This article is a US Government work and, as such, is in the public domain in the United States of America.
Production of recombinant proteins by vaccinia virus in a microcarrier based mammalian cell perfusion bioreactor†
Article first published online: 27 APR 2005
Published 2005 Wiley Periodicals, Inc.
Biotechnology and Bioengineering
Volume 90, Issue 6, pages 663–674, 20 June 2005
How to Cite
Bleckwenn, N. A., Golding, H., Bentley, W. E. and Shiloach, J. (2005), Production of recombinant proteins by vaccinia virus in a microcarrier based mammalian cell perfusion bioreactor. Biotechnol. Bioeng., 90: 663–674. doi: 10.1002/bit.20423
- Issue published online: 16 MAY 2005
- Article first published online: 27 APR 2005
- Manuscript Accepted: 3 DEC 2004
- Manuscript Received: 13 MAY 2004
- NIDDK, National Institutes of Health, DHHS, Bethesda, MD
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 1.5 × 106 to 4.4 × 106 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 μg/106 infected cells, was obtained at 50% DO and 31°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 μg/mL, which is comparable to previous reports. Published 2005 Wiley-Periodicals, Inc.