Purification of hepatitis B surface antigen virus-like particles from recombinant Pichia pastoris and in vivo analysis of their immunogenic properties

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.

Non-conventional expression systems for the production of vaccine proteins and immunotherapeutic molecules

“…It shows low viscosity morphology, allowing easy, large-scale, high-cell-density growth in fermenters using inexpensive substrates. Furthermore, this fungus supports broad ranges of pH (4.5-7.0) and temperature (25)(26)(27)(28)(29)(30)(31)(32)(33)(34)(35)(36)(37)(38)(39)(40)(41)(42)(43)(44) C) for growth. A C1 genetic toolbox is available, and the 3eight megabase pair genome has been sequenced, enabling the development of various gene expression strategies.…”

“…In 2002, P. pastoris was approved in 2002 by the World Health Organization (WHO) for the production of an HBV vaccine biosimilar by Shantha Biotechnologies (a Sanofi company), and this biosimilar has recently been shown to be potentially more effective than vaccines produced in S. cerevisiae in particular patient groups. 33,34 Hyper-mannosylation, a well-known limitation of conventional yeast systems, is weaker in P. pastoris, which has been engineered to produce human-like glycosylation of recombinant proteins by knocking out 4 glycosylationassociated genes and introducing more than 14 heterologous genes. 35,36 In 2004, this technique resulted in the development of Glycoswitch Ò from Merck, a technology allowing for uniform Man5 N-glycosylation and development of a set of vectors to express glycan-modifying enzymes in yeast strains not limited to P. pastoris.…”

Assessing stability and assembly of the hepatitis B surface antigen into virus-like particles during down-stream processing

“…The first VLP-like structures appeared after elution of proteins bound to colloidal silica ( Figure 1D). Binding of HBsAg to colloidal silica (Aerosil-380) in clarified PEG extracts at neutral pH through hydrophobic adsorption represents an important step during purification as the elution of bound proteins from colloidal silica already leads to a HBsAg preparation with a purity of 60-70% [37]. An electron microscopic investigation of this eluate revealed that the size of these irregular VLP-like structures were in the range of HBsAg VLPs but clearly neither uniform in size nor in shape ( Figure 1D).…”

“…However, the size-exclusion chromatography step removed remaining host cell proteins as the HBsAg VLPs elute with the void volume and host cell contaminants elute afterwards [37].…”

“…The layering order of HBsAg in these lamellar structures strongly suggested the presence of well-ordered HBsAg subunits [28], which should be solubilizable without getting structurally disordered to reassemble into VLPs under appropriate conditions. Purification of Pichia-derived HBsAg using the protocol also employed in this study was finally leading to well-defined VLPs [7;28;37] with excellent immunogenic properties [37].…”