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Evaluation of Production Parameters with the Vaccinia Virus Expression System Using Microcarrier Attached HeLa Cells
Author(s) -
Bleckwenn Nicole A.,
Bentley William E.,
Shiloach Joseph
Publication year - 2008
Publication title -
biotechnology progress
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.572
H-Index - 129
eISSN - 1520-6033
pISSN - 8756-7938
DOI - 10.1021/bp0498443
Subject(s) - microcarrier , vaccinia , hela , virology , virus , biology , production (economics) , cell culture , microbiology and biotechnology , chemistry , recombinant dna , genetics , gene , macroeconomics , economics
Parameters that affect production of the recombinant reporter protein, EGFP, in the T7 promoter based VOTE vaccinia virus‐HeLa cell expression system were examined. Length of infection phase, inducer concentration, and timing of its addition relative to infection were evaluated in 6‐well plate monolayer cultures. One hour infection with 1.0 mM IPTG added at the time of infection provided a robust process. For larger scale experiments, anchorage‐dependent HeLa cells were grown on 5 g/L Cytodex 3 microcarriers. The change to this dynamic culture environment, with cell‐covered microcarriers suspended in culture medium in spinner flasks, suggested a re‐examination of the multiplicity of infection (MOI) for this culture type that indicated a need for an increase in the number of virus particles per cell to 5.0, higher than that needed for complete infection in monolayer tissue flask culture. Additionally, dissolved oxygen level and temperature during the protein production phase were evaluated for their effect on EGFP expression in microcarrier spinner flask culture. Both increased dissolved oxygen, based on surface area to volume (SA/V) adjustments, and decreased temperature from 37 to 31 °C showed increases in EGFP production over the course of the production phase. The level of production achieved with this system reached approximately 17 μg EGFP/10 6 infected cells.