Evaluation of Production Parameters with the Vaccinia Virus Expression System Using Microcarrier Attached HeLa Cells Nicole A. Bleckwenn, †,‡,§ William E. Bentley, ‡,§ and Joseph Shiloach* ,† Biotechnology Unit, NIDDK, National Institutes of Health, DHHS, Bldg 14A Rm 176, 9000 Rockville Pike, Bethesda, Maryland 20892 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. Introduction Vaccinia virus infection of HeLa cell culture has shown potential as a recombinant protein production method (1). The current literature on growth and infection of HeLa cells in large scale culture is limited (2, 3). This study investigates several parameters that may affect produc- tion of recombinant protein in small scale monolayer tissue plate culture and then extends these results into microcarrier-based culture of HeLa cells in spinner flasks to obtain basic information on how to best cultivate and infect these cells to express recombinant proteins on a larger scale. Microcarriers provide solid support onto which anchorage-dependent cells attach and grow (4-7), but also the carriers can be suspended in culture medium and treated similarly to suspension culture, which makes this method of cultivation amenable to scale-up in conventional stirred tank bioreactors. Enhanced green fluorescent protein (EGFP) was used as a model protein with this viral expression system. Green fluorescent protein has previously been used as a model for studying protein production with other expres- sion methods, such as E. coli (8, 9), the insect cell or insect larvae-baculovirus system (10-14), and mammalian cell systems (15, 16) because its expression levels are easily quantified via fluorescence spectrophotometry. The VOTE vaccinia expression system (17) was used to control expression of the EGFP reporter gene, where the gene is introduced into the cytoplasm of the cell via the viral infection and protein is made upon induction with isopropyl--D-thiogalactopyranoside (IPTG). The VOTE construct is a T7 promoter based system (18) where all of the control components are present in a single virus, unlike other vaccinia constructs that require dual infec- tion (3, 19). Expression is tightly controlled by the addition of the inducing agent, IPTG, allowing for controlled expression of the protein of interest, which is advantageous when the protein to be produced is detri- mental to the cell. Previous work in our lab evaluated several basic infection characteristics with this reporter virus in small scale tissue flask experiments, which included multiplic- ity of infection (MOI) and both volume of medium and serum concentration during the infection phase. With this viral expression method, there are three main phases to consider: growth phase (where the cells are seeded and grown to sufficient density prior to infection), infection phase (where the cells are exposed to virus in a reduced volume, serum-free medium environment in order to synchronously infect the entire population of cells), and production phase (the post-infection incubation where production occurs after adjusting the medium volume and serum concentration back to their full levels). The current work examines parameters in each of these phases in order to enhance the production capability of the vaccinia- HeLa expression method. We present an evaluation of several parameters in small scale monolayer culture in well plates, increasing the number of conditions tested. For these experiments, parameters including MOI, vol- ume during infection, and serum concentration during * To whom correspondence should be addressed. Email: yossi@ nih.gov. † National Institutes of Health. ‡ Dept. of Chemical Engineering, University of Maryland, College Park, MD. § Center for Biosystems Research, University of Maryland Biotechnology Institute, College Park, MD. 554 Biotechnol. Prog. 2005, 21, 554-561 10.1021/bp0498443 This article not subject to U.S. Copyright. Published 2005 by the American Chemical Society and American Institute of Chemical Engineers Published on Web 01/22/2005