Modeling and Analysis of the Affinity Filtration Process, Including Broth Feeding, Washing, and Elution Steps

Abstract

Affinity filtration is a developing protein purification technique that combines the high selectivity of affinity chromatography and the high processing speed of membrane filtration. In this work a lumped kinetic model was developed to describe the whole affinity filtration process, including broth feeding, contaminant washing, and elution steps. Affinity filtration experiments were conducted to evaluate the model using bovine serum albumin as a model protein and a highly substituted Blue Sepharose as an affinity adsorbent. The model with nonadjustable parameters agreed fairly to the experimental results. Thus, the performance of the affinity filtration in processing a crude broth containing contaminant proteins was analyzed by computer simulations using the lumped model. The simulation results show that there is an optimal protein loading for obtaining the maximum recovery yield of the desired protein with a constant purity at each operating condition. The concentration of a crude broth is beneficial in increasing the recovery yield of the desired protein. Using a constant amount of the affinity adsorbent, the recovery yield can be enhanced by decreasing the solution volume in the stirred tank due to the increase of the adsorbent weight fraction. It was found that the lumped kinetic model was simple and useful in analyzing the whole affinity filtration process.

A Predictive Model for Salt Effects on the Dye-Ligand Affinity Adsorption Equilibrium of Protein

“…Using the Langmuir equation, He et al studied the adsorption of bovine serum albumin (BSA) to CB-modified Sepharose CL-6B over extensive salt concentrations and gave empirical correlations between the Langmuir model parameters and the salt concentration. The empirical correlations were employed to simulate the dye-ligand affinity separation process of protein. , However, the affinity constants determined using the Langmuir model are not adequate for predicting the protein adsorption behavior as a function of ionic strength. , Furthermore, the protein adsorption to dye affinity adsorbents does not follow the Langmuir model because of the following two important phenomena. First, the binding of most proteins to the multifunctional dye ligand is not monovalent but rather by a combination of specific and nonspecific interactions between a protein and several ligand molecules, , especially when the ligand density is high .…”

Para-aminobenzamidine linked regenerated cellulose membranes for plasminogen activator purification: Effect of spacer arm length and ligand density

“…Alternatively such membranes might be used as sensory devices in PA production. As has been discussed in several reviews and reports, in addition to improved module designs, there is a significant need for the development of activated, stable membranes that can be coupled by the end-user to provide the specific ligate capacity as per need [8, 11]. The study described in this manuscript is a step forward in this direction by providing a deeper understanding of issues that are critical in development of functional affinity membranes.…”

“…Affinity membranes in particular have been a very promising alternative to affinity bead based chromatographic media. Modification of membranes with ligands that have affinity and selectivity for the target protein provides a very effective tool for the separation of proteins that belong to a narrow size range [4, 11, 12]. In the process of making available new membranes for separation processes, several things need to be considered, such as the type of membrane, the length of spacer arm (SA), and the type of ligand.…”

Affinity ultrafiltration: Effects of ligand binding on selectivity and process optimization

“…The ®rst application of anity ultra®ltration was for the puri®cation of concanavalin A using residues on the membrane of heat-killed Saccharomyces cerevisae as the anity ligand (Mattiasson and Ramstrop, 1989). Subsequent studies have examined the separation of trypsin from chymotrypsin using a high-MW polymer bearing m-aminobenzamidine (Luong et al, 1988), the isolation of urokinase using N-acryloyl-m-aminobenzamide copolymerized with acrylamide (Male et al, 1990), the puri®cation of avidin using biotinylated liposomes (Powers et al, 1990), the isolation of human serum albumin and lysozyme using cibacron blue bound to agarose as the``anity escort'' or macroligand (Herrak and Merrill, 1989), and the recovery of bovine serum albumin using highly substituted blue sepharose (He et al, 1998). Anity ultra®ltration can also be used for the separation of chiral molecules using a stereoselective macroligand, e.g., the optical resolution of tryptophan can be eected using bovine serum albumin (Higuchi et al, 1993;.…”

“…Powers et al (1990) extended this basic approach to consider reversible trypsin binding to the anity-modi®ed liposome, but the mathematical development was limited to cases where the anity ligand was in very large excess. Both Ghosh et al (1996) and He et al (1998) considered the actual binding equilibrium between the target and the anity ligand, but their analyses were limited to conditions where the impurity had no interactions of any kind with the anity ligand. Such an analysis is clearly inappropriate for applications of af-®nity ultra®ltration for chiral separations (Higuchi et al, 1993;Garnier et al, 1999, Poncet et al, 1997, and will likely give erroneous results for many protein systems in which the impurity may have speci®c or nonspeci®c binding to the macroligand.…”