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.
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.
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