Modeling of salt and pH gradient elution in ion‐exchange chromatography

The separation of proteins by internally and externally generated pH gradients in chromatofocusing on ion‐exchange columns is a well‐established analytical method with a large number of applications. In this work, a stoichiometric displacement model was used to describe the retention behavior of lysozyme on SP Sepharose FF and a monoclonal antibody on Fractogel SO₃ (S) in linear salt and pH gradient elution. The pH dependence of the binding charge B in the linear gradient elution model is introduced using a protein net charge model, while the pH dependence of the equilibrium constant is based on a thermodynamic approach. The model parameter and pH dependences are calculated from linear salt gradient elutions at different pH values as well as from linear pH gradient elutions at different fixed salt concentrations. The application of the model for the well‐characterized protein lysozyme resulted in almost identical model parameters based on either linear salt or pH gradient elution data. For the antibody, only the approach based on linear pH gradients is feasible because of the limited pH range useful for salt gradient elution. The application of the model for the separation of an acid variant of the antibody from the major monomeric form is discussed.     

Purification of His-tagged hepatitis B core antigen from unclarified bacterial homogenate using immobilized metal affinity-expanded bed adsorption chromatography

Hepatitis B core antigen (HBcAg) is used as a diagnostic reagent for the detection of hepatitis B virus infection. In this study, immobilized metal affinity-expanded bed adsorption chromatography (IMA-EBAC) was employed to purify N-terminally His-tagged HBcAg from unclarified bacterial homogenate. Streamline Chelating was used as the adsorbent and the batch adsorption experiment showed that the optimal binding pH of His-tagged HBcAg was 8.0 with a binding capacity of 1.8 mg per ml of adsorbent. The optimal elution condition for the elution of His-tagged HBcAg from the adsorbent was at pH 7 in the presence of 500 mM imidazole and 1.5 M NaCl. The IMA-EBAC has successfully recovered 56% of His-tagged HBcAg from the unclarified E. coli homogenate with a purification factor of 3.64. Enzyme-linked immunosorbent assay (ELISA) showed that the antigenicity of the recovered His-tagged HBcAg was not affected throughout the IMA-EBAC purification process and electron microscopy revealed that the protein assembled into virus-like particles (VLP).     

Hydrophobic interaction chromatography of proteins. I. The effects of protein and adsorbent properties on retention and recovery

The contributions of protein and adsorbent properties to retention and recovery were examined for hydrophobic interaction chromatography (HIC) using eight commercially available phenyl media and five model proteins (ribonuclease A, lysozyme, alpha-lactalbumin, ovalbumin and BSA). The physical properties of the adsorbents were determined by inverse size exclusion chromatography (ISEC). The adsorbents examined differ from each other in terms of base matrix, ligand density, porosity, mean pore radius, pore size distribution (PSD) and phase ratio, allowing systematic studies to understand how these properties affect protein retention and recovery in HIC media. The proteins differ in such properties as adiabatic compressibility and molecular mass. The retention factors of the proteins in the media were determined by isocratic elution. The results show a very clear trend in that proteins with high adiabatic compressibility (higher flexibility) were more strongly retained. For proteins with similar adiabatic compressibilities, those with higher molecular mass showed stronger retention in Sepharose media, but this trend was not observed in adsorbents with polymethacrylate and polystyrene divinylbenzene base matrices. This observation could be related to protein recovery, which was sensitive to protein flexibility, molecular size, and conformation as well as the ligand densities and base matrices of the adsorbents. Low protein recovery during isocratic elution could affect the interpretation of protein selectivity results in HIC media. The retention data were fitted to a previously published retention model based on the preferential interaction theory, in terms of which retention is driven by release of water molecules and ions upon protein-adsorbent interaction. The calculated number of water molecules released was found to be statistically independent of protein retention strength and adsorbent and protein properties.     

