An Integrated Process for Biomolecule Isolation and Purification

Abstract

Biomolecule isolation and purification from a fermentation broth usually involve centrifugation, filtration, adsorption, and chromatography steps. Each step contributes to the product cost and product loss. In this research, a cyclic process integrating commercially available ultrafiltration membranes and chromatographic resin beads was developed to achieve the same goal in one device. The device consisted of ion exchange beads on the shell side of a hollow fiber ultrafiltration module. Loading of proteins on the stationary phase on the shell side was carried out for a period of 5-20 min from the permeate on the shell side produced from tube-side feed in ultrafiltration. The eluent was then introduced either from the shell-side inlet or tube-side inlet; the chromatographic fractions were collected from the shell-side outlet. The column was regenerated/washed next to start a new cycle. Systems studied in this cyclic process include the following binary mixtures: myoglobin and beta-lactoglobulin; hemoglobin and bovine serum albumin; and myoglobin and alpha-lactalbumin. Excellent resolutions of the proteins were obtained. A yeast-based cellular suspension containing a mixture of myoglobin and alpha-lactalbumin was also applied to this device. The target proteins were recovered and purified successfully. The cyclic process-based device integrates clarification, concentration, and chromatographic purification of biomolecules and is suitable for both extracellular and intracellular products.

Protein loading, elution, and resolution behavior in a novel device that integrates ultrafiltration and chromatographic separation

“…We emphasize that such a cyclic process has already been demonstrated experimentally for a cellular broth entering the fiber bore (Dai et al, 1999). Furthermore, to begin, an ultrafiltration membrane is adopted.…”

“…Because each process step involves a certain amount of product loss in the particular device in which it is carried out, an integrated device and process would be expected to reduce such losses. We have recently developed one such integrated process in a single device in which ion-exchange resin beads were introduced into the shell side of a hollow fiber-based microfiltration/ultrafiltration module (Dai et al, 1999;Molinari et al, 1990). The device and the cyclic process were found to be capable of elution chromatographic separation of two proteins in a simulated fermentation broth.…”

“…In this integrated process, shown schematically in Figure 1 (Dai et al, 1999), there are three basic steps. Figure 1a shows that the cellular feed is first introduced into the bores of hollow fiber membranes in the first step, namely loading of biomolecules in the resin beads on the shell side for a given period of time.…”

“…1b); the retentate outlet of the hollow fiber bores is closed during this elution step, so that all of the eluent introduced exits through the permeate exit on the shell side. In the first study (Dai et al, 1999), stepwise elution was carried out in two steps, involving solutions with two different NaCl concentrations, resulting in two distinct peaks in the solution appearing in the permeate exit streams. After completion of the elution step, the next step, washing and bed regeneration, is undertaken (step c) to prepare the system for another cycle of operation with the cellular feed being introduced through the fiber bore as the first step in the next cycle.…”

“…Dai et al (2000) recently developed a model to describe such profiles in a bead-filled hollow fiber membrane module. This model was versatile enough to describe successfully the following modes of operations: conventional ultrafiltration/microfiltration; pure Starling flow; permeate-restricted conventional ultrafiltration/microfiltration with tendencies toward Starling flow; and elution chromatographic flow mode for tube-side elution (as investigated in Dai et al, 1999). The present investigation adopts this model.…”