Comparative studies on the isothermal characteristics of proteins adsorbed under batch equilibrium conditions to ion-exchange, immobilised metal ion affinity and dye affinity matrices with different ionic strength and temperature conditions.

In these investigations, the influence of a range of experimental parameters on the isothermal characteristics of hen egg white lysozyme (HEWL) and human serum albumin (HSA) adsorbed to several different adsorbents has been examined. The adsorbents were selected to encompass the same basic types of silica support matrices, but with the ligand properties and surface characteristics adjusted so that the dominant mode of interaction between the protein and the ligand involved either electrostatic binding (i.e. as ion-exchange interaction with polyaspartic acid immobilised onto glycidoxypropyl-modified Fractosil 1000), mixed-mode binding with both hydrophobic and electrostatic effect contributing to the protein-ligand interaction (i.e. as dye-affinity interactions with Cibacron Blue F3G-A immobilised onto Lichroprep DIOL or onto glycidoxypropyl-modified Fractosil 1000), or lone pair coordination binding (i.e. as immobilised metal ion affinity (IMAC) interactions with Cu2+ ions complexed with iminodiacetic acid immobilised onto glycidoxypropyl-modified Fractosil 1000). In each case, the adsorbents exhibited similar ligand densities and had the same particle size ranges and silica surface pretreatment. The effect of the ionic strength of the adsorption buffer and temperature on the isothermal adsorption behaviour under batch equilibrium binding conditions of the two test proteins were determined. Consistent with previous observations with soft gel ion exchangers and triazine dye-based adsorbents that are used in packed bed chromatographic systems, the capacities of the silica-based ion-exchange adsorbents, as well as the Cibacron Blue F3G-A dye affinity adsorbents, for both HSA and HEWL were reduced as the salt concentration was increased under batch equilibrium binding conditions. Moreover, with both of these classes of adsorbents, as the ionic strength was increased under constant temperature conditions, the isothermal adsorption dependencies progressively approximated more closely a Langmuirean model of independent binding site interactions, typical of a mono-layer binding process. In contrast, with the silica-based immobilised metal ion affinity adsorbents as the ionic strength was increased the adsorption behaviour appeared to follow a Freundlich model, indicative of positive cooperativity in the binding process. In parallel experiments, the effect of changes in temperature under iso-ionic strength conditions was examined. With increasing temperature, different patterns of isothermal adsorption behaviour for both test proteins were observed, with the magnitude of these trends depending on the type of interaction involved between the immobilised ligand and the protein. Utilising first order Van't Hoff relationships to analyse the experimental data for these protein-ligand interactions, the apparent changes in enthalpy and entropy for these interactions have been derived from the dependency of the change in the apparent Gibbs free energy on 1/T.

Studies on the expansion characteristics of fluidised beds with silica-based adsorbents used in protein purification.

In these investigations, a detailed examination of the fluid dynamic characteristics of expanded beds containing silica-based chromatographic adsorbents has been carried out. In particular, the effects of the column accessories such as distributor design and the flow-rate on the dispersion coefficient of the adsorbent particles have been examined. The experimental data have been analysed in terms of residency time effects, fluid flow characteristics and physical properties of the adsorbent particles using several different theoretical models. In common with experience of packed-bed systems, the results confirm that the optimisation of the dynamic capacities as well as the dynamic adsorption rates of adsorbents in expanded-bed systems must take into account column design characteristics as well as the physical/chemical features of the adsorbents, if the highest productivities of expanded-bed/fluidisation procedures are to be achieved with crude feedstocks from biotechnological applications.

Optimization considerations for the purification of alpha1-antitrypsin using silica-based ion-exchange adsorbents in packed and expanded beds.