4-(1H-imidazol-1-yl) aniline: A new ligand of mixed-mode chromatography for antibody purification

4-(1H-imidazol-1-yl) aniline (AN) was immobilized on Sepharose CL-6B (AN-Sepharose) for use as a new ligand of mixed-mode chromatography. Adsorption equilibria of immunoglobulin G (IgG) and bovine serum albumin (BSA) to AN-Sepharose were studied at extensive pH values (4.0–8.8) and salt concentrations (0–1.0 mol/L). Static binding studies indicated that AN-Sepharose had a good salt-tolerance property for IgG adsorption up to 1.0 mol/L NaCl. This was attributed to the combined ligand–protein interactions (hydrophobic interaction, hydrogen bonding and charge transfer interaction). By contrast with BSA, AN-Sepharose showed a high binding selectivity for IgG at NaCl > 0.2 mol/L. Dynamic binding capacities (DBC) of IgG and BSA at 10% breakthrough were measured at pH 4.0–8.8 by frontal analysis chromatography. IgG had DBC values over 40 mg/mL at pH 7.0–8.8, and the maximum reached 59 mg/mL at pH 8.0. At pH 5.0, a distinct drop in DBC to 8.5 mg/mL was observed, but that for BSA kept over 22 mg/mL. The result suggested that IgG could be selectively desorbed from AN-Sepharose by decreasing pH to about 5. Therefore, compared to BSA, AN-Sepharose exhibited a dual-selectivity for IgG in both adsorption and elution. Purification of IgG from bovine serum also confirmed the dual-selectivity. IgG purity of the pooled fractions by elution at pH 4.0, 4.5 and 5.0 reached 55% and the highest purity, 80%, was obtained at pH 4.5. The average purification factor of IgG was over 25. The results indicate that AN is a promising ligand of mixed-mode chromatography for antibody purification from a complex feedstock.     

Microcalorimetric study of adsorption of glycomacropeptide on anion-exchange chromatography adsorbent

The adsorption of glycomacropeptide (GMP) from cheese whey on an anion-exchange adsorbent was investigated using isothermal titration microcalorimetry to measure thermodynamic information regarding such processes. Isotherms data were measured at temperatures of 25 and 45 °C, pH 8.2 and various ionic strengths (0–0.08 mol L⁻¹ NaCl). The equilibrium data were fit using the Langmuir model and the process was observed to be reversible. Temperature was observed to positively affect the interaction of the protein and adsorbent. Microcalorimetric studies indicated endothermic adsorption enthalpy in all cases, except at 45 °C and 0.0 mol L⁻¹ NaCl. The adsorption process was observed to be entropically driven at all conditions studied. It was concluded that the increase in entropy, attributed to the release of hydration waters as well as bounded ions from the adsorbent and protein surface due to interactions of the protein and adsorbent, was a major driving force for the adsorption of GMP on the anion-exchange adsorbent. These results could allow for design of more effective ion-exchange separation processes for proteins.     

Fast cation-exchange separation of proteins in a plastic microcapillary disc

A novel disposable adsorbent material for fast cation-exchange separation of proteins was developed based on plastic microcapillary films (MCFs). A MCF containing 19 parallel microcapillaries, each with a mean internal diameter of 142 μm, was prepared using a melt extrusion process from an ethylene-vinyl alcohol copolymer (EVOH). The MCF was surface functionalised to produce a cation-exchange adsorbent (herein referred as MCF-EVOH-SP). The dynamic binding capacity of the new MCF-EVOH-SP material was experimentally determined by frontal analysis using pure protein solutions in a standard liquid chromatography instrument for a range of superficial flow velocities, u_LS = 5.5–27.7 cm s⁻¹. The mean dynamic binding capacity for hen-egg lysozyme was found to be approximately 100 μg for a 5 m length film, giving a ligand binding density of 413 ng cm⁻². The dynamic binding capacity did not vary significantly over the range of u_LS tested. The application of this novel material to subtractive chromatography was demonstrated for anionic BSA and cationic lysozyme at pH 7.2. The chromatographic separation of two cationic proteins, lysozyme and cytochrome-c, was also performed with a view to applying this technology to the analysis or purification of proteins. Future applications might include separation based on anion exchange and other modes of adsorption.     

