William Lee

Protein binding to polymer brush, based on ion-exchange, hydrophobic, and affinity interactions

The major limitations associated with conventional packed bed chromatography for protein separation and purification can be overcome by using adsorptive microporous membranes as chromatographic media. Microporous membranes have advantages as support matrices in comparison to conventional bead supports because they are not compressible and they eliminate diffusion limitations. As a result, higher throughput and shorter processing times are possible using these membrane systems. In this paper, we review the current state of development in the area of attaching functionalized polymer brushes onto a microporous membrane to form a novel chromatographic medium for protein separation and purification. The functionalized polymer brushes were appended onto the pore surface of a microporous hollow-fiber membrane uniformly across the membrane thickness by radiation-induced graft polymerization and subsequent chemical modifications. We review various applications of this adsorptive membrane chromatography by focusing on polymer brushes bearing ion-exchange, hydrophobic and affinity groups. Proteins were captured in multilayers by the ion-exchange group-containing polymer brushes due to the formation of a three-dimensional space for protein binding via the electrostatic repulsion of the polymer brushes. In contrast, proteins were captured in a monolayer at most by the polymer brushes containing hydrophobic or affinity ligands. By permeating a protein solution through the pores rimmed by the polymer brushes, an ideal capturing rate of the proteins with a negligible diffusional mass-transfer resistance was achieved by the functionalized polymer brushes, based on ion-exchange, hydrophobic, and affinity interactions.

Proton Transport Through Polyethylene‐Tetrafluoroethylene‐Copolymer‐Based Membrane Containing Sulfonic Acid Group Prepared by RIGP

A novel sulfonic acid (SO 3 H) group containing membrane (SH membrane) was prepared by grafting glycidyl methacrylate onto an ethylene-tetrafluoroethylene copolymer film with subsequent sulfonation. The graft chains introduced on the SH membrane form a flexible brush-type structure capable of binding water molecules (30% more than Nafion 117 membrane) and transporting protons. Proton conductivity of the SH membrane was evaluated as a function of SO 3 H group density. The SH membrane with the SO 3 H group conversion of 97% and the thickness of 70 μm had a specific resistivity of 24 Ω cm at 303 K, which was comparable to that of Nafion 117 membrane. A uniform distribution of the SO 3 H group across the SH membrane was verified using an electron probe x-ray microanalyzer.

Design of urea-permeable anion-exchange membrane by radiation-induced graft polymerization

Abstract Strong base anion-exchange membranes were prepared by radiation-induced graft polymerization. Quaternary ammonium salts were introduced onto nonporous polyethylene films by grafting of vinyl monomers such as glycidyl methacrylate (GMA), diethylaminoethyl methacrylate (DEAEMA), and vinyl benzyl trimethyl ammonium chloride (VBTAC), followed by subsequent chemical modifications. Hydroxyethyl methacrylate (HEMA) was cografted to (a) enhance the diffusion of the VBTAC into the polymeric matrix, and (b) initiate the grafting of the VBTAC. The VBTAC/HEMA-cografted membrane was selected because of its practical physical strength. Urea permeability across the resulting anion-exchange membrane was evaluated with a diffusion dialysis cell. Anion-exchange membranes with degrees of VBTAC/HEMA grafting higher than 70% exhibited urea and phosphate ion permeation. When the VBTAC/HEMA-cografted membrane was compared with a conventional anion-exchange membrane based on a styrene-divinylbenzene matrix, higher hydrophilicity and permeability were observed.

Capture of microbial cells on brush-type polymeric materials bearing different functional groups

A brush-type microbial-cell-capturing polymeric material was prepared by radiation-induced grafting of an epoxy-group-containing monomer, glycidyl-methacrylate (GMA), onto a polyethylene-based fiber. The epoxy ring (EO) of GMA was opened with different degrees of introduction of diethylamine (DEA). The residual epoxy group was hydrophilized by ethanolamine (EA). The prepared DEA membranes with coexisting EO or EA groups were tested for their ability to capture Staphylococcus aureus and Escherichia coli cells. The DEA membrane (2.7 mol/kg of product of DEA group density) with coexisting EO groups (DEA-EO membrane) exhibited good S. aureus-cell-capturing ability with a capturing rate constant of 1.82 x 10(-6) m/s, whereas the DEA membrane with coexisting EA groups (DEA-EA membrane) retarded capturing abilities for both S. aureus and E. coli cells. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 53: 523-528, 1997.

Highly Multilayered Urease Decomposes Highly Concentrated Urea

Urease was immobilized at a density of 1.2 g of urease per gram of a matrix via ion‐exchange binding of urease to an anion‐exchange polymer chain grafted onto a pore surface of a porous hollow‐fiber membrane and subsequent cross‐linking of urease with transglutaminase. Urea was hydrolyzed during the permeation of a urea solution, the concentration of which ranged from 2 to 8 M, through the pores of the resultant membrane with a thickness of approximately 1 mm. Quantitative hydrolysis of 4 M urea was achieved at a permeation rate lower than 1 mL/h, i.e., a residence time longer than 5.1 min, at ambient temperature. This performance is ascribed to convective transport of urea through the pores rimmed by the urease‐immobilized polymer chains at a high density. Urease was denatured in the presence of urea at concentrations higher than 6 M while hydrolyzing urea.

Adsorption Kinetics of Microbial Cells onto a Novel Brush‐Type Polymeric Material Prepared by Radiation‐Induced Graft Polymerization

A novel brush‐type microbial‐cell‐adsorbing material was prepared by radiation‐induced graft polymerization. A vinyl monomer containing an epoxy group, glycidyl methacrylate (GMA), was first grafted onto a polyethylene‐based fiber before the introduction of a tertiary amino group, i.e., diethylamine (DEA). The grafted‐type DEA fibers prepared were then tested for their microbial‐cell‐adsorbing activity by contacting the fibers with a Staphylococcus aureus suspension. The DEA fibers showed adsorption ability of S. aureus, and the adsorption rate increased with increasing density of the DEA group. On the other hand, DEA was also introduced onto cross‐linked‐type GMA beads and its adsorption rate of S. aureus cells was compared with that of the grafted‐type DEA fibers. The grafted‐type DEA fibers exhibited an adsorption rate constant 1000‐fold greater than that of the cross‐linked‐type DEA beads. Observation by scanning electron microscopy showed that the shape of S. aureus cells adsorbed on the grafted‐type DEA fibers was intact whereas the shape of those adsorbed on the cross‐linked‐type DEA beads was difficult to identify.

Validation of a sensitive assay for thiocoraline in mouse plasma using liquid chromatography-tandem mass spectrometry.

A sensitive high-performance liquid chromatography-tandem mass spectrometry assay for thiocoraline, an anti-tumor depsipeptide, in mouse plasma is described. Echinomycin, a quinoxaline peptide, was used as an internal standard. Thiocoraline was recovered from the mouse plasma using protein precipitation with acetonitrile and followed by solid-phase extraction of the supernatant. The mobile phase consisted of methanol (0.1% formic acid)-water (0.1% formic acid) (90:10, v/v). The analytical column was a YMC C(18). The standard curve was linear from 0.1 to 50 ng/ml (R(2)>0.99). The lower limit of quantitation was 0.1 ng/ml. The assay was specific based on the multiple reaction monitoring transitions at m/z 1157-->215 and m/z 1101-->243 for thiocoraline and the internal standard, echinomycin, respectively. The mean intra- and inter-day assay accuracies remained below 5 and 12%, respectively, for all calibration standards and quality control (QC) samples. The intra- and inter-day assay precisions were less than 11.4 and 9.5% for all QC levels, respectively. The utility of the assay was demonstrated by a pharmacokinetic study of i.v. (bolus) thiocoraline on CD-1 mice. Thiocoraline was stable in mouse plasma in an ice-water bath for 6 h and for three freeze-thaw cycles. The reconstituted thiocoraline after extraction and drying sample process was stable in the autosampler for over 24 h. The assay was able to quantify thiocoraline in plasma up to 48 h following dose. Pharmacokinetic analysis showed that thiocoraline has distinct pharmacokinetic profiling when dosed in different formulation solutions. The assay is currently used to measure thiocoraline plasma concentrations in support of a project to develop a suitable formulation with a desirable pharmacokinetic profile.