Continuous Matrix‐assisted Refolding of Inclusion‐body Proteins: Effect of Recycling

Abstract

A recombinant therapeutic protein expressed in Escherichia coli in form of inclusion bodies was used as a model system for comparative studies on the refolding efficiencies obtained by continuous matrix-assisted refolding or batch-dilution in a stirred tank reactor. Due to the strong aggregation tendency the refolding yield after dilution into an appropriate renaturation buffer was only 10 % at a protein concentration of 0.1 mg/mL. To improve the yield of active protein matrix-assisted refolding using size-exclusion chromatography was tested. A preparative continuous annular chromatography system (PCAC) equipped with a size-exclusion resin was used for continuous processing of the solubilized unfolded protein. As the protein passed through the column, it was separated from the chaotropic agent and started to regain its native structure. Tightly bound aggregates produced during the refolding reaction were continuously removed from the rotating bed by 6 M guanidine-hydrochloride. The aggregate fraction was recycled to the feed stream after concentrating by ultradiafiltration to increase the amount of refolded protein. The refolding yield of 27 % without recycling was increased to 45 % at a recycling rate of 0.51 at a protein concentration of 1 mg/mL. The stability of the reactor under continuous operation was shown for 15 hours without any significant loss of refolding efficiency or chromatographic resolution. Interdependencies of operational parameters such as protein concentration, loading factor and recycling rate on throughput of refolded protein are shown.

Review of the current state of protein aggregation inhibition from a materials chemistry perspective: special focus on polymeric materials

“…The size exclusion and adsorption-based chromatographic refolding processes can be used at high protein concentrations compared to the dilution technique. Schlegl et al described a continuous matrix-assisted refolding process for bovine-a-lactalbumin, 494 and recombinant therapeutic proteins, 495 using annular chromatography at different operational conditions.…”

Buffer recycling in downstream processing of biologics

“…However, the system showed instability due to inhomogeneous flow through the column and hence peak wobbling, an effect in annular chromatography when the eluate periodically change outlet ports. A similar approach, but using size exclusion resins instead for continuous annular chromatography (CAC), was demonstrated by Schlegl et al [17]. Although size exclusion chromatography (SEC) offers the advantage that denaturing agents can be removed completely upon separation, this technique is limited by the feed volume and dilution rate (up to 10-fold) (Figure 1c).…”

“…The simplest but also most time consuming method is by batch dilution [8,9 ,10 ,11-13]. Strategies to improve refolding productivity and efficiency include crossflow ultrafiltration processes [14], continuous stirred tank reactors (CSTR) [15], and annular [16,17] and simulated moving bed chromatography [18 ]. Intensification of these processes has 2 Biotechnology and bioprocess engineering been achieved by different recycling strategies.…”

Chromatographic and electrophoretic characterization of protein variants

“…The dense protein aggregates can be easily removed from the cell homogenate by centrifugation, and the protein can be recovered in a very pure form. The simple recovery is followed by a time-consuming solubilization and refolding [6].…”