Nevra Öztürk

Phenylalanine containing hydrophobic nanospheres for antibody purification

In this study, novel hydrophobic nanospheres with an average size of 158 nm utilizing N‐methacryloyl‐(L)‐phenylalanine methyl ester (MAPA) as a hydrophobic monomer were produced by surfactant free emulsion polymerization of 2‐hydroxyethyl methacrylate (HEMA) and MAPA conducted in an aqueous dispersion medium. MAPA was synthesized using methacryloyl chloride and L‐phenylalanine methyl ester. Specific surface area of the nonporous nanospheres was found to be 1874 m2/g. Poly(HEMA–MAPA) nanospheres were characterized by Fourier transform infrared spectroscopy (FTIR) and scanning electron microscopy (SEM). Average particle size, size distribution, and surface charge measurements were also performed. Elemental analysis of MAPA for nitrogen was estimated as 0.42 mmol/g polymer. Then, poly(HEMA–MAPA) nanospheres were used in the adsorption of immunoglobulin G (IgG) in batch system. Higher adsorption values (780 mg/g) were obtained when the poly (HEMA–MAPA) nanospheres were used from both aqueous solutions and human plasma. The adsorption phenomena appeared to follow a typical Langmuir isotherm. It was observed that IgG could be repeatedly adsorbed and desorbed without significant loss in adsorption amount. These findings show considerable promise for this material as a hydrophobic support in industrial processes.

High capacity binding of antibodies by poly(hydroxyethyl methacrylate) nanoparticles.

Poly(hydroxyethyl methacrylate) (PHEMA) nanoparticles with an average size of 150 nm in diameter and with a poly-dispersity index of 1.171 were produced by a surfactant free emulsion polymerization. Specific surface area of the PHEMA nanoparticles was found to be 1779 m(2)/g. Reactive imidazole containing 3-(2-imidazoline-1-yl)propyl(triethoxysilane) (IMEO) was used as a pseudo-specific ligand. IMEO was attached covalently onto the nanoparticles. PHEMA-IMEO nanoparticles were used for the affinity binding of immunoglobulin-G (IgG) from human plasma. To evaluate the degree of IMEO loading, the PHEMA nanoparticles were subjected to Si analysis by using flame atomizer atomic absorption spectrometer and it was estimated as 64.5 mg/g of polymer. The nanoparticles were characterized by transmission electron microscopy (TEM) and Fourier transform infrared spectroscopy (FTIR). IgG binding onto the PHEMA nanoparticles was found to be 5.2 mg/g. Much higher binding values (up to 843 mg/g) were obtained for the PHEMA-IMEO nanoparticles. IgG could be repeatedly bound and eluted on PHEMA-IMEO nanoparticles without noticeable loss in the IgG binding capacity.

Selective separation of human serum albumin with copper(II) chelated poly(hydroxyethyl methacrylate) based nanoparticles.

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.

Silane‐Modified Magnetic Beads: Application to Immunoglobulin G Separation

The magnetic poly(2‐hydroxyethyl methacrylate ethylene glycol dimethacrylate) [m‐poly(HEMA‐EGDMA)] beads (150–250‐μm diameter in spherical form) were prepared by a radical suspension polymerization technique. The pseudo‐specific ligand, reactive imidazole containing 3–(2‐imidazoline‐1‐yl)propyl (triethoxysilane) (IMEO) was selected as a silanization agent. IMEO was covalently immobilized onto the magnetic beads. IMEO‐immobilized m‐poly(HEMA‐EGDMA) beads were used for the affinity adsorption of immunoglobulin‐G (IgG) from aqueous solutions and human plasma. To evaluate the degree of IMEO attachment, the m‐poly(HEMA‐EGDMA) beads were subjected to Si analysis by using flame atomizer atomic absorption spectrometer, and it was estimated as 36.6 mg IMEO/g of polymer. The nonspecific IgG adsorption onto the plain m‐poly(HEMA‐EGDMA) beads was very low (about 0.4 mg/g). Higher adsorption values (up to 55 mg/g) were obtained when the m‐poly(HEMA‐EGDMA)/IMEO beads were used from both aqueous solutions and human plasma. The maximum IgG adsorption on the m‐poly(HEMA‐EGDMA)‐IMEO beads was observed at pH 7.0. The IgG molecules could be repeatedly adsorbed and desorbed with m‐poly(HEMA‐EGDMA)‐IMEO beads without noticeable loss in the IgG adsorption capacity. The adsorption capacity from human plasma in magnetically stabilized fluidized bed decreased drastically from 78.9 to 19.6 mg/g with the increase of the flow rate from 0.2 to 3.5 mL/min.

A novel support for antibody purification: fatty acid attached chitosan beads.

Linoleic acid attached chitosan beads [poly(LA-Ch)] (1.25 microm in diameter) are obtained by the formation of amide linkages between linoleic acid and chitosan. Poly(LA-Ch) beads are characterized by FTIR, TEM, and swelling studies. Poly(LA-Ch) beads are used for the purification of immunoglobulin-G (IgG) from human plasma in a batch system. The maximum IgG adsorption is observed at pH 7.0 for HEPES buffer. IgG adsorption onto the plain chitosan beads is found to be negligible. Adsorption values up to 136.7 mg/g from aqueous solutions are obtained by poly(LA-Ch) beads. IgG adsorption saw an increase as a result of increasing temperature. Higher amounts of IgG are adsorbed from human plasma (up to 390 mg/g) with a purity of 92%. The adsorption phenomena appeared to follow a typical Langmuir isotherm. It is observed that IgG could be repeatedly adsorbed and desorbed without significant loss when we take into account the adsorption amount. It is concluded that the poly(LA-Ch) beads allowed one-step purification of IgG from human plasma.