Two-component protein adsorption kinetics in porous ion exchange media

This work provides a theoretical analysis of multicomponent adsorption kinetics for conditions typical of protein adsorption in porous ion exchangers as well as experimental results for the adsorption of lysozyme/cytochrome c mixtures in the cation exchanger SP-Sepharose-FF. The theory predicts the formation of overshoots in the intraparticle concentration profiles and in the total amount adsorbed for the more weakly adsorbed component. An analytical solution valid for the case where the isotherms are rectangular is developed and found to be in good agreement with the limiting behavior of the general numerical solution of the model equations. The experimental results show that the two proteins are competitively adsorbed and that an overshoot of adsorbed cytochrome c occurs during simultaneous adsorption. Model predictions based on the assumption that the adsorption isotherms are rectangular and that lysozyme completely displaces cytochrome c are in qualitative and quantitative agreement with the experimental kinetics suggesting that the overshoot phenomena observed with multicomponent systems in these resins can be explained with a diffusion model without the need to account for flux coupling or electrophoretic contributions to transport.     

A Novel Model for Protein Immobilization on Microspheres

The immobilization of proteins, especially receptor proteins commonly used in high through-put screening of drugs (HTS), have received great attention in recent years. There are many successful isothermal models for describing the adsorption of protein onto solid surface, such as Langmuir model, Bi-Langmuir model, Fowler model, Freundlich model, Freundlich-Langmuir model and Tekmin model etc. In all these models, Langmuir model was the most favorable one model accepted by many researchers, but the experimental results showed that it was not entirely fit to all adsorption behaviors. So new models were required for describing protein adsorption onto microspheres in different conditions.In our research, a novel isothermal model, including Langmuir and other adsorbing behaviors was presented basing on the holding degree of surface active sites and the interaction styles of protein immobilization. In Langmuir model, the adsorbing amount of protein was described as [PS] =Km[P]/1 + K[P], where [PS] was the concentration of adsorbed protein, [P] was the concentration of freeprotein at equilibrium state, and Km and K was constant. According to the interactions of protein and ligands, there were three patterns in the interactions of protein and ligands. On the similar assumption that the interaction of protein and microspheres were three styles, and based on the definition of the holding degree of surface active sites (Y), three adsorption behaviors could be described as Y K[ P ]φ/ K[P]φ+1 or ln K + φ ln[P] =ln(Y/1-Y) in which [P] was the concentration of free protein at equilibrium state, and φ and K was constant. Different scale of φ presented different adsorption behaviors, especially when φ was 1, the adsorption behavior was Langmuir adsorbing model. Figure I indicated the different adsorbing results in different adsorption behaviors (φ＞1, φ＜1,and φ=1).     

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.     

Protein interactions with solid surfaces following contact with plasma and blood

Two aspects of blood protein interactions with solid surfaces are discussed. The first is competitive adsorption among the many proteins in the blood. The general factors which determine the composition of the adsorbed layer in any multicomponent protein system are considered and then related to current knowledge of competitive adsorption in blood. The latter pertains largely to the so-called Vroman effect whereby proteins are adsorbed and displaced from the surface in an ordered sequence. Secondly the possible transformation of adsorbed enzyme precursors into active enzymes by the agency of the surface is discussed. The example of the plasminogen-plasmin system is used to illustrate this discussion.     

Spreading of polymer molecules on solid surfaces

When a homopolymer adsorbs from dilute solution onto a solid surface it first attaches and then maximises its number of contacts with the substrate by means of a spreading process. Evidence for this spreading process can be obtained from experiments on the adsorption kinetics. We report on a case where the adsorption kinetics depend on the rate at which the polymer was supplied to the surface (a protein adsorbing onto silica). Also, we discuss competitive adsorption experiments in which one kind of chain molecule attempts to displace another one from the surface. In these experiments, the desorption rate of the displaced species reflects the spreading rate of the displacer. When this rate is slower than the supply of displacer molecules, oversaturated layers result that spontaneously eject polymer. We have measured the rate of these displacement-driven desorption processes in various cases and conclude that it depends strongly on the energy of the segment-surface bond. A model involving the diffusion of defects over the surface may account for this finding.     

