Quantitative assessment of LPS-HBsAg interaction by introducing a novel application of immunoaffinity chromatography

Lipopolysaccharide (LPS), as a stubborn contamination, should be monitored and kept in an acceptable level during the pharmaceutical production process. Recombinant hepatitis B surface antigen (r-HBsAg) is one of the recombinant biological products, which is probable to suffer from extrinsic endotoxin due to its long and complex production process. This research aims to assess the potential interaction between LPS and r-HBsAg by recruiting immunoaffinity chromatography (IAC) as a novel tool to quantify the interaction. Molecular modeling was performed on the HBsAg molecule to theoretically predict its potential binding and interaction sites. Then dynamic light scattering (DLS) analysis was implemented on HBsAg, LPS, and mixtures of them to reveal the interaction. The virus-like particle (VLP) structure of HBsAg and the ribbon-like structure of LPS were visualized by transmission electron microscopy (TEM). Finally, the interaction was quantified by applying various LPS/HBsAg ratios ranging from 1.67 to 120 EU/dose in the IAC. Consequently, the LPS/HBsAg ratios in the eluate were measured from 1.67 to a maximum of 92.5 EU/dose. The results indicated that 77 to 100% of total LPS interacted with HBsAg by an inverse relationship to the incubated LPS concentration. The findings implied that the introduced procedure is remarkably practical in the quantification of LPS interaction with a target recombinant protein.