Development and Optimization of EX-CELL™ EBx™ Serum-Free Medium

for Virus and Recombinant Protein Production

Coleen McCormick1, Chas Hernandez1, Sandy McNorton1, James S. Ross1, Matthew V. Caple1, Stephane Olivier2 Majid Mehtali2 and Jeanette Hartshorn1
1
Cell Sciences & Development, SAFC Biosciences, Lenexa, Kansas 66215 2Vivalis, Saint-Herblain, Nantes 44800, France
ESACT 2007 Poster#1121

Abstract Results Human Influenza Hemagglutinin Production in EX-CELL™ EBx™

Although egg-based and primary chicken embryonic
fibroblast (CEF) systems are commonly used for human
Growth and Viability of EB14 Cells in EX-CELL™ EBx™ A
and Medium X in Stirred Tank Bioreactors 1.60E+07
and veterinary vaccine production, many limitations exist B/Jiansu (3L)
for these systems, including cumbersome handling and 1.40E+07
7.00E+06 100 A/H3N2/New York (3L)
scaling procedures, supply and contamination issues

Viable Cell Density/mL

90

Viable Cell Density/mL

and the inability to replicate highly virulent strains of 6.00E+06 1.20E+07 A/H1N1/N C (20L)
influenza that are lethal to chickens. With the recent 80

Perfect Viability
5.00E+06 70 1.00E+07
release of updated guidelines for cell-based vaccine
development by the United States Food and Drug 4.00E+06 60 8.00E+06
Administration (FDA), continuous cell lines are becoming 50
an increasingly promising platform to overcome some of 3.00E+06 40 6.00E+06
these limitations associated with traditional methods for 2.00E+06 30
vaccine production. 4.00E+06
20
1.00E+06
A scientific partnership was formed between SAFC 10 2.00E+06
Biosciences and Vivalis. Utilizing EBx™ cells from Vivalis, 0.00E+00 0
Day 0 Day 1 Day 2 Day 3 Day 4 Day 5 0.00E+00
SAFC Biosciences sought to develop a serum-free cell- Pre Post Day 1 Day 2 Day 3 Day 4 Day 5 Day 6 Day 7 Day 8
based platform that would be capable of supporting Days Post-Seeding
the propagation of a diverse range of human and
animal viruses. EBx™ is a novel suspension cell line Bioreactor 1 (0.4e6/m L) Bioreactor 1 (0.4e6/m L) B
derived from avian embryonic stem cells and is highly Bioreactor 2 (0.2e6/m L) Bioreactor 2 (0.2e6/m L) 50
susceptible to human and animal viruses such as Bioreactor 3 (0.1e6/m L) Bioreactor 3 (0.1e6/m L)
poxviruses and influenza A and B viruses. This cell line is 45
Competitor X (0.1e6/m L) Competitor X (0.1e6/m L)
well-characterized, diploid, genetically stable and non- 40

HA Antigen µg/mL
tumorigenic. Further, EBx™ cells can be cultured using Figure 1: EB14 cells reached a significantly higher maximum cell density and demonstrated greater longevity in 35
industrial processes, including stirred-tank bioreactor EX-CELL™ EBx™ Medium when compared to Medium X. EB14 cells seeded in Medium X had a significant drop
systems. 30
in cell viability by day 5 (followed by a drop in cell density on day 6, data not shown)
™
25
Recently, EBx cells have also been shown to be
amenable to genetic engineering which allows 20
the expression of human proteins and monoclonal Infectious MVA Production in EX-CELL™ EBx™ 15
antibodies. EBx™ cells readily adapt from adherent to with and without Feeds Compared to Medium X 10
suspension which eases the selection of the expressing
5
cells and rapid expansion in serum-free conditions. This 10
study will highlight the development and optimization 9 0
B/Jiansu/10/2003 (3L) A/H3N2/New A/H1N1/New
of serum-free media formulations, feed and processes. York/55/2004 (3L) Caledonia/20/99 (20L)
As a result, EX-CELL™ EBx™ Serum-Free Medium was 8
Day 3 Day 4 Day 5 Day 6 Day 7 Day 8
LogTCID50/mL

developed specifically for robust cell growth, viral and 7

recombinant protein production utilizing EBx™ cells for
6 Figure 5: As shown in panel A, all three bioreactors reached at least 8e6 cells/mL following infection and
vaccine and therapeutic manufacturing.
produced at least 30 µg/mL of HA as determined by SRID (panel B)..
5
4
3 Antibody Production in EX-CELL™ EBx™ in Stirred Tank Bioreactors
Introduction
2 1.80E+07 300
SAFC Biosciences and Vivalis formed a scientific
1
partnership to develop a novel cell culture-based Cell Density
1.60E+07
platform for vaccine and recombinant protein IgG
Base Feed 1 Feed 2 Competitor X 250
production. Vivalis has taken advantage of its expertise
in avian biology and embryonic stem cells to develop Production Media 1.40E+07
fully characterized and documented cell lines that Day 1 Day 3 Day 5 Day 6 Day 7 Day 9
are permissive to a variety of viruses and expression 1.20E+07 200
of recombinant proteins. EB14 is a novel suspension

igG mg/ML
Figure 2: Feed 1 increased the viral titer over the base formulation by approximately 1 log, but more
cell line that was derived from avian embryonic importantly, the addition of Feed 2 increased the viral titer by 2 logs. 1.00E+07
stem (ES) cells using a proprietary process by Vivalis.
mL

