SAFCB 2006

Optimization of Serum-Free Media for EB14 Cell Growth and Viral Production
SAFC Biosciences
Coleen McCormick, Jeanette Hartshorn, Chas Hernandez, Sandy McNorton, 13804 W. 107th Street
Lenexa, KS 66215
Gaurav Chauhan, Elisa Atarod, Majid Mehtali1, and Matt Caple USA

Abstract
Many human and animal vaccines are currently being developed and produced in eggs Single Radial Immunodiffusion Assay (SRID) B/Jiansu/10/2003 (3L) A/H3N2/New York/55/2004 (3L) A/H1N1/New Caledonia/20/99 (20L)
Day 1 Day 2 Day 3 Day 4 Day 5 Day 6
or primary chicken embryonic fibroblasts (CEF). However, there are known limitations SRID analysis was performed following standard protocols to determine hemmaglutinin
to these platforms, including challenges in both handling and scaling. Consequently, (H.A.) concentration (Wood, et. al., 1977). Human influenza anti-sera was obtained 10 1.60E+07
continuous cell lines and the use of animal-component free media show exciting promise and used at concentrations recommended by NIBSC. Dose-response curves of antigen 9
1.40E+07
for vaccine manufacturers. EB14 is a novel suspension cell line derived from avian dilutions against the surface were constructed and the results were calculated according 8
1.20E+07
embryonic stem cells and has been identified as a permissive host for pox and influenza to standard slope-ratio assay methods.
7
viruses in cell culture. EB14 is a well characterized, genetically stable cell line which 1.00E+07
Analytical Methods 6
has been adapted to serum-free media and can be cultured using industrial processes
A 8.00E+06
including stirred tank bioreactors. The present study identifies optimized serum-free Cell density and viability was determined by Cedex (Innovatis, Bielefeld, Germany) and 5
media formulations, feed strategies and cell culture processes that support robust growth by trypan blue exclusion method with hemacytometer. Metabolic consumption and 6.00E+06
4

Log TCID 50 /mL

and viral productivity in the EB14 cell line. production in uninfected cultures was monitored offline with the BioProfile® 100 (Nova 4.00E+06

Viable Cell Density/mL

3
Biomedical Corporation, Waltham, MA; data not shown).
2 2.00E+06

1 0.00E+00
Introduction 0 -4 -3 -2 -1 n n 1 2 3 4 5 6 7 8
Results y y y y tio tio y y y y y y y y
SAFC Biosciences (www.safcbiosciences.com) and Vivalis (www.vivalis.com) formed a Da Da Da Da il u il u Da Da Da Da Da Da Da Da
Feed 2 (n=2) Com plete (n=2) Com petitor X (n=3) D -D
e- st
scientific partnership to develop a novel cell culture-based vaccine platform. Vivalis has Pr Po
7.00E+06 100 Production M e dia
taken advantage of its expertise in avian biology and embryonic stem cells to develop 90
6.00E+06
Figure 3: Infectious MVA Production in EX-CELLTM EBxTM as a Complete Medium Compared to Day 3 Day 4 Day 5 Day 6 Day 7 Day 8
fully characterized and documented cell lines that are permissive to a variety of viruses. Competitor X Medium.
80
SAFC Biosciences has drawn on its expertise in media development to generate an EB14 cells were seeded at 0.2E6 cells/mL in EX-CELLTM EBxTM or competitor X media. After 48 hours, the
5.00E+06 70 50
offering of serum-free media that support robust growth and viral production in the cells were infected with MVA at and M.O.I. of 10-2 and production medium was added. The supplements
EB14 cell line. The EX-CELLTM EBxTM media allows consistently high growth and viral titers 60 contained in Feed 2 can be incorporated directly into the production medium at the time of manufacture, 45
4.00E+06
resulting in a similar increase in viral titers.
in scales ranging from flasks to bioreactors. This cell line provides a novel serum-free, 50 40
animal component-free, cell culture-based platform for vaccine production. 3.00E+06
40 35

