Analytical Characterization of Interleukin-6 Aggregation During Refolding

Interleukin-6 (IL-6) is a multifunctional cytokine involved in essential biological processes, including cell proliferation, differentiation, and maturation, making it a key target in therapeutic development, particularly for COVID-19 treatments. Given its complex biological properties, a thorough understanding of IL-6’s aggregation behavior is critical to optimize the production and stability of anti-IL-6 therapeutic candidates. Recombinant human IL-6 (hIL-6) is typically expressed in Escherichia coli, resulting in significant formation of inclusion bodies, which presents challenges in solubility and refolding. Furthermore, IL-6 exhibits a strong propensity for aggregation, reducing the overall yield of biologically active protein after refolding. This study aimed to elucidate the underlying mechanisms of IL-6 aggregation and to identify refolding conditions that mitigate this phenomenon. Using a combination of SDS-PAGE, size-exclusion chromatography (SEC), Dynamic light scattering (DLS), and circular dichroism (CD) spectroscopy, we analyzed the dimerization and aggregation behavior of IL-6 under varying denaturation and refolding conditions. Our results suggest that IL-6 dimerizes primarily through non-covalent interactions, with its secondary structure largely preserved in the dimeric state. Comparative refolding studies under acidic and guanidine-induced denaturation conditions revealed that the rate of solvent exchange is a critical factor influencing aggregation propensity. These findings provide mechanistic insights into IL-6 aggregation during refolding and highlight strategic parameters for reducing aggregation, thereby enhancing the production yield of refolded IL-6. This work establishes a foundation for refining refolding protocols for IL-6 and potentially other aggregation-prone proteins used in therapeutic applications.