TY - JOUR

T1 - Assessing stability and assembly of the hepatitis B surface antigen into virus-like particles during down-stream processing

AU - Zahid, Maria

AU - Lünsdorf, Heinrich

AU - Rinas, Ursula

N1 - Funding Information: Maria Zahid wishes to express her gratitude to the Deutscher Akademischer Austauschdienst (DAAD) of Germany and the Higher Education Commission (HEC) of Pakistan for her fellowship. Publisher Copyright: © 2015 Elsevier Ltd.

PY - 2015/7/17

Y1 - 2015/7/17

N2 - The hepatitis B surface antigen (HBsAg) is a recombinant protein-based vaccine being able to form virus-like particles (VLPs). HBsAg is mainly produced using yeast-based expression systems, however, recent results strongly suggest that VLPs are not formed within the yeast cells during the cultivation but are formed in a gradual manner during the following down-stream procedures. VLPs are also not detectable during the first down-stream steps including mechanical and EDTA/detergent-assisted cell destruction. Moreover, VLPs are not detectable in the cell lysate treated with polyethylene glycol and colloidal silica. The first VLP resembling structures appear after elution of HBsAg from colloidal silica to which it binds through hydrophobic interaction. These first VLP resembling structures are non-symmetrical as well as heterodisperse and exhibit a high tendency toward cluster formation presumably because of surface exposed hydrophobic patches. More symmetrical and monodisperse VLPs appear after the following ion-exchange and size-exclusion chromatography most likely as the result of buffer changes during these purification steps (toward more neutral pH and less salt). Final treatment of the VLPs with the denaturant KSCN at moderate concentrations with following KSCN removal by dialysis does not cause unfolding and VLP disassembly but results in a re- and fine-structuring of the VLP surface topology.

AB - The hepatitis B surface antigen (HBsAg) is a recombinant protein-based vaccine being able to form virus-like particles (VLPs). HBsAg is mainly produced using yeast-based expression systems, however, recent results strongly suggest that VLPs are not formed within the yeast cells during the cultivation but are formed in a gradual manner during the following down-stream procedures. VLPs are also not detectable during the first down-stream steps including mechanical and EDTA/detergent-assisted cell destruction. Moreover, VLPs are not detectable in the cell lysate treated with polyethylene glycol and colloidal silica. The first VLP resembling structures appear after elution of HBsAg from colloidal silica to which it binds through hydrophobic interaction. These first VLP resembling structures are non-symmetrical as well as heterodisperse and exhibit a high tendency toward cluster formation presumably because of surface exposed hydrophobic patches. More symmetrical and monodisperse VLPs appear after the following ion-exchange and size-exclusion chromatography most likely as the result of buffer changes during these purification steps (toward more neutral pH and less salt). Final treatment of the VLPs with the denaturant KSCN at moderate concentrations with following KSCN removal by dialysis does not cause unfolding and VLP disassembly but results in a re- and fine-structuring of the VLP surface topology.

KW - Aerosil-380

KW - Hepatitis B surface antigen virus-like particles

KW - Ion-exchange chromatography

KW - Purification

KW - Size-exclusion chromatography

KW - Transmission electron microscopy

KW - Vaccine

UR - http://www.scopus.com/inward/record.url?scp=84937635544&partnerID=8YFLogxK

U2 - 10.1016/j.vaccine.2015.05.066

DO - 10.1016/j.vaccine.2015.05.066

M3 - Article

C2 - 26079614

AN - SCOPUS:84937635544

VL - 33

SP - 3739

EP - 3745

JO - Vaccine

JF - Vaccine

SN - 0264-410X

IS - 31

ER -