This study evaluated the simultaneous recovery of recombinant 503 antigen and endotoxin removal from E. coli homogenate by immobilized metal affinity- expanded bed adsorption chromatography (EBAC), using the non-ionic surfactant Triton X-114 during the washing step. The strategy employed was able to remove more than 99% of endotoxin in all conditions tested, and the lowest residual concentration obtained was 10.22 EU/mL, with a recovery of 503 antigen ranging from 41.4% to 61.1%. Therefore, the procedure of integrating the clarification, recovery of protein, and endotoxin removal into a unit operation exploiting the EBAC was successful.