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-ex clusion chromatography (SEC). For this purpose, we used a multidimensional approach based on SEC fractionation of purified HBsAg followed by the indiv idual analysis of SEC fractions by a battery of assays, such as SEC, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, enzyme-linked immunoso rbent assay and transmission electron microscopy. As a result, HBsAg partic les were shown to be heterogeneous in terms of particle assembly. In order to elucidate the origin of HBsAg heterogeneity, we included here the denatu ring SEC into a multidimensional approach. The data from denaturing SEC evi denced the fragmentation of protein monomers within the HBsAg particle that , probably, occurs during fermentation broth, rather than during in vitro H BsAg processing. The fractions isolated from widely separated regions of HB sAg peak differed in the extent of protein fragmentation, suggesting that t he variable extent of protein degradation within HBsAg particles may be one of the factors responsible for broadening of the HBsAg peak in SEC. (C) 19 99 Elsevier Science B.V. All rights reserved.