Continuous refolding of L-asparaginase inclusion bodies using periodic counter-current chromatography

Chromatography-based refolding is emerging as a promising alternative to dilution-refolding of solubilized inclusion bodies (IBs). The advantages of this matrix-assisted refolding (MAR) lie in its ability to reduce aggregate formation, leading to better recovery of active protein, and enabling refolding at higher protein concentration. However, batch chromatography has the disadvantage of ineffective solvent utilization, under-utilization of resin, and low throughput. In this work, we overcome these challenges by using a 3-column Periodic Counter-current Chromatographic (PCC) system for continuous refolding of IBs, formed during the production of L-asparaginase by recombinant E. coli cultures. Initial experiments were conducted in batch processes using single-column immobilized metal-affinity chromatography. Different gradient operations were designed to improve the protein loading for the single-column, batchMAR processes. Optimized conditions, based on the batch-MAR experiments, were used for designing the continuous-MAR processes using the PCC system. The continuous-MAR experiments were carried out over 3 cycles (similar to 30 h) in the PCC system. A detailed quantitative comparison based on recovery, throughput, buffer consumption, and resin utilization was made for the three modes of operation: pulse-dilution, single-column batch-MAR, and 3-Column PCC-based continuous-MAR processes. While recovery (73%) and throughput (11 mg/h) were the highest in PCC, specific buffer consumption (6.9 ml/mg) was the least. Also, during PCC operation, resin utilization improved by 92% in comparison to the single-column batch-MAR process. These quantitative comparisons clearly establish the advantages of the continuous-MAR process over the batch-MAR and other conventional refolding techniques. (C) 2021 Elsevier B.V. All rights reserved.

Automated summary

Chromatography-based refolding offers advantages over dilution-refolding in terms of reducing aggregate formation and enabling refolding at higher protein concentration. By using a 3-column Periodic Counter-current Chromatographic system for continuous refolding, it is possible to improve resin utilization and reduce specific buffer consumption, ultimately leading to higher recovery and throughput.