Bioaffinity chromatography of yeast-derived recombinant Hepatitis B virus major surface antigen

Chronic Hepatitis B virus infection claims millions of lives across the world every year. Vaccination with recombinant Hepatitis B virus major surface antigen (rHBsAg) is the only key to prevent the spread of virus thereby reducing the occurrence of virus-induced hepatocellular carcinoma. Conventional purification methods for the vaccine antigen follow a laborious and expensive downstream processing. Bioaffinity chromatography using monoclonal antibodies (MAbs) as ligand and NHS-activated Sepharose as support has been optimized in this study for a simple, one-step purification of rHBsAg. With a maximum coupling strength achieved at pH 8.3, an average recovery of ≥ 90% purified rHBsAg protein was eluted at acidic pH followed by a quick neutralization, without affecting its biological activity. The operational binding capacity of the coupled gel was estimated to be 50µg of rHBsAg per ml. The purified antigen was demonstrated for its antigen-specificity, protein purity and insignificant levels of leached ligand and host cell proteins. Permissible levels of endotoxin, DNA and carbohydrate contents were recorded, while the lipid content was slightly more than the recommendations for a Hepatitis B vaccine dose. Further improvement of the bioaffinity purification protocol with a delipidation step or detergent treatment can be explored to enhance the quality requirements of the purified rHBsAg as a vaccine. The maximum antigen recovery and the number of purification cycles supported by the chromatographic resin prepared in this study will throw light over the optimized purification method.