Respiration Activity Monitoring System
© HiTec Zang GmbH - HRE
S. 1
Bioprocessoptimisation
The RAMOS® System
Online – respiration
activity measurement
Respiration Activity Monitoring System
© HiTec Zang GmbH - HRE
in shaking flasks
S. 2
(OTR, CTR, RQ)
© HiTec Zang GmbH - HRE
S. 3
The Tray
Fields of Application
© HiTec Zang GmbH - HRE
S. 4
 Online-tracing of the metabolic activity
of pro- and eukaryotic cultures in shaking flasks
Possibilities
Easy Determination of parameters:
- oxygen transfer rate (OTR)
- carbon dioxide transfer rate (CTR)
- respiration quotient (RQ)
- maximum growth rate (µmax)
…,
S. 5
- volumetric oxygen transfer coefficient (kLa)
© HiTec Zang GmbH - HRE
which afford a safe Scale-Up.
Possibilities
 Detection of characteristic biological phenomena (OTR)
Substrate limitation
(except C-source)
Diauxic growth
Time of fermentation
Oxygen transfer rate
= Total oxygen
consumption [mol/l]
Time of fermentation
S. 6
Product inhibition
( e.g. pH)
maximum
oxygen transfer
capacity
© HiTec Zang GmbH - HRE
Time of fermentation
Oxygen transfer rate
Time of fermentation
Time of fermentation
Oxygen limitation
Oxygen transfer rate
Oxygen transfer rate
Oxygen transfer rate
Unlimited growth on
minimal media
Possibilities
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S. 7
 Detection of characteristic
biological phenomena
CTR development:
Possibilities
 Recognition of suitable conditions for conventional
mass screening
(operation duration, culture media, operation conditions …)
 Optimisation of substrate concentrations and
reduction of media development time
 Growth control under sterile conditions
 Targeted sampling depending on oxygen transfer rate
 Quality control
© HiTec Zang GmbH - HRE
S. 8
 Fermentation balancing (cytotoxycity- and
proliferation assays)
State of the Art
online-exhaust
gas analytik
?
stirred
bioreactor
shaking
bioreactor
© HiTec Zang GmbH - HRE
S. 9
online
OTR
CTR
RQ
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.“
Hilton, 1999
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„Weakness of small-scale liquid
fermentations:
discontinuous monitoring“
S. 10
Payne et al., 1990
What kind of Online Signal?
carbon source
(glutamine, glucose, ...)
trace elements,
vitamins
nitrogen source
(ammonia sulfate, urea,
yeast extract, peptone, ...)
product
(proteins, alcohol
amino acids, ...)
Carbon dioxide
S. 11
sulfate source
(sulfate, cysteine, ...)
Oxygen
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phosphorus source
(phosphate, phytin)
Unknown Fermentation Process
normal shaking flask:
culture process
A
?
Time
end of
experiment
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S. 12
B
Known Fermentation Process
culture process
A
B
B
Time
end of
experiment
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S. 13
A
Solution
measures online the
respiration activities (OTR, CTR, RQ)
of aerobic biological systems
in shaking flasks under
© HiTec Zang GmbH - HRE
S. 14
sterile conditions
Distinct Advantages
 more information about microbiological processes
in shaking flasks
 rapid characterisation and targeted optimisation of media
 replaces expensive experiments in the fermenter
 parallel technology (time, comparability ...)
 casily handling
 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
© HiTec Zang GmbH - HRE
 visualising the perfect inoculation point
S. 15
 creates optimal repoducabilty options
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S. 16
Graduated flask
Sample Fermentations
Determination of the optimal inoculation- and
fed-batch starting time
OTR/CTR [mol/(L·h)]
Mammalian cell culture Hybridoma
(50 ml liquid volume)
glutamine- and
glucose consumption
0
50
glucose consumption
100
150
200
Time of Fermentation [h]
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S. 17
Cell density [N/mL]
OTR
CTR
cell density
Sample Fermentations
Media optimisation
Example: optimum of osmolarity
Growth rate µ [h-1]
Mammalian cell culture Hybridoma
(50 ml liquid volume)
0,03
optimum of osmolarity
at 0,318 osmol/kg
0,025
0,02
0,01
0,005
0
0,22
0,24
0,26
0,28
0,3
0,32
0,34
0,36
0,38
Osmolarity [osmol/kg]
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S. 18
0,015
Sample Fermentations
Comparison of RAMOS to a stirred reactor with
online exhaust gas analytics
OTR [mol/(L·h)]
Mammalian cell culture Hybridoma
Dipl.-Ing. M. Canzoneri
stirred tank reactor (2 litre culture volume)
0
20
40
60
80
Time of Fermentation [h]
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S. 19
RAMOS (0,05 litre culture volume)
Sample Fermentations
Effect of different liquid volumes
