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Biotechnol Bioeng 2005 Jun 20;90(6):663-74

Production of recombinant proteins by vaccinia virus in a microcarrier based mammalian cell perfusion bioreactor.

Bleckwenn NA, Golding H, Bentley WE, Shiloach J

Bleckwenn NA, NIDDK, Biotechnol Unit, DHHS, NIH, Bldg 14A Rm 173,MSC 5522,9000 Rockville Pike, Bethesda, MD USA NIDDK, Biotechnol Unit, DHHS, NIH, Bethesda, MD USA US FDA, Div Viral Prod, Ctr Biol Evaluat & Res, Bethesda, MD USA Univ Maryland, Dept Chem Engn, College Pk, MD 20742 USA Univ Maryland, Inst Biotechnol, Ctr Biosyst Res, College Pk, MD USA

Abstract

The HeLa cell-vaccinia virus expression system was evaluated for the production of recombinant proteins (enhanced green fluorescent protein (EGFP) and HIV envelope coat protein, gp120) using microcarriers in 1.5 L perfused bioreactor cultures. Perfusion was achieved by use of an alternating tangential flow device (ATF), increasing the length of the exponential phase by 50 h compared to batch culture and increasing the maximum cell density from 1.5x10(6) to 4.4x10(6) cell/mL. A seed train expansion method using cells harvested from microcarrier culture and reseeding onto fresh carriers was developed. EGFP was first used as a model protein to study process parameters affecting protein yield, specifically dissolved oxygen (DO) and temperature during the production phase. The highest level of EGFP, 12+/-1.5 microg/10(6) infected cells, was obtained at 50% DO and 31 degrees C. These setpoints were then used to produce glycoprotein, gp120, which was purified and deglycosylated, revealing a significant amount of N-linked glycosylation. Also, biological activity was assayed, resulting in an ID50 of 3.1 microg/mL, which is comparable to previous reports.


Category: Journal Article
PubMed ID: #15858791
Includes FDA Authors from Scientific Area(s): Biologics
Entry Created: 2011-10-04 Entry Last Modified: 2012-08-29
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