Incorporating water-release and lateral protein interactions in modeling equilibrium adsorption for ion-exchange chromatography

The equilibrium adsorption of two albumin proteins on a commercial ion exchanger has been studied using a colloidal model. The model accounts for electrostatic and van der Waals forces between proteins and the ion exchanger surface, the energy of interaction between adsorbed proteins, and the contribution of entropy from water-release accompanying protein adsorption. Protein-surface interactions were calculated using methods previously reported in the literature. Lateral interactions between adsorbed proteins were experimentally measured with microcalorimetry. Water-release was estimated by applying the preferential interaction approach to chromatographic retention data. The adsorption of ovalbumin and bovine serum albumin on an anion exchanger at solution pH>pI of protein was measured. The experimental isotherms have been modeled from the linear region to saturation, and the influence of three modulating alkali chlorides on capacity has been evaluated. The heat of adsorption is endothermic for all cases studied, despite the fact that the net charge on the protein is opposite that of the adsorbing surface. Strong repulsive forces between adsorbed proteins underlie the endothermic heat of adsorption, and these forces intensify with protein loading. It was found that the driving force for adsorption is the entropy increase due to the release of water from the protein and adsorbent surfaces. It is shown that the colloidal model predicts protein adsorption capacity in both the linear and non-linear isotherm regions, and can account for the effects of modulating salt.     

A high-throughput methodology for liquid phase adsorption experimentation

This article describes a novel application of high-throughput experimentation, namely in the field of liquid mixture separation through adsorption. Two separate setups are designed and extensively used to study multicomponent liquid phase adsorption: the first setup performs batch adsorption in static conditions to obtain adsorption isotherms while the latter carries out breakthrough experiments in dynamic conditions, which yield multicomponent breakthrough curves. The obtained data serves as an indicator of the separative qualities of an adsorbent exposed to a particular liquid mixture. The reliability of the obtained measurements is assessed using different validation techniques. Case studies pertaining to the competitive adsorption of binary alkane/alkene/aromatic mixtures on faujasites complete the validation process. A new model for batch adsorption isotherms is proposed based on the equilibrium conditions in liquid phase.     

Characterizing the adsorption of proteins on glass capillary surfaces using electrospray-differential mobility analysis

We quantify the adsorption and desorption of a monoclonal immunoglobulin-G antibody, rituxamab (RmAb), on silica capillary surfaces using electrospray-differential mobility analysis (ES-DMA). We first develop a theory to calculate coverages and desorption rate constants from the ES-DMA data for proteins adsorbing on glass capillaries used to electrospray protein solutions. This model is then used to study the adsorption of RmAb on a bare silica capillary surface. A concentration-independent coverage of ≈4.0 mg/m(2) is found for RmAb concentrations ranging from 0.01 to 0.1 mg/mL. A study of RmAb adsorption to bare silica as a function of pH shows maximum adsorption at its isoelectric point (pI of pH 8.5) consistent with literature. The desorption rate constants are determined to be ≈10(-5) s(-1), consistent with previously reported values, thus suggesting that shear forces in the capillary may not have a considerable effect on desorption. We anticipate that this study will allow ES-DMA to be used as a "label-free" tool to study adsorption of oligomeric and multicomponent protein systems onto fused silica as well as other surface modifications.     

Ion exchange using poorly activated supports, an easy way for purification of large proteins

Ion-exchange chromatography using commercial ionic supports is a commonly used technique for protein purification. However, selective adsorption of a target protein from a given extract onto commercial ion exchangers seems to be quite complex since they are designed to adsorb the maximum percentage of proteins with the opposite charge. In this paper, ion-exchanger supports with different activation degrees (from 1 to 40 micromol of amino groups per g of agarose) have been prepared and used for the purification of large proteins. These kinds of proteins have large surfaces to interact by many points with the support. Therefore, it was possible to purify large proteins as beta-galactosidase from Thermus sp. strain T2 from a crude extract from Escherichia coli or bovine liver catalase from a commercial preparation, with tailor-made ion-exchanger supports. A simple step of adsorption/desorption on lowly activated supports rendered both enzymes rather pure as confirmed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis. Moreover, this strategy makes also easy the desorption step that requires rather low NaCl concentrations, which may become a serious problem for desorption of large proteins when using conventional supports, due to their ability of generating a very strong adsorption.     

Competitive adsorption of toxic metals on activated carbon

Competitive adsorption of zinc and copper on activated carbon is studied in this article. Main aim was to suggest an advanced model for competitive adsorption of both metals considering pH influence and precipitation. A surface-complexation approach was employed for the modeling. Two models were considered: simple adsorption and ion exchange. System “The Geochemists Workbench” was used for calculation of both static and dynamic adsorption tasks. From the batch experiments, concentration of four types of sorbing sites on the carbon surface and its protonation and sorption constants were deduced. Then, batch competitive adsorption experiments were compared with the models’ results. Finally, a column experiment (fixed bed adsorption) was carried out. It was observed that the model of ion exchange can satisfyingly predict both chromatographic effect and increase of zinc concentration in effluent over its initial value, although a quantitative agreement between the model and the experiment was not totally precise.     

