Process-analytical study of IL-6 refolding during batch buffer exchange

Abstract

Interleukin-6 (IL-6) is an aggregation-prone cytokine whose recombinant production from Escherichia coli inclusion bodies remains challenging because of inefficient refolding. Here, we present a limited batch-mode process-analytical study comparing two practical buffer-exchange operations, “fast” and “slow”, during batch dilution refolding of IL-6. Using SDS-PAGE, size-exclusion chromatography (SEC), dynamic light scattering (DLS), and far-UV circular dichroism (CD) spectroscopy, we examined dimerization and aggregation under acidic and guanidine denaturation workflows. The data suggest that IL-6 forms predominantly non-covalent dimers and that batch buffer-exchange operation and timescale are associated with aggregation outcome. Because the acidic and guanidine workflows differed in starting solvent composition, residual denaturant, and final protein concentration, the results should be interpreted as comparisons of complete workflow conditions rather than isolated denaturant effects. Overall, the combined use of SEC, DLS, and far-UV CD provides a practical framework for monitoring IL-6 refolding outcomes, while mechanistic assignments remain beyond the scope of this study.