Generation of equally sized particle plaques using solid-liquid suspensions.

A device is presented for the generation of equally sized plaques of sensitive particles in a 96-well format. The resulting particle plaques can be used for the measurement of adsorption isotherms and uptake kinetics in protein chromatography or for immobilization reactions. The particle plaques are formed from suspensions with a vacuum device that is designed as a reusable sandwich module. The particles are retained by a mesh while the solvent is removed by the vacuum. As most particles used for protein chromatography are sensitive to mechanical stress and dehydration, the vacuum device is gentle enough to allow the use of these particles, thus eliminating the uncertainty of slurry preparation and pipetting. Apparatus characteristics and preparation procedures are described precisely. The physical intactness of the particles after the preparation procedure is proved by microscopic analysis. Data on the uniformity of the obtained resin plaques with respect to the reproducibility of their adsorption performance is given. Finally, adsorption isothermal and kinetic data of BSA on an ordinary HIC system obtained by high-throughput measurements are shown as an application example.

Competitive adsorption of labeled and native protein in confocal laser scanning microscopy.

Confocal laser scanning microscopy has been previously applied to the study of protein uptake in porous chromatography resins. This method requires labeling the protein with a fluorescent probe. The labeled protein is then diluted with a large quantity of native protein so that the fluorescence intensity is a linear function of the labeled protein concentration. Ideally, the attachment of a fluorescent probe should not affect the affinity of the protein for the stationary phase; however, recent experimental work has shown that this assumption is difficult to satisfy. In the present study, we present a mathematical model of protein diffusion and adsorption in a single adsorbent particle. The differences in adsorption behavior of labeled and native protein are accounted for by treating the system as a two-component system (labeled and native protein) described by the steric mass action isotherm (SMA). SMA parameters are regressed from experimental linear gradient elution data for lysozyme and lysozyme-dye conjugates (for the fluorescent dyes Cy3, Cy5, Bodipy FL, and Atto635). When the regressed parameters are employed in the model, an overshoot in the labeled lysozyme concentration is predicted for Cy5- and Bodipy-labeled lysozyme, but not for Atto635-labeled lysozyme. The model predictions agree qualitatively well with recent work showing the dependence of the concentration overshoot on the identity of the attached dye and provide further evidence that the overshoot is likely caused by the change of binding characteristics due to the fluorescent label.

Direct quantification of intraparticle protein diffusion in chromatographic media.

Diffusion coefficients of proteins in chromatographic media are important parameters for the rational design of stationary phases and purification schemes. In contrast to free diffusion, intraparticle diffusion is hindered by the porous structure of the media. Direct intraparticle diffusion analysis (IDA) is a novel approach for the determination of intraparticle protein diffusion coefficients. IDA is based on the evaluation of spatially and temporally resolved intraparticle concentration profiles. To prevent adsorption and to study diffusion only, the chromatographic media are investigated in underivatized form. With IDA, intraparticle concentration profiles are measured in a microcolumn by confocal laser scanning microscopy (CLSM). From this dynamic data, the diffusion coefficients are determined by parameter estimation, using a spheric diffusion model. The boundary condition is given by the measured protein concentration in the bulk phase. IDA is applied to determine intraparticle diffusion coefficients of seven different proteins in Sepharose 6 FF. The results show excellent congruence of experimental data and simulation results. Moreover, the determined diffusion coefficients lie well within the range of data published in the literature. Given that the material in question allows optical analysis, IDA is a general approach for studying protein diffusion in porous particles and is easily adapted to different proteins, solution conditions and stationary phases.