HSP65‐MUC1 is a fusion protein between BCG (Bacille Calmette‐Guerin)‐derived HSP65 (heat‐shock protein 65) and human MUC1 (mucin 1) VNTR (variable number of tandem repeats)‐domain peptides that has shown antitumour efficacy. China's Food and Drug Administration has recently approved a Phase I clinical trial using HSP65‐MUC1 for the treatment of MUC1‐positive breast cancer. In order to produce sufficient quantities of clinical‐grade HSP65‐MUC1, we established a pilot‐scale purification scheme comprising two steps of column chromatography: HIC (hydrophobic‐interaction chromatography) and IEX (ion‐exchange chromatography). The pH values of the buffers used in homogenization and HIC were adjusted to pH 9.0 to maintain protein stability and prevent protein degradation. Using this manufacturing process, we obtained clinical‐grade HSP65‐MUC1 with a yield of 400 mg per 70 g of wet cell pellet and >96% purity.