金属螯合亲和色谱中固定金属与蛋白质的作用

在不同PHNaCl的磷酸缓冲体系，比较了牛血清蛋白（BSA）、核糖核酸酶（RNase)、细色素C（Cyt-C)和溶菌酶（Lys)在IDA裸柱和一些金属螯合柱上的保留特性，考察了固定金属对蛋白质保留行为的影响，指出蛋白质在强结合IDA－Cu柱上的保留主要受固定金属和蛋白质间配位作用支配，在弱亲和的IDA－Ni,IDA-Co和IDA－Zn柱上的保留主要受静电作用控制，配位作用为辅，讨论了金属螯合亲和色谱中影响蛋白质和金属配位的主要因素，金属离子的电荷和半径，配位原子对中心离子外层d轨道的影响，以及蛋白质表面配位的组氨酸数目，离解常数和取向，影响金属螯合配体和蛋白质静电作用的主要因素为溶液的PH和蛋白质的等电点pI.     

Solubility and binding properties of PEGylated lysozyme derivatives with increasing molecular weight on hydrophobic-interaction chromatographic resins

Dynamic binding capacities and resolution of PEGylated lysozyme derivatives with varying molecular weights of poly (ethylene) glycol (PEG) with 5 kDa, 10 kDa and 30 kDa for HIC resins and columns are presented. To find the optimal range for the operating conditions, solubility studies were performed by high-throughput analyses in a 96-well plate format, and optimal salt concentrations and pH values were determined. The solubility of PEG-proteins was strongly influenced by the length of the PEG moiety. Large differences in the solubilities of PEGylated lysozymes in two different salts, ammonium sulfate and sodium chloride were found. Solubility of PEGylated lysozyme derivatives in ammonium sulfate decreases with increased length of attached PEG chains. In sodium chloride all PEGylated lysozyme derivatives are fully soluble in a concentration range between 0.1 mg protein/ml and 10 mg protein/ml. The binding capacities for PEGylated lysozyme to HIC resins are dependent on the salt type and molecular weight of the PEG polymer. In both salt solutions, ammonium sulfate and sodium chloride, the highest binding capacity of the resin was found for 5 kDa PEGylated lysozyme. For both native lysozyme and 30 kDa mono-PEGylated lysozyme the binding capacities were lower. In separation experiments on a TSKgel Butyl-NPR hydrophobic-interaction column with ammonium sulfate as mobile phase, the elution order was: native lysozyme, 5 kDa mono-PEGylated lysozyme and oligo-PEGylated lysozyme. This elution order was found to be reversed when sodium chloride was used. Furthermore, the resolution of the three mono-PEGylated forms was not possible with this column and ammonium sulfate as mobile phase. In 4 M sodium chloride a resolution of all PEGylated lysozyme forms was achieved. A tentative explanation for these phenomena can be the increased solvation of the PEG polymers in sodium chloride which changes the usual attractive hydrophobic forces in ammonium sulfate to more repulsive hydration forces in this hydrotrophic salt.     

New alpha-amino phenylalanine tetrazole ligand for immobilized metal affinity chromatography of proteins

