Chinese hamster ovary (CHO) cells are often used to produce therapeutic monoclonal antibodies (mAbs). CHO cells express many host cell proteins (HCPs) required for their growth. Interactions of HCPs with mAbs can sometimes result in co-purification of trace levels of hitchhiker' HCPs during the manufacturing process. Purified mAb-1 product produced in early stages of process optimization had high HCP levels. In addition, these lots formed delayed-onset particles containing mAb-1 and its heavy chain C-terminal fragments. Studies were performed to determine the cause of the observed particle formation and to optimize the purification for improved HCP clearance. Protease activity and inhibitor stability studies confirmed that an aspartyl protease was responsible for fragmentation of mAb-1 resulting in particle formation. An affinity resin was used to selectively capture aspartyl proteases from the mAb-1 product. Mass spectrometry identified the captured aspartyl protease as CHO cathepsin D. A wash step at high pH with salt and caprylate was implemented during the protein A affinity step to disrupt the HCP-mAb interactions and improve HCP clearance. The product at the end of purification using the optimized process had very low HCP levels, did not contain detectable protease activity, and did not form particles. Spiking of CHO cathepsin D back into mAb-1 product from the optimized process confirmed that it was the cause of the particle formation. This work demonstrated that process optimization focused on removal of HCPs was successful in eliminating particle formation in the final mAb-1 product. (c) 2015 American Institute of Chemical Engineers Biotechnol. Prog., 31:1360-1369, 2015