“Prevention of structural perturbation and aggregation of hepatitis B surface antigen: screening of various additives”

Abstract

The instability of protein and antigen(s) during encapsulation in biodegradable polymers by water-in-oil-in-water (w/o/w) encapsulation is well established. The aim of present study is to screen various additives to prevent the inactivation and loss of immunogenicity of HBsAg upon its exposure to the water/CH₂Cl₂ (methylene chloride) interface by simulating the formulation steps involved in the preparation of microspheres. The secondary structure of HBsAg, recovered under different conditions after primary emulsification, was investigated by FTIR spectroscopy and Circular Dichorism. Subsequently, PLGA microspheres were formulated and characterized for their size, shape, incorporation efficiency, antigen integrity, and immunogenicity. The immunogenicity and the HBsAg recovery under different conditions were tested in BALB/c mice. Inulin and trehalose were found to be better stabilizing agents to prevent the aggregation, the structural perturbations and immunogenicity of HBsAg. This study substantiated that inulin could overcome the aggregation and denaturing effects of the water/CH₂Cl₂ interface upon HBsAg during emulsification step and upon encapsulation.

Keywords::

• Antibody titer
• antigen delivery
• hepatitis B surface antigen
• protein stabilization

Acknowledgements

The authors are grateful to Serum Institute of India for providing the gift sample of HBsAg.

Declaration of interest

All authors report no conflicts of interest. The authors do not have any commercial or other association that might pose a conflict of interest in this publication. The authors alone are responsible for the content and writing of the paper.