The future of cell based vaccine production
Mats Lundgren, Customer Applications Director, GE Healthcare
Platform technologies applied where possible (e.g., cell expansion on microcarriers and
purification by chromatography)
Updated cell substrates—from eggs and diploid cells to continuous cell lines
Single-use technologies and automated solutions
Live viral vector production—need for efficient platforms
Vaccine
bioprocess
technology
Process economy modelling implemented early in process development
Focus on analytical technologies driven by increased regulatory requirements
Vaccine production technology trends
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Processes developed decades ago Processes difficult to scale up
Old cell substrates or eggs
Limited purification
Significant expertise required
Unfavorable process economyIncreased regulatory requirements
Vaccine production today
Centrifugation
Fixed installations
Roller bottles
Low yields
Long process times
Labor-intense processes
Dedicated facilities
Open handling
Batch variability
Serum supplementation
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Processes developed decades ago Processes difficult to scale up
Old cell substrates or eggs
Limited purification
Significant expertise required
Unfavorable process economyIncreased regulatory requirements
Vaccine production tomorrow
Centrifugation
Fixed installations
Roller bottles
Low yields
Long process times
Labor-intense processes
Dedicated facilities
Open handling
Batch variability
Serum supplementation
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Platform cell lines
Efficient purification
based on
chromatography
Scalable technologies
enabled by, e.g.,
single-use
technologies
Efficient and rational
process design
Flexible facilites
Closed handling
Quality by design (QbD)
Chemically defined cell
culture media
4
Cell substrates for vaccines
Vero
MRC-5CECCB. anthracis
V. cholerae
S.typhi
N. meningitidisS. cerevisiae
B. pertussis
C. tetani
S. pneumoniae
C. diphtheriae
H. influenzae
Infectious
agent
Vaccine
type
PER.C6™
Production
systemEGGS
Sf9
WI-38
MDCK
Viral Bacterial
Attenuated SubunitInactivated VLP Toxoid
CPS
CELLS BACTERIA
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YEAST
PS
Attenuated Inactivated
CPS = conjugated polysaccharides, PS = polysaccharides, VLP = virus-like particle
6
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40 vaccines still to be developed
Where would this trend lead?
Vero
MRC-5CECCB. anthracis
V. cholerae
S.typhi
N. meningitidisS. cerevisiae
B. pertussis
C. tetani
S. pneumoniae
C. diphtheriae
H. influenzae
Infectious
agent
Vaccine
type
PER.C6™
Production
systemEGGS
Sf9
WI-38
MDCK
Viral Bacterial
Attenuated SubunitInactivated VLP Toxoid
CPS
CELLS BACTERIAYEAST
PS
Attenuated Inactivated
CPS = conjugated polysaccharides, PS = polysaccharides, VLP = virus-like particle
7
Cell substrate evolution from primary to diploid to continuous cell lines
Modern options: Vero, MDCK, EBx, AGE, PER.C6™…
Requirements
• Suitable for GMP production
• Good safety track record
• Animal-origin free media preferred
• Good virus propagation
• Broadly and highly permissive
• Scalable to high-volume production
Selecting a cell line for virus production
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Serum—ensure quality, traceability, and origin
Classical medium
Animal-origin free media
Complex media containing hydrolysates
Chemically defined media
Cell culture medium and serum
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Scale-up of adherent and suspension cells
Cell growth is limited by surface area
Need enzymatic passaging
More complex scale-up
Higher virus production/cell
Microcarriers increase volumetric output by maximizing the surface to volume ratio for adherent cells
Cell growth is limited by cell concentration in medium
Easier passage and scale-up
Lower virus production/cell
Adherent cells Suspension cells
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Delivered gamma-sterilized and ready to use. Supplied dry to save storage space and facilitate transportation.
