Physico-chemical characterization of recombinant hepatitis B surface antigen by a multidimensional approach

Initially, our work was directed to respond to the question: why hepatitis B surface antigen (HBsAg) produces a very broad peak in preparative size-exclusion chromatography (SEC). For this purpose, we used a multidimensional approach based on SEC fractionation of purified HBsAg followed by the individual analysis of SEC fractions by a battery of assays, such as SEC, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, enzyme-linked immunosorbent assay and transmission electron microscopy. As a result, HBsAg particles were shown to be heterogeneous in terms of particle assembly. In order to elucidate the origin of HBsAg heterogeneity, we included here the denaturing SEC into a multidimensional approach. The data from denaturing SEC evidenced the fragmentation of protein monomers within the HBsAg particle that, probably, occurs during fermentation broth, rather than during in vitro HBsAg processing. The fractions isolated from widely separated regions of HBsAg peak differed in the extent of protein fragmentation, suggesting that the variable extent of protein degradation within HBsAg particles may be one of the factors responsible for broadening of the HBsAg peak in SEC.