J Bacteriol Virol.  2004 Sep;34(3):213-221.

Evaluation of JEV and BVDV Clearance During the Purification of Recombinant HPV-16 L1 Virus-Like Particles

Affiliations
  • 1Department of Biologics Evaluation, Korea Food and Drug Administration, 5 Nokbun-dong Eunpyung-gu, Seoul, 122-704, Korea. suenie@kfda.go.kr

Abstract

Insect cell-derived biotechnological products have a potential for viral contamination from cell line sources themselves or from adventitious introduction of virus during production. The objective of this study was to establish techniques for viral clearance validation of insect cell-derived recombinant human papillomavirus (HPV)-16 type L1 virus-like particles (VLPs) using Japanese encephalitis virus (JEV) and bovine viral diarrhea virus (BVDV) as relevant viruses. The downstream process for the production of recombinant HPV-16 L1 VLPs was sequentially carried out employing detergent lysis (NP-40/PBS), sonication, sucrose cushion centrifugation, and cesium chloride (CsCl) equilibrium density centrifugation. Recombinant HPV-16 L1 capsid protein (56 kD) expressed in Sf9 cell culture was clearly detected by SDS-PAGE and Western blotting analysis. Each purification step was evaluated to determine reduction factor for viral clearance by infectivity assay. In individual purification steps, detergent treatment (0.50% v/v, NP-40/PBS) and CsCl equilibrium density centrifugation were found to be effective in JEV and BVDV clearance. Overall cumulative reduction factors of JEV and BVDV infectivity titer for the purification procedure implemented in this study were 12.53 and 10.05 log TCID(50)/pool, respectively. The results suggest that the purification procedure employed in this study for the HPV-16 L1 VLPs produced from recombinant baculovirus-infected Sf9 cells will be effective over 10 log TCID(50)/pool reduction factor in the clearance of enveloped, adventitious viruses with a buoyant density lower than approximately 1.23 g/ml.

Keyword

Recombinant HPV-16 L1 VLPs; Viral clearance; JEV; BVDV

MeSH Terms

Blotting, Western
Capsid Proteins
Cell Line
Centrifugation
Cesium
Detergents
Diarrhea
Electrophoresis, Polyacrylamide Gel
Encephalitis Virus, Japanese
Human papillomavirus 16*
Humans
Insects
Sf9 Cells
Sonication
Sucrose
Capsid Proteins
Cesium
Detergents
Sucrose
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