title: Production of recombinant proteins by vaccinia virus in a microcarrier based mammalian cell perfusion bioreactor.
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.
authors.affiliation: Biotechnology Unit, NIDDK, National Institutes of Health, DHHS, Bldg. 14A Rm. 173, MSC 5522, 9000 Rockville Pike, Bethesda, Maryland, USA.
authors.forename: Nicole A
authors.initials: NA
authors.lastname: Bleckwenn
authors.forename: Hana
authors.initials: H
authors.lastname: Golding
authors.forename: William E
authors.initials: WE
authors.lastname: Bentley
authors.forename: Joseph
authors.initials: J
authors.lastname: Shiloach
mesh_terms: D019149:Bioreactors; D018929:Cell Culture Techniques / Q000295:instrumentation / Q000379:methods*; D005240:Feasibility Studies; D015699:HIV Envelope Protein gp120 / Q000032:analysis / Q000096:biosynthesis* / Q000235:genetics; D006367:HeLa Cells; D006801:Humans; D015202:Protein Engineering / Q000379:methods*; D011994:Recombinant Proteins / Q000096:biosynthesis*; D014616:Vaccinia virus / Q000502:physiology*; D014764:Viral Proteins / Q000096:biosynthesis / Q000235:genetics; D014776:Virus Cultivation / Q000379:methods
chemical_list: D015699:HIV Envelope Protein gp120; D011994:Recombinant Proteins; D014764:Viral Proteins