Universite de Montreal Veng 2011 RV

7/25/2019 Universite de Montreal Veng 2011 RV

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Cours BCM 6013 Techniques en instrumentationModule 1 Cellular experimentation

Universit de Montral, May 2-6, 2011

Bioreactors and cultivation modes

Presenter:

Robert Voyer, B. Ing., M. Sc. A.

My academic background

Bachelor degree in Chemical Engineering, cole Polytechnique deMtl, 1989 Microbial fermenter: production of a biopolymer

Applied cultivation modes: batch and chemostat

Master in Applied Sciences, cole 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 NationalResearch 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 andhuman cells).

Design, and set-up of laboratories dedicated to mammalian cell culturein bioreactors.

Project Leader: In charge of bioreactor scale-up infrastructure foranimal cell culture and Large Scale Biosafety (Containment) Level 2facilities.

Project Manager with industrial partner for the production and thepurification of an oncolytic virus.

Active member of the Biosafety Committee.

Objectives of this session

1. Initiation to the operation of bioreactors andfamiliarization with their components andaccessories, including the required steps toits preparation for cell cultivation and itsoperation.

2. Learn the different cultivation modes usedfor animal cell culture in bioreactors.


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What is a bioreactor?

General definition: A bioreactor is a vesselused to achieve a biochemical processinvolvingorganismsor active components derivedfromthese organisms.

Specifically, a bioreactor allows the control ofthe 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 anincubator, the bench scale bioreactor allows for more accurate control ofthe cell cultivation conditions (aeration, monitoring, sampling).

Recent trends:

Miniaturization of bioreactors

Advantages: 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 -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 L

Controls: pH, temperature,pO2,agitation rate Monitoring of optical density

Usefulness of bioreactors

Production: Bioreactors allow and facilitate the scale-upof biochemical production process up to thousandsof liters (~ 20,000 L for cell culture process and above100,000 L for microbial fermenters).

Scale-up capability of a process is generally a criticalstep 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 thecell 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)

in situ sterilization: volume > ~ 3L (Stainless Steel vessels)

Mini-bioreactors: volume < 50 mL (high-throughput screening)

Single-use bioreactors: volumes up to

2000 L!!!


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Types and sizes of bioreactors configuredfor animal cell culture

Single-use stirred tank bioreactors Available working volume range: 50 L to 2000 L

Sterile gamma-irradiated bags Optical or traditional sensors with aseptic insertion device Built-in elements for mixing and sparging

http://www.xcellerex.com/platform-xdr-single-use-bioreactors.htm

http://www.hyclone.com/bpc/sub_info.php

Types and sizes of bioreactors configured

for animal cell culture

Wave Bioreactor Available working volume (100 mL to 500L)

Uses custom gamma-irradiated bags equipped with single-usesensors.

Increasing use in biopharmaceutical manufacturing for theproduction of small clinical lots and as a cell expansion vessel.

Not a preferred tool for R & D due to control and monitoringlimitations and scalability.

www.wavebiotech.comSystme 20/50


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Other types of bioreactors

Air-lift or bubble columnbioreactors HL/DT limit its scalability Cell death due to bubbles bursting at

the gas-liquid interface Foaming issues depending on

selected culture medium

http://electrolab.co.ukFerMac Air Lift Bioreactor


Single-use Air-lift bioreactor

http://www.cellexusbiosystems.comCellMaker LiteTM


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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 availabledissolved oxygen within the bed


www.nbsc.comCelligen Plus

www.corning.comE-CubeTMculture system

www.biovest.com/BiovestInstruments.htm

Bioreactors for micro carrierbased processes Micro carriers allow cultivation of

adherent cells in traditionalstirred tank bioreactors withdesign adjustments tohydrodynamic stress tolerances

Cell density can be estimatedthrough traditional sampling

Limitation: diffusion of gas and

nutrients through multilayers ofcells


www.hyclone.comHyQ Sphere


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Stirred tank bioreactor designed foranimal 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 STB

