vaccine upstream processing –an overview
TRANSCRIPT
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Vaccine Upstream Processing –an overviewDCVMN 10 March 2017
Dr. Mats Lundgren, GE Healthcare Life Sciences
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Outline of presentation
March 13, 2017Presentation Title 2
• Cell substrates for virus production
• Processing overview — batch, fed-batch, perfusion
• Cell culture using Microcarriers
• Scale up of Microcarrier cultures
• Conclusions
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Cell substrates for virus production
March 13, 2017Presentation Title 3
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Diversification of technology – low efficiency
Vero
MRC-5CECCB.anthracis
V.cholerae
S.typhi
N.meningitidisS.cerevisiae
B.pertussis
C.tetani
S.pneumoniae
C.diphtheriae
H.influenzae
Infectiousagent
Vaccinetype
Per.C6
Productionsystem
EGGS
Sf9
WI-38
MDCK
Viral Bacterial
Live-attenuated
Live
Inactivated VLP
Inactivated
Live-attenuated Toxoid
CPS/PS
CELL CULTURE MICROBIAL CELL CULTURE
13 mars 2017Building Efficiencies 4
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Selecting a cell line for virus production
March 13, 2017Presentation Title 5
• 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
• Good virus propagation
• Broadly and highly permissive
• Scalable to high volume production
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Vero cells
March 13, 2017Presentation Title 6
• Accepted by regulatory authorities for viral vaccine production
• Used for production of live attenuated viral vaccines
• Long track record for production of polio and rabies vaccine
• The cell line was derived in 1962 from kidney epithelial cells of the African Green Monkey
• Available from ATCC at passage level 121
• Most vaccine manufacture is performed with cells at passage levels in the 130’s or 140’s
• Non-tumorigenic at vaccine production passage levels
• Anchorage dependent, can be expanded on CytodexTM microcarries
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MDCK and Vero cells
March 13, 2017Presentation Title 7
MDCK Vero
+
Higher productivity
Technically easier
Less risk for propagation ofadventitious viruses
Platform cell line (can be used for several virus vaccines)
Good safety record
Used for several marketed vaccines
-
Potential tumorigenicity/ oncogenicity
New cell substrate
Restricted to influenza
Lower productivity
Technically challenging
Potential propagation of adventitious viruses
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Processing overview—batch, fed-batch, perfusion
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All culture systems have certain needs in common
9
• Absence of a competing organism
• Suitable temperature
• Suitable pH
• Suitable osmolality
• Well-mixed environment
• Gas transfer
• Not too much shear
• Nutrients to consume
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A bioreactor provides a well-controlled environment to meet these needs
Online measurement and control of:
pH
dO2
Temp
dCO2
Sparge
Agitation
Overlay
CO2 and Air/O2
Bioreactors may also have the ability to measure and control glucose concentration, as well
as to measure biomass (viable cell density)
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What factors contribute to batch failure?
11
Nutrient depletion• Cells have nothing left to “eat” (i.e., glucose, essential amino acids, vitamins)
Accumulation of toxic metabolites• Could be inhibitory to cell growth or protein production
Shear sensitivity of the cells• Cell death, or lysis that releases HCP and other “garbage” into media• Often advisable to stop batch and purify a “cleaner” harvest stream before too much
viability drop and cell lysis
Contamination
Loss of stability of production clone• Cells do not produce product anymore; sometimes seen with increasing generations
HCP = host cell protein
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Batch process―all nutrients added into base medium along with inoculum at start of batch
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Cell density
Nutrients
Metabolites
time
[c]
A high cell/product density is challenging to reach―osmolality constraints, because nutrients are added at start
Process duration ~ 3-4 d seed cultures
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Harvest day
Fed-batch process―concentrated nutrients added over time as cells grow
13
Inoculation day
Q
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Fed-batch process―feeding over time allows stable nutrient concentration
14
Q
Growth of cells prolonged, resulting in higher titer
time
Cell density
Nutrients
Metabolites
[c]
Process duration ~ 2 wk
Metabolites could reach levels toxic to cell growth, yielding lower titers (assuming no change in cell-specific productivity)
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Freshmedia
Q1Permeate/Centrate
pump
Permeate/Centrate• Permeate is the
spent media waste if purpose of perfusion is to increase cell density in bioreactor
• Permeate is the product if protein is secreted into media in production bioreactor
Q2
**P
erf
usi
on
de
viceRecirculation
pump
Recirculation
loop
Retentate
** Multiple options for perfusion devices
Perfusion―rate is in vessel volumes per day (VVD) Reactor volume remains stable
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Perfusion without Cell
Bleed
Perfusion with Cell Bleed
time time time[c] [c] [c]
Cell density
Nutrients
Metabolites
time
[c]
time
[c]
time
[c] [c]
time
Summary of batch, fed-batch, and perfusion processing for product manufacture
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Bioreactors – Fixed vs Disposabled
March 13, 2017Presentation Title 17
Control and scalability
WAVE
XDR10L
2000L
Stainless steel
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Cell culture using Microcarriers
March 13, 2017Presentation Title 18
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Scale up of adherent cell cultures
Increase volume Increase number of units
March 13, 2017Presentation Title 19
Genetic Engineering News, 2007
One 2500 L bioreactor with a carrier concentration of 3 g/L (Cytodex™ 1) provides the same surface area as 40 000 roller bottles (850 cm2/bottle)
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Why Microcarriers in vaccine production?