A new chelating compound has been developed for use in the immobilized metal affinity chromatographic (IMAC) separation of proteins. The bidentate ligand, alpha-amino phenylalanine tetrazole, 4, was synthesized via a five-step synthesis from N-fluorenylmethoxycarbonyl phenylalanine and then immobilized onto silica through the epoxide coupling procedure. The binding behavior of the resulting IMAC sorbent, following chelation with Zn2+ to a density of 183 micromol Zn2+ ions/g silica, was characterized by the retention of proteins in the pH range of 5.0-8.0, and by the adsorption behavior of lysozyme with frontal chromatography at pH 6.0 and 8.0. The prepared column showed the separation ability to four test proteins and the retention time of these proteins increased with an increase in pH. From the derived isotherms, the adsorption capacity, qm, for the binding of lysozyme to immobilized Zn2+-alpha-amino phenylalanine tetrazole-silica was found to be 1.21 micromol/g at pH 6.0 and 1.20 micromol/g sorbent at pH 8.0, respectively, whilst the dissociation constants KD at these pH values were 5.22x10(-6) and 3.49x10(-6) M, respectively, indicating that the lysozyme was retained more stable under alkaline conditions, although the binding capacity in terms of micromole protein per gram sorbent remained essentially unchanged.     

Surface properties of Reactive Yellow 2 immobilised pHEMA and HEMA/chitosan membranes: characterisation of their selectivity to different proteins

Poly(hydroxyethyl methacrylate), pHEMA, and a composite pHEMA/chitosan networks were synthesized in the membrane form via UV initiated photo-polymerisation in the presence of an initiator ,′-azoisobutyronitrile. Reactive Yellow 2 (RY-2) was covalently immobilised as a dye–ligand onto both membranes. The polarity and surface energy of the investigated membranes were determined by contact angle measurement. The incorporation of chitosan in the pHEMA networks produced more hydrophilic surface, as indicated by contact angle analysis. The binding characteristics of lysozyme, γ-globulins, human serum albumin (HSA) and bovine serum albumin (BSA) to pHEMA-RY-2 and pHEMA/chitosan-RY-2 affinity membranes have been investigated from aqueous solution and their dye–ligand free forms were used as control systems. When chitosan was incorporated in the pHEMA network as a cationic polymer led to higher adsorption capacity for the lysozyme. Selective adsorption behaviour was also observed in the case of pHEMA/chitosan-RY-2 membrane for the lysozyme. The non-specific adsorptions of the lysozyme on the pHEMA and pHEMA/chitosan membranes were about 1.9 and 7.2 mg/ml, respectively. These were negligible for all others investigated proteins. The lysozyme adsorption data was analysed using the first-order and the second-order models. The first-order equation in both affinity membrane systems is the most appropriate equation to predict the adsorption capacities of the adsorbents. The adsorption isotherms well fitted the combined Langmuir–Freundlich model. A theoretical analysis has been conducted to estimate the thermodynamic contributions (changes in enthalpy, entropy and Gibbs free energy) for the adsorption of lysozyme to both dye–ligand immobilised membranes. The adsorption capacities of both dye–ligand immobilised membranes increased with increasing the temperature while decreased with increasing the NaCl concentration. Both affinity membranes are stable when subjected to sanitization with sodium hydroxide after repeated separation–elution cycles.     

Modeling protein binding and elution over a chromatographic surface probed by surface plasmon resonance

Surface plasmon resonance (SPR) spectroscopy is used as a scaled-down, analytical, pseudo-chromatography tool for analyzing protein binding and elution over an ion-exchange surface under cyclic sorption conditions. A micrometric-scale adsorption surface was produced by immobilizing a typical ion exchange ligand – diethylaminoethyl (DEAE) – onto commercially available planar gold sensor chip surfaces pre-derivatized with a self-assembled monolayer of 11-mercaptoundecanoic acid with known density. An explicit mathematical formulation is provided for the deconvolution and interpretation of the SPR sensorgrams. An adsorption rate model is proposed to describe the SPR sensorgrams for bovine serum albumin, used here as model protein, when the DEAE surface is subjected to a cyclic series of binding and elution steps. Overall, we demonstrate that the adsorption rate model is capable of quantitatively describing BSA binding and elution for protein titers from dilute conditions up to overloaded conditions and a broad range of salt concentrations.     

