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

verfasst von

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.

Organisationseinheit(en)

Institut für Technische Chemie

Externe Organisation(en)

Helmholtz-Zentrum für Infektionsforschung GmbH (HZI)
International Centre for Genetic Engineering and Biotechnology
University of Maryland Baltimore County

Typ

Artikel

Journal

Journal of Chromatography B: Analytical Technologies in the Biomedical and Life Sciences

Band

940

Seiten

104-111

Anzahl der Seiten

8

ISSN

1570-0232

Publikationsdatum

01.12.2013

Publikationsstatus

Veröffentlicht

Peer-reviewed

Ja

ASJC Scopus Sachgebiete

Analytische Chemie, Biochemie, Klinische Biochemie, Zellbiologie

Ziele für nachhaltige Entwicklung

SDG 3 – Gute Gesundheit und Wohlergehen

Elektronische Version(en)

https://repository.helmholtz-hzi.de/bitstream/handle/10033/305192/Gurramkonda%20et%20al_final.pdf?sequence=1 (Zugang: Geschlossen)
https://doi.org/10.1016/j.jchromb.2013.09.030 (Zugang: Geschlossen)