Download - S. 1 © HiTec Zang GmbH - HRE Respiration Activity Monitoring System Bioprocessoptimisation
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RRespirationespiration AActivityctivity MoMonitoringnitoring SSystemystem
Bioprocessoptimisation
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Online – respiration
activity measurement
(OTR, CTR, RQ)
in shaking flasks
RRespirationespiration AActivityctivity MoMonitoringnitoring SSystemystem
The RAMOS® System
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The Tray
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Fields of Application
Online-tracing of the metabolic activityof pro- and eukaryotic cultures in shaking flasks
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Easy Determination of parameters:
- oxygen transfer rate (OTR)
- carbon dioxide transfer rate (CTR)
- respiration quotient (RQ)
- maximum growth rate (µmax)
- volumetric oxygen transfer coefficient (kLa)
…,
which afford a safe Scale-Up.
Possibilities
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Unlimited growth on minimal media Oxygen limitation
Product inhibition( e.g. pH) Diauxic growth
Time of fermentation
Oxy
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Time of fermentation
Oxy
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Time of fermentation
Oxy
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Time of fermentation
Oxy
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maximum oxygen transfer
capacity
Total oxygenconsumption [mol/l]
=
Substrate limitation(except C-source)
Time of fermentation
Oxy
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Possibilities
Detection of characteristic biological phenomena (OTR)
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Possibilities
Detection of characteristicbiological phenomenaCTR development:
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Recognition of suitable conditions for conventional
mass screening
(operation duration, culture media, operation conditions …)
Optimisation of substrate concentrations and
reduction of media development time
Fermentation balancing (cytotoxycity- and
proliferation assays)
Growth control under sterile conditions
Targeted sampling depending on oxygen transfer rate
Quality control
Possibilities
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?
online-exhaustgas analytik
stirredbioreactor
OTRCTRRQ
online
shakingbioreactor
State of the Art
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Motivation
„The disadvantage of the shake flask as an experimental system is that the
experimenter has only limited capabilities for on-line
monitoring and control.“Payne et al., 1990
„Weakness of small-scale liquid fermentations:
discontinuous monitoring“Hilton, 1999
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What kind of Online Signal?
carbon source(glutamine, glucose, ...)
nitrogen source(ammonia sulfate, urea,
yeast extract, peptone, ...)
phosphorus source(phosphate, phytin)
sulfate source(sulfate, cysteine, ...)
trace elements, vitamins
Carbon dioxide
Oxygen
product(proteins, alcohol amino acids, ...)
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Unknown Fermentation Process
Time
culture process
end of experiment
A
B
normal shaking flask:
?
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B
A
Time
culture process
end of experiment
A
B
Known Fermentation Process
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measures online the
respiration activities (OTR, CTR, RQ)
of aerobic biological systems
in shaking flasks under
sterile conditions
Solution
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more information about microbiological processesin shaking flasks
rapid characterisation and targeted optimisation of media
replaces expensive experiments in the fermenter
creates optimal repoducabilty options
virtual non-stop operation by very short set-up time
reduction of experimental time to the actually required time
distinction of process-related and biological effects
casily handling
Distinct Advantages
parallel technology (time, comparability ...)
visualising the perfect inoculation point
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Graduated flask
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Sample Fermentations
Mammalian cell culture Hybridoma (50 ml liquid volume)
Determination of the optimal inoculation- and fed-batch starting time
Time of Fermentation [h]
OTR
/CTR
[m
ol/
(L·h
)]
Cell d
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N/m
L]
0 50 100 150 200
OTRCTRcell density
glutamine- andglucose consumption
glucose consumption
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Media optimisationExample: optimum of osmolarity
0
0,005
0,01
0,015
0,02
0,025
0,03
0,22 0,24 0,26 0,28 0,3 0,32 0,34 0,36 0,38
Gro
wth
rate
µ [
h-1]
Osmolarity [osmol/kg]
optimum of osmolarityat 0,318 osmol/kg
Mammalian cell culture Hybridoma (50 ml liquid volume)
Sample Fermentations
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Mammalian cell culture Hybridoma
Comparison of RAMOS to a stirred reactor with online exhaust gas analytics
OTR
[m
ol/
(L·h
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0 20 40 60 80
stirred tank reactor (2 litre culture volume)
RAMOS (0,05 litre culture volume)
Dipl.-Ing. M. Canzoneri
Sample Fermentations
Time of Fermentation [h]
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Bacterium Corynebacterium glutamicum
Effect of different liquid volumes
Sample Fermentations
OTR
[m
ol/
(L·h
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Time of Fermentation [h]
