Step-Gradient Twin-Column Recycling Chromatography for Efficient Integrated Purification of Fidaxomicin Based on Complementary Binary Solvent Selectivity

Crude fidaxomicin contains difficult-to-separate impurities, and conventional dual-step purification usually requires intermediate concentration and transfer, which increases process complexity and may aggravate product loss or degradation. To address this challenge, this study exploits the complementary selectivity of methanol/water (80/20, v/v) and acetonitrile/water (70/30, v/v) binary mobile phases and proposes two purification processes based on step-gradient twin-column recycling chromatography, namely spatial integration and system integration. In the spatial integration strategy, dual-stage separations that are conventionally performed in separate chromatographic systems are sequentially integrated into a single twin-column recycling system in combination with on-line heart-cutting, thereby eliminating intermediate off-line processing steps. In contrast, the system integration strategy merges the two binary mobile phases in defined proportions to construct a single ternary mobile phase composed of methanol/acetonitrile/water (37.5/37.5/25, v/v/v), enabling one-step complete separation. The results demonstrate that the spatial integration strategy, employing binary mobile-phase switching, produces fidaxomicin with a purity of 99.9%, recoveries ranging from 75.27% to 78.77%, and productivities ranging from 307.22 to 328.82 g·L−1·day−1, regardless of the switching sequence. The system integration strategy, based on one-step elution with the ternary mobile phase, achieves the same product purity of 99.9% without mobile-phase switching, with a recovery of 70.41% and a productivity of 246.33 g·L−1·day−1. These results confirm the applicability and flexibility of both integrated strategies for fidaxomicin purification, while indicating that the spatial integration strategy provides better overall preparative performance and the system integration strategy offers a simpler one-step operation.