Merck

Development of EV71 virus-like particle purification processes.

Vaccine (2015-05-06)
Shih-Yeh Lin, Hsin-Yi Chiu, Bor-Luen Chiang, Yu-Chen Hu
ABSTRACT

Enterovirus 71 (EV71) causes the outbreaks of hand-foot-and-mouth disease and results in deaths of hundreds of young children. EV71 virus-like particles (VLPs) are empty capsids consisting of viral structural proteins and can elicit potent immune responses, thus holding promise as an EV71 vaccine candidate. However, an efficient, scalable production and purification scheme is missing. For mass production of EV71 VLPs, this study aimed to develop a production and chromatography-based purification process. We first demonstrated the successful EV71 VLPs production in the stirred-tank bioreactor in which High Five™ cells were infected with a recombinant baculovirus co-expressing EV71 structural polyprotein P1 and protease 3CD. The culture supernatant containing the VLPs was subjected to tangential flow filtration (TFF) for concentration/diafiltration, which enabled the removal of >80% of proteins while recovering >80% of VLPs. The concentrated VLPs were next subjected to hydroxyapatite chromatography (HAC) in which the VLPs were mainly found in the flow through. After another TFF concentration/diafiltration, the VLPs were purified by size-exclusion chromatography (SEC) and concentrated/diafiltered by a final TFF. The integrated process yielded an overall VLPs recovery of ≈ 36% and a purity of ≈ 83%, which was better or comparable to the recovery and purity for the purification of live EV71 virus particles. This process thus may move the EV71 VLPs vaccine one step closer to the clinical applications.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
3,3′,5,5′-Tetramethylbenzidine, ≥98.0% (NT)
Sigma-Aldrich
3,3′,5,5′-Tetramethylbenzidine, ≥99%
Sigma-Aldrich
Nitrotetrazolium Blue chloride, powder, electrophoresis grade
Sigma-Aldrich
Nitrotetrazolium Blue chloride, ≥90.0% (HPLC)
Sigma-Aldrich
3,3′,5,5′-Tetramethylbenzidine, ≥98% (TLC)
Sigma-Aldrich
3,3′,5,5′-Tetramethylbenzidine, tablet, 1 mg substrate per tablet