Autoclavable STB

http://www.nbsc.com/bf110_cc.aspx


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Main elements and accessories of an STB


Helical ribbon impellerPitched blade impeller


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:

37.0C 0.1C Tshift strategy

Sensing device: Pt-100 probe (variation of electrical resistance proportional to

temperature; 100 Ohms at 0C)

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 thevessel

www.endress.comRTD model TH17

Control of basic environmental parameters

in STB for mammalian cell culture

pH Control Typical value :

7.0 7.2

Sensing device: Gel pH electrode

http://us.mt.comDPA model

Reference electrode

Compares the external surface potential with that of the internalreference electrode using Nernst Equation:

E = E1 + (2.3RT/nF) log (unknown[H+

]/internal[H+])

E: Change in potential n: number of electrons E1: Reference electrode potential F: Faraday ConstantR: Perfect gas constant [H +]: Hydrogen ions concentrationT: Temperature (K)


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pH Control (cont.) Control strategy

Increase of CO2 gas fraction in supplied gas mixture to acidifythe culture

Addition of a base solution of bicarbonate (NaHCO3 7.5%) tobasify the culture (mixture of 9%NaHCO3/4%NaOH used toincrease pH buffering capacity)

Use a dead band = no controller action

CO2(g)

CO2(l) HCO3-

H+

CO2(g) CO2(l) pCO2 = He[CO2](l) ;f(T, N, P)

CO2(l) + H2O HCO3- + H+

Henrys Law


in STB for mammalian cell culture pH control example

Production virale - Rgulation du pH

0

5

10

15

20

25

0.00 20.00 40.00 60.00 80.00 100.00 120.00 140.00 160.00 180.00

Temps de culture (h)

YC

O2(%),cellulesviables(E6cellules/mL)etpH

0

40

80

120

160

200

Tempscumuld'additiondebase(min)

pHYCO2XvNaHCO3

Infection



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Agitation rate control Typical value at small scale with PBI:

90 150 revolution/min (rpm)

Scale-up: Impeller size Agitation rate (~30 rpm @ 500L)

Measuring device: Tachometer

Control strategy: Impellers mounted on an internal shaft that is driven by an

external motor using an aseptic coupling seal (mechanical,magnetic)

Rocking platform (Wave Bioreactor)



Dissolved oxygen control (pO2) Typical value:

20 60% of air saturation

Sensing device: Polarographic electrode (Clark cell)

Control devices: Rotameter, solenoid valve, Mass Flow Controller

http://us.mt.comInPro model

www.emersonprocess.com/brooks www.burkert.ca6013 model www.emersonprocess.com/brooks


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Polarographic electrode V applied between anode and cathode Electrolyte: KCl Solution

Membrane

Anode (Ag)

Elect ro lyte Insula tion

Cathode (Pt)

Anode reaction: Ag + Cl- AgCl + e-

Cathode reaction : O2 + H2O + 2e- 2OH-Pt

Electron motion generate a small current (nA) proportional to dissolvedO2 molecules



Dissolved oxygen control strategy will varybased on cell line tolerance to stress

Vessel overlay aeration Air supplemented with oxygen (pO2(g)) Baffles at the gas-li quid 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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Dissolved oxygen control exampleTransfection transitoire l'chelle 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)

Agitation(rpm),pO2(%),temprature(C),pH*10

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1

QO2(L/min)

TEMP( C)

Agitation (rpm)

DO2 (%)

pH*10

QO2 (L/min)

Aration en tte

de bioracteur

Aration submerge


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 culturewith an alkaline detergent (manual at small scale andautomated at larger scale), including proper rinsing (PBS,RO)

2. Calibration of pH sensor with adequate buffers and testresponse of pO2 sensor (change electrolyte if need be)

3. Vessel set-up: assembly of internal components(agitating shaft, impellers, gas sparger, others), insertionof sensors (side-wall or lid), visual inspection of seals(replace if needed), lid assembly and installation ofexternal components (filter, condenser, ports and plugs)

