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Cryogels with embedded natural adsorbent are new trend of chromatographic media for separation of biomolecules. In this report, experimental determination of immunoglobulin G (IgG) purification by Cu2+-attached pumice particles unified cryogel (Cu2+-PPUC) was performed. For this purpose, after preparation of Cu2+-attached pumice particles, they were unified with 2-hydroxyethyl methacrylate monomers to produce Cu2+-PPUC through polymerization of gel-forming precursors at subzero temperatures. IgG separation experiments were accomplished in a continuous column system. The highest binding capacity (596.8 mg/g) was obtained by working with 0.02 M phosphate buffer at pH 6.0. The chemical analysis of pumice was examined by X-ray fluorescence spectrometer. Scanning electron microscopy was performed to identify the morphology of Cu2+-PPUC. Langmuir adsorption model was best fitted to interaction when compared to Freundlich model. Temkin model was utilized to characterize adsorption, energetically. Purification ability of Cu2+-PPUC for IgG was shown with high selectivity via reducing SDS–PAGE electrophoresis.

Cu(2+) -immobilized magnetic poly(hydroxyethylmethacrylate-N-methacryloyl-(l)-histidinemethylester) (mPHEMAH) nanoparticles were prepared by surfactant-free emulsion polymerization for cytochrome C (cyt C) purification from rat liver. Elemental analysis, atomic force microscopy, zeta sizer, and vibrating sample magnetometer were used to characterize mPHEMAH nanoparticles. In addition to these characterization steps, surface area, average particle size, and size distribution of mPHEMAH nanoparticles were determined. Quantity of immobilized Cu(2+) was measured using atomic absorption spectrophotometry. N-Methacryloyl-(l)-histidinemethylester and Cu(2+) content of mPHEMAH nanoparticles were 0.18 mmol/g polymer and 0.11 mmol/g polymer, respectively. Specific surface area of Cu(2+) -immobilized mPHEMAH nanoparticles was 1180 m(2) /g. Effect of initial cyt C concentration, pH, temperature, and ionic strength on cyt C adsorption onto Cu(2+) -immobilized mPHEMAH nanoparticles was investigated. Maximum cyt C adsorption capacity of Cu(2+) -immobilized mPHEMAH nanoparticles was 311.9 mg/g polymer. Maximum adsorption was obtained at pH 8.0 and 4 °C. Cu(2+) -immobilized mPHEMAH nanoparticles were used ten times with 4.1% decrease in adsorption capacity. In the last stage, Cu(2+) -immobilized mPHEMAH nanoparticles were used to purify cyt C from rat liver tissue, and the purity of desorbed fractions was controlled by SDS-PAGE.

The aim of this study is to prepare poly(hydroxyethyl methacrylate-N-methacryloyl-(L)-histidine) [PHEMAH] magnetic nanoparticles for plasmid DNA (pDNA) purification from Escherichia coli (E. coli) cell lysate. Magnetic nanoparticles were produced by surfactant free emulsion polymerization. mPHEMAH nanoparticles were characterized by elemental analysis, Fourier transform infrared spectroscopy (FTIR), atomic force microscopy (AFM), vibrating sample magnetometer (VSM), electron spin resonance (ESR), thermogravimetric analyses (TGA) and transmission electron microscopy (TEM). Surface area, average particle size and size distribution were also performed. Specific surface area of the mPHEMAH nanoparticles was found to be 1180 m2/g. Elemental analysis of MAH for nitrogen was estimated as 0.18 mmol/g polymer. The amount of pDNA adsorbed onto the mPHEMAH nanoparticles first increased and then reached a saturation value at around 1.0 mg/mL of pDNA concentration. Compared with the mPHEMA nanoparticles (50 μg/g polymer), the pDNA adsorption capacity of the mPHEMAH nanoparticles (154 mg/g polymer) was improved significantly due to the MAH incorporation into the polymeric matrix. The maximum pDNA adsorption was achieved at 25 °C. The overall recovery of pDNA was calculated as 92%. The mPHEMAH nanoparticles could be used six times without decreasing the pDNA adsorption capacity significantly. The results indicate that the PHEMAH nanoparticles promise high selectivity for pDNA.

