Abstract：bjective To develop the separation and purification process of B0 component in the A-40926 mixture (here in after referred to as A-40926 B0). Methods Using medium pressure chromatography system, the polymer microspheres were used as carriers and the yield of A-40926 B0 as indexes. The types of polymer microspheres were screened and the elution conditions were optimized. Results The type of UniPS30-300 polymer particles were selected as the best chromatographic media. The optimal conditions were as follows: The sample loading amount was 10mg (per 1mL filler), and 30% and 45% ethanol solution was used as the elution solution at a flow rate of 1.5BV/h (BV: column volume). The purity of the product was higher than 95% and the yield was above 75%. Conclusion The established purification process of A-40926 B0 was simple to operate and the yield was high and feasible. Thus the process was suitable for industrialized production.