Pseudobiospecific Affinity Research Articles

Development of l‐histidine immobilized CIM® monolithic disks for purification of immunoglobulin G

The pseudobiospecific affinity ligand l-histidine was immobilized on epoxy, carbonyldiimidazole (CDI), and ethylenediamine (EDA) convective interaction media (BIA Separations, Slovenia) monolithic disks to obtain the histidyl affinity column for purification of immunoglobulin G (IgG). The kinetics and the mass transfer properties of the affinity columns were studied to determine the optimum buffer condition, flow rate, and concentration of IgG for maximum IgG adsorption. The binding capacities of all the three affinity columns were higher with zwitterionic buffer morpholinopropanesulfonic acid than with charged buffers such as tris-HCl and phosphate buffers, and the optimum pH was 6.5. The interaction of IgG with histidine immobilized CDI and epoxy disks was found to be predominantly driven by ionic interaction, while the interaction with EDA-histidine disk could be partially governed by multiple non-covalent forces of interaction. The maximum binding capacity (Qm ) of l-histidine immobilized on EDA-, CDI-, and epoxy-activated convective interaction media disks were 19.83 ± 0.25, 15.85 ± 0.18 and 12.11 ± 0.17 mg/ml of support, respectively, and the dissociation constant (Kd ) were calculated to be in the micromolar range for all the three histidyl monolithic columns. Purification of IgG from untreated human serum was also attempted, and the results indicate the high potential of this method for purification of total IgG from complex biological sources and also for separation of IgG1 from other subclasses.

Adsorption of lysozyme from aqueous solutions by a novel bentonite–tyrptophane (Bent–Trp) microcomposite affinity sorbent

This study deals with developing bentonite–tryptophan (Bent–Trp) microcomposite affinity sorbent (38–105μm) for lysozyme adsorption from aqueous solutions. The structural analysis results of Bent–Trp microcomposites obtained from X-ray diffraction (XRD), Fourier Transform Infrared (FTIR), thermal (TG-DTG/DTA) and elemental analysis techniques clearly pointed out that the pseudo-biospecific affinity ligand L-Tryptophan was penetrated into interlayer space of the bentonite by diplacing water molecules and an elemental analysis of immobilized L-Tryptophan for nitrogen was 541.3μmolg−1 bentonite. The effects of initial concentration, pH, and temperature on the adsorption efficiency of this sorbent were also studied in a batch system. The maximum amount of lysozyme adsorption from aqueous solution was determined as 365.16mg/g at pH 10.0 and 1.25mg/mL initial concentration of lysozyme, while the non-specific adsorption of lysozyme was very low (4.21mgg−1 sorbent). We concluded that Bent-Trp microcomposite affinity sorbents could be repeatedly applied for lysozyme adsorption without significant losses in the adsorption capacity.

Click chemistry: A route to designing and preparing pseudo-biospecific immunoadsorbent for IgG adsorption

l-histidine is a promising alternative to expensive protein ligands for the adsorption of IgG due to its high selectivity, no toxicity and low cost; while click chemistry can improve the reaction selectivity between the ligands and the support matrix under mild reaction conditions. Thus, using l-histidine as a ligand and original sepharose gel as a support, a novel immunoadsorbent possessing pseudo-biospecific affinity for IgG from human plasma, Sep-triazole-His, was designed and prepared according to the principle of Click-reaction between alkyne and azide functional groups; while both sepharose-based control samples Sep-His and Sep-PA were prepared by a conventional method using l-histidine and protein A as a ligand, respectively. The ligand density and IgG adsorption performance of Sep-triazole-His from human plasma were measured and evaluated. The influences of click chemistry on the preparation, structure and performance of sepharose-based immunoadsorbent were also investigated. The results indicate that the ligand density immobilized on Sep-triazole-His is 319.1μmol/g sepharose gel, almost 4-fold as high as that on Sep-His; the IgG adsorption capacity of Sep-triazole-His from human plasma reaches 16.49mg/g at pH 7.0, or increases 5.72-fold with respect to Sep-His, and does not decrease noticeably after being repeatedly used for 10 times; and Sep-triazole-His can exhibit high adsorption selectivity for IgG comparable to Sep-PA. The further studies prove that the 1,2,3-triazole ring in the spacer-arm of Sep-triazole-His, can facilitate the binding of IgG without non-specific adsorption.