Flask 2 : 15 mL
Flask 3 : 20 mL
Flask 4 : 30 mL
Flask 5 : 40 mL
Flask 6 : 50 mL
S. 20
oxygen limitation
Flask 1 : 10 mL
Time of Fermentation [h]
© HiTec Zang GmbH - HRE
OTR [mol/(L·h)]
Bacterium Corynebacterium glutamicum
Sample Fermentations
Effect of different substrate concentrations
S. 21
1x concentrated
2x concentrated
4x concentrated
fermentation time [h]
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OTR [mol/(L·h)]
Bacterium Pseudomonas fluorescens
Sample Fermentations
Media- and process optimisation
Media with 100% comp. 1,
30 ml liquid
Media with 200% comp. 1,
30 ml liquid
S. 22
Media with 200% comp. 1,
20 ml liquid
Time of Fermentation [h]
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OTR [mol/(L·h)]
Yeast Hansenula polymorpha
Sample Fermentations
Cell-growth within a
RAMOS experiment
S. 23
Dipl.-Ing. M. Canzoneri
© HiTec Zang GmbH - HRE
Mammalian cell cultures Hybridoma
Sample Fermentations
Cell proliferation within a RAMOS experiment
Mammalian cell culture Hybridoma
8-time parallel measurement
0
40
80
120
160
Time of Fermentation [h]
© HiTec Zang GmbH - HRE
S. 24
Cell density [N/ml]
Dipl.-Ing. M. Canzoneri
Easy Handling
 little required space –
RAMOS fits to normal bench top
 virtual non-stop operation by very short set-up time
© HiTec Zang GmbH - HRE
 fully automated user software
S. 25
 easy and fast-learnable appliance
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S. 26
Operating Interface
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Flask Overview
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S. 28
Oxygen Transfer Rate (OTR)
Detail View for each Flask
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(OTR, CTR, RQ)
O2-, CO2 - Transfer
Oxygen transfer (OT)
Carbon dioxide transfer (CT)
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S. 30
 Balancing of the total
oxygen transfer during the fermentation process
maximum Growth Rate µ
growth rate µ
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S. 31
maximum growth rate µ
Shedding light on your process
S. 32
CTR
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OTR
OTR [mol/(L·h)]
Economic efficiency consideration
Media with 100% comp. 1,
30 ml liquid
Media with 200% comp. 1,
30 ml liquid
 The variation of the media concentration led to an
reduction of the time of fermentation of ca. 37 %
 Time of amortisation: ca. 6 months
© HiTec Zang GmbH - HRE
Time of Fermentation [h]
S. 33
Media with 200% comp. 1,
20 ml liquid
Cell culture (Hybridoma)
© HiTec Zang GmbH - HRE
S. 34
• Dosing
FTT® Fluid-Train System
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S. 35
• Dosing and automated samplin
FTT® Fluid-Train System
© HiTec Zang GmbH - HRE
S. 36
• controlled loop dosing
RQFeed™ - Feeding algorithm
S. 37
determination of RQ by OUR, CER online measurement
exact feeding of cultures
significant increase in production rates
shortening of the fermentation periods
© HiTec Zang GmbH - HRE
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CellDrum™ - Cell force measurement
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
© HiTec Zang GmbH - HRE
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HiSense™ - Precision Gas Analysis
S. 39
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
© HiTec Zang GmbH - HRE
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Cell culture (Hybridoma)
© HiTec Zang GmbH - HRE
S. 40
• Without dosing
Cell culture (Hybridoma)
© HiTec Zang GmbH - HRE
S. 41
• Dosing according to OTR controlled loop starting at RQ<1
Cell culture (Hybridoma)
© HiTec Zang GmbH - HRE
S. 42
• Dosing program
Cell culture (Hybridoma)
© HiTec Zang GmbH - HRE
S. 43
• Parameterisation of taking samples
Cooperations and Publications
Cooperations:
Prof. Dr. Manfred Biselli
Aachen University of Applied Science, Division Jülich
Faculty of Biotechnology
Prof. Dr.-Ing. Jochen Büchs
RWTH Aachen University,
Faculty of Bioprocess Engineering
 Stöckmann Ch., Maier U., Anderlei T., Knocke Ch., Gellissen G., Büchs J.,
The Oxygen Transfer Rate as Key Parameter for the Characterisation of Hansenula polymorpha
Screening 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 culture
conditions in shaking flasks, Biochem. Eng. J. 17(3), 195-204, 2004
 Losen M., Lingen B., Pohl M., BüchsJ., Effect of oxygen-limitation and medium composition
on Escherichia coli in small-scale cultures, Biotechnol. Progress. (accepted)
© HiTec Zang GmbH - HRE
 Anderlei T., Büchs J., Device for sterile online measurement of the oxygen transfer
rate in shaking flasks, Biochem. Eng. J. 7(2), 157-162, 2001
S. 44
Publications: