Handan Yavuz

Poly(glycidyl methacrylate) beads embedded cryogels for pseudo-specific affinity depletion of albumin and immunoglobulin G

Depletion of high abundant proteins like albumin and immunoglobulin G (IgG) can be beneficial in the analysis of serum proteins. For this purpose, Cibacron Blue F3GA and iminodiacetic acid (IDA)-Cu2+ containing poly(glycidyl methacrylate) (PGMA) beads (1.6µm in diameter) were embedded into the poly(hydroxyethyl methacrylate) (PHEMA) cryogel. The PGMA beads were prepared by dispersion polymerization. The PGMA beads were modified with Cibacron Blue F3GA and iminodiacetic acid (IDA)-Cu2+ for simultaneous albumin and IgG depletion, respectively. The PHEMA cryogel was synthesized by free radical polymerization in the presence of the modified PGMA beads. The PHEMA and PHEMA/PGMA composite cryogels were characterized by swelling measurements and scanning electron microscopy (SEM). Protein depletion studies were carried out in a continuous experimental set-up in a stacked column. Albumin adsorption capacity of the PGMA-Cibacron Blue F3GA beads embedded PHEMA cryogel (PHEMA/PGMA-Cibacron Blue F3GA) was 342mg/g and IgG adsorption capacity of the PGMA-IDA-Cu2+ beads embedded PHEMA cryogel (PHEMA/PGMA-IDA-Cu2+) was 257mg/g. The composite cryogels depleted albumin and IgG from human serum with 89.4% and 93.6% efficiency, respectively. High desorption values (over 90% for both modified cryogels) were achieved with 0.05M phosphate buffer containing1.0M NaCl.

Human serum albumin chromatography by Cibacron Blue F3GA-derived microporous polyamide hollow-fiber affinity membranes

An affinity dye ligand, Cibacron Blue F3GA was covalently attached onto commercially available microporous polyamide hollow-fibre membranes for human serum albumin (HSA) adsorption from both aqueous solutions and human plasma. Different amounts of Cibacron Blue F3GA were incorporated on the polyamide hollow-fibres by changing the dye attachment conditions, i.e. initial dye concentration, addition of sodium carbonate and sodium chloride. The maximum amount of Cibacron Blue F3GA attachment was obtained at 42.5 micromol g(-1) when the hollow-fibres were treated with 3 M HCI for 30 min before performing the dye attachment. HSA adsorption onto unmodified and Cibacron Blue F3GA-derived polyamide hollow-fibre membranes was investigated batchwise. The non-specific adsorption of HSA was very low (6.0 mg g(-1) hollow-fibre). Cibacron Blue F3GA attachment onto the hollow-fibres significantly increased the HSA adsorption (147 mg g(-1) hollow-fibre). The maximum HSA adsorption was observed at pH 5.0. Higher HSA adsorption was observed from human plasma (230 mg HSA g(-1) hollow-fibre). Desorption of HSA from Cibacron Blue F3GA derived hollow-fibres was obtained using 0. 1 M Tris-HCl buffer containing 0.5 M NaSCN or 1.0 M NaCl. High desorption ratios (up to 98% of the adsorbed HSA) were observed. It was possible to reuse Cibacron Blue F3GA derived polyamide hollow-fibre without significant decreases in the adsorption capacities.

Selective removal of 17β-estradiol with molecularly imprinted particle-embedded cryogel systems

The selective removal of 17β-estradiol (E2) was investigated by using molecularly E2 imprinted (MIP) particle embedded poly(hydroxyethyl methacrylate) (PHEMA) cryogel. PHEMA/MIP composite cryogel was characterized by FTIR, SEM, swelling studies, and surface area measurements. E2 adsorption studies were performed by using aqueous solutions which contain various amounts of E2. The specificity of PHEMA/MIP cryogel to recognition of E2 was performed by using cholesterol and stigmasterol. PHEMA/MIP cryogel exhibited a high binding capacity (5.32 mg/gpolymer) and high selectivity for E2 in the presence of competitive molecules, cholesterol (k(E2/cholesterol) = 7.6) and stigmasterol (k(E2/Stigmasterol) = 85.8). There is no significant decrease in adsorption capacity after several adsorption-desorption cycles.

