ASSAY FOR RECOMBINANT HEPATITIS-B SURFACE-ANTIGEN USING REVERSED-PHASE HIGH-PERFORMANCE LIQUID-CHROMATOGRAPHY

Several HPLC assays are reported for monitoring the mass of recombinan t hepatitis B surface antigen (rHBsAg) in yeast cell lysates. The assa ys utilized either a polymeric resin column containing a phenyl ligate or a silica-based octadecyl micropellicular column. Prior to chromato graphy on the polymeric column, the samples were derivatized with the thiolspecific fluorescent probe monobromobimane to discriminate the rH BsAg from nonfluorescent cellular components. Using a dual gradient of acetic acid and acetonitrile the derivatized rHBsAg eluted with a ret ention time equal to 17 min. Chromatography on the micropellicular col umn did not require prederivatization and utilized an isopropanol grad ient with increasing amounts of acetonitrile. Operating this column at elevated temperature with a high flow rate resolved the rHBsAg from y east components within 5 min and allowed a new sample injection every 10 min. AU the assays displayed useful linear ranges for analyzing rHB sAg in cell lysates and had detection limits for rHBsAg between 10 and 50 ng per injection. In 1995 Academic Press, Inc.