Mixology of protein solutions and the Vroman effect

Mixing rules stipulating both concentration and distribution of proteins adsorbed to the liquid-vapor (LV) interphase from multicomponent aqueous solutions are derived from a relatively straightforward protein-adsorption model. Accordingly, proteins compete for space within an interphase separating bulk-vapor and bulk-solution phases on a weight, not molar, concentration basis. This results in an equilibrium weight-fraction distribution within the interphase that is identical to bulk solution. However, the absolute interphase concentration of any particular protein adsorbing from an m-component solution is 1/mth that adsorbed from a pure, single-component solution of that protein due to competition with m - 1 constituents. Applied to adsorption from complex biological fluids such as blood plasma and serum, mixing rules suggest that there is no energetic reason to expect selective adsorption of any particular protein from the mixture. Thus, dilute members of the plasma proteome are overwhelmed at the hydrophobic LV surface by the 30 classical plasma proteins occupying the first 5 decades of physiological concentration. Mixing rules rationalize the experimental observations that (i) concentration-dependent liquid-vapor interfacial tension, gammalv, of blood plasma and serum (comprised of about 490 different proteins) cannot be confidently resolved, even though serum is substantially depleted of coagulable proteins (e.g., fibrinogen), and (ii) gammalv of plasma is startlingly similar to that of purified protein constituents. Adsorption-kinetics studies of human albumin (66.3 kDa) and IgM (1000 kDa) binary mixtures revealed that relatively sluggish IgM molecules displace faster-moving albumin molecules adsorbing to the LV surface. This Vroman-effect-like process leads to an equilibrium gammalv reflecting the linear combination of weight/volume concentrations at the surface predicted by theory. Thus, the Vroman effect is interpreted as a natural outcome of protein reorganization to achieve an equilibrium interphase composition dictated by a firm set of mixing rules.     

Comparison of agarose and dextran-grafted agarose strong ion exchangers for the separation of protein aggregates

The control of aggregate levels in recombinant protein based drugs is a primary concern during process development and manufacture. In recent years, a novel class of dextran-grafted ion exchange matrices has gained popularity for process scale protein purification due to increased mass transfer rates and higher dynamic binding capacity compared to conventional matrices. Using bovine serum albumin and a monoclonal antibody as model proteins, we studied Sepharose FF and Sepharose XL ion exchangers for the separation of protein aggregates. Experimental results comparing linear gradient elution, stepwise elution, and flow-through chromatography for aggregate separation are described. Differences in performance for the various ion exchangers are discussed and modeled. Strategies for the optimization of protein aggregate separation are provided.     

Effect of pH on protein adsorption capacity of strong cation exchangers with grafted layer

The effect of pH on the static adsorption capacity of immunoglobulin G, human serum albumin, and equine myoglobin was investigated for a set of five strong cation exchangers with the grafted tentacle layer having a different ligand density. A sharp maximum of adsorption capacity with pH was observed for adsorbents with a high ligand density. The results were elucidated using the protein structure and calculations of pK(a) of ionizable groups of surface basic residues. Inverse size-exclusion experiments were carried out to understand the relation between the adsorption capacity and pore accessibility of the investigated proteins.     

Automated coating procedures to produce poly(ethylene glycol) brushes in fused‐silica capillaries

Many bioanalytical methods rely on electrophoretic separation of structurally labile and surface active biomolecules such as proteins and peptides. Often poor separation efficiency is due to surface adsorption processes leading to protein denaturation and surface fouling in the separation channel. Flexible and reliable approaches for preventing unwanted protein adsorption in separation science are thus in high demand. We therefore present new coating approaches based on an automated in‐capillary surface‐initiated atom transfer radical polymerization process (covalent coating) as well as by electrostatically adsorbing a presynthesized polymer leading to functionalized molecular brushes. The electroosmotic flow was measured following each step of the covalent coating procedure providing a detailed characterization and quality control. Both approaches resulted in good fouling resistance against the four model proteins cytochrome c, myoglobin, ovalbumin, and human serum albumin in the pH range 3.4−8.4. Further, even samples containing 10% v/v plasma derived from human blood did not show signs of adsorbing to the coated capillaries. The covalent as well as the electrostatically adsorbed coating were both found to be stable and provided almost complete suppression of the electroosmotic flow in the pH range 3.4−8.4. The coating procedures may easily be integrated in fully automated capillary electrophoresis methodologies.     

Characteristic and mechanism of Cr(Ⅵ) adsorption by ammonium sulfamate-bacterial cellulose in aqueous solutions

A new adsorbent, ammonium sulfamate-bacterial cellulose (ASBC), was prepared through chemical modifications of bacterial cellulose. The process of adsorbing Cr(Ⅵ) including its isotherm and kinetics, was measured and studied. The results showed that pH value was a very important parameter to the adsorbing efficiency. The adsorption kinetics can be described by a pseudo-second rate model and a particle diffusion equation. Both physical and chemical adsorptions existed in the adsorption process, but chemical adsorption was more dominatant. And particles internal diffusion was not the only rate controlling step. The adsorption equilibrium can be described by the Langmuir type, which indicated that a typical single-molecule layer adsorption of Cr(Ⅵ) by ASBC could be described. And the rate of adsorption followed the Slips model well, which indicated that ASBC had some multiphase and asymmetry. The coordination adsorption and ion exchange effect were the main mechanisms of chemical adsorption. The absorbed Cr(Ⅵ) can be desorbed effectively by 0.5 mol/L EDTA or HCl from the adsorbent, which could make it be reusable.     

