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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.

Hemoglobin binding from human blood hemolysate with poly(glycidyl methacrylate) beads.

Metal-chelating affinity beads have attracted increasing interest in recent years for protein purification. In this study, iminodiacetic acid (IDA) was covalently attached to the poly(glycidyl methacrylate) [PGMA] beads (1.6 μm in diameter). Cu(2+) ions were chelated via IDA groups on PGMA beads for affinity binding of hemoglobin (Hb) from human blood hemolysate. The PGMA beads were characterized by scanning electron microscopy (SEM). The PGMA-Cu(2+) beads (628 μmol/g) were used in the Hb binding-elution studies. The effects of Hb concentration, pH and temperature on the binding efficiency of PGMA-Cu(2+) beads were performed in a batch system. Non-specific binding of Hb to PGMA beads in the absence of Cu(2+) ions was very low (0.39 mg/g). The maximum Hb binding was 130.3 mg/g. The equilibrium Hb binding increased with increasing temperature. The negative change in Gibbs free energy (ΔG°<0) indicated that the binding of Hb on the PGMA-Cu(2+) beads was a thermodynamically favorable process. The ΔS and ΔH values were 102.2 J/mol K and -2.02 kJ/mol, respectively. Significant amount of the bound Hb (up to 95.8%) was eluted in the elution medium containing 1.0 M NaCl in 1 h. The binding followed Langmuir isotherm model with monolayer binding capacity of 80.3-135.7 mg/g. Consecutive binding-elution experiments showed that the PGMA-Cu(2+) beads can be reused almost without any loss in the Hb binding capacity. To test the efficiency of Hb depletion from blood hemolysate, eluted portion was analyzed by fast protein liquid chromatography. The depletion efficiency for Hb was above 97.5%. This study determined that the PGMA-Cu(2+) beads had a superior binding capacity for Hb compared to the other carriers within this study.

Methacryloylamidoglutamic acid having porous magnetic beads as a stationary phase in metal chelate affinity chromatography

We have prepared a novel magnetic metal-chelate adsorbent utilizing methacryloylamidoglutamic acid (MAGA) as a metal-chelating ligand. MAGA was synthesized by using methacryloyl chloride and L-glutamic acid dihydrochloride. Magnetic beads with an average diameter of 50–100 μm were produced by suspension polymerization of 2-hydroxyethyl methacrylate (HEMA) and MAGA in the presence of Fe3O4 particles carried out in an aqueous dispersion medium. Magnetic beads were charged with the Cu2+ ions directly via MAGA for the adsorption of cytochrome c (cyt c) from aqueous solutions. The maximum cyt c adsorption capacity of the Cu2+-chelated beads (0.86 mmol/g Cu2+ loading) was found to be 37 mg/g at pH 8.0 in phosphate buffer. Cyt c adsorption on the poly(HEMA-MAGA) beads was 15.4 mg/g. Cu2+ charging increased the cyt c adsorption significantly (37 mg/g). Cyt c adsorption decreased with increasing temperature. Cyt c molecules could be adsorbed and desorbed five times with these adsorbents without noticeable loss in their cyt c adsorption capacity. The resulting magnetic chelator beads posses excellent long term storage stability.

Efficient Removal of Bilirubin from Human Serum by Monosize Dye Affinity Beads

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.

Concanavalin a Immobilized Monosize and Magnetic Poly(glycidyl Methacrylate) Beads for Use in Yeast Invertase Adsorption

Concanavalin A (Con A) immobilized monosize and magnetic poly(glycidyl methacrylate)[m-poly(GMA)] beads were investigated for specific adsorption of yeast invertase from aqueous solutions. m-Poly(GMA) beads (1.6 μ m in diameter) were prepared by dispersion polymerization in the presence of Fe3O4 nanopowder. The epoxy groups of m-poly(GMA) beads were opened by base catalyses. Then, Con A was immobilized by covalent binding onto the beads. Con A immobilization amount was 12.5 mg/g. The invertase adsorption capacity of the m-poly(GMA)/Con A beads was 111 mg/g. The maximum invertase adsorption on the m-poly(GMA)/Con A beads was observed at pH 5.5. The optimum activity for both free and adsorbed invertase was observed at 50°C. Vmax values were determined as 330 U/mg and 292 U/mg enzyme, for free and adsorbed invertase, respectively. KM values were found to be the same for free and adsorbed invertase (20 mM). Adsorption of invertase via Con A improved the pH stability of invertase. Thermal stability also increased with adsorption. The adsorbed enzyme activity was found to be quite stable in repeated experiments.