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

authored by: Chandrasekhar Gurramkonda, Maria Zahid, Satish Kumar Nemani, Ahmad Adnan, Satheesh Kumar Gudi, Navin Khanna, Thomas Ebensen, Heinrich Lünsdorf, Carlos A. Guzmán, Ursula Rinas
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 ~600. bars 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.
Organisation(s): Institute of Technical Chemistry
External Organisation(s): Helmholtz Centre for Infection Research (HZI)
International Centre for Genetic Engineering and Biotechnology
University of Maryland Baltimore County
Type: Article
Journal: Journal of Chromatography B: Analytical Technologies in the Biomedical and Life Sciences
Volume: 940
Pages: 104-111
No. of pages: 8
ISSN: 1570-0232
Publication date: 01.12.2013
Publication status: Published
Peer reviewed: Yes
ASJC Scopus subject areas: Analytical Chemistry, Biochemistry, Clinical Biochemistry, Cell Biology
Sustainable Development Goals: SDG 3 - Good Health and Well-being
Electronic version(s): https://repository.helmholtz-hzi.de/bitstream/handle/10033/305192/Gurramkonda%20et%20al_final.pdf?sequence=1 (Access: Closed)
https://doi.org/10.1016/j.jchromb.2013.09.030 (Access: Closed)