Monosize and non-porous p(HEMA-co-MMA) microparticles designed as dye- and metal-chelate affinity sorbents

Abstract Congo red was immobilised onto monosize and non-porous poly(2-hydroxyethylmethacrylate-co-methylmethacrylate) [p(HEMA-co-MMA)] copolymer microparticles (4.0 μm in diameter). Then Fe(III) ions were complexed by chelation with the immobilised congo red molecules. Different amounts of Fe(III) ions were loaded on the dye-derived microparticles by changing the concentration of Fe(III) ions and pH of the reaction medium. Congo red-derived and Fe(III)-complexed microparticles were used in the adsorption of glucose oxidase, catalase, lysozyme and bovine serum albumin. The maximum adsorption capacities of these microparticles were determined by changing pH and the concentration of the proteins in the adsorption medium. Their adsorption behavior can be described at least approximately with the Langmuir equation. Glucose oxidase, catalase, lysozyme and bovine serum albumin adsorption capacities of the Fe(III) complexed microparticles (165.1, 135.2, 67.6 and 44.5 mg g−1) were higher than those of the congo red-immobilised microparticles (125.9, 94.2, 35.8 and 21.2 mg g−1, respectively). The non-specific adsorption of the proteins on the p(HEMA-co-MMA) microparticles was negligible. The resulting dye- and metal-chelate affinity microparticles have excellent reusability and long term storage stability.

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.

Albumin adsorption from aqueous solutions and human plasma in a packed-bed column with Cibacron Blue F3GA-Zn(II) attached poly(EGDMA-HEMA) microbeads

Affinity dye-ligand Cibacron Blue F3GA, was covalently coupled with poly(EGDMA-HEMA) microbeads via nucleophilic reaction between the chloride of its triazine ring and the hydroxyl groups of the HEMA under alkaline conditions. The microbeads carrying 16.5 μmol Cibacron Blue F3GA per gram polymer was incorporated with Zn(II) ions. Zn(II) loading was 189.6 μmol/g. Cibacron Blue F3GA-Zn(II) attached affinity sorbent was used for albumin adsorption from aqueous solutions and human plasma in a packed-bed column. BSA adsorption capacity of the microbeads decreased with an increase in the recirculation rate. High adsorption rates were observed at the beginning, then equilibrium was gradually achieved in about 60 min. The BSA concentration in the mobile phase also effected adsorption. BSA adsorption was first increased with BSA concentration, then reached a plateau which was about 128 mg BSA/g. The maximum adsorption was observed at pH 5.0 which is the isoelectric pH of BSA. Higher human serum albumin adsorption was observed from human plasma (215 mg HSA/g). High desorption ratios (over 90% of the adsorbed albumin) were achieved by using 1.0 M NaSCN (pH 8.0) in 30 min.

A new metal chelate affinity adsorbent for cytochrome c

We have prepared a novel metal‐chelate adsorbent utilizing N‐methacryloyl‐l‐histidine methyl ester (MAH) as a metal‐chelating ligand. MAH was synthesized by using methacryloyl chloride and l‐histidine methyl ester dihydrochloride. Spherical beads with an average diameter of 75–125 μm were produced by suspension polymerization of 2‐hydroxyethyl methacrylate (HEMA) and MAH carried out in an aqueous dispersion medium. Then, Cu2+ ions were chelated directly on the chelating beads. Cu2+‐chelated beads were used in the adsorption of cytochrome c (cyt c) from aqueous solutions. The maximum cyt c adsorption capacity of the Cu2+‐chelated beads (658.2 μmol/g Cu2+ loading) was found to be 31.7 mg/g at pH 10 in phosphate buffer. The nonspecific cyt c adsorption on the naked PHEMA beads was 0.2 mg/g. Cyt c adsorption increased with increasing Cu2+ loading. Cyt c adsorption capacity was demonstrated for the buffer types with the effects in the order phosphate > HEPES > MOPS > MES > Tris‐HCl. Cyt c molecules could be adsorbed and desorbed five times with these adsorbents without noticeable loss in their cyt c adsorption capacity.

