1 Cours BCM 6013 – Techniques en instrumentation Module 1 – Cellular experimentation Université de Montréal, May 2-6, 2011 Bioreactors and cultivation modes Presenter: Robert Voyer, B. Ing., M. Sc. A. My academic background Bachelor degree in Chemical Engineering, École Polytechnique de Mtl, 1989 Microbial fermenter: production of a biopolymer Applied cultivation modes: batch and chemostat Master in Applied Sciences, École Polytechnique de Mtl, 1993 Extraction and purification of biopolymers produced by microbial fermentation Applied cultivation mode: fed-batch at the 40 L and 750 L scales
Transcript
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Cours BCM 6013 – Techniques en instrumentationModule 1 – Cellular experimentation
Université de Montréal, May 2-6, 2011
Bioreactors and cultivation modes
Presenter:
Robert Voyer, B. Ing., M. Sc. A.
My academic background
� Bachelor degree in Chemical Engineering, École Polytechnique de Mtl, 1989� Microbial fermenter: production of a biopolymer� Applied cultivation modes: batch and chemostat
� Master in Applied Sciences, École Polytechnique de Mtl, 1993� Extraction and purification of biopolymers produced by microbial
fermentation� Applied cultivation mode: fed-batch at the 40 L and 750 L scales
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My Work Experience
� Employee of the Biotechnology Research Institute of the National Research Council (NRC) since 1994� Design, configuration and implementation of a control and monitoring
system to support bioreactors operation (3L to 500L scales).
� Operation of animal cell culture bioreactors (insect, mammalian and human cells).
� Design, and set-up of laboratories dedicated to mammalian cell culture in bioreactors.
� Project Leader: In charge of bioreactor scale-up infrastructure for animal cell culture and Large Scale Biosafety (Containment) Level 2 facilities.
� Project Manager with industrial partner for the production and the purification of an oncolytic virus.
� Active member of the Biosafety Committee.
Objectives of this session
1. Initiation to the operation of bioreactors and familiarization with their components and accessories, including the required steps to its preparation for cell cultivation and its operation.
2. Learn the different cultivation modes used for animal cell culture in bioreactors.
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What is a bioreactor?
� General definition: A bioreactor is a vessel used to achieve a biochemical process involving organisms or active components derived from these organisms.
� Specifically, a bioreactor allows the control of the cultivation conditions to support the yield optimization of a bioproduct.
Usefulness of bioreactors
� R & D: Though often more complex to operate than a shaker flask in an incubator, the bench scale bioreactor allows for more accurate control of the cell cultivation conditions (aeration, monitoring, sampling).
Recent trends:
Miniaturization of bioreactorsAdvantages:� High throughput screening
� Small working volume allows for evaluation of expensive culture media� Allows to evaluate more clones that may not be the best producers but may happen
to be more robust and better suited to more stressful bioreactor cultivation condition
� Simplifies scale-up
Limitations:� Gas supply mode � Control strategies� Cultivation mode � Batch� Sampling volume � Kinetic studies
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Examples of mini-bioreactors
www.applikon-bio.com µ-24 Bioreactor
Commercially available:� Working Volume: 10 mL � Controls: pH, temperature and pO2
Szita et al. Lab Chip, 2005, 5, 819 - 826
Prototype:� Working Volume: 150 µL � Controls: pH, temperature,pO2,
agitation rate� Monitoring of optical density
Usefulness of bioreactors
� Production: Bioreactors allow and facilitate the scale-
up of biochemical production process up to thousands of liters (~ 20,000 L for cell culture process and above 100,000 L for microbial fermenters).
Scale-up capability of a process is generally a critical step towards the commercialization of a product
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Which bioreactor design for my process?
� The choice of bioreactor design will vary based on the cell line and the targeted volumetric scale:� Adherent cells or cells adapted for free suspension culture
(with or without serum)?� Cell tolerance to hydrodynamic stress (sparging and mixing)?� Maximum targeted cell density to support?� Required sensors to monitor and control the process?
Types and sizes of bioreactors configured
for animal cell culture
� Stirred Tank Bioreactors (most broadly used)� Working volume range: 10 mL to 20,000 L
� Suspension cell culture, including micro carriers
� Autoclave sterilization: generally volume < 20L (typically glass vessels)
� Fixed-bed bioreactors� Used for adherent cell lines where
the product of interest is secreted� Typically used in perfusion mode� Difficult to assess the cell density
and viability as well as available dissolved oxygen within the bed
Other types of bioreactors
www.nbsc.com Celligen Plus®
www.corning.com E-CubeTM culture system
www.biovest.com/BiovestInstruments.htm
� Bioreactors for micro carrier based processes� Micro carriers allow cultivation of
adherent cells in traditional stirred tank bioreactors with design adjustments to hydrodynamic stress tolerances
� Cell density can be estimated through traditional sampling
� Limitation: diffusion of gas and nutrients through multilayers of cells
Other types of bioreactors
www.hyclone.com HyQ® Sphere™
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Stirred tank bioreactor designed for
animal cell cultivation
� Typical internal configuration:
HL / DT = 1.0 to 1.5
Di / DT = 0.4 to 0.6
Sparging = 0.002 to 0.02 vvm
Number of mixers : scale dependent!
pO2(l)
DT
HL
Di
Process scale-up in STB
125 mL 500 mL 2000 mL
20 L
100 L500 L2000 L
4x – 8x
1 mL 10 mL
Dilution ratio 1/4 – 1/510000 L
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Main elements and accessories of an STBIn situ Sterilization STB
NRC – Biotechnology Research Institute
Exhaust gas – Filter and condenser
Inlet gas filter(s)
Heating/Cooling Jacket
Sensors
Sampling device
Harvest valve
Transfer bottle and tubingInjection ports
Control unit
Drive coupling
Main elements and accessories of an STBAutoclavable STB
http://www.nbsc.com/bf110_cc.aspx
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Main elements and accessories of an STB
NRC – Biotechnology Research Institute
Helical ribbon impellerPitched blade impeller
NRC – Biotechnology Research Institute
Gas sparger
� Utilities� Cooling water supply (optional for autoclavable STB)� Autoclave (sterilization) or steam supply for in situ sterilization (steam
quality to be considered!)
� Process and instrumentation (dryness) compressed air supplies� Process gas (O2, CO2, N2 distributed from tanks)� Continuous power supply (emergency power and UPS)!!!� Drains (effluent segregation: sanitary and contaminated)
Other peripherals required for the
operation of a STB
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Control of basic environmental parametersin STB for mammalian cell culture
� Temperature Control� Typical value:
� 37.0°C ± 0.1°C� T°shift strategy
� Sensing device: � Pt-100 probe (variation of electrical resistance proportional to
temperature; 100 Ohms at 0°C)
� Control operation strategy:� Cooling: Addition of cold water to a water circulation loop
within a jacket surrounding the vessel or in a submerged coil� Heating: Electrical heater or steam addition within the
circulation loop or electrical heating blanket surrounding the vessel
www.endress.com RTD model TH17
Control of basic environmental parametersin STB for mammalian cell culture
� pH Control� Typical value :
� 7.0 – 7.2
� Sensing device: � Gel pH electrode
http://us.mt.com DPA model
Reference electrode
Compares the external surface potential with that of the internal reference electrode using Nernst Equation:
E = E1 + (2.3RT/nF) log (unknown[H+]/internal[H+])
� Control devices:� Rotameter, solenoid valve, Mass Flow Controller
http://us.mt.com InPro model
www.emersonprocess.com/brooks www.burkert.ca 6013 model www.emersonprocess.com/brooks
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Control of basic environmental parametersin STB for mammalian cell culture
� Polarographic electrode� ∆ V applied between anode and cathode� Electrolyte: KCl Solution
Membrane
Anode (Ag)
Electrolyte Insulation
Cathode (Pt)
Anode reaction: Ag + Cl- AgCl + e-
Cathode reaction : ½O2 + H2O + 2e- 2OH-Pt
� Electron motion generate a small current (nA) proportional to dissolved O2 molecules
Control of basic environmental parametersin STB for mammalian cell culture
� Dissolved oxygen control strategy will vary based on cell line tolerance to stress
� Vessel overlay aeration� Air supplemented with oxygen (pO2(g))� Baffles at the gas-liquid interface to increase
