Elias Klein

Affinity membranes: a 10-year review

Abstract A review of affinity membrane publications from 1989 to 1999 is presented containing six major divisions: (a) modification of existing membranes for affinity adsorption applications; (b) membranes specifically prepared for affinity binding; (c) module designs for immuno- and other affinity sorption processes; (d) the kinetics of the affinity sorption process; (e) a selection of interesting applications that have appeared during the period and (f) a summary of reviews published during the period. Although there have been a large number of membrane preparation and process applications described in the literature during the period covered by the review, the commercialization of affinity membranes remains meager. One purpose of the review is to point out opportunities for more applied development of affinity membranes, including the design of appropriate modules to house them. From the available literature it should be possible to produce high efficiency separation devices based on the combination of sieving and adsorption characteristics of affinity membranes.

Microfiltration isolation of human urinary exosomes for characterization by MS

Purpose: The purpose of this study was to address the hypothesis that small vesicular urinary particles known as exosomes could be selectively microfiltered using low protein‐binding size exclusion filters, thereby simplifying their use in clinical biomarker discovery studies.

Comparison of experimental and calculated permeability and rejection coefficients for hemodialysis membranes

Abstract The permeability coefficients of four membranes suitable for use in hemodialysis are reported. Experimental values are compared to values calculated from application of a hydrodynamic theory based on pore structure models. Relatively good agreement between experimental and computed values is demonstrated. The computed values are based on effective pore area and average pore dimensions derived from HTO (tritiated water) permeability and hydraulic conductivity for each membrane. The same values are used in predicting reflection coefficients (σ) which are equated to limiting solute rejections (R∞). The computed rejections agree with experimental values for Cuprophan, but do not correlate as well for the larger-pored membranes. The probable effect of heteroporosity is discussed in terms of the predicted values.

Transport and mechanical properties of hemodialysis hollow fibers

Abstract Methods for characterizing the mechanical and mass transfer properties of hollow fibers and the experimental requirements for dealing with fluid boundary resistances are described. Ultrafiltration measurements, diffusive solute transport and the contribution of convective solute transport are examined. Three hollow fibers are evaluated in detail: cellulosic fibers made by Dow Chemical Co. and by Enka Glanzstoff, and polysulfone fibers manufactured by Amicon Corp. All these fibers have mechanical and solute transport properties useful for fabrication into hemodialyzers.

AFFINITY MEMBRANES PREPARED FROM HYDROPHILIC COATINGS ON MICROPOROUS POLYSULFONE HOLLOW FIBERS

Abstract Microporous polysulfone hollow fibers were modified by reacting terminal phenol groups with ethylene glycol diglycidyl ether (EGDGE) to produce terminal epoxy groups. These were used to covalently link hydroxyethyl cellulose (HEC) polymers to the membrane surface. Further epoxidation of the HEC and reaction with hexane-diamine produced amine-terminated leashes on the microporous fibers. Recombinant protein A was linked to the amine functions and the resulting affinity membranes were evaluated for IgG capacities. The EGDGE reactions were studied under both acidic and basic catalysis conditions. IgG capacities of the acid-catalyzed reaction products reached as high as 20 mg/ml bed volume. Comparisons of equilibrium and dynamic capacities show that dynamic capacities are somewhat lower than the equilibrium capacities.

Microporous poly(caprolactam) hollow fibers for therapeutic affinity adsorption

Abstract Microporous nylon-6 hollow fibers were modified for use as affinity fibers. The initial amine end-group concentration of 21 μmoles/g fiber was amplified by reacting the polymer with lysine, using standard peptide synthetic methods. The carbohydrate side chain of a rabbit polyclonal anti-BSA IgG was oxidized to facilitate linkage of the Ab to the hollow fibers. The covalent links were either to terminal amine groups [from lysine of the poly(caprolactam)] or to hydrazide groups. The latter were produced by coupling adipic acid dihydrazide to the amine groups via a glutaraldehyde bridge. Coupling of the oxidized Ab to amine groups of required a pH⪢8.0, whereas the same Ab could react with the hydrazide groups at pH 5.5. The higher pH coupling conditions led to crosslinking of the IgG, presumably between side chain amine groups on the protein and the carbohydrate aldehyde groups. Although significant amounts of IgG could be coupled to the amine groups, the recognition of antigen by such Ab was markedly reduced. In contrast, the coupling of oxidized Ab to hydrazide groups, carried out at pH 5.5, gave bound IgG which exhibited the theoretical number of binding sites for its antigen. Equilibrium binding coefficients for BSA showed values ranging from 5,2×10 6 M −1 to 9.7×10 5 M −1 . The molar ratio of BSA to calculated anti-BSA binding sites was generally 2:1. Although stripping experiments with SDS at ⪢85°C indicated none of the BSA was covalently linked to either the IgG or the fiber substrate, less than 60% of the adsorbed BSA could be eluted with pH change, salt, chaotropic agent, etc. The same responses were observed with hydrazide modified Sepharose carried through the experiments for comparison.

Affinity adsorption devices prepared from microporous poly(amide) hollow fibers and sheet membranes

Microporous poly(caprolactam) hollow fibers and flat sheet membranes have been modified by covalent linkage to produce affinity matrices. Using linkages developed in the past for the chromatographic media, it is shown that a number of biological ligands can be bonded covalently to these membranes to produce effective affinity-separation devices. We report here, results for immobilized antibodies, r-Pr A, and DEAE-functionalized copolymers produced on the membranes. In addition, we show how metal-chelate chromatography can be adapted to these microporous materials. The physical configurations tested included modules prepared with multiple layers of flat sheet membrane; with three-layer pleated sheets; with cross-flow hollow fiber modules; and with dead-end hollow fiber modules. Each of these geometries offers some unique advantage, principally in the types of frontal chromatography suitable for various applications. The chromatographic results illustrate how high-speed separations can be accomplished when ligand-modified membranes are used with gradient-elution separations.

CHITOSAN MODIFIED SULFONATED POLY(ETHERSULFONE) AS A SUPPORT FOR AFFINITY SEPARATIONS

Abstract Microporous hollow fiber membranes covalently modified with protein ligands offer the possibility of high speed affinity isolation of biologicals. Although using coordination chemistries developed for chromatographic bead separations, affinity membranes are not mass transfer rate limited. A problem in their preparation is that mechanically stable microporous fibers do not, generally, contain functional groups suitable for linking the ligands. This paper presents a method for depositing and cross-linking a chitosan film on the surface of partially sulfonated poly(ethersulfone) hollow fibers. The amine and hydroxyl groups of the coating are further modified to link ligands. Results are presented in terms of equilibrium and dynamic capacities of IgG captured by immobilized recombinant protein A.

Dialysate contamination and back filtration may limit the use of high-flux dialysis membranes.

Endotoxins, or fragments thereof, can reach the blood stream of dialysis patients, transported by diffusion and connection across the intact high-flux membrane. This transfer depends upon the phenomenon of back filtration. Back filtration generally occurs under conventional high-flux dialysis conditions with membranes having an ultrafiltration coefficient in blood (UF-C) above 20 ml/hr/m2/mmHg. The clinical consequences of back filtration vary from center to center depending primarily on the quality of dialysate. We therefore surveyed the bacterial and endotoxin levels of purified water and effluent dialysate in a cross section of dialysis centers in the central United States. Using a high recovery medium, we found that 53% of the centers had bacterial counts above the Association for the Advancement of Medical Instruments standard in water (20% cfu/ml) and 35% above the standard in dialysate (2,100 cfu/ml). Endotoxin concentrations higher than 5.0 EU/ml in both water and dialysate were found in 4% and 11.8% of the centers, respectively. Since high-flux membranes are believed to be of benefit for long-term dialysis patients, manufacturers will have to offer dialysate preparation systems with additional safety features. The proper membrane design will be a key to the success of such systems.

Immobilisation of pig liver esterase in hollow fibre membranes

Different methods were evaluated to immobilise Pig Liver Esterase (PLE) in hollow fibre membranes. Four covalent bonding techniques (using epoxy, imidazol, amino and carboxylic acid terminal groups) were tested to link the enzyme to microporous nylon membranes. Physical immobilisation was also studied, by entrapment of the enzyme inside the microporous structure of a polysulfone asymmetric ultrafiltration membrane. The entrapment method lead to a higher retention of enzymatic activity for a longer period of time. This technique was selected to be used in a biphasic membrane bioreactor where the microporous hydrophilic membrane, containing the enzyme, is used to separate an aqueous from an organic phase, in which the substrate is dissolved. Different enzyme loading procedures were studied in the biphasic reactor and the resulting axial and radial enzyme distribution in the hollow fibre module were related to the global enzymatic activity.