Percent Viablity
2.00E+06 30 30

Viable Cell Density/m L

20 B 25
Materials and Methods 1.00E+06
10 20
Cells 0.00E+00 0
15
HA Antigen µg/mL

EB14 is a novel suspension cell line that was derived from avian embryonic stem (ES) cells Day 0 Day 1 Day 2 Day 3 Day 4 Day 5
using a proprietary process by Vivalis. EB14 cells maintain some of the unique features of Da ys Post-Se e ding 10
ES cells, such as a strong constitutive expression of telomerase. Furthermore, EB14 cells Bior e actor 1 (0.4e 6/m L) Bior e actor 2 (0.2e 6/m L) Bior e actor 3 (0.1e 6/m L) Com pe titor X (0.1e 6/m L)
5
are diploid, undifferentiated, non-tumorigenic and genetically stable. Stock cultures 0
Bior e actor 1 (0.4e 6/m L) Bior e actor 2 (0.2e 6/m L) Bior e actor 3 (0.1e 6/m L) Com pe titor X (0.1e 6/m L)
were maintained in a 37 ºC, 7.5% CO2 humidified incubator. EB14 cells were cultured B/Jiansu/10/2003 (3L) A/H3N2/New A/H1N1/New
TM TM
in 125 mL Erlenmeyer flasks (25 mL culture volumes) at 90 rpm. Figure 1: Growth and Viability of EB14 Cells in EX-CELL EBx and Competitor X Medium in York/55/2004 (3L) Caledonia/20/99 (20L)
Stirred Tank Bioreactors.
Cell Culture Media EB14 cells were seeded into 3L Applikon stirred tank bioreactors at 0.1E6, 0.2E6, and 0.4E6 cells/mL. Cell
Figure 6: Human Influenza Hemagglutinin Production in EX-CELLTM EBxTM.
density and viability were monitored for five days following seeding. EB14 cells reached a significantly Figure 4: MVA-GFP Infected EB14 Cells in EX-CELLTM EBxTM in Flasks.
Stock cultures were maintained in EX-CELLTM EBxTM (SAFCB Item No. 63066) and Medium EB14 cells were seeded at 0.4E6 cells/mL in EX-CELLTM EBxTM in 3L or 20L bioreactors as indicated in the
higher maximum cell density and demonstrated greater longevity in EX-CELLTM EBxTM when compared EB14 cells were seeded at 0.4E6 cells/mL in EX-CELLTM EBxTM. After 48hrs, cells were infected with MVA-
X. Both media were supplemented with 2.5 mM L-glutamine (Catalog No. 59202C). legends. After 72 to 96 hours, cells were infected with the indicated strains of human influenza at an
to Medium X. EB14 cells seeded in competitor Medium X had a significant drop in cell viability by day 5 GFP at an M.O.I. of 10-2 and production medium was added. A sample was removed and examined for M.O.I. of 10-4. Feed 2 was added 1 hour later and samples were pulled daily to monitor viable cell density
Base medium (SAFCB Item No. 65421) and complete medium (SAFCB Item No. 65946) (followed by a drop in cell density on day 6, data not shown). fluorescence at 10X magnification 4 days post-infection. (Panel A) and production of H.A. antigen (Panel B). All three bioreactors reached at least 8E6 cells/mL
were used for viral production. All items except Medium X are from SAFC Biosciences, following infection and produced at least 30 µg/mL of HA as determined by SRID.
Lenexa, Kansas.
Day 1 Day 3 Day 5 Day 6 Day 7 Day9
10 A/H3N2/Johannesburg
10 /33/94
Process Development 9 A/H3N2/Wyom ing
9 /3/2003
Bioreactor runs were conducted in 3L stirred tank bioreactors (Applikon® Biotechnology, 8 A/H3N2/New York Conclusions
Sciedam, Holland). Bioreactors were seeded at 0.1, 0.2, and 0. cells/mL in EX-CELLTM /55/2005
8 7 A/H1N1/New Caledonia • EX-CELLTM EBxTM is a serum-free, animal-component free medium for EB14 cells in
EBxTM and Medium X. DO, pH and temperature were monitored and controlled. /20/99
7 6 A/H1N1/Beijing/262/95
suspension culture.
Uninfected samples were collected daily to monitor cell density, viability and metabolic
consumption/production (data not shown). Infected samples were collected daily to 6 5 B/Yam anashi/166/88
• EX-CELLTM EBxTM supports higher cell density, improved viability and increased culture
4 B/Shandong/7/97

Log TCID50/mL
monitor viral production by TCID50 endpoint dilution, hemmaglutinin production, and 5 longevity when compared with a competitor medium.
fluorescent microscopy. 3 B/Harbin7/94
4 • EX-CELLTM EBxTM supports MVA and Influenza Virus production

LogTCID50/mL
2 B/Jiangsu/10/2003
Viral Infection 3
1 B/Johannesburg/5/99 • Optimization of key components, feeds, and metabolic analysis has led to the
EB14 cells were seeded at 0.1 to 0.4E6 cells/mL forty eight to seventy two hours prior to
2 0 A/H1N1/Texas/36/9 development of media that exhibit robust growth and viral productivity.
infection in 125 mL Erlenmeyer flasks or 3L stirred tank bioreactors. Cells were inoculated
0h 24h 48h 72h 96h 120h 144h 168h 192h 216h
with an multiplicity of infection (M.O.I.) of 10-2 for MVA and 10-4 for the indicated strains 1
Days Post-Infection
of human influenza and allowed to adsorb for 1 hour at 37 °C or 34 °C, respectively.
0
After 1 hour, production media (with or without feeds) was added and the cultures were Base (n=5) Feed 1 (n=2) Feed 2 (n=2) Competitor X (n=3) Figure 5: Infectious Human Influenza Production in EX-CELLTM EBxTM in Flasks.
EB14 cells were seeded at 0.4E6 cells/mL in EX-CELLTM EBxTM. After 48 hrs, cells were infected with the
Acknowledgements
incubated at the appropriate temperature for an additional 5 to 9 days. Production Media
indicated strains of human influenza at an M.O.I. of 10-4. Feed 2 was added 1 hour later and samples The authors would like to thank Cell Sciences and Development (SAFC Biosciences) and
Figure 2: Infectious MVA Production in EX-CELLTM EBxTM with and without Feeds Compared to were pulled daily to monitor viable cell density (data not shown) and production of infectious virus. High the Research and Development (Vivalis) teams, for without their dedicated efforts and
Titration of Viruses by TCID50 Endpoint Dilution Competitor X Medium. viral titers were obtained with all strains of human influenza tested. expertise this project would not have been possible.
TCID50 analysis was performed following standard protocols (Reed and Muench, 1938). EB14 cells were seeded at 0.2E6 cells/mL in EX-CELLTM EBxTM or competitor X media. After 48 hours,
Briefly, 100 µL of viral sample serial dilutions (10-4 to 10-11) were dispensed in replicates the cells were infected with MVA at an M.O.I. of 10-2 and production medium was added. The feeds
were also added at the time of infection. Feed 1 increased the viral titer over the base formulation titer
and one row was left as a control in which no virus was added. The titer was then
by 2 logs.
calculated by the Reed-Muench method. DF-1 and MDCK cells were used for MVA and References
human influenza strains, respectively. EX-CELLTM is a trademark of SAFC Biosciences.
EBxTM is a trademark of Vivalis.

Footnote
1. Vivalis, Nantes, France

001214