the oxygen transfer into the liquid phase
� Gas sparging� Ring or L-shaped perforated SS tube� Porous diffuser� Silicone tubes (bubble-free)
� Agitation (manual)� Pressure
pO2(g)
pO2(l)
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Control of basic environmental parametersin STB for mammalian cell culture
� Dissolved oxygen control exampleTransfection transitoire à l'échelle de 45L
0
20
40
60
80
100
120
140
0.00 50.00 100.00 150.00 200.00 250.00
Temps de culture (h)
Ag
itat
ion
(rp
m),
pO
2 (%
), t
emp
érat
ure
(°C
), p
H*1
0
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1
QO
2 (L
/min
)
TEMP (°C)
Agitation (rpm)
DO2 (%)
pH*10
QO2 (L/min)
Aération en têtede bioréacteur
Aération submergée
NRC – Biotechnology Research Institute
� Cell density quantification (viable and total)� Microscope (manual or automated cell counts)� Particle counter (Coulter counter; total cells only)� Absorbance/turbidity (external or in situ; total cells only)� Capacitance (in situ; viable cells’ biovolume)
� Substrates and metabolites quantification� Glucose, lactate, glutamine, glutamate and ammonia
(Enzymatic) or NH4+ (electrode)� Amino acids (HPLC)� Osmolality (freezing point, vapor pressure)� Increasingly used in manufacturing: BioProfile (Nova Biomedical)
� Combines up to 10 measurements with a single sample injection…$$$
� Products� Western Blot, SDS-Page, ELISA, FACS, HPLC, Electrophoresis
(2-D, 3-D), etc
Additional off-line and on-line monitoring
tools for mammalian cell culture
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Typical steps for the preparation and operation ofa bioreactor for animal cell cultivation
1. Cleaning of all parts coming in contact with the culture with an alkaline detergent (manual at small scale and automated at larger scale), including proper rinsing (PBS, RO)
2. Calibration of pH sensor with adequate buffers and test response of pO2 sensor (change electrolyte if need be)
3. Vessel set-up: assembly of internal components (agitating shaft, impellers, gas sparger, others), insertion of sensors (side-wall or lid), visual inspection of seals (replace if needed), lid assembly and installation of external components (filter, condenser, ports and plugs)
4. Pressure test (only for in situ sterilization bioreactor).
Typical steps for the preparation and operation ofa bioreactor for animal cell cultivation
5. Autoclave sterilization of addition lines and bottles, including base solution, and of gas inlet filter/s (autoclavable vessels are sterilized with these items already installed)
6. Bioreactor sterilization (121°C, 30-40 minutes*)7. Connection of inlet gas filter and addition lines during
post-sterilization cool-down phase (only for in situsterilization bioreactor) followed with air supplied to overlay to maintain positive pressure for final cool-down phase
8. Calibration of pO2 sensor after at least 6 hours to allow for polarization (0% can be set during sterilization at 121°C or post-sterilization with pure N2; 100% is calibrated in air)
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Typical steps for the preparation and operation ofa bioreactor for animal cell cultivation
9. Withdrawal of sterile water and aseptic addition of medium (when prepared from powder, medium is filtered through a 0.22 µm; when practical, pre-heat medium to cultivation temperature before addition)
10. Start control and monitoring system to establish environmental conditions to desired set points
11. Bioreactor seeding12. Sampling during growth and production13. Harvest once set criteria is reached (duration, viability,
others) and transfer to DSP team for product recovery14. Vessel inactivation (typical: 60°C for 1 hr)15. Cleaning, rinsing, disassembly and dry storage
Specific growth rate estimation in batch cultivation mode
� Mass balanceHypotheses:
• no environmental limitations
• stable biomass composition
VL p
xvs
VL
S
Xv
P
: Bioreactor working volume
: Substrate concentration at time t
: Viable cell density at time t
: Product concentration at time t
VLdxv
dt=
VL µxv
µ: specific growth rate
dxv
dt=
µxv
� Fed-Batch cultivation mode operation� Started as a batch culture except for a lower starting working
volume. Once the limiting substrate is identified, a concentrated boost solution can be added to alleviate this growth limitation.
