Abstract: Apolipoprotein A-1 Milano (ApoA-1M), the protein component of a high-density lipoprotein (HDL) mimic with promising potential for reduction of atherosclerotic plaque, is produced on a large scale by expression in E. coli. Significant difficulty with clearance of host cell proteins (HCPs) was experienced in the original manufacturing process, despite lengthy purification. Analysis of purified protein solutions and intermediate process samples led to the identification of several major HCPs, and a bacterial protease causing the production of a truncated species of ApoA-1M co-purifying with the product. Deletions in these genes from the original host strain succeeded in substantially reducing the levels of HCPs without adversely affecting overall fermentation productivity, contributing to a much more efficient and robust new manufacturing process.