Investigation of protein binding affinity in multimodal chromatographic systems using a homologous protein library

A library of cold shock protein B mutant variants was employed to examine differences in protein binding behavior in ion exchange and multimodal chromatography. Single site mutations introduced at charged amino acids on the protein surface resulted in a homologous protein set with varying charge density and distribution. The retention times of the mutants varied significantly during linear gradient chromatography in both systems. The majority of the proteins were more strongly retained on the multimodal cation exchange resin as compared to the traditional cation exchanger. Further, the elution order of the mutants on the multimodal resin was different from that obtained with the ion exchanger. Quantitative structure–property relationship models generated using a support vector regression technique were shown to provide good predictions for the retention times of protein mutants on the multimodal resin. A coarse-grained ligand docking package was employed to examine the various interactions between the proteins and ligands in free solution. The multimodal ligand was shown to utilize multiple interaction types to achieve stronger retention on the protein surface. The use of this protein library in concert with the qualitative and quantitative analyses presented in this paper provides an improved understanding of protein behavior in multimodal chromatographic systems.     

High-performance liquid chromatography of amino acids, peptides and proteins. CIX. Investigations on the relation between the ligand density of cibacron blue immobilized porous and non-porous sorbents and protein-binding capacities and association constants.

A porous silica of nominal 5 microns particle diameter and 30 nm pore size (Nucleosil 300-5) and a non-porous silica of nominal 1.5 microns particle diameter were activated with 3-mercaptopropyltriethoxysilane (MPTS), followed by the immobilization of the triazine dye, Cibacron Blue F3GA. Various biomimetic dye sorbents with graduated ligand densities between 1 mumol/m2 and 0.01 mumol/m2 were prepared. The capacities and the association constants associated with the binding of lysozyme to these sorbents were determined by frontal analysis experiments [J. Chromatogr., 476 (1989) 205-225]. Due to the ability of the Cibacron Blue F3GA-modified silicas to act as mixed mode coulombic and hydrophobic interaction sorbents and the highly charged nature of the surface structure of lysozyme (pl 11), two mobile phase conditions were examined. In one case a 0.1 M phosphate buffer, pH 7.8, was used as the equilibration and loading buffer, in the second case 1 M sodium chloride-0.1 M phosphate buffer, pH 7.8 was employed as the equilibration and loading buffer to monitor the influence of ionic interactions. The elution was performed in each case with a 2.5 M potassium thiocyanate solution. With the porous silica dye sorbents and 1 M NaCl present in the loading buffer, the highest capacity was achieved when Cibacron Blue F3GA was immobilised to the level of 0.1 mumol/m2. In the case of the non-porous silica dye sorbents, the maximum protein capacity was achieved when 0.5 mumol/m2 dye were immobilised onto the support. Evaluation of the frontal breakthrough curves confirmed that the kinetics of adsorption of lysozyme onto the non-porous sorbent were substantially faster than the adsorption of lysozyme onto the porous sorbent due to the absence of pore diffusion effects in case of the non-porous support. Furthermore, the adsorption of lysozyme on both sorbents was faster when no salt was added to the loading buffer, indicating that there is either conformational or reorientation effects operating during the specific binding of the protein to the dye ligand, or that the interaction is proceeding through the participation of a second class of binding sites. The magnitude of the association constants, Ka, for the lysozyme-Cibacron Blue F3GA systems were found to be dependent on the ligand density of the sorbent. With decreasing ligand density, the protein-ligand interaction became stronger, e.g. Ka values became larger. These results confirm earlier observations on the effect of ligand steric compression on the affinate-ligand association constant, e.g. the protein needs sufficient space to interact with the ligand in an optimum way.(ABSTRACT TRUNCATED AT 400 WORDS)     

Experimental study of albumin and lysozyme adsorption onto acrylic acid (AA) and 2-hydroxyethyl methacrylate (HEMA) surfaces

Many commercial soft contact lenses are based on poly-2-hydroxyethyl methacrylate (HEMA) and acrylic acid (AA) hydrogels. The adsorption of proteins, albumin and lysozyme, on such contact lens surfaces may cause problems in their applications. In this work the adsorption of proteins, albumin and lysozyme, on hydrogel surfaces, AA and HEMA, was investigated as a function of concentration of protein. Also the effects of pH and ionic strength of protein solution on the adsorption of protein were examined. The obtained results indicated that the degree of adsorption of protein increased with the concentration of protein, and the adsorption of albumin on HEMA surface at the studied pHs (6.2-8.6) was higher than AA surface, whereas the adsorption of lysozyme on AA surface at the same pHs was higher than HEMA. The change in ionic strength of protein solution affected the proteins adsorption on both AA and HEMA surfaces. Also, the amount of sodium ions deposited on the AA surface was much higher than HEMA surface. This effect can be related to the negative surface charge of AA and its higher tendency for adsorption of sodium ions compared to the HEMA surface.     

Influence of protein and stationary phase properties on protein-matrix-interaction in cation exchange chromatography

A large number of different stationary phases for ion-exchange chromatography from different manufacturers are available, which vary significantly in a number of chemical and physical properties. As a consequence, binding mechanisms may be different as well. In the work reported here, the retention data of model proteins (lysozyme, cytochrome c and two monoclonal antibodies) were determined for nine commercially available cation-exchange adsorbents. The linear gradient elution model in combination with a thermodynamic approach was used to analyse the characteristic parameters of the protein-stationary phase-interactions. Based on the pH dependency of the characteristic charge and the equilibrium constant for binding the differences between the standard Gibbs energies in the adsorbed and the solute state for the protein ΔG(P)° and the salt ΔG(S)° were calculated. The characteristic charge B of the proteins strongly depends on the molecular mass of the protein. For small proteins like lysozyme there is almost no influence of the stationary phase chemistry on B, while for the Mabs the surface modification strongly influences the B value. Surface extenders or tentacles usually increase the B values. The variation of the characteristic charge of the MABs is more pronounced the lower the pH value of the mobile phase is, i.e. the higher the negative net charge of the protein is. The standard Gibbs energy changes for the proteins ΔG(P)° are higher for the Mabs compared to lysozyme and more strongly depend on the stationary phase properties. Surface modified resins usually show higher ΔG(P)° and higher B values. A correlation between ΔG(P)° and B is not observed, indicating that non-electrostatic interactions as well as entropic factors are important for ΔG(P)° while for the B values the accessibility of binding sites on the protein surface is most important.     

Three‐Dimensional Ultralarge‐Pore Ia3d Mesoporous Silica with Various Pore Diameters and Their Application in Biomolecule Immobilization

Highly ordered mesoporous three‐dimensional Ia3d silica (KIT‐6) with different pore diameters has been synthesized by using pluronic P123 as surfactant template and n‐butanol as cosolvent at different synthesis temperatures in a highly acidic medium. The materials were characterized by XRD and N₂ adsorption. The synthesis temperature plays a significant role in controlling the pore diameter, surface area, and pore volume of the materials. The material prepared at 150 °C, KIT‐6‐150, has a large pore diameter (11.3 nm) and a high specific pore volume (1.53 cm³ g^?1). We also demonstrate immobilization of lysozyme, which is a stable and hard protein, on KIT‐6 materials with different pore diameters. The amount of lysozyme adsorbed on large‐pore KIT‐6 is extremely large (57.2 μmol g^?1) and is much higher than that observed for mesoporous silicas MCM‐41, SBA‐15, and KIT‐5, mesoporous carbons, and carbon nanocages. The effect of various parameters such as buffer concentration, adsorption temperature, concentration of the lysozyme, and the textural parameter of the adsorbent on the lysozyme adsorption capacity of KIT‐6 was studied. The amount adsorbed mainly depends on solution pH, ionic strength, adsorption temperature, and pore volume and pore diameter of the adsorbent. The mechanism of adsorption on KIT‐6 under different adsorption conditions is discussed. In addition, the structural stability of lysozyme molecules and the KIT‐6 adsorbent before and after adsorption were investigated by XRD, nitrogen adsorption, and FTIR spectroscopy.     

