments by indirect measurements of the protein concentration in the fluid phase. A novel quantitative approach was undertaken by modifying the simple kinetic rate model traditionally used to determine the kinetic rate constant, k₁, for batch uptake experiments, in order to describe batch uptake kinetics based on CLSM data. Although BLG results were in good agreement, there was a discrepancy in ALA results.     

Optimization of adsorption conditions of BSA on thermosensitive magnetic composite particles using response surface methodology

Thermosensitive core-shell magnetic composite particles with a magnetic silica core and a rich poly (N-vinylcaprolactam) (PNVCL) shell layer were developed for studying the adsorption of bovine serum albumin (BSA) in a batch system. Various analytical and spectroscopic techniques including SEM, FT-IR, VSM and DSC were used to characterize the adsorbents prepared in this study. The combined effects of operating parameters such as initial temperature, pH and initial BSA concentration on the adsorption were analyzed using response surface methodology. The optimum conditions were 40°C, pH 4.68, and initial BSA concentration 2.0 mg/mL. Desorption experiments were conducted by altering the system temperature where a high recovery rate of protein was obtained. The separation process developed here indicates that the dual-responsive smart adsorbent could be an ideal candidate for the separation of protein.     

Developing a chromatographic column model for bovine serum albumin on strong anion-exchanger Source30Q using data from confocal laser scanning microscopy

A systematic approach for the development of a column model for protein purification is introduced. The approach includes phenomenological investigations of mass transfer and adsorption behaviour applying confocal laser scanning microscopy (CLSM). Insights from CLSM measurements are then implemented mathematically into a single particle model which is coupled afterwards with a column model (i.e. general rate model). Finally, the general rate model is used to predict the results from pulse and frontal analysis in a chromatographic column. The applied exemplary chromatographic system is bovine serum albumin (BSA) on strong anion-exchanger Source30Q at neutral pH value.     

Mechanism and kinetics of protein transport in chromatographic media studied by confocal laser scanning microscopy. Part I. The interplay of sorbent structure and fluid phase conditions

An experimental study on the interplay of sorbent structure and fluid phase conditions (pH) has been carried out examining adsorption and transport of bovine serum albumin (BSA) and a monoclonal antibody (IgG 2a) on SP Sepharose Fast Flow and SP Sepharose XL. SP Sepharose Fast Flow is characterised by a relatively open pore network, while SP Sepharose XL is a composite structure with ligand-carrying dextran chains filling the pore space. Both adsorbents have similar ionic capacity. Protein transport and adsorption profiles were evaluated using confocal laser scanning microscopy. Under all investigated conditions, BSA uptake could be adequately explained by a pore diffusion mechanism. The adsorption profiles obtained for IgG 2a, however, indicated that changes in fluid phase conditions as well as a change in the solid phase structure could result in a more complex uptake mechanism as compared to pore diffusion alone. This mechanism results in a fast transport of proteins into the adsorbent, followed by an overshoot of protein in the center of the sorbent and a setback towards a homogeneous adsorption profile.     

Expanded Bed Chromatography as a Tool for Nanoparticulate Separation: Kinetic Study and Adsorption of Protein Nanoparticles

Expanded bed adsorption was investigated together with its suitability for the practical recovery of nanoparticulate mimics of products such as plasmid DNA and viruses as putative gene therapy vectors. The study assessed the binding of protein nanoparticles fabricated from bovine serum albumin (BSA) with average size of 80 nm as a model system and viral size/charge mimic to the streamline DEAE adsorbent in the expanded bed column chromatography. The adsorption kinetics and adsorption mechanism for the BSA nanoparticles on the adsorbent were studied. In batch adsorption studies, the factors nanoparticle concentration, contact time and adsorbent amount, affecting adsorption isotherms were investigated. Subsequently the data were regressed against the Lagergren equation, which represents a first-order kinetics equation and also against a pseudo-second-order kinetics equation. The results demonstrated that the adsorption process followed a Langmuir isotherm equation. The kinetics of the adsorption process followed a pseudo-second-order kinetics model with a rate constant value of 0.025 g mg^?1 min^?1. The dynamic binding capacity of the BSA nanoparticles on an expanded bed was calculated. The recovery of the nanoparticles was more than 85%.     