4. Pressure test (only for in situsterilization bioreactor).

Typical steps for the preparation and operation of

a bioreactor for animal cell cultivation5. Autoclave sterilization of addition lines and bottles,

including base solution, and of gas inlet filter/s(autoclavable vessels are sterilized with these itemsalready installed)

6. Bioreactor sterilization (121C, 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 tooverlay to maintain positive pressure for final cool-downphase

8. Calibration of pO2 sensor after at least 6 hours to allowfor polarization (0% can be set during sterilization at121C or post-sterilization with pure N2; 100% iscalibrated 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 a0.22 m; when practical, pre-heat medium to cultivationtemperature before addition)

10. Start control and monitoring system to establishenvironmental 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: 60C for 1 hr)

15. Cleaning, rinsing, disassembly and dry storage

Then its ready to start over again!

Bioreactor cultivation modes

Batch Fed-Batch

Fresh Medium

Spent medium + cells

Chemostat

Filtrate = spent medium

with product

Perfusion

Ht=0

Hfinal

Fresh Medium Fresh Medium

Substrate boost


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Cultivation modes comparison:

Chemostat

Fed-Batch

Batch

Perfusion

Cultivation Time

ViableCells

Batch cultivation mode operation I: Lag phase II: Accelerating growth phase III: Exponential growth phase IV:Decelerating growth phase V: Stationary phase VI: Death phase

I II III IV V VI

Cultivation Time

X (biomass)

S (limiting substrate)

Product non growth associated(secondary metabolites)

Product growth associated



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Specific growth rate estimation in batchcultivation mode

Mass balanceHypotheses:

no environmental limitations

stable biomass composition

VL p

xvs

VLS

XvP

: Bioreactor working volume: Substrate concentration at time t

: Viable cell density at time t

: Product concentration at time t

VLdxvdt

=VLxv

: specific growth rate

dxvdt

= xv

Fed-Batch cultivation mode operation Started as a batch culture except for a lower starting working

volume. Once the limiting substrate is identified, a concentratedboost 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


Substrate boost


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VL p

xvs

VLs

xvp

Fisi

: 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

dxvdt

= (- F/V)xv

Fi, si

d(Xv)

dt= Xv

where Xv: number of total viable cells at time t.

For a constant feed flow rate, replacing Xv with Vxv,

in above equation gives :

xv = x0(V0/V) etExponential:

Specific growth rate estimation in fed-batchcultivation mode

Mass balanceHypotheses:

no environmental limitations

stable biomass composition

Medium or batch replacement cultivation modeoperation (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 thevessel 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


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-productor due to inhibitory osmolality). The perfusion cultivation mode preventssuch inhibitors accumulation since the spent medium is continuouslyfiltered and removed from the cultivation vessel while cells are kept orreturned to the bioreactor. The total number of viable cells in thebioreactor eventually reaches a plateau that is dictated by the flow of freshmedium fed combined with the capacity of the filtration system.

Fed-Batch

Perfusion

Viab

lecells

Fresh medium


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 theculture. After a few days of batch culture, fresh medium perfusion is initiated(generally of the same recipe as the starting medium). The filtration system isstarted and spent medium is removed at the same rate as the fresh mediumfeed rate: the perfusion rate (expressed as volume perfused/culture workingvolume/day [vvd])

Once the viable cell density is in equilibrium with the limit ing substrate, viablecells 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


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= Vxv - Vkdxv - Foxv,o

d(xv)

dt= ( - kd)xv - Dxv,o

Plateau: dxv/dt = 0 kd for xv>>>xv,o

Beginning: kd and xv,o 0 d(xv)/dt = xv

Specific growth rate estimation in perfusioncultivation mode

Mass BalanceHypothesis:

External loop volume is much smaller than the

culture operating working volume

Cultivation mode performance comparisonBatch Fed-Batch Perfusion

Maximum celldensity

28 E6 cells/mL 10- 20 E6 cells/mL 20 - 35 E6 cells/mL

Duration 4 - 6 days 8 - 12 days 100 180 days

Specificproductivity

20 pg/cell/d 20 pg/cell/d 20 pg/cell/d

Volumetricproductivity

14 mg/L/d 36 mg/L/d 600 mg/L/d



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Pros and Cons of different cultivation mode:


