Safc Biosciences Scientific Posters - Development And Optimization Of Ex-cell™ Ebx™ Serum-free Medium For Virus And Recombinant Protein Production
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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
Human Influenza Hemagglutinin Production in EX-CELL™ EBx™
Growth and Viability of EB14 Cells in EX-CELL™ EBx™ and Medium X in Stirred Tank Bioreactors
1.60E+07 B/Jiansu (3L)
7.00E+06
100
6.00E+06
90 80
5.00E+06
3.00E+06
70 60 50 40
2.00E+06
30
1.00E+06
20 10
0.00E+00
0
4.00E+06
Day 0
Day 1
Day 2
Day 3
Day 4
Day 5
1.40E+07
Bioreactor 1 (0.4e6/m L)
Bioreactor 1 (0.4e6/m L)
Bioreactor 2 (0.2e6/m L)
Bioreactor 2 (0.2e6/m L)
Bioreactor 3 (0.1e6/m L)
Bioreactor 3 (0.1e6/m L)
Competitor X (0.1e6/m L)
Competitor X (0.1e6/m L)
Materials and Methods Cells Stock cultures were maintained in a 37 oC, 7.5% CO2 humidified incubator. EB14 cells were cultured in 125 mL Erlenmeyer flasks (25 mL culture volumes) at 90 rpm. A subclone of EB14 was derived to stably express recombinant human IgG1 by nucleofection of a plasmid encoding the gene of interest. In order to maintain stable expression of recombinant human IgG1, cells were cultured with 200 μg/mL G418. Cell Culture Media Stock cultures were maintained in EX-CELL™ EBx™ Medium (Cat. No. 63066) and competitor Medium X. Both were supplemented with 2.5 mM L-glutamine (Cat. No. 59202C). Base medium (Item No. 66307C) and complete medium (Item No. 65946) were used for viral production. All items except Medium X are from SAFC Biosciences. Process Development Bioreactor runs were conducted in 3 L stirred tank bioreactors (Applikon® Biotechnology, Sciedam, Holland). Bioreactors were seeded at 0.1, 0.2, and 0.4e6 cells/mL in EX-CELL™ EBx™ Medium and Medium X. DO, pH and temperature were monitored and controlled. Uninfected samples were collected daily to monitor cell density, viability and metabolic consumption/production (data not shown). Infected samples were collected daily to monitor viral production by TCID50 endpoint dilution(1), hemmaglutinin production(2) and fluorescent microscopy. Viral Infection 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.
6.00E+06 4.00E+06 2.00E+06 Pre
Post Day 1 Day 2 Day 3 Day 4 Day 5 Day 6 Day 7 Day 8
50 45
Infectious MVA Production in EX-CELL™ EBx™ with and without Feeds Compared to Medium X
40 35 30 25 20 15 10
10
5
9
0
B/Jiansu/10/2003 (3L) Day 3
7 6
A/H3N2/New York/55/2004 (3L)
Day 4
Day 5
Day 6
A/H1N1/New Caledonia/20/99 (20L) Day 7
Day 8
Figure 5: As shown in panel A, all three bioreactors reached at least 8e6 cells/mL following infection and produced at least 30 µg/mL of HA as determined by SRID (panel B)..
5 4
Antibody Production in EX-CELL™ EBx™ in Stirred Tank Bioreactors
3 2
1.80E+07
300
1
Cell Density IgG
1.60E+07 Base
Feed 1
Feed 2
Competitor X
Production Media Day 1
Day 3
Day 5
Day 6
250
1.40E+07
Day 7
Day 9 200
1.20E+07 Figure 2: Feed 1 increased the viral titer over the base formulation by approximately 1 log, but more importantly, the addition of Feed 2 increased the viral titer by 2 logs.
1.00E+07
™
™
Infectious MVA Production in EX-CELL EBx as a Complete Medium Compared to Medium X 10 9
150 8.00E+06 6.00E+06
igG mg/ML
LogTCID50/mL
8
mL
SAFC Biosciences and Vivalis formed a scientific partnership to develop a novel cell culture-based platform for vaccine and recombinant protein production. Vivalis has taken advantage of its expertise in avian biology and embryonic stem cells to develop fully characterized and documented cell lines that are permissive to a variety of viruses and expression of recombinant proteins. EB14 is a novel suspension cell line that was derived from avian embryonic stem (ES) cells using a proprietary process by Vivalis. EB14 cells maintain some of the unique features of ES cells, such as a strong constitutive expression of telomerase. Furthermore, EB14 cells are diploid, undifferentiated, non-tumorigenic and genetically stable. SAFC Biosciences has drawn on its expertise in media development to generate an offering of serumfree media that support robust growth and production in the EBx™ cell lines. EX-CELL™ EBx™ Medium allows consistently high growth and viral and protein titers in scales ranging from flasks to bioreactors.
