The thermal stability of HBc particles was systematically investigated for efficient removal of host cell proteins (HCP) by heat treatment before chromatographic step. The HBc particles were found stable up to 80 degrees C for 30 min without any noticeable change in circular dichroism spectra, fluorescence spectra and transmission electron microscope observation. When heating was applied to precipitate the HCP in the cell disruption supernatant of HBc fermentation, the HCP removal effect was more obvious as the temperature went higher. However, a phenomenon was found beyond 70 degrees C where the recovered HBc particles had larger than normal size and molecular weight as observed by dynamic light scattering and multi-angle laser light scattering. Analysis found that the HBc particles possess nanopores which expand with temperature. When the temperature was above 70 degrees, the pores were large enough for some HCP to penetrate in, but not being able to get out after cooling down. To fully utilize the thermal stability and avoid the interference of HCP entering, a two-step heat treatment strategy was designed. The supernatant was firstly heated up to 60 degrees C for 30 min to precipitate most HCP, then another 30 min at 70 degrees C was used to remove the rest impurities. The two-step heat treatment effectively avoided the HCP entering problem, achieving 85.8% particle recovery and 74.7% purity. With further one-step hydrophobic interaction chromatography, the purity was increased to 99.0% with overall process recovery of 77.7%, considerably higher than those reported in the literature. The same process design was applied to purify three HBc-related products, including OVA-HBc, M2e-HBc and NP-HBc. All recoveries were higher than 50% with purity greater than 97%. (C) 2018 Elsevier B.V. All rights reserved.