Reversible immobilization of catalase by using a novel bentonite–cysteine (Bent–Cys) microcomposite affinity sorbents

This study focused on developing bentonite–cysteine (Bent–Cys) microcomposite affinity sorbents (38–105μm) for catalase adsorption from aqueous solutions. Pseudo-biospecific affinity ligand l-cysteine was covalently binded onto the bentonite structures. X-ray diffraction and FTIR analysis of Bent–Cys composite affinity sorbents were performed. According to X-Ray diffraction data, cysteine molecule is parallel to the tetrahedral–octahedral–tetrahedral layer (TOT silicate layer) and is a monolayer in the inner layer space of the structure. The surface areas of the Bentonite and Bent–Cys microcomposite structures were found as 33.00±0.30 and 22.80±0.30, respectively. An elemental analysis of immobilized l-cysteine for nitrogen was estimated to be 541.3μmolg−1 bentonite. Catalase adsorption onto the microcomposite affinity sorbents from aqueous solutions containing different amounts of catalase at different pH was investigated in a batch system. The maximum catalase adsorption capacity of the Bent–Cys microcomposite affinity sorbents was 175mgg−1. The non-specific catalase adsorption onto the bentonite was very low (about 2.7mgg−1). The activity yield decreased with the increase of the enzyme loading. It was observed that there was a significant change between Vmax value of the free catalase and Vmax value of the adsorbed catalase on the Bent–Cys microcomposite affinity sorbents. The Km value of the immobilized enzyme was higher 1.1 times than that of the free enzyme. Optimum operational temperature was 10°C higher than that of the free enzyme and was significantly broader. It was observed that enzyme could be repeatedly adsorbed and desorbed without loss of adsorption capacity or enzyme activity.

Newly synthesized bentonite–histidine (Bent–His) micro-composite affinity sorbents for IgG adsorption

Bentonite has several advantages for use as a support, including its lack of toxicity, chemical reactivity and hydrophilicity allowing easy fixation of biomolecules. A new approach was developed to obtain an efficient and cost effective purification of IgG from human plasma. This study focused on developing bentonite–histidine micro-composite affinity sorbents (Bent–His) (38–105 μm) for immunoglobulin-G (IgG) adsorption from human plasma. The pseudo-biospecific affinity ligand l-histidine was reacted with covalent binding onto the bentonite structures. X-ray diffraction and FT-IR analysis of Bent–His composite affinity sorbents were performed. The surface area of the bentonite and Bent–His micro-composite structures were found as 33.00 ± 0.30 and 24.70 ± 0.30, respectively. An elemental analysis of immobilized l-histidine for nitrogen was estimated to be 541.3 μmol/g bentonite. The non-specific IgG adsorption onto bentonite structures was negligible (ca. 0.3 mg/g). Higher adsorption values up to 15.6 mg/g were obtained in which Bent–His micro-composite affinity sorbents were used from aqueous solutions. Isotherm studies were carried out to evaluate the effect of initial concentration of IgG, pH and the desorption characteristics of Bent–His micro-composite affinity sorbents. The maximum amount of IgG adsorption from aqueous solution in phosphate buffer was 15.6 mg/g at pH 7.0. A higher adsorption value was obtained from human plasma (up to 89.6 mg/g) with a purity of 94.5%. IgG molecules could be repeatedly adsorbed and desorbed with these micro-composite affinity sorbent systems without noticeable loss in their IgG adsorption capacity.

Separation of human-immunoglobulin-G from human plasma with l-histidine immobilized pseudo-specific bioaffinity adsorbents