Introduction to Cytodex™ 1 and 3 Gamma microcarriers
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Upstream Downstream Analysis
Virus titer
% infected cells: flow cytometry
Virus infectious titer
TCID50
Automated fluoresence microscopy
Total virus titer
qPCR
Nanosight™
Biacore™ system
Amersham™ WB system
Host cell
DNA: qPCR
Protein: ELISA
Protein pattern: Amersham WB system
AV vaccine production process
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Seed train
Virus production
XcellerexTM XDR-10WCB
WVSS
2–3 weeks
Shaker
flask
ReadyToProcess WAVE™ 25
Clarification
Conc. and buffer exchange
Capture
Polishing
Conc. and buffer exchange
Sterile filtration
Cell lysis
DNA fragmentation
TCID50 = 50% tissue culture infective dose, WCB = working cell bank, WVSS = working viral seed stockc
13
AdV productivity in CCM B (CDM4HEK293) vs E (competitor)
1429254440 AA I 14 March 2017
TOI: 1 × 106 cells/mL
TOH: 42 h
GFP expression
at TOH
AdV = adenovirus
CCM = cell culture medium
GFP = green fluorescent protein
MOI = multiplicity of infection
TOI = time of infection
TOH = time of harvest
Consistent adenovirus production in single-use Xcellerex™ XDR-10 bioreactor system
15KA747131017PP
1,0E+05
1,0E+06
1,0E+07
1,0E+08
1,0E+09
1,0E+10
1,0E+11
Run 1 Run 2 Run 3 Control
IN Cell (ivp/mL) qPCR (vp/mL)
• Common cause of diarrheal disease in young children
• 200 000 deaths in children under 5 years of age annually, majority in Africa and Asia (data estimated from 2013)
• Vaccines on the market: eg. Merck, GSK, Bharat and Lanzhou
• Limited efficacy in developing countries
• Live attenuated oral vaccines produced in Vero cells
• Vaccines produced by old technology in T-flasks / Roller bottles using animal derived components (serum and trypsin)
Rotavirus vaccines
17KA747131017PP
0,8x105
8,5x105
0
1
2
3
4
5
6
7
8
9
FFA
x10
5/
mL
Rotavirus titer in Spinner flask cultivation
OptiProSFM
VaccineXpress
Rotavirus can be propagated on Cytodex 1 Gamma using VaccineXpress
VaccineXpress medium
Competitor medium
Rotavirus expression (IN Cell)
Clarification
Cell culture
Harvest
Clarification
Primary purification
Secondary purification
Formulation
Filtration
• Normal flow
• Tangential flow (TFF)—hollow fiber filters
Centrifugation
Process flow Avaliable techniques
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Purification
TFF—hollow fiber filters
Density gradient centrifugation
Selective precipitation
Chromatography
• IEX, MM, AC, HIC, SEC
• Resin format (packed bed)
• Membrane format (capsule), ReadyToProcess™ Adsorber Q
Process flow Avaliable techniques
Cell culture
Harvest
Clarification
Primary purification
Secondary purification
Formulation
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AC = affinity chromatography, HIC = hydrophobic interaction chromatography,
IEX = ion exchange chromatography, MM = multimodal chromatography, SEC = size exclusion chromatography
22
Upstream Downstream Analysis
Virus titer
% infected cells: flow cytometry
Virus infectious titer
TCID50
Automated fluoresence microscopy
Total virus titer
qPCR
Nanosight™
Biacore™ system
Amersham™ WB system
Host cell
DNA: qPCR
Protein: ELISA
Protein pattern: Amersham WB system
AV vaccine production process
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Seed train
Virus production
XcellerexTM XDR-10WCB
WVSS
2–3 weeks
Shaker
flask
ReadyToProcess WAVE™ 25
Clarification
Conc. and buffer exchange
Capture
Polishing
Conc. and buffer exchange
Sterile filtration
Cell lysis
DNA fragmentation
TCID50 = 50% tissue culture infective dose, WCB = working cell bank, WVSS = working viral seed stock
23
Modern alternative to SEC
Easily scalable and suitable for single-use chromatography
Core bead chromatography: host cell proteins and DNA fragments bind to the core and viruses stay in the void
Virus
Host cell proteins
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SEC = size exclusion chromatography
24