8.00E+06
B
Figure 1: EB14 cells reached a significantly higher maximum cell density and demonstrated greater longevity in EX-CELL™ EBx™ Medium when compared to Medium X. EB14 cells seeded in Medium X had a significant drop in cell viability by day 5 (followed by a drop in cell density on day 6, data not shown)
LogTCID50/mL
Introduction
A/H1N1/N C (20L)
1.00E+07
0.00E+00
™
Recently, EBx cells have also been shown to be amenable to genetic engineering which allows the expression of human proteins and monoclonal antibodies. EBx™ cells readily adapt from adherent to suspension which eases the selection of the expressing cells and rapid expansion in serum-free conditions. This study will highlight the development and optimization of serum-free media formulations, feed and processes. As a result, EX-CELL™ EBx™ Serum-Free Medium was developed specifically for robust cell growth, viral and recombinant protein production utilizing EBx™ cells for vaccine and therapeutic manufacturing.
A/H3N2/New York (3L)
1.20E+07
Days Post-Seeding
HA Antigen µg/mL
A scientific partnership was formed between SAFC Biosciences and Vivalis. Utilizing EBx™ cells from Vivalis, SAFC Biosciences sought to develop a serum-free cellbased platform that would be capable of supporting the propagation of a diverse range of human and animal viruses. EBx™ is a novel suspension cell line derived from avian embryonic stem cells and is highly susceptible to human and animal viruses such as poxviruses and influenza A and B viruses. This cell line is well-characterized, diploid, genetically stable and nontumorigenic. Further, EBx™ cells can be cultured using industrial processes, including stirred-tank bioreactor systems.
A
Viable Cell Density/mL
Although egg-based and primary chicken embryonic fibroblast (CEF) systems are commonly used for human and veterinary vaccine production, many limitations exist for these systems, including cumbersome handling and scaling procedures, supply and contamination issues and the inability to replicate highly virulent strains of influenza that are lethal to chickens. With the recent release of updated guidelines for cell-based vaccine development by the United States Food and Drug Administration (FDA), continuous cell lines are becoming an increasingly promising platform to overcome some of these limitations associated with traditional methods for vaccine production.
Perfect Viability
Results
Viable Cell Density/mL
Abstract
100
4.00E+06 50
8
2.00E+06
7 6
0
0.00E+00
5
Day Day Day Day Day Day Day Day Day Day Day Day Day Day 0 1 2 3 4 5 6 7 8 9 10 11 12 13
4 3
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 approximately 240 mg/L. The specific protein productivity was calculated as 10 – 20 pg/cell/day.
2 1 0
Feed 2
Complete
Competitor X
Production Media Day 1
Day 2
Day 3
Day 4
Day 5
Day 6
Conclusions
Figure 3: The supplements contained in Feed 2 can be incorporated directly into the production medium at the time of manufacture, resulting in a significant increase in viral titers.
™
™
MVA-GFP Infected EB14 Cells in EX-CELL EBx
• EX-CELL™ EBx™ is a serum-free, animal-component free medium for EBx™ cells in suspension culture. • EX-CELL™ EBx™ supports higher cell density, improved viability and increased culture longevity when compared with a competitor medium. • EX-CELL™ EBx™ supports the production of a variety of viruses, including MVA and Influenza.
A
B
C
• EX-CELL™ EBx™ supports transient and stable expression of recombinant proteins such as IgG. • Optimization of key components, feeds, and metabolic analysis has led to the development of EX-CELL™ EBx™ Serum-Free Medium, which exhibits robust growth, as well as high viral and protein productivity.
D
E
F
Acknowledgements The authors would like to thank Cell Sciences and Development (SAFC Biosciences) and the Research and Development (Vivalis) teams, for without their dedicated efforts and expertise, this project would not have been possible.
Figure 4: Panel A represents an uninfected culture by examined by bright field microscopy. Samples were removed and examined for fluorescence at 10X magnification from day 1 (B) through day 5 (F). By day 4 postinfection almost all cells show fluorescence indicating a highly productive infection.