March 13, 2017Presentation Title 20
• Necessary for adherent cell lines
• Proven scalable technology (1000’s of L)
• Large volume to surface ratio (less waste
problem)
• Cost effective surface supply/m2
• Separates cells from secreted products
• Microporous carriers allow polarization &
differentiation
• Increased productivity of functional
product
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CytodexTM specifications
March 13, 2017Presentation Title 21
Cytodex 1 Cytodex 3
Matrix Sephadex™ Sephadex
Particle diameter (µm) 200 175
Effective surface area (m2/g dry)
0.44 0.27
Relative density 1.03 1.04
Swelling volume (mL/g dry weight)
18 14
Surface modification DEAE Gelatine
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Introduction to Cytodex™ 1 and 3 Gamma
March 13, 201729236656 AA 22
• Delivered gamma sterilized and ready to use. Supplied dry to save storage space and facilitate transportation.
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Viruses produced in microcarrier cultures
March 13, 2017Presentation Title 23
• Adenovirus
• Bovine rhinotrachteritis
• Endogenous C type
• Equine rhinopneumonitis
• Foot and mouth
• Group B arboviruses
• HAV
• Herpes
• Influenza
• Japaneese encephalitis
• Marek’s
• Papova virus
• Polio
• Polyoma
• Pseudorabies
• Rabies
• RSV
• Rous sarcoma
• Rubella
• Sendai
• SV40
• Sindbis
• Small pox
• Vaccinia
• Vesicular stomatitis
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Cell culture media and serum
March 13, 2017Presentation Title 24
Five questions to ask before selecting a raw materials 5supplier | 18 September 2014
Media Supplements Buffers and process liquidsSera
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Cell culture media and serum
March 13, 2017Presentation Title 26
• Serum - Ensure quality, traceability and origin
• Classical media
• Animal origin free media
• Complex media containing hydrolysates
• Chemically defined media
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The effect of cell culture media
Medium 1 Medium 2
March 13, 2017Presentation Title 27
Medium 3
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Serum-free expansion of Vero cells
March 13, 2017Presentation Title 28
No supplements Supplemented with Soy peptone
CytodexTM 1(DEAE surface)
Cytodex 3(collagen surface
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The effect of medium supplementation
-Supplement +Supplement
March 13, 2017Presentation Title 29
Comparison
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Scale up of microcarrier cultures
March 13, 2017Presentation Title 30
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Subcultivation – Scale up
March 13, 2017Presentation Title 31
Procedure
• Wash culture
• Add Trypsin. Optimal concentration and time of incubation need to be tested
• Inhibit trypsin when 90% of cells are detached
• Easy CytodexTM retention by using 100µm stainless steel sieve
Start detachment 20% detached 70% detached 90% detached
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Scale-Up
March 13, 2017Presentation Title 32
2000 L
Receiving tank containing fresh Cytodex
CytodexTM
retention by 100 µm sieve
Minimise shear stress transfer by pressure overlay
At 90% detachment inhibit trypsine
Wash culture, add trypsin, extensive
sampling to determine cell detachment
Bead to bead
transfer
Transfer to
2000 L tank
400 L
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Change in workflow with presterilized microcarriersConventional method Simplified process
29203492 AA | April 2016 33
Add cell culture medium
Drain buffer
Sterilize (autoclave / fermentor)
Wash with buffer
Swell in buffer
Weigh in
Dry standard microcarriers
Add cell culture medium
Add to fermentor
Presterilized microcarriers
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Propagation of virus
March 13, 2017Presentation Title 34
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Cell Culture
March 13, 2017Presentation Title 35
Cell line: Vero (ATCC-CCL81)Cell culture media: DMEM/Hams F-12Medium supplements: 5% FCS, 0.2% Soy peptone, 0.2% Pluronic F-68Microcarrier: Cytodex™ 1 at 3 g/LCell detatchment: Trypsin
Virus: Influenza A/Solomon Island/3/2006(H1N1)MOI: 0.004
Equipment:Cell factory, 2 layers, 10 layersWAVE Bioreactor™ system 20/50WAVE Bioreactor system 200
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Overview
March 13, 2017Presentation Title 36
5 days
VeroWCB 225 ml
T-flask 2-layercell factory
4 days
5 daysDSP
4 days
1x10-layercell factory 3x10-layer
cell factory
Harvest
4 days3 days
3 days
Seed Train
10LCell expansion to
confluent microcarriers
Bead to bead scale-up 50L
Cell expansion on microcarriers 50L
Virus infection
Cell Expansion and Infection
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Cell Growth
Cell concentration
March 13, 2017Presentation Title 37
• Cell growth in seed and production reactor
• Upstream process in disposable bioreactors
• Procedure for bead to bead scale-up
• Comparable cell growth rate at 10 L and 50 L scale
0.0E+00
5.0E+05
1.0E+06
1.5E+06
2.0E+06
2.5E+06
3.0E+06
3.5E+06
0 2 4 6 8 10
Cell Conc. [c/ml]
Time [d]
10 L #1
10 L #2
50 L #1
50 L #2
Growth rate
0
0.2
0.4
0.6
0.8
Avg. Growth Rate [1/d]
10 L 50 L
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0.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
0 1 2 3 4 5
Conc. [g/L]
Process Time [d]
Glc
Lac
Cell Metabolism
March 13, 2017Presentation Title 38
Depletion of glucose and glutamine prevented by medium exchange
0.0
1.0
2.0
3.0
4.0
5.0
6.0
0 1 2 3 4 5Conc. [mM/L]
Process Time [d]
Gln
NH4
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Virus growth kinetics
HA concentration and virus titer during culture Cytopathic effect at harvest
March 13, 2017Presentation Title 39
HA, µg/mL
Log 10,TCID50/mL
Log 10,Virus counter
• HA concentration at harvest was close to 12 µg/mL • ViroCyt™ virus counter data correlates well with TCID50
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