Abstract
Following earlier studies on high-level intracellular production of hepatitis B surface antigen (HBsAg) using recombinant Pichia pastoris, we present here in detail an enhanced method for the purification of recombinant HBsAg virus-like particles (VLPs). We have screened various detergents for their ability to promote the solubilization of recombinant intracellular HBsAg. In addition, we have analyzed the effect of cell disruption and extraction regarding their impact on the release of HBsAg. Our results show that introduction of the mild nonionic detergent Tween 20 in the initial process of cell lysis at ∼600bars by high pressure homogenization leads to the best results. The subsequent purification steps involved polyethylene glycol precipitation of host cell contaminants, hydrophobic adsorption of HBsAg to colloidal silica followed by ion-exchange chromatography and either isopycnic density ultracentrifugation or size exclusion chromatography for the recovery of the VLPs. After final KSCN treatment and dialysis, a total yield of ∼3% with a purity of >99% was reached. The pure protein was characterized by electron microscopy, showing the presence of uniform VLPs which are the pre-requisite for immunogenicity. The intramuscular co-administration of HBsAg VLPs, with either alum or a PEGylated-derivative of the toll-like receptor 2/6 agonist MALP-2, to mice resulted in the elicitation of significantly higher HBsAg-specific IgG titers as well as a stronger cellular immune response compared to mice vaccinated with a gold standard vaccine (Engerix™). These results show that P. pastoris derived HBsAg VLPs exhibit a high potential as a superior biosimilar vaccine against hepatitis B.
Highlights
The development of a safe recombinant hepatitis B vaccine has led to the inclusion of hepatitis B vaccination in the national infant immunization schedules of approximately 160 countries [1]
Recently it was shown that hepatitis B surface antigen (HBsAg) – when produced in yeast, e.g. P. pastoris – does not assemble into virus-like particles (VLPs) within the cell as was assumed previously nor does it insert in significant amounts into endoplasmic reticulum (ER) membranes as was proposed
Evidence has been presented that the HBsAg remains in the endoplasmic reticulum (ER) where it does not form VLPs but where a major fraction assembles into well-ordered multi-layered lamellar structures [24]
Summary
The development of a safe recombinant hepatitis B vaccine has led to the inclusion of hepatitis B vaccination in the national infant immunization schedules of approximately 160 countries [1]. Recombinant DNA technology was used to produce hepatitis B surface antigen (HBsAg) in form of virus-like particles (VLPs) using the yeast Saccharomyces cerevisiae leading to the development of a so-called “second”. Generation hepatitis B vaccine and the first recombinant subunit vaccine available [2]. This formulation of the hepatitis B vaccine has been on the market since 1986. “third” generation “biosimilar” recombinant HBsAg VLP-based vaccines are being introduced into the market by a variety of new manufacturers which try to make the vaccine more affordable to developing countries [2]. The purification of recombinant HBsAg from yeast cultures is well documented [3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25],
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