Effective diffusivities of BSA in cellulosic fiber beds measured with magnetic resonance imaging

The temporal and spatial evolution of concentration profiles of bovine serum albumin (BSA) in various cellulosic fiber beds is measured using magnetic resonance imaging. Effective diffusivities are calculated using a numerical one dimensional Fickian model to match experimental concentration profiles. Experimental values of the diffusivities are compared with predictions from a simple diffusion-adsorption model which accounts for porosity, tortuosity, and surface adsorption. BSA was found to have negligible adsorption in the concentration range studied, resulting in a simplified diffusion model based on fiber characteristics and geometry. Effective diffusivities agreed well with the predicted values and were within an order of magnitude of the estimated bulk diffusivity of BSA.     

Hydrophobic interaction chromatography of proteins. IV. Protein adsorption capacity and transport in preparative mode

The adsorption isotherms of four model proteins (lysozyme, α-lactalbumin, ovalbumin, and BSA) on eight commercial phenyl hydrophobic interaction chromatography media were measured. The isotherms were softer than those usually seen in ion-exchange chromatography of proteins, and the static capacities of the media were lower, ranging from 30 to 110 mg/mL, depending on the ammonium sulfate concentration and the protein and adsorbent types. The protein-accessible surface area appears to be the main factor determining the binding capacity, and little correlation was seen with the protein affinities of the adsorbents. Breakthrough experiments showed that the dynamic capacities of the adsorbents at 10% breakthrough were 20-80% of the static capacities, depending on adsorbent type. Protein diffusivities in the adsorbents were estimated from batch uptake experiments using the pore diffusion and homogeneous diffusion models. Protein transport was affected by the adsorbent pore structures. Apparent diffusivities were higher at lower salt concentrations and column loadings, suggesting that adsorbed proteins may retard intraparticle protein transport. The diffusivities estimated from the batch uptake experiments were used to predict column breakthrough behavior. Analytical solutions developed for ion-exchange systems were able to provide accurate predictions for lysozyme breakthrough but not for ovalbumin. Impurities in the ovalbumin solutions used for the breakthrough experiments may have affected the ovalbumin uptake and led to the discrepancies between the predictions and the experimental results.     

Removal of bovine serum albumin using solid-phase extraction with in-situ polymerized stationary phase in a microfluidic device

Serum albumin, one of the most abundant serum proteins, blocks the expression of other important biomarkers. The objective of this study is to remove serum albumin effectively by using solid-phase extraction (SPE) in microfluidic devices. Photo-polymerized adsorbent as a stationary phase of SPE was used to remove bovine serum albumin (BSA). The adsorption capacity was examined with the effect of pH and concentration in BSA solution, and adjustment of monomer concentration such as hydrophilic 2-acrylamido-2-methyl-1-propanesulfonic acid and acrylamide in the adsorbent. The effect of hydrophobic butyl methacylate on BSA adsorption was also studied. Selective removal in a bicomponent with BSA and bovine gamma-globulin was performed by adjusting the pH as required.     

A NOVEL METAL CHELATE AFFINITY ADSORBENT FOR PROTEIN UPTAKE

1. INTRODUCTION Chitosan is a hydrolyzed derivative of chitin and belongs to a family of linear unbranched polysaccharides which contain large amounts of 1,4-linked-2-amino-2-deoxy-β-D-glucan residues. The presence of free amine groups in chitosan enhances the solubility and reactivity of this polymer. Interest in modifying chitosan by using glutaraldehyde has recently increased. The derivatized polymers have been employed for many applications [1~2], including protein immobilization…     

Profiling protein-surface interactions of multicomponent suspensions via flow cytometry

