This investigation focused on the structure change of Hepatitis B virus surface antigen (HBsAg) in the process of ultratiltration (UF). Based on the assay of high performance size exclusion chromatography combining with on-line multi-angle laser light scattering (HPSEC-MALLS) and enzyme-linked immunosorbent assay (ELISA), the HBsAg assemblies were found to aggregate into large-size HBsAg aggregation with only about 20% HBsAg activity of the normal HBsAg assembly. The secondary structure of large size HBsAg aggregation was monitored by circular dichroism spectroscopy (CD) and demonstrated that the content of a-helix in HBsAg decreased from 48.2% to 34.4% and the content of gamma-turn increased from 29.6% to 38.7% due to aggregation. The lipid structure of large size HBsAg aggregation was also changed markedly by the assay of infrared spectroscopy (IR) at the wavenumber 1750 cm(-1) which is corresponding to ester acyl. (c) 2006 Elsevier Ltd. All rights reserved.