Application examples core beads
Influenza
• Egg-based
• Cell-based
Dengue, Zika, and other flaviviruses
Lentivirus
Adenovirus
Cytomegolavirus
Respiratory syncytial virus
Poxvirus vectors
Polysaccharide conjugates
VLPs, etc., dependent on size
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VLPs = virus-like particles
25
• Find unit operations for AV purification
• Define suitable running conditions Litterature search
• Set up different process alternatives
• Investigate different production scalesProcess modeling in Biosolve™
• Identify economically feasible unit operations to evaluate experimentally
Evaluation of results
• Start to experimentally evaluate low cost alternatives
• Evaluate only high cost alternatives if needed for required purityProcess development
Economical considerations in early development
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Process alternatives Process
1–8
1
2
3
4
5
6
7
8
B/E = bind-elute mode, FT = flow-through mode, NFF = normal flow filtration,
Upstream process
Detergent
Nuclease treatment
Clarification NFF
TFF
Q Sepharose™ XL Sepharose 4 FF TFF
Capto Q ImpRes Capto Core 700 TFF
ReadyToProcess™ Adsorber Q B/E
ReadyToProcess Adsorber Q FT
TFF
Sampleconditioning
ReadyToProcessAdsorber Q B/E
ReadyToProcessAdsorber Q FT
TFF
ReadyToProcessAdsorber Q B/E
Capto Core 700 TFF
Capto Q ImpResReadyToProcess
Adsorber Q FTTFF
Capto Q ImpRes Capto Core 700 TFF
Clarification NFF TFF Capto™ Q ImpResNuclease
treatmentCapto Core 700 TFF
0
10 000
20 000
30 000
40 000
50 000
60 000
70 000
80 000
90 000
0
10 000
20 000
30 000
40 000
50 000
60 000
70 000
80 000
90 000
US
D/b
atc
h
Capital Materials Consumables Labour Other Total
Contributing cost factors
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Labor
29
Evaluation of productivity for modernizing a vaccine process with a different purification technique
Evaluate the effect on productivity by replacing a SEC step with a core bead chromatography step in a vaccine process at different production scales
Study objectives
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SEC = size exclusion chromatography
31
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Principle of SEC
Exluded from pores
Enter a fraction of the pores
Enter all pores
Ab
sorb
an
ce
Sample injection
High molecular
weight
Intermediate molecular
weight
Low molecular
weight
Sample injection
High
molecular
weight
Equilibration
Intermediate
molecular weight
Column volume (CV)
Ab
sorb
an
ce
Low
molecular weight
SEC = size exclusion chromatography
32
Productivity for SEC and core bead chromatography
0
100
200
300
400
500
600
700
800
Core beadchromatography, SS
column
Core beadchromatography, SU
column
Size exclusionchromatography, SS
column
Infl
uen
za a
nti
gen
(H
A m
g) /
tim
e (h
)
0
100
200
300
400
500
600
700
800
Core beadchromatography, SS
column
Core beadchromatography, SU
column
Size exclusionchromatography, SS
column
Infl
uen
za a
nti
gen
(H
A m
g) /
tim
e (h
)
200-L scale 2000-L scale
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HA = hemagglutinin, SEC = size exclusion chromatography, SS = stainless steel, SU = single-use
33
• Paradigm shift for vaccine production—from lab bench process to rational design incorporating process economy calculations early
• A combination of single-use membrane and resin technologies seems to yield beneficial economy overall
• Core bead technology can increase productivity as compared to SEC
Conclusion
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Acknowledgement
36KA747131017PP
Florence Vicaire
Günter Jagschies
Björn Lundgren
Rotavirus team
Christine Sund Lundström
Eva Blanck
Ann-Christin Magnusson
Acknowledgement – Adenovirus team
37KA747131017PP
Gustaf Ahlén
Sara Häggblad-Sahlberg
Pelle Sjöholm
Anna Åkerblom
Magnus Bergman
Maria Soultsioti
Elisabeth Wallby
Åsa Hagner McWhirter
Eva Blanck
Åsa Lagerlöf
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