References 1. Reed and Muench, 1938 2. Wood et al., 1977
02946-21104
Cell Sciences & Development, SAFC Biosciences, Lenexa, Kansas 66215 2Vivalis, Saint-Herblain, Nantes 44800, France ESACT 2007 Poster#1121
Human Influenza Hemagglutinin Production in EX-CELL™ EBx™
Growth and Viability of EB14 Cells in EX-CELL™ EBx™ and Medium X in Stirred Tank Bioreactors
1.60E+07 B/Jiansu (3L)
7.00E+06
100
6.00E+06
90 80
5.00E+06
3.00E+06
70 60 50 40
2.00E+06
30
1.00E+06
20 10
0.00E+00
0
4.00E+06
Day 0
Day 1
Day 2
Day 3
Day 4
Day 5
1.40E+07
Bioreactor 1 (0.4e6/m L)
Bioreactor 1 (0.4e6/m L)
Bioreactor 2 (0.2e6/m L)
Bioreactor 2 (0.2e6/m L)
Bioreactor 3 (0.1e6/m L)
Bioreactor 3 (0.1e6/m L)
Competitor X (0.1e6/m L)
Competitor X (0.1e6/m L)
Materials and Methods Cells Stock cultures were maintained in a 37 oC, 7.5% CO2 humidified incubator. EB14 cells were cultured in 125 mL Erlenmeyer flasks (25 mL culture volumes) at 90 rpm. A subclone of EB14 was derived to stably express recombinant human IgG1 by nucleofection of a plasmid encoding the gene of interest. In order to maintain stable expression of recombinant human IgG1, cells were cultured with 200 μg/mL G418. Cell Culture Media Stock cultures were maintained in EX-CELL™ EBx™ Medium (Cat. No. 63066) and competitor Medium X. Both were supplemented with 2.5 mM L-glutamine (Cat. No. 59202C). Base medium (Item No. 66307C) and complete medium (Item No. 65946) were used for viral production. All items except Medium X are from SAFC Biosciences. Process Development Bioreactor runs were conducted in 3 L stirred tank bioreactors (Applikon® Biotechnology, Sciedam, Holland). Bioreactors were seeded at 0.1, 0.2, and 0.4e6 cells/mL in EX-CELL™ EBx™ Medium and Medium X. DO, pH and temperature were monitored and controlled. Uninfected samples were collected daily to monitor cell density, viability and metabolic consumption/production (data not shown). Infected samples were collected daily to monitor viral production by TCID50 endpoint dilution(1), hemmaglutinin production(2) and fluorescent microscopy. Viral Infection 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.
6.00E+06 4.00E+06 2.00E+06 Pre
Post Day 1 Day 2 Day 3 Day 4 Day 5 Day 6 Day 7 Day 8
50 45
Infectious MVA Production in EX-CELL™ EBx™ with and without Feeds Compared to Medium X
40 35 30 25 20 15 10
10
5
9
0
B/Jiansu/10/2003 (3L) Day 3
7 6
A/H3N2/New York/55/2004 (3L)
Day 4
Day 5
Day 6
A/H1N1/New Caledonia/20/99 (20L) Day 7
Day 8
Figure 5: As shown in panel A, all three bioreactors reached at least 8e6 cells/mL following infection and produced at least 30 µg/mL of HA as determined by SRID (panel B)..
5 4
Antibody Production in EX-CELL™ EBx™ in Stirred Tank Bioreactors
3 2
1.80E+07
300
1
Cell Density IgG
1.60E+07 Base
Feed 1
Feed 2
Competitor X
Production Media Day 1
Day 3
Day 5
Day 6
250
1.40E+07
Day 7
Day 9 200
1.20E+07 Figure 2: Feed 1 increased the viral titer over the base formulation by approximately 1 log, but more importantly, the addition of Feed 2 increased the viral titer by 2 logs.
1.00E+07
™
™
Infectious MVA Production in EX-CELL EBx as a Complete Medium Compared to Medium X 10 9
150 8.00E+06 6.00E+06
igG mg/ML
LogTCID50/mL
8
mL
SAFC Biosciences and Vivalis formed a scientific partnership to develop a novel cell culture-based platform for vaccine and recombinant protein production. Vivalis has taken advantage of its expertise in avian biology and embryonic stem cells to develop fully characterized and documented cell lines that are permissive to a variety of viruses and expression of recombinant proteins. EB14 is a novel suspension cell line that was derived from avian embryonic stem (ES) cells using a proprietary process by Vivalis. EB14 cells maintain some of the unique features of ES cells, such as a strong constitutive expression of telomerase. Furthermore, EB14 cells are diploid, undifferentiated, non-tumorigenic and genetically stable. SAFC Biosciences has drawn on its expertise in media development to generate an offering of serumfree media that support robust growth and production in the EBx™ cell lines. EX-CELL™ EBx™ Medium allows consistently high growth and viral and protein titers in scales ranging from flasks to bioreactors.
