Premium
Continuous Matrix‐assisted Refolding of Inclusion‐body Proteins: Effect of Recycling
Author(s) -
Schlegl R.,
Necina R.,
Jungbauer A.
Publication year - 2005
Publication title -
chemical engineering and technology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.403
H-Index - 81
eISSN - 1521-4125
pISSN - 0930-7516
DOI - 10.1002/ceat.200500121
Subject(s) - chemistry , chaotropic agent , chromatography , dilution , yield (engineering) , size exclusion chromatography , guanidine , matrix (chemical analysis) , inclusion bodies , protein aggregation , fraction (chemistry) , escherichia coli , biochemistry , materials science , physics , gene , metallurgy , thermodynamics , enzyme
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.