Cibacron Blue F3GA (CB) was covalently attached onto poly(glycidyl methacrylate) (PGMA) monosize beads for removal of bilirubin from hyperbilirubinemia human serum. PGMA beads were produced by dispersion polymerization (1.6 μm in diameter). CB loading was 1.73 mmol/g. Bilirubin adsorption experiments were performed by stirred-batch adsorption. The non-specific adsorption of bilirubin was low (0.4 mg/g polymer). CB attachment onto the PGMA beads significantly increased the bilirubin adsorption (241.5 mg/g) from aqueous solutions. The maximum bilirubin adsorption was observed at pH 6.0. With an increase of the aqueous phase concentration of sodium chloride, the adsorption amount of bilirubin decreased drastically. The equilibrium adsorption of bilirubin significantly increased with increasing temperature. Much higher adsorption values up to 332 mg bilirubin/g were achieved in the case of the PGMA/CB beads from human plasma.

WOS: 000246089000002, PubMed ID: 17222902, The aim of this study is to prepare cholesterol-imprinted polymeric particles. N-Methacryloyl-(L)-tyrosinemethylester (MAT) was chosen as the complexing monomer. In the first step, functional monomer MAT was synthesized by the reaction Of L-tyrosine methylester and methacryloyl chloride and characterized by FTIR and NMR. Then, cholesterol was complexed with MAT in different mol ratios and the cholesterol-imprinted poly(2-hydroxyethyl methacrylate-N-methacrylOyl-(L)-tyrosine methylester) [MIP] particles were synthesized by bulk polymerization. After that, the template molecules (i.e., cholesterol) were removed using chloroform. MIP particles were characterized by elemental analysis, FTIR, SEM, swelling tests and surface area measurements. Cholesterol adsorption experiments were performed in a batch experimental set-up. Adsorption medium was methanol or intestinal mimicking solution. Stigmasterol and estradiol were used as competing molecules in selectivity tests. Obtained results were as follows: swelling ratio of MIP and non-imprinted (NIP) particles were 60.8% and 44.1% in water. With the increase in the amount of MAT in the polymerization medium, incorporation of MAT was increased (16.6-78.0 mu mol/g). SEM photographs showed the surface roughness and porosity. Specific surface area of NIP and MIP particles were found as 19.2 and 31.5 m(2)/g, respectively. Template molecules (i.e., cholesterol) were removed from the polymer structure in the ratio of 76-84% of the initial concentration. Cholesterol adsorption increased with the increase in cholesterol concentration up to 1.5 mg/mL. MIP particles prepared using higher amounts of cholesterol exhibit significantly higher capacity to the NIP particles (i.e., control polymer). MIP particles were 3.09 and 3.60 times selective with respect to the stigmasterol and estradiol, respectively. Reusability of MIP particles was also investigated. MIP particles showed negligible loss in the cholesterol adsorption capacity after five adsorption-desorption cycles with the same adsorbent

Cu(2+) -immobilized magnetic poly(hydroxyethylmethacrylate-N-methacryloyl-(l)-histidinemethylester) (mPHEMAH) nanoparticles were prepared by surfactant-free emulsion polymerization for cytochrome C (cyt C) purification from rat liver. Elemental analysis, atomic force microscopy, zeta sizer, and vibrating sample magnetometer were used to characterize mPHEMAH nanoparticles. In addition to these characterization steps, surface area, average particle size, and size distribution of mPHEMAH nanoparticles were determined. Quantity of immobilized Cu(2+) was measured using atomic absorption spectrophotometry. N-Methacryloyl-(l)-histidinemethylester and Cu(2+) content of mPHEMAH nanoparticles were 0.18 mmol/g polymer and 0.11 mmol/g polymer, respectively. Specific surface area of Cu(2+) -immobilized mPHEMAH nanoparticles was 1180 m(2) /g. Effect of initial cyt C concentration, pH, temperature, and ionic strength on cyt C adsorption onto Cu(2+) -immobilized mPHEMAH nanoparticles was investigated. Maximum cyt C adsorption capacity of Cu(2+) -immobilized mPHEMAH nanoparticles was 311.9 mg/g polymer. Maximum adsorption was obtained at pH 8.0 and 4 °C. Cu(2+) -immobilized mPHEMAH nanoparticles were used ten times with 4.1% decrease in adsorption capacity. In the last stage, Cu(2+) -immobilized mPHEMAH nanoparticles were used to purify cyt C from rat liver tissue, and the purity of desorbed fractions was controlled by SDS-PAGE.