150
EB14 cells maintain some of the unique features
8.00E+06
of ES cells, such as a strong constitutive expression Infectious MVA Production in EX-CELL EBx ™ ™
of telomerase. Furthermore, EB14 cells are diploid,
as a Complete Medium Compared to Medium X 6.00E+06 100
undifferentiated, non-tumorigenic and genetically
stable. SAFC Biosciences has drawn on its expertise in 10
media development to generate an offering of serum- 9 4.00E+06
free media that support robust growth and production 50
in the EBx™ cell lines. EX-CELL™ EBx™ Medium allows 8
LogTCID50/mL

2.00E+06
consistently high growth and viral and protein titers in 7
scales ranging from flasks to bioreactors. 6
0.00E+00 0
5 Day Day Day Day Day Day Day Day Day Day Day Day Day Day
4 0 1 2 3 4 5 6 7 8 9 10 11 12 13

3
Materials and Methods 2 Figure 6: IgG expression was measured by ELISA in a subclone of EB14 cells stably transfected to express the
recombinant human protein. Peak cell density reached 1.65e7 cells/mL and maximum protein expression was
Cells 1 approximately 240 mg/L. The specific protein productivity was calculated as 10 – 20 pg/cell/day.
Stock cultures were maintained in a 37 oC, 7.5% 0
Feed 2 Complete Competitor X
CO2 humidified incubator. EB14 cells were cultured
in 125 mL Erlenmeyer flasks (25 mL culture volumes) Production Media
at 90 rpm. A subclone of EB14 was derived to stably Day 1 Day 2 Day 3 Day 4 Day 5 Day 6
express recombinant human IgG1 by nucleofection Conclusions
of a plasmid encoding the gene of interest. In order Figure 3: The supplements contained in Feed 2 can be incorporated directly into the production medium at the • EX-CELL™ EBx™ is a serum-free, animal-component free medium for EBx™ cells in suspension
to maintain stable expression of recombinant human time of manufacture, resulting in a significant increase in viral titers. culture.
IgG1, cells were cultured with 200 μg/mL G418.
• EX-CELL™ EBx™ supports higher cell density, improved viability and increased culture longevity
Cell Culture Media
MVA-GFP Infected EB14 Cells in EX-CELL EBx ™ ™ when compared with a competitor medium.
Stock cultures were maintained in EX-CELL™ EBx™
Medium (Cat. No. 63066) and competitor Medium • EX-CELL™ EBx™ supports the production of a variety of viruses, including MVA and Influenza.
X. Both were supplemented with 2.5 mM L-glutamine
(Cat. No. 59202C). Base medium (Item No. 66307C)
A B C • EX-CELL™ EBx™ supports transient and stable expression of recombinant proteins such as IgG.
and complete medium (Item No. 65946) were used • Optimization of key components, feeds, and metabolic analysis has led to the development of
for viral production. All items except Medium X are EX-CELL™ EBx™ Serum-Free Medium, which exhibits robust growth, as well as high viral and
from SAFC Biosciences. protein productivity.
Process Development
Bioreactor runs were conducted in 3 L stirred tank
bioreactors (Applikon® Biotechnology, Sciedam, D E F
Holland). Bioreactors were seeded at 0.1, 0.2, Acknowledgements
and 0.4e6 cells/mL in EX-CELL™ EBx™ Medium
and Medium X. DO, pH and temperature were The authors would like to thank Cell Sciences and Development (SAFC Biosciences) and the
monitored and controlled. Uninfected samples were Research and Development (Vivalis) teams, for without their dedicated efforts and expertise, this
collected daily to monitor cell density, viability and project would not have been possible.
metabolic consumption/production (data not shown).
Infected samples were collected daily to monitor
Figure 4: Panel A represents an uninfected culture by examined by bright field microscopy. Samples were
viral production by TCID50 endpoint dilution(1),
removed and examined for fluorescence at 10X magnification from day 1 (B) through day 5 (F). By day 4 post-
hemmaglutinin production(2) and fluorescent infection almost all cells show fluorescence indicating a highly productive infection. References
microscopy.
1. Reed and Muench, 1938
Viral Infection
2. Wood et al., 1977
EB14 cells were seeded at 0.1 to 0.4e6 cells/mL
for 48 to 96 hours prior to infection in 125 mL
Erlenmeyer flasks or stirred tank bioreactors. Cells
were inoculated with a multiplicity of infection (MOI)
of 10-2 for MVA and 10-4 for the indicated strains of
human influenza and allowed to adsorb for 1 hour at
37 oC or 34 oC, respectively. Production media (with
or without feeds) was added and the cultures were
incubated at the appropriate temperature.

02946-21104