Flask 1 : 10 mL
Flask 2 : 15 mL
Flask 3 : 20 mL
Flask 4 : 30 mL
Flask 5 : 40 mL
Flask 6 : 50 mL
oxygen limitation
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Effect of different substrate concentrations
Bacterium Pseudomonas fluorescens
Sample Fermentations
OTR
[m
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(L·h
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fermentation time [h]
1x concentrated2x concentrated4x concentrated
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Media- and process optimisation
OTR
[m
ol/
(L·h
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Time of Fermentation [h]
Media with 100% comp. 1,30 ml liquid
Media with 200% comp. 1,30 ml liquid
Media with 200% comp. 1,20 ml liquid
Yeast Hansenula polymorpha
Sample Fermentations
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Mammalian cell cultures Hybridoma
Cell-growth within aRAMOS experiment
Dipl.-Ing. M. Canzoneri
Sample Fermentations
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Mammalian cell culture Hybridoma
Time of Fermentation [h]
Cell d
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N/m
l]
0 40 80 120 160
8-time parallel measurement
Dipl.-Ing. M. Canzoneri
Cell proliferation within a RAMOS experiment
Sample Fermentations
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Easy Handling
little required space –
RAMOS fits to normal bench top
easy and fast-learnable appliance
fully automated user software
virtual non-stop operation by very short set-up time
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Operating Interface
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Flask Overview
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Oxygen Transfer Rate (OTR)
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Detail View for each Flask(OTR, CTR, RQ)
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O2-, CO2 - Transfer
Oxygen transfer (OT) Carbon dioxide transfer (CT)
Balancing of the total
oxygen transfer during the fermentation process
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growth rate µ
maximum Growth Rate µ
maximum growth rate µ
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OTR CTR
Shedding light on your processShedding light on your process
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Economic efficiency consideration
The variation of the media concentration led to an
reduction of the time of fermentation of ca. 37 %
Time of amortisation: ca. 6 months
OTR
[m
ol/
(L·h
)]
Time of Fermentation [h]
Media with 100% comp. 1,30 ml liquid
Media with 200% comp. 1,30 ml liquid
Media with 200% comp. 1,20 ml liquid
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Cell culture (Hybridoma)
• Dosing
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FTT® Fluid-Train System
• Dosing and automated samplin
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FTT® Fluid-Train System
• controlled loop dosing
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● determination of RQ by OUR, CER online measurement● exact feeding of cultures● significant increase in production rates ● shortening of the fermentation periods
RQFeed™ - Feeding algorithm
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● reproducable biomechanical measurement● personalised drug and toxin research● alternative to animal experiments● integrated, fully automated and heat sterilisable pipetting unit● 24 - 96 Multiwell units with integrated sensorics
CellDrum™ - Cell force measurement
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● 1 to 8(5) Measurement Channels for 1 to 4 Fermenters ● High Resolution Measurement● Humidity Compensation (-c Version)● "True" OUR, CER and RQ Measurements (-c Version)● Low Interference ● Possible Overpressure ● Wear-resistant Sensor System ● Compact Design● Additional Functions can be integrated● Optionally free Programmability● Numerous Coupling Options● Data Export is possible
HiSense™ - Precision Gas Analysis
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Cell culture (Hybridoma)
• Without dosing
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Cell culture (Hybridoma)
• Dosing according to OTR controlled loop starting at RQ<1
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Cell culture (Hybridoma)
• Dosing program
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Cell culture (Hybridoma)
• Parameterisation of taking samples
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Cooperations and Publications
Cooperations:
Publications:Anderlei T., Büchs J., Device for sterile online measurement of the oxygen transferrate in shaking flasks, Biochem. Eng. J. 7(2), 157-162, 2001
Stöckmann Ch., Maier U., Anderlei T., Knocke Ch., Gellissen G., Büchs J.,The Oxygen Transfer Rate as Key Parameter for the Characterisation of Hansenula polymorphaScreening Cultures, J. Ind. Microbiol. Biotechnol. 30, 613-622, 2003 Anderlei T., Zang W., Büchs J., Online respiration activity measurement (OTR, CTR, RQ)in shake flasks, Biochem. Eng. J. 17(3), 187-194, 2004 Lotter St., Büchs J. Utilization of power input measurements for optimisation of cultureconditions in shaking flasks, Biochem. Eng. J. 17(3), 195-204, 2004 Losen M., Lingen B., Pohl M., BüchsJ., Effect of oxygen-limitation and medium compositionon Escherichia coli in small-scale cultures, Biotechnol. Progress. (accepted)
Prof. Dr. Manfred BiselliAachen University of Applied Science, Division JülichFaculty of Biotechnology
Prof. Dr.-Ing. Jochen BüchsRWTH Aachen University,Faculty of Bioprocess Engineering