Interferons are potent biologically active proteins synthesized and secreted by somatic cells of all mammalian species. Dye-affinity adsorption is increasingly used for protein separation. Hollow fibers have several advantages as adsorbents in comparison to conventional bead supports because they are not compressible and they eliminate internal diffusion limitations. 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 recombinant interferon-α (rHuIFN-α). The hollow fiber was characterized by scanning electron microscopy. These dye-carrying hollow fibers (35.8 μmol/g) were used in the rHuIFN-α adsorption-elution studies. The effects of initial concentration of rHuIFN-α, medium pH, ionic strength and temperature on the adsorption efficiency of dye-attached hollow fibers were studied in a batch system. The non-specific adsorption of rHuIFN-α on the hollow fibers was 1.2 mg/g. Cibacron Blue F3GA attachment significantly increased the rHuIFN-α adsorption up to 99.8 mg/g. Significant amount of the adsorbed rHuIFN-α (up to 94.8%) was eluted in 1 h in the elution medium containing 1.0 M NaCl. In order to determine the effects of adsorption conditions on possible conformational changes of rHuIFN-α structure, fluorescence spectrophotometry was employed. We resulted that dye-affinity hollow fibers can be applied for rHuIFN-α adsorption without causing any significant conformational changes. Repeated adsorption/elution processes showed that these dye-attached hollow fibers are suitable for rHuIFN-α adsorption.

The selective binding of albumin on dye-affinity nanospheres was combined with magnetic properties as an alternative approach for albumin depletion from human plasma. Magnetic poly(hydroxyethyl methacrylate) (mPHEMA) nanospheres were synthesized using mini-emulsion polymerization method in the presence of magnetite powder. The specific surface area of the mPHEMA nanospheres was found to be 1302 m2/g. Subsequent to Cibacron Blue F3GA (CB) immobilization onto mPHEMA nanospheres, a serial characterization processing was implemented. The quantity of immobilized CB was calculated as 800 μmol/g. Ultimately, albumin adsorption performance of the CB-attached mPHEMA nanospheres from both aqueous dissolving medium and human plasma were explored.

The preparation of affinity membranes for application in antibody purification studies is described here. Protein-A-attached poly(hydroxyethyl methacrylate-N-methacryloyl-L-alanine) (PHEMAAL) membranes were produced by a photopolymerization technique and then characterized by swelling tests, surface area measurements, contact angle and scanning electron microscopy (SEM) studies. The water swelling ratio of the PHEMAAL membrane was 133.2%. PHEMAAL membranes have large pores with a size in the range of 5-10 μm. Protein A was covalently attached onto the PHEMAAL membranes via cyanogen bromide (CNBr) activation. Maximum protein A loading was 4.7 mg/g. There was a very low non-specific IgG adsorption onto the PHEMAAL membranes, about 0.38 mg/g. The maximum IgG adsorption on the PHEMAAL-protein A membrane was found to be 9.8 mg/g at pH 7.4 from aqueous solutions. Higher adsorption amount was observed from human plasma (up to 37.3 mg/g). Adsorbed IgG was eluted using 0.1 M glycine-HCl buffer (pH 3.5) with a purity of 93%. PHEMAAL-protein A membrane was used for repetitive adsorption/elution of IgG without noticeable loss in IgG adsorption amount after 10 cycles. The PHEMAAL-protein A membrane showed several advantages, such as simpler preparation procedure, good selectivity for IgG purification from human plasma and good stability throughout repeated adsorption-elution cycles.

Poly(hydroxyethyl methacrylate) (PHEMA) nanoparticles with an average size of 300 nm in diameter and with a polydispersity index of 1.156 were produced by surfactant free emulsion polymerization. Specific surface area of the PHEMA nanoparticles was found to be 996 m(2)/g. Metal-chelating ligand 3-(2-imidazoline-1-yl)propyl(triethoxysilane) (IMEO) was covalently attached to the PHEMA nanoparticles. IMEO content was 0.97 mmol IEMO/g. The morphology and properties of these nanoparticles were characterized with scanning electron microscopy, Fourier transform infrared spectroscopy and atomic force microscopy. The Cu2+-chelated PHEMA-IMEO nanoparticles were used in the adsorption-elution studies of human serum albumin (HSA) in a batch system. Maximum HSA adsorption amount of the Cu2+ chelated nanoparticles was 680 mg HSA/g. The PHEMA-IMEO-Cu2+ nanoparticles exhibited a quite high adsorption capacity and fast adsorption rate due to their high specific surface area and the absence of internal diffusion resistance.