聚(N-异丙基丙烯酰胺)改性硅表面与血浆蛋白质的相互作用

通过表面引发原子转移自由基聚合(ATRP)在硅表面接枝了聚(N-异丙基丙烯酰胺)(PNIPAAm)聚合物刷,并考察了PNIPAAm改性表面在单一蛋白质溶液以及血浆中与血浆蛋白质之间的相互作用.蛋白质吸附测试表明,与未改性的硅表面相比,改性后的表面对纤维蛋白原的吸附量大大降低,特别是在血浆中纤维蛋白原吸附量小于5ng/c...     

Synergistic effects in competitive adsorption of carbohydrates on an ion-exchange resin

Adsorption of the three carbohydrates sucrose, glucose and fructose from aqueous solutions was investigated on an ion-exchange resin. The adsorption equilibrium of single components, binary and ternary mixtures was quantified by frontal analysis and the adsorption-desorption method. The experiments covered a concentration range up to 600 g/L at 60 degrees C and 80 degrees C. Within this range the adsorption isotherms of carbohydrates exhibited anti-Langmuirian behavior. Data of mixture adsorption revealed reversed competitive (synergistic or cooperative) effects, i.e., an increase of the concentration of one component of the mixture enhanced the adsorption of others. To model such an adsorption behavior the anti-Langmuir model has been used. The isotherm parameters determined for single components were used to simulate the competitive adsorption equilibria through the IAS (ideal adsorbed solution) theory. Finally, dynamic concentration profiles of multicomponent mixtures have been recorded. The shapes of adsorption and desorption curves confirmed the observed competitive effects found in the equilibrium studies. The breakthrough curves measured were simulated using the equilibrium theory as well as a numerical solution of the equilibrium dispersive model.     

Microcalorimetric studies of the interaction mechanisms between proteins and Q-sepharose at pH near the isoelectric point (pI) effects of NaCl concentration, pH value, and temperature

This study examined the interaction mechanisms of beta-lactoglobulins A and B (Lg A, Lg B) with an anion exchanger, Q-Sepharose at pH near the isoelectric point at which the proteins are expected to be electrically neutralized under various NaCl concentrations and temperatures by the equilibrium binding analysis and the adsorption enthalpy directly measured by isothermal titration calorimetry. The data evaluated from isotherms fitted by the Langmuirean model reveal that the addition of NaCl considerably reduced the binding affinities and capacities of both the proteins with Q-Sepharose at pH 5.2, indicating that electrostatic forces are dominant during the adsorption. However, the hydrophobic interaction seems to be involved in adsorption as well at a higher NaCl concentration, and the adsorption enthalpies confirm this suggestion. In addition, the effects of temperature on the equilibrium binding behaviors for Lg A or Lg B with Q-Sepharose were found to be salt concentration-dependent, probably due to their different binding mechanisms at 0.03 M and 0.3 M NaCl. Where, at 0.3 M NaCl, the hydrophobic interaction plays a more pronounced role. This implication was again supported by the adsorption enthalpies. The presented data provide further insight to the interaction mechanisms between proteins and ion exchangers, facilitating the optimization of protein separations.     

Adsorption and viscoelastic properties of fractionated mucin (BSM) and bovine serum albumin (BSA) studied with quartz crystal microbalance (QCM-D)

The adsorption profile and viscoelastic properties of bovine submaxillary gland mucin (BSM) and bovine serum albumin (BSA), extracted from a commercial mucin preparation, adsorbing to polystyrene surfaces has been studied using quartz crystal microbalance with dissipation monitoring (QCM-D). A significant difference in the adsorption properties of the different proteins was detected; with the BSA adsorbing in a flat rigid layer whilst the mucin adsorbed in a diffuse, highly viscoelastic layer. Subsequent addition of BSA to the preadsorbed mucin layer resulted in stiffening of the protein layer which was attributed to complexation of the mucin by BSA. In contrast, a preadsorbed layer of BSA prevented mucin adsorption altogether. Combined mixtures of mucin and BSA in well defined ratios revealed intermediate properties between the two separate protein species which varied systematically with the protein ratios. The results shed light on the synergistic effects of complexation of lower molecular weight biomolecular species with mucin. The possibility to selectively control protein uptake and tailor the physical properties of the adsorbed layer makes mucin an attractive option for application in biomaterial coatings.