Cultivation Time
X, S, P Batch
X, S, P Fed-Batch
Initiation of feeding
Fresh medium
Bioreactor cultivation modes
Substrate boost
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VL p
xvs
VL
s
xv
p
Fi
si
: Bioreactor working volume
: Substrate concentration at time t
: Viable cell density at time t
: Product concentration at time t
: Feed flow rate at time t
: Substrate concentration in feed
dxv
dt=
(µ- F/V)⋅xv
Fi, si
d(Xv)
dt= µXv
where Xv: number of total viable cells at time t.
For a constant feed flow rate, replacing Xv with V⋅xv,
in above equation gives :
xv = x0⋅(V0/V)⋅ eµtExponential:
Specific growth rate estimation in fed-batch cultivation mode
Mass balanceHypotheses:
• no environmental limitations
• stable biomass composition
� Medium or batch replacement cultivation mode operation (non growth associated product)
� Culture starts as a standard batch culture� Prior to substrate limitation, the whole or partial volume of the culture
broth is aseptically centrifuged and concentrated cells are returned to the vessel with fresh medium.
� Production medium could be different than growth medium� Limiting substrate could also be different� Production triggered by new component
X
S
P
Bioreactor cultivation modes
Cultivation Time
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� Perfusion cultivation mode operation� The fed-batch cultivation mode eventually reach a limitation due to the
accumulation of inhibitors (inhibitory concentration of a specific by-product or due to inhibitory osmolality). The perfusion cultivation mode preventssuch inhibitors accumulation since the spent medium is continuously filtered and removed from the cultivation vessel while cells are kept or returned to the bioreactor. The total number of viable cells in the bioreactor eventually reaches a plateau that is dictated by the flow of fresh medium fed combined with the capacity of the filtration system.
Fed-Batch
Perfusion
Via
ble
cel
ls
Fresh medium
Bioreactor cultivation modes
Cultivation Time
Spent medium
with product
� Perfusion cultivation mode operation (cont.)� Here again, the culture is initiated in batch cultivation mode. The difference with
the fed-batch is that the working volume remains constant throughout the culture. After a few days of batch culture, fresh medium perfusion is initiated (generally of the same recipe as the starting medium). The filtration system is started and spent medium is removed at the same rate as the fresh medium feed rate: the perfusion rate (expressed as volume perfused/culture working volume/day [vvd])
� Once the viable cell density is in equilibrium with the limiting substrate, viable cells reach a plateau. The product titer also reaches a plateau a few days later. Total cell density keeps increasing and eventually reach a plateau as well.
Xv
P
S
Xt
Bioreactor cultivation modes
Cultivation Time
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VLp
xvs
xv,0
F0
: Viable cell density in the spent medium
leaving the system at time t
: Flow of spent medium leaving the
system at time t
Fi, si
Where kd is the specific cell death rate. Dividing each side
of the equation by the volume:Fo, s, xv,o, p
Vd(xv)
dt= V⋅µ⋅xv - V⋅kd⋅xv - Fo⋅xv,o
d(xv)
dt= (µ - kd)⋅xv - D⋅xv,o
Plateau: dxv/dt = 0 µ ≈ kd for xv>>>xv,o
Beginning: kd and xv,o ≈ 0 d(xv)/dt = µ⋅xv
Specific growth rate estimation in perfusion cultivation mode