The pseudo-biospecific affinity ligand l-histidine immobilized poly(2-hydroxyethylmethacrylate) (PHEMA) in spherical form (100–150 μm in diameter) was used for the affinity chromatographic separation of human-immunoglobulin-G (HIgG) from aqueous solutions and human plasma. The PHEMA adsorbents were prepared by a radical suspension polymerization technique. Reactive aminoacid-ligand l-histidine was then immobilized by covalent binding onto these adsorbents. Elemental analysis of immobilized l-histidine for nitrogen was estimated as 62.3 mg l-histidine/g of PHEMA. The maximum HIgG adsorption on the l-histidine immobilized PHEMA adsorbents was observed at pH 7.4. The non-specific HIgG adsorption onto the plain PHEMA adsorbents was very low (about 0.167 mg/g). Higher adsorption values (up to 3.5 mg/g) were obtained when the l-histidine immobilized PHEMA adsorbents were used from aqueous solutions. Much higher amounts of HIgG were adsorbed from human plasma (up to 44.8 mg/g). Adsorption capacities of other blood proteins were obtained as 2.2 mg/g for fibrinogen and 2.8 mg/g for albumin. The total protein adsorption was determined as 52.1 mg/g. The affinity microbeads allowed the one-step separation of HIgG from human plasma. The HIgG molecules could be repeatedly adsorbed and desorbed with these l-histidine-immobilized PHEMA adsorbents without noticeable loss in their HIgG adsorption capacity.

Aprotinin recovery: comparison between biospecific and pseudobiospecific affinity adsorptions

Two adsorption techniques were studied as potential methods for the recovery of aprotinin: a biospecific adsorption (using immobilized trypsin and chymotrypsin as ligants) and a pseudobiospecific adsorption of aprotinin-trypsin complex onto IMAC matrix. These studies indicated that ionic strength has an important effect on aprotinin adsorption on immobilized trypsin, and the pH was the most significant variable in aprotinin desorption from both immobilized enzymes. The aprotinin-trypsin complex adsorption onto the IMAC matrix was not as significantly affected by changes in pH as it was for changes in ionic strength. For the desorption step, both variables significantly affected the complex recovery. However, the effect of ionic strength was markedly stronger for this desorption step.

Experimental kinetic aspects of hollow fiber membrane-based pseudobioaffinity filtration: process for IgG separation from human plasma

The pseudobiospecific affinity ligand l-histidine was immobilized through an ether linkage onto poly(ethylene vinyl alcohol) hollow fiber cartridge to obtain an affinity support for IgG purification or IgG removal from human plasma with a view to clinical apheresis for the treatment of immune disorders, alloimmunization and cancer. The kinetics and mass transfer aspects were studied in order to determine the optimum loading flow rate for IgG adsorption. Experiments were run at various filtrate flow rates (various residence times) in cross flow mode with recirculation of both the retentate and the permeate in the reservoir. The results showed that this affinity membrane had the potential of fractionating plasma proteins at fast flow rates (residence times below 10 s). However, protein trapping into the membrane was enhanced by residence times below 5 s. Comparison of equilibrium and dynamic maximum capacities determined using Langmuir isotherms showed that dynamic capacity (190 μg/cm 2) was somewhat higher than the equilibrium capacity (148 μg/cm 2). The dissociation constants for IgG were determined using the Langmuir isotherm equation to be 3.7 × 10 −6 M (dynamic mode) and 9.8 × 10 −6 M (equilibrium binding analysis), indicating medium affinity, which was typical for pseudobiospecific affinity ligands. The cartridge was loaded with human serum or human plasma to test its ability to fractionate these protein solutions. The affinity membrane showed high selectivity for IgG from two fold diluted human plasma or serum. IgM was also adsorbed to a certain extent. When the serum or plasma was not diluted with equilibration buffer before loading the cartridge, the amount of adsorbed proteins was significantly (50%) smaller than with the 2 or 10 fold dilutions, but the specificity remained.

In Vitro Removal of Human Igg by Pseudobiospecific Affinity Membrane Filtration on a Large Scale. A Preliminary Report

We have developed a pseudobiospecific affinity membrane device for selective removal of human IgG from plasma or serum in vitro for clinical apheresis application. The pseudobiospecific affinity ligand L-histidine was immobilized through an ether linkage onto poly(ethylenevinyl alcohol) hollow fiber cartridge. The obtained affinity membranes showed high selectivity for IgG adsorption from untreated human serum. These membranes are able to adsorb IgG1, IgG2, IgG3 if Mops buffer is used, and more selectively IgG1, and IgG3 in Tris-HCl buffer. With respect to the binding capacity, the pseudobiospecific affinity membrane used showed a higher capacity as compared to protein A-membranes described in the literature. Due to the high capacity, specificity and stability of the histidine affinity membranes, in addition to their lower cost, the approach proposed in this paper may offer a useful alternative to protein A based devices in the treatment of immune-related diseases.