Protein adsorption and transport in dextran-modified ion-exchange media. III. Effects of resin charge density and dextran content on adsorption and intraparticle uptake

Custom-synthesized variants of the commercial Capto S resin were used to examine the effects of resin charge density and dextran content on protein adsorption and intraparticle uptake. For the small protein lysozyme, resin charge density had the greatest effect on equilibrium capacity, consistent with calculations suggesting that lysozyme capacity should be limited by the available charge on the resin. Isocratic retention data and confocal microscopy imaging for this protein revealed a consistent ordering of the resins linking stronger protein-resin interactions with higher static capacities but slower intraparticle uptake rates over the range of properties studied. For the larger protein lactoferrin, it was found that increasing dextran content led to increased protein exclusion from the dextran layer, but that increasing resin charge density helped overcome the exclusion, presumably due to the increased electrostatic attraction between the resin and protein. Collectively examining the lysozyme and lactoferrin data along with information from previous studies suggests that a trade-off in maximizing dynamic capacities should exist between static capacities that increase to a finite extent with increased resin charge density and uptake rates that decrease with increased charge density. Column breakthrough data for lysozyme and lactoferrin appear to support the hypothesis, though it appears that whether a resin charge density is low or high must be considered in relation to the protein charge density. Using these trends, this work could be useful in guiding resin selection or design.     

Bioinert surface to protein adsorption with higher generation of dendrimer SAMs

Interactions between proteins and biomaterial surfaces correlate with many important phenomena in biological systems. Such interactions have been used to develop various artificial biomaterials and applications, in which regulation of non-specific protein adsorption has been achieved with bioinert properties. In this research, we investigated the protein adsorption behavior of polymer brushes of dendrimer self-assembled monolayers (SAMs) with other generations. The surface adsorption properties of proteins with different pI values were examined on gold substrates modified with poly(amidoamine) dendrimer SAMs. The amount of fibrinogen adsorption was greater than that of lysozyme, potentially because of the surface electric charge. However, as the generations increased, protein adsorption decreased regardless of the surface charge, suggesting that protein adsorption was also affected by density of terminal group.     

Comparison of adsorption performances of metal–chelated polyamide hollow fibre membranes in lysozyme separation

Commercially available microporous polyamide hollow fibres are modified by acid hydrolysis to activate the reactive groups and subsequently binding of the ligand, i.e. Cibacron Blue F3GA. Then the Cibacron Blue F3GA-derived hollow fibres were loaded with different metal ions (i.e. Zn(II), Cu(II), Ni(II)) to form the metal chelate. The internal polymer matrix was characterised by scanning electron microscopy. The effects of pH, initial concentration of lysozyme, metal type and temperature on the adsorption of lysozyme to the metal–chelated hollow fibres were examined in a batch reactor. The non-specific adsorption of lysozyme onto the polyamide hollow fibres was 1.8 mg/g. Cibacron Blue F3GA immobilisation increased the lysozyme adsorption up to 62.3 mg/g. Metal–chelated hollow fibres showed a significant increase of the adsorption efficiency. Lysozyme adsorption capacities of Zn(II), Cu(II) and Ni(II)-chelated hollow fibres were different. The maximum capacities of Zn(II), Cu(II) or Ni(II)-chelated hollow fibres were 144.2, 75.2 and 68.6 mg/g, respectively. Significant amount of the adsorbed lysozyme (up to 97%) was eluted in 1 h in the elution medium containing 1.0 M NaSCN at pH 8.0 and 25 mM EDTA at pH 4.9. Repeated adsorption–desorption process showed that this novel metal–chelated polyamide hollow fibres are suitable for lysozyme adsorption.