Glucose oxidase and catalase adsorption onto Cibacron Blue F3GA-attached microporous polyamide hollow-fibres

Abstract The aim of this study was to explore in detail the performance of polyamide hollow fibers to which Cibacron Blue F3GA was attached for adsorption of proteins. Model proteins were glucose oxidase, as a flavo-enzyme and contains two tightly bound flavine adenine dinucleotide cofactor, and catalase as a heme-containing metallo-enzyme. The hollow fiber structure was characterized by scanning electron microscopy. These dye-carrying hollow-fibers (35.8 μmol/g) were used in the glucose oxidase and catalase adsorption–elution studies. The non-specific adsorption values were 1.25 mg/g for glucose oxidase and 1.97 mg/g for catalase. Cibacron Blue F3GA attachment increased the adsorption capacity up to 248 mg/g. Langmuir adsorption model was found to be applicable in interpreting glucose oxidase and catalase adsorption by Cibacron Blue F3GA-attached hollow fibers. Significant amount of the adsorbed proteins (up to 97%) was eluted in 1 h in the elution medium containing 1.0 M NaSCN at pH 8.0. In order to determine the effects of adsorption and elution conditions on possible conformational changes of studied protein structures, fluorescence spectrophotometry was employed. It was concluded that polyamide dye-affinity hollow-fibers can be applied for glucose oxidase and catalase adsorption without causing any significant conformational changes. Repeated adsorption/elution processes showed that these dye-attached hollow-fibers are suitable for glucose oxidase and catalase separation.

Nonporous monosize polymeric sorbents: Dye and metal chelate affinity separation of lysozyme

Lysozyme adsorption onto dye-attached nonporous monosize poly(2-hydroxyethyl-methacrylate-methylmethacrylate) [poly(HEMA-MMA)] microspheres was investigated. Poly(HEMA-MMA) microspheres were prepared by dispersion polymerization. The monochloro-triazine dye, Cibacron Blue F3GA, was immobilized covalently as dye-ligand. These dye-affinity microspheres were used in the lysozyme adsorption-desorption studies. The effect of initial concentration of lysozyme and medium pH on the adsorption efficiency of dye-attached and metal-chelated microspheres were studied in a batch reactor. Effect of Cu(II) chelation on lysozyme adsorption was also studied. The nonspecific adsorption of lysozyme on the poly(HEMA-MMA) microspheres was 3.6 mg/g. Cibacron Blue F3GA attachment significantly increased the lysozyme adsorption up to 247.8 mg/g. Lysozyme adsorption capacity of the Cu(II) incorporated microspheres (318.9 mg/g) was greater than that of the Cibacron Blue F3GA-attached microspheres. Significant amount of the adsorbed lysozyme (up to 97%) was desorbed in 1 h in the desorption medium containing 1.0M NaSCN at pH 8.0 and 25 mM EDTA at pH 4.9. In order to examine the effects of separation conditions on possible conformational changes of lysozyme structure, fluorescence spectrophotometry was employed. We conclude that dye- and metal-chelate affinity chromatography with poly(HEMA-MMA) microspheres can be applied for lysozyme separation without causing any significant changes and denaturation. Repeated adsorption/desorption processes showed that these novel dye-attached monosize microspheres are suitable for lysozyme adsorption.

Molecularly imprinted composite cryogel for albumin depletion from human serum

A new composite protein‐imprinted macroporous cryogel was prepared for depletion of albumin from human serum prior to use in proteom applications. Polyhydroxyethyl‐methacylate‐based molecularly ımprinted polymer (MIP) composite cryogel was prepared with high gel fraction yields up to 83%, and its morphology and porosity were characterized by Fourier transform ınfrared, scanning electron microscopy, swelling studies, flow dynamics, and surface area measurements. Selective binding experiments were performed in the presence of competitive proteins human transferrin (HTR) and myoglobin (MYB). MIP composite cryogel exhibited a high binding capacity and selectivity for human serum albumin (HSA) in the presence of HTR and MYB. The competitive adsorption amount for HSA in MIP composite cryogel is 722.1 mg/dL in the presence of competitive proteins (HTR and MYB). MIP composite cryogel column was successfully applied in the fast protein liquid chromatography system for selective depletion of albumin in human serum. The depletion ratio was highly increased by embedding beads into cryogel (85%). Finally, MIP composite cryogel can be reused many times with no apparent decrease in HSA adsorption capacity. Copyright