The influences of the fluid superficial velocity, sample concentration, loading volume, and wash cycle on the recovery and corresponding purification factors for alpha1-antitrypsin [syn. alpha1-proteinase inhibitor (alpha1-PI)] from crude mixtures of human plasma proteins were investigated using packed and expanded beds of DEAE-Spherodex LS. As part of this study, the effect of fluid superficial velocity on the bed dispersion number (Dv) and dispersion coefficient (D) for this adsorbent in expanded beds was determined with feedstocks containing human serum albumin (HSA), the most abundant of the contaminating proteins in human plasma protein preparations used for the isolation of alpha1-PI. When multicomponent protein feedstocks prepared from human plasma were examined with DEAE-Spherodex LS, reduced chromatographic productivity was observed for alpha1-PI as the extent of column utilization and the superficial velocity were increased, yet the opposite trend was evident for HSA. In particular, higher adsorption capacities and recoveries were obtained for alpha1-PI at lower fluid superficial velocities with both packed and expanded bed conditions. These findings indicate that for process scale purifications of alpha1-PI from multicomponent feedstocks with expanded beds containing this silica-based ion-exchange adsorbent, the optimal range of superficial velocities to achieve the highest bed productivity will not be synonymous with maximally fluidized modes of operation. Rather, the results confirm that the adsorbent has an optimum operational performance when fluidization procedures corresponding to plug flow expansion are employed for the capture of alpha1-PI. These findings also indicate that advantage can be taken of displacement effects between closely related protein species with packed and expanded bed systems containing the DEAE-Spherodex LS type of ion-exchange porous silicas.

Adsorption behavior of multicomponent protein mixtures containing alpha 1-proteinase inhibitor with the anion exchanger, 2-(diethylamino)ethyl-Spherodex.

The equilibrium binding behavior of alpha 1-proteinase inhibitor (alpha 1-PI) in the presence of human serum albumin (HSA) has been determined in packed bed systems with the anion exchanger, 2-(diethylamino)ethyl (DEAE)-Spherodex. Experimental data derived for the individual proteins were compared with the corresponding data obtained from batch adsorption studies as well as studies in which mixtures of these two proteins were loaded at different concentration ratios onto columns of the same anion exchange adsorbent. The results confirm that alpha 1-PI has a greater affinity for the anion exchanger, although competitive adsorption was observed as the inlet concentration of HSA was increased. Under these conditions, decreased binding capacities and lower dynamic adsorption rates were observed for alpha 1-PI with the DEAE-Spherodex anion exchange adsorbent. The results are discussed in terms of the influence which various contaminants that occur in multicomponent mixtures of proteins from human plasma can have on the equilibrium binding characteristics of a target protein with weak or strong ion exchange adsorbents under conditions approaching concentration overload in preparative chromatographic systems. These investigations have also addressed, as the first part of an iterative approach for the simulation of the adsorption behavior of multicomponent mixtures of human plasma proteins with ion exchange and affinity chromatographic adsorbents, the ability of noncompetitive and competitive Langmuirean models to simulate the adsorption of alpha 1-PI in the presence of different concentrations of HSA to DEAE-Spherodex.

Examination of protein adsorption in fluidized bed and packed bed columns at different temperatures using frontal chromatographic methods.

The influences of various experimental parameters on the dynamic adsorption capacity (DAC) and the dynamic adsorption rate (DAR) of a biomimetic affinity silica-based adsorbent in fluidized and packed bed columns operated under plug flow conditions and at different temperatures have been investigated with different inlet concentrations of hen egg white lysozyme (HEWL) and human serum albumin (HSA). The DACs as well as the DARs of both the fluidized and packed beds were examined at 10% saturation (i.e., at the QB value) and the experimental data compared with the corresponding data obtained from batch equilibrium adsorption procedures. Parameters examined included the fluid superficial velocity and protein concentration and their effect on the binding capacity and column efficiency. Consistent with various results reported from this and other laboratories on the behavior of biospecific affinity adsorbents derived from porous silica and zirconia particles, adsorbents prepared from Fractosil 1000 were found to exhibit appropriate rheological characteristics in fluidized bed systems under the experimental conditions. Moreover, changes in temperature resulted in a more significant effect on the breakthrough profiles of HSA compared to HEWL with the immobilized Cibacron Blue F3G-A with Fractosil 1000 adsorbent. This result suggests that temperature effects can possibly be employed profitably in some processes as part of a strategy to enhance column performance with fluidized bed systems for selective recovery of target proteins. At relatively low superficial velocities of the feed, the DARs with HEWL and HSA were similar for both the fluidized and packed bed column systems, whereas, at high superficial velocities, the DARs for these proteins were larger with the packed bed columns.