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A new continuous refolding method using a four-zone simulated moving bed process based on the size exclusion mechanism was developed in order to overcome the disadvantages associated with inclusion body refolding in a batch dilution and chromatography. Protein refolding using size exclusion SMB enables refolded protein to be obtained continuously with a high productivity, low consumption of the refolding buffer, and a high efficiency of the size exclusion medium. The size exclusion factors and kinetic parameters for the SMB operation were estimated from the best-fit values by comparing the simulation and experimental chromatography results. The SMB operating conditions were obtained from the standing wave design. Computer simulations and several SMB experiments showed that the standing wave design and the proposed process could achieve a high level of protein recovery (96%), a high specific protein refolding yield (96%), and low degree of protein aggregation. |
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