|Title||Production of recombinant proteins by vaccinia virus in a microcarrier based mammalian cell perfusion bioreactor.|
|Publication Type||Journal Article|
|Year of Publication||2005|
|Authors||Bleckwenn, NA, Golding, H, Bentley, WE, Shiloach, J|
|Date Published||2005 Jun 20|
|Keywords||Bioreactors, Cell Culture Techniques, Feasibility Studies, HeLa Cells, HIV Envelope Protein gp120, Humans, Protein Engineering, Recombinant Proteins, Vaccinia virus, Viral Proteins, Virus Cultivation|
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.
|Alternate Journal||Biotechnol. Bioeng.|