Whole Broth Adsorptive Extraction

Integrated fermentation and recovery may enable reduction of feedback inhibition resulting from product accumulation in the bioreactor, thus providing for an overall more productive system (41). Whereas integrated solvent extraction requires intermittent phase separation and culture recycle, integrated adsorption can be accomplished either by inclusion of adsorbents within the bioreactor or by passing filter-clarified broth through an external contactor with continuous recycle to the bioreactor.

An improved system for direct-contact broth extraction was developed by immobilizing finely divided affinity adsorbent particles in a large hydrogel bead (42). The large bead size facilitates separation from broth components, and the reversible Ca2+ hydrogel facilitates efficient recovery of the costly affinity ligand for recycle and reuse. Hydrogels, by virtue of their extremely high water content (>90%), offer limited diffusional resistance to the desired product while protecting the affinity ligand from fouling components of the broth. Addition of adsorptive particles to a cell culture or fermentation broth can also remove those components from the liquid medium that are responsible for low filtration fluxes frequently encountered in clarification of untreated streams (43). Even in those cases where flocculants thus formed raise the viscosity ofthe medium, the resulting flux is still less significantly impaired than that decreased by the presence of a significant concentration polarization layer.

Incorporation of an external fluidized bed contactor minimizes attrition to both adsorbent particles and cells encountered in direct inclusion systems while allowing circulation of whole broth to avoid frequent membrane fouling associated with broth clarification. Single-stage recirculating fluidized beds are less susceptible to time-based changes in biomass and viscosity over the course of a fermentation cycle than expanded beds, which are best suited to single-pass batch treatments. The greatest limitation on implementation of such integrated fermentation and recovery systems, as in the case of fluidized-bed purification systems, has been the lack of available supports. Because of the strict sterility requirements in fermentation or cell culture applications, any such adsorbents must also withstand sterilization.

Continuous Affinity Recycle Extraction

A variant of batch adsorption referred to as continuous affinity-recycle extraction (CARE) was developed in the late 1980s. It combines adsorption purification with membrane filtration for the continuous separation of proteins (44). The method employs two continuous stirred-tank reactors (CSTRs), one for adsorption and the other for desorption, between which the adsorbent is recirculated. The feed solution is continuously added to the adsorption tank, where the desired product is contacted with the selectively adsorbing resin. Simultaneously, contaminants are diluted with the addition of wash buffer and removed by passage through a screen filter, which allows even crude lysates to be processed. A stream of the product-containing adsorbent is pumped into the second CSTR along with the de-sorbing buffer. The product is eluted in the tank and recovered through an ultrafiltration membrane in the permeate stream. The regenerated adsorbent is recycled to the adsorption tank. By controlling the relative rates of the feed, product, and recycle streams, which are dictated by the rates of adsorption in CSTR-1 and desorption in

CSTR-2, the system can be operated continuously at a steady state.

This scheme was demonstrated for both ion exchange and affinity modes of purification for the recovery of j-galactosidase from a partially clarified Escherichia coli ly-sate. A recovery yield of 70% was achieved with a 35-fold purification factor, although the product was substantially diluted (45).

Ultrafiltration-Coupled Adsorption Systems

Mattiasson et al. (46) described a method of ultrafiltration affinity purification to combine purification based on affinity interactions with membrane separation. By selecting membranes with pore sizes large enough to freely pass the protein of interest but then introducing recirculating (membrane-rejected) macromolecular ligands with specific affinity for this protein, all molecules except the target protein are rapidly washed from the feed pool. When all proteins not bound to the ligand are removed, the affinity complex may be dissociated, and the liberated material can pass through the membrane for collection. In batch experiments capturing concanavalin A using yeast cells as li-gands, yields up to 70% of electrophoretically homogeneous product have been obtained. As in column-affinity chromatography, the adsorption, wash, and desorption steps can be optimized independently.

Fletcher and Deley (47) applied a similar concept using recirculating fine adsorbent particles, such as Biocryl bio-processing aids, to purification of a peptide from highly colored cell culture streams. After pH adjustment for optimal binding to the adsorbent resin and a short incubation, the bound peptide was concentrated twofold and then diafiltered to remove remaining color bodies. The permeate contained colored material but minimal peptide. The loaded particles were then diafiltered against elution buffer, and product peptide was collected in the permeate. This preliminary removal of load-limiting contaminant allowed a subsequent reverse phase chromatography step to be used for polishing purposes, with a 100-fold increase in column capacity.

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