8.00E+06
B
Figure 1: EB14 cells reached a significantly higher maximum cell density and demonstrated greater longevity in EX-CELL™ EBx™ Medium when compared to Medium X. EB14 cells seeded in Medium X had a significant drop in cell viability by day 5 (followed by a drop in cell density on day 6, data not shown)
LogTCID50/mL
Introduction
A/H1N1/N C (20L)
1.00E+07
0.00E+00
™
Recently, EBx cells have also been shown to be amenable to genetic engineering which allows the expression of human proteins and monoclonal antibodies. EBx™ cells readily adapt from adherent to suspension which eases the selection of the expressing cells and rapid expansion in serum-free conditions. This study will highlight the development and optimization of serum-free media formulations, feed and processes. As a result, EX-CELL™ EBx™ Serum-Free Medium was developed specifically for robust cell growth, viral and recombinant protein production utilizing EBx™ cells for vaccine and therapeutic manufacturing.
A/H3N2/New York (3L)
1.20E+07
Days Post-Seeding
HA Antigen µg/mL
A scientific partnership was formed between SAFC Biosciences and Vivalis. Utilizing EBx™ cells from Vivalis, SAFC Biosciences sought to develop a serum-free cellbased platform that would be capable of supporting the propagation of a diverse range of human and animal viruses. EBx™ is a novel suspension cell line derived from avian embryonic stem cells and is highly susceptible to human and animal viruses such as poxviruses and influenza A and B viruses. This cell line is well-characterized, diploid, genetically stable and nontumorigenic. Further, EBx™ cells can be cultured using industrial processes, including stirred-tank bioreactor systems.
A
Viable Cell Density/mL
Although egg-based and primary chicken embryonic fibroblast (CEF) systems are commonly used for human and veterinary vaccine production, many limitations exist for these systems, including cumbersome handling and scaling procedures, supply and contamination issues and the inability to replicate highly virulent strains of influenza that are lethal to chickens. With the recent release of updated guidelines for cell-based vaccine development by the United States Food and Drug Administration (FDA), continuous cell lines are becoming an increasingly promising platform to overcome some of these limitations associated with traditional methods for vaccine production.
Perfect Viability
Results
Viable Cell Density/mL
Abstract
100
4.00E+06 50
8
2.00E+06
7 6
0
0.00E+00
5
Day Day Day Day Day Day Day Day Day Day Day Day Day Day 0 1 2 3 4 5 6 7 8 9 10 11 12 13
4 3
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 approximately 240 mg/L. The specific protein productivity was calculated as 10 – 20 pg/cell/day.
2 1 0
Feed 2
Complete
Competitor X
Production Media Day 1
Day 2
Day 3
Day 4
Day 5
Day 6
Conclusions
Figure 3: The supplements contained in Feed 2 can be incorporated directly into the production medium at the time of manufacture, resulting in a significant increase in viral titers.
™
™
MVA-GFP Infected EB14 Cells in EX-CELL EBx
• EX-CELL™ EBx™ is a serum-free, animal-component free medium for EBx™ cells in suspension culture. • EX-CELL™ EBx™ supports higher cell density, improved viability and increased culture longevity when compared with a competitor medium. • EX-CELL™ EBx™ supports the production of a variety of viruses, including MVA and Influenza.
A
B
C
• EX-CELL™ EBx™ supports transient and stable expression of recombinant proteins such as IgG. • Optimization of key components, feeds, and metabolic analysis has led to the development of EX-CELL™ EBx™ Serum-Free Medium, which exhibits robust growth, as well as high viral and protein productivity.
D
E
F
Acknowledgements The authors would like to thank Cell Sciences and Development (SAFC Biosciences) and the Research and Development (Vivalis) teams, for without their dedicated efforts and expertise, this project would not have been possible.
Figure 4: Panel A represents an uninfected culture by examined by bright field microscopy. Samples were removed and examined for fluorescence at 10X magnification from day 1 (B) through day 5 (F). By day 4 postinfection almost all cells show fluorescence indicating a highly productive infection.
References 1. Reed and Muench, 1938 2. Wood et al., 1977
02946-21104
