TY - JOUR

T1 - Purification of hepatitis B surface antigen virus-like particles from recombinant Pichia pastoris and in vivo analysis of their immunogenic properties

AU - Gurramkonda, Chandrasekhar

AU - Zahid, Maria

AU - Nemani, Satish Kumar

AU - Adnan, Ahmad

AU - Gudi, Satheesh Kumar

AU - Khanna, Navin

AU - Ebensen, Thomas

AU - Lünsdorf, Heinrich

AU - Guzmán, Carlos A.

AU - Rinas, Ursula

N1 - Funding Information: This work was supported by institutional core funds of the Helmholtz Centre for Infection Research and ICGEB and an Indo-German collaborative grant ( International Bureau of the BMBF, DLR , IND 03/009). Maria Zahid and Ahmad Adnan wish to express their gratitude to the Deutscher Akademischer Austauschdienst (DAAD) of Germany and the Higher Education Commission (HEC) of Pakistan for their fellowships.

PY - 2013/12/1

Y1 - 2013/12/1

N2 - 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.

AB - 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.

KW - Aerosil-380

KW - Electron microscopy

KW - Hepatitis B surface antigen virus-like particles

KW - Ion-exchange chromatography

KW - Size-exclusion chromatography

KW - Ultracentrifugation

KW - Vaccine

UR - http://www.scopus.com/inward/record.url?scp=84886255487&partnerID=8YFLogxK

U2 - 10.1016/j.jchromb.2013.09.030

DO - 10.1016/j.jchromb.2013.09.030

M3 - Article

C2 - 24141044

AN - SCOPUS:84886255487

VL - 940

SP - 104

EP - 111

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

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

SN - 1570-0232

ER -