This study presents the use of flow cytometry as a high-throughput quantifiable technique to study multicomponent adsorption interactions between proteins and surfaces. Flow cytometry offers the advantage of high-throughput analysis of multiple parameters on a very small sampling scale. This enables flow cytometry to distinguish between individual adsorbent particles and adsorbate components within a suspension. As a proof of concept study, the adsorption of three proteins--bovine serum albumin (BSA), bovine immunoglobulin gamma (IgG) and fibrinogen--onto five surface-modified organosilica microsphere surfaces was used as a model multicomponent system for analysis. By uniquely labeling each protein and solid support type with spectrally distinguishable fluorescent dyes, the adsorption process could be "multiplexed" allowing for simultaneous screening of multiple adsorbate (protein) and adsorbent (particle surface) interactions. Protein adsorption experiments quantified by flow cytometry were found to be comparable to single-component adsorption studies by solution depletion. Quantitative distribution of the simultaneous competitive adsorption of BSA and IgG indicated that, at concentrations below surface saturation, both proteins adsorbed onto the surface. However, at concentrations greater than surface saturation, BSA preferentially adsorbed. Multiplexed particle suspensions of optically encoded particles were modified to produce a positively and negatively charged surface, a grafted 3400 MW poly(ethylene glycol) layer, or a physisorbed BSA or IgG layer. It was observed that adsorption was rapid and irreversible on all of the surfaces, and preadsorbed protein layers were the most effective in preventing further protein adsorption.     

Probe surface chemistry dependence and local polymer network structure in F-actin microrheology

We investigate the dependence of F-actin microrheology on probe surface chemistry using diffusing wave spectroscopy. Polystyrene probe particles exhibit subdiffusive mean-squared displacements, where Deltar(2)(t) approximately t(0.77)(+/-)(0.03) consistent with previous experiments and theory. However, polystyrene probes preadsorbed with bovine serum albumin (BSA) interact weakly with the surrounding polymer network and exhibit a scaling exponent similar to pure diffusion Deltar(2)(t) approximately t, which decreases as particle size and actin concentration increases. Using models of particle diffusion in locally heterogeneous viscoelastic microenvironments, we find that the microrheological response of BSA-treated particles is consistent with the formation of a polymer-depleted shell surrounding the probes. The shell thickness scales with particle size but not polymer concentration. These results suggest that the depletion is caused by exclusion or orientation of actin filaments near probes due to their long length and rigidity.     

A NOVEL METAL CHELATE AFFINITY ADSORBENT FOR PROTEIN UPTAKE

In this article,a spherical chitosan gel crosslinked by epichlorohydrin was prepared.It was then loaded with copper ions to produce a metal chelate affinity adsorbent for protein.The uptake of bovine serum albumin(BSA)by the affinity adsorbent was investigated.and the adsorption capacity for BSA as high as 40mg/g-wet beads was observed.The adsorption equilibrium data was well correlated by the Langmuir equation.The adsorption was considerably affected by pH.In additio.The amount of BSA adsorbed onto the beads decreased with the increasing of aqueous phase ionic strength,so adsorbed BAS can be desorbed by adjusting pH orionic strength of the solution.     

Bovine serum albumin adsorption onto colloidal Al2O3 particles: a new model based on zeta potential and UV-vis measurements

We investigated the adsorption of bovine serum albumin (BSA) on colloidal Al2O3 particles in an aqueous environment. Changes in the zeta potential of the Al2O3 particles upon the adsorption of BSA were measured using an electro-acoustic technique. The mass of protein adsorbed was determined by using UV-vis spectroscopy. The change of the isoelectric point of the Al2O3 powder-protein suspension was found to be a function of adsorbed protein mass. It was shown that approximately one monolayer of BSA was needed to fully mask the surface and to compromise the charge of Al2O3. From titration experiments it follows that about 30-36% of the negatively charged groups of the protein form bonds with the protonated and charged Al2O3 surface. On the basis of our observations we introduced a new adsorption model for BSA on Al2O3 particles.     

