A process based on reversed-phase high-performance liquid chromatography (RP-HPLC) has been developed for the purification of biosynthetic human insulin (BHI). The RP-HPLC procedure has been successfully integrated into the multimodal chromatographic production process used to purify kilogram quantities of BHI. Axial compression column technology was used in the scale-up process. The RP-HPLC procedure yields an insulin product having high chemical purity and full biological activity.