Batch Medium (batch)replacement

Fed-Batch Perfusion

Pros SimpleFast

Optimal mediumcomposition forproduction optimalgrowth mediumRapid removal ofundesirable metabolites

High cell densityLarger volumetricproductivity vs. batchmodeFairly simple toimplement

Increasedproductivity over along periodSignificantreduction in requiredscale ($)Reduction incleaning frequency

Cons Sub-OptimalManpowerrequirement

Scale needed

Aseptic mediumreplacement operationand time for itscompletion limits scale-up

Accumulation ofinhibitors and increasein osmolality

Working volumelimitations

ComplexRisk forcontamination

Drift of sensorsand other measuringdevices

Cultivation strategy examples

Example 1: Insect cells fed-batch culture

0

10

20

30

40

50

60

0 24 48 72 96 120 144 168 192 216 240 264

Time h

CelldensityxE6cells/mL

200

300

400

500

600

Osmolality(mOsm)

total cells

viable cells

Osmolality



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0

10

20

30

40

50

60

70

80

90

0 5 10 15 20 25 30 35 40

Time (d)

On-lineGFP

fluorescence(RFU)

0

5

10

15

20

25

30

35

40

Totalandviablecells

(106/mL)

GFP Viable cells Total cells

Cultivation strategy example

Example 2: Insect cells perfusion culture


Addendum: Specific rate calculations for batchand fed-batch cultivation modes

Addendum


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Examplevolution de la densit cellulaire viable

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

1.8

0 20 40 60 80 100 120 140

Temps (h)

Xv(Mcellules/mL

Slope = dxv/dt

dxvdt

= xv

Estimation of specific growth rate in batchcultivation mode

volution du taux de croissance cellulaire

-5.00E-03

0.00E+00

5.00E-03

1.00E-02

1.50E-02

2.00E-02

2.50E-02

3.00E-02

3.50E-02

0 20 40 60 80 100 120 140

Temps (h)

(1/h)

= dxv/dt 1/xv

Example (cont.)

Estimation of specific growth rate in batch

cultivation mode


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Exponential growth phase: = constant

dxv = dt

xvxv = x0e

t

volution du taux de croissance

-5.00E-03

0.00E+00

5.00E-03

1.00E-02

1.50E-02

2.00E-02

2.50E-02

3.00E-02

3.50E-02

0 20 40 60 80 100 120 140

Tiemps (h)

(1/h)

Estimation par lissage de courbe

Estimation du taux de croissance exponentiel

Estimation of specific growth rate in batchcultivation mode

Exponential growth phase: = constant

xv = 2x0 = x0et

volution du taux de croissance

-5.00E-03

0.00E+00

5.00E-03

1.00E-02

1.50E-02

2.00E-02

2.50E-02

3.00E-02

3.50E-02

0 20 40 60 80 100 120 140

Tiemps (h)

(1/h)

Estimation par lissage de courbe

Estimation du taux de croissance exponentiel

Estimation of specific growth rate in batch

cultivation mode

td = 2/ln()


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ds

dt=

-qsxv As for , qs can be estimatedfrom the slope of s vs t.

qs = -ds/dt 1/xv

YieldYx/s = -dxv/ds

Yield can be obtained from the graph of

xv vs s.

Estimation of substrate consumption rate andyield in batch cultivation mode

dp

dt=

qpxv As for and qs , qp value can be estimatedfrom the slope of p vs t.

qp = dp/dt 1/xv

Estimation of the specific production rate in

batch cultivation mode


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ds

dt=

qsxv + (si s)F/V

Specific growth rate:

Substrate specific consumption rate:

dp

dt =q

px

v (F/V)p

Estimation of specific growth, consumption andproduction rates in fed-batch cultivation mode

Specific production rate:

dxvdt

=(- F/V)xv

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