title: 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.
authors.affiliation: Department of Structural Biology, Helmholtz Centre for Infection Research, Braunschweig, Germany; International Centre for Genetic Engineering and Biotechnology, New Delhi, India; Technology Research Centre, Department of Chemical, Biochemical and Environmental Engineering, University of Maryland Baltimore County, Baltimore, USA. Electronic address: chskrg@umbc.edu.
authors.forename: Chandrasekhar
authors.initials: C
authors.lastname: Gurramkonda
authors.forename: Maria
authors.initials: M
authors.lastname: Zahid
authors.forename: Satish Kumar
authors.initials: SK
authors.lastname: Nemani
authors.forename: Ahmad
authors.initials: A
authors.lastname: Adnan
authors.forename: Satheesh Kumar
authors.initials: SK
authors.lastname: Gudi
authors.forename: Navin
authors.initials: N
authors.lastname: Khanna
authors.forename: Thomas
authors.initials: T
authors.lastname: Ebensen
authors.forename: Heinrich
authors.initials: H
authors.lastname: Lünsdorf
authors.forename: Carlos A
authors.initials: CA
authors.lastname: Guzmán
authors.forename: Ursula
authors.initials: U
authors.lastname: Rinas
mesh_terms: D000818:Animals; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D005260:Female; D006514:Hepatitis B Surface Antigens / Q000737:chemistry / Q000276:immunology* / Q000302:isolation & purification* / Q000378:metabolism; D008807:Mice, Inbred BALB C; D010843:Pichia / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000276:immunology / Q000302:isolation & purification / Q000378:metabolism; D012822:Silicon Dioxide; D018709:Statistics, Nonparametric; D013861:Thiocyanates; D014461:Ultracentrifugation; D014771:Virion / Q000737:chemistry / Q000276:immunology / Q000302:isolation & purification / Q000378:metabolism
chemical_list: D006514:Hepatitis B Surface Antigens; D011994:Recombinant Proteins; D013861:Thiocyanates; D012822:Silicon Dioxide; C009941:potassium thiocyanate
keywords: Aerosil-380; Electron microscopy; Hepatitis B surface antigen virus-like particles; Ion-exchange chromatography; Size-exclusion chromatography; Ultracentrifugation; Vaccine