Separation of serum proteins with uncoupled microsphere particles in a stirred cell

Submicron microspheres were used directly without ligand coupling for the batch and continuous separations of proteins. In the batch experiments for separating BSA (bovine serum albumin) from BHb (bovine hemoglobin), introducing both hydrophobic effects for BSA and electrostatic repulsion for BHb (and vice versa) was required for high selectivity, and microspheres with low number density of surface groups were advantageous. For the continuous experiments, the utilization of a stirred cell was successful, where the microspheres were in the form of latex with good dispersion of particles. The flow rate without a pump was 0.5–1.3 ml min⁻¹, and the ratio of BSA and BHb was varied. In the experiments for eliminating BHb from BSA, elution curves of BHb corresponded to the single component breakthrough curves, while those for BSA did not. The latter is believed to be due to the interference by BHb in the adsorption of BSA.     

Fabrication and characterization of a rigid magnetic matrix for protein adsorption

This article describes the fabrication and characterization of a novel magnetic poly(glycidyl methacrylate-triallyl isocyanurate-divinylbenzene) matrix containing magnetite colloids. The results showed that the matrix was superparamagnetic and could be separated magnetically from a suspension in a few seconds. Protein adsorption properties of diethylamine-derivatized matrix were characterized with bovine serum albumin (BSA) as a model protein. The static capacity determined by batch adsorption was 79 mg/ml wet matrix. Kinetic study gave an effective diffusivity of BSA of 5.0 x 10(-13) m2/s in the matrix at an initial BSA concentration in the liquid phase of 1.0 mg/ml. Stability of the matrix was confirmed by recycling of the matrix in protein adsorptions.     

Adsorption of Bovine Serum Albumin and Lysozyme on Hydrophobic Calcium Hydroxyapatites

The adsorption of bovine serum albumin (BSA) and lysozyme (LSZ) to oleyl phosphate(OP)-grafted calcium hydroxyapatite (OP-CaHAP) with different degrees of hydrophobicity, ranging the number of surface oleyl group per unit nm2 (nO) from 0 to 2.60, was investigated. The pronounced effects of the hydrophobic moiety of adsorbent on protein adsorption were observed. The saturated amount of adsorbed BSA (ns) was increased up to nO = 0.6 by an enlargement of hydrophobic interaction between hydrophobic CaHAP particle and proteins. However, ns decreased at nO >/= 1.3 by increasing the electrostatic repulsive force between negatively charged BSA and OP-CaHAP particles. On the other hand, the ns value of LSZ was continuously increased up to nO = 2.0 and saturated by increasing either the hydrophobic interaction or the electrostatic attraction of positively charged LSZ and negatively charged OP-grafted CaHAPs. The BSA adsorption experiment revealed that the effect of positively charged adsorption sites on the exposed ac or bc crystal faces (C-sites) of the CaHAPs is screened by the OP-groups grafted on their particle surfaces. Copyright 1999 Academic Press.     

Fabrication and Characterization of a Dye-Immobilized Yttria-Stabilized Zirconia Pellicular Adsorbent for Expanded Bed Adsorption Chromatography

This study deals with the fabrication and characterization of a pellicular adsorbent appropriate for the expanded bed adsorption (EBA) process. The synthesized adsorbent has an yttria-stabilized zirconia nucleus coated with agarose. Morphological analysis of the coated particles was performed by light-scattering microscopy and showed an average diameter of 197.54 and 202.25 µm, for the nucleus and coated particle, respectively. A screening for the reactive dyes reactive blue 19 (RB19), reactive blue 21 (RB21) and reactive orange 107 (RO107) was performed after immobilization onto the pellicular adsorbent by changing the pH, aiming at finding the binding capacity of these to adsorb bovine serum albumin (BSA). The reactive orange 107 was selected and it was more stable at pH 4.5. Study of the kinetics between BSA and the dye-immobilized particle showed that equilibrium is reached before 1 h. The adsorption isotherm of BSA onto RO107-immobilized adsorbent fitted the Langmuir model showing a q_m = 102.328 mg BSA/mL of adsorbent. The pellicular adsorbent also showed good expansion even at a high operating flow rate. Therefore, at a linear velocity as high as 2725 cm/h, a dynamic capacity of 15.7 mg of BSA/mL of adsorbent was obtained.