
9a63243b0e1b98be0d167ada84f19267.ppt
- Количество слайдов: 44
Respiration Activity Monitoring System © Hi. Tec Zang Gmb. H - HRE Bioprocessoptimisation
The RAMOS® System Online – respiration activity measurement (OTR, CTR, RQ) Respiration Activity Monitoring System © Hi. Tec Zang Gmb. H - HRE in shaking flasks
© Hi. Tec Zang Gmb. H - HRE The Tray
Fields of Application © Hi. Tec Zang Gmb. H - HRE 4 Online-tracing of the metabolic activity of pro- and eukaryotic cultures in shaking flasks
Possibilities 4 Easy Determination of parameters: - oxygen transfer rate (OTR) - carbon dioxide transfer rate (CTR) - respiration quotient (RQ) - maximum growth rate (µmax) - volumetric oxygen transfer coefficient (k. La) which afford a safe Scale-Up. © Hi. Tec Zang Gmb. H - HRE …,
Possibilities 4 Detection of characteristic biological phenomena (OTR) Substrate limitation (except C-source) Oxygen limitation Oxygen transfer rate Unlimited growth on minimal media Time of fermentation Product inhibition ( e. g. p. H) Diauxic growth Time of fermentation © Hi. Tec Zang Gmb. H - HRE Time of fermentation = Total oxygen consumption [mol/l] Oxygen transfer rate Time of fermentation maximum oxygen transfer capacity
Possibilities © Hi. Tec Zang Gmb. H - HRE 4 Detection of characteristic biological phenomena CTR development:
Possibilities 4 Recognition of suitable conditions for conventional mass screening (operation duration, culture media, operation conditions …) 4 Optimisation of substrate concentrations and reduction of media development time 4 Growth control under sterile conditions 4 Targeted sampling depending on oxygen transfer rate 4 Quality control © Hi. Tec Zang Gmb. H - HRE 4 Fermentation balancing (cytotoxycity- and proliferation assays)
State of the Art online-exhaust gas analytik ? stirred bioreactor shaking bioreactor © Hi. Tec Zang Gmb. H - HRE 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. “ „Weakness of small-scale liquid fermentations: discontinuous monitoring“ Hilton, 1999 © Hi. Tec Zang Gmb. H - HRE Payne et al. , 1990
What kind of Online Signal? carbon source (glutamine, glucose, . . . ) trace elements, vitamins nitrogen source (ammonia sulfate, urea, yeast extract, peptone, . . . ) sulfate source (sulfate, cysteine, . . . ) product (proteins, alcohol amino acids, . . . ) Oxygen Carbon dioxide © Hi. Tec Zang Gmb. H - HRE phosphorus source (phosphate, phytin)
Unknown Fermentation Process normal shaking flask: culture process A ? Time end of experiment © Hi. Tec Zang Gmb. H - HRE B
Known Fermentation Process culture process A B B Time end of experiment © Hi. Tec Zang Gmb. H - HRE A
Solution measures online the respiration activities (OTR, CTR, RQ) of aerobic biological systems in shaking flasks under © Hi. Tec Zang Gmb. H - HRE sterile conditions
Distinct Advantages 4 more information about microbiological processes in shaking flasks 4 rapid characterisation and targeted optimisation of media 4 replaces expensive experiments in the fermenter 4 parallel technology (time, comparability. . . ) 4 casily handling 4 creates optimal repoducabilty options 4 virtual non-stop operation by very short set-up time 4 reduction of experimental time to the actually required time 4 distinction of process-related and biological effects © Hi. Tec Zang Gmb. H - HRE 4 visualising the perfect inoculation point
© Hi. Tec Zang Gmb. H - HRE Graduated flask
Sample Fermentations Determination of the optimal inoculation- and fed -batch starting time (50 ml liquid volume) Cell density [N/m. L] OTR CTR cell density glutamine- and glucose consumption 0 50 glucose consumption 100 150 200 Time of Fermentation [h] © Hi. Tec Zang Gmb. H - HRE OTR/CTR [mol/(L·h)] Mammalian cell culture Hybridoma
Sample Fermentations Media optimisation Example: optimum of osmolarity (50 ml liquid volume) optimum of osmolarity at 0, 318 osmol/kg Osmolarity [osmol/kg] © Hi. Tec Zang Gmb. H - HRE Growth rate µ [h-1] Mammalian cell culture Hybridoma
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] © Hi. Tec Zang Gmb. H - HRE RAMOS (0, 05 litre culture volume)
Sample Fermentations Effect of different liquid volumes oxygen limitation Flask 1 : 10 m. L Flask 2 : 15 m. L Flask 3 : 20 m. L Flask 4 : 30 m. L Flask 5 : 40 m. L Flask 6 : 50 m. L Time of Fermentation [h] © Hi. Tec Zang Gmb. H - HRE OTR [mol/(L·h)] Bacterium Corynebacterium glutamicum
Sample Fermentations Effect of different substrate concentrations OTR [mol/(L·h)] Bacterium Pseudomonas fluorescens fermentation time [h] © Hi. Tec Zang Gmb. H - HRE 1 x concentrated 2 x concentrated 4 x concentrated
Sample Fermentations Media- and process optimisation OTR [mol/(L·h)] Yeast Hansenula polymorpha Media with 100% comp. 1, 30 ml liquid Media with 200% comp. 1, 30 ml liquid Time of Fermentation [h] © Hi. Tec Zang Gmb. H - HRE Media with 200% comp. 1, 20 ml liquid
Sample Fermentations Cell-growth within a RAMOS experiment 23 Dipl. -Ing. M. Canzoneri © Hi. Tec Zang Gmb. H - 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] © Hi. Tec Zang Gmb. H - HRE Cell density [N/ml] Dipl. -Ing. M. Canzoneri
Easy Handling 4 little required space – RAMOS fits to normal bench top 4 virtual non-stop operation by very short set-up time 4 easy and fast-learnable appliance © Hi. Tec Zang Gmb. H - HRE 4 fully automated user software
© Hi. Tec Zang Gmb. H - HRE Operating Interface
© Hi. Tec Zang Gmb. H - HRE Flask Overview
© Hi. Tec Zang Gmb. H - HRE Oxygen Transfer Rate (OTR)
Detail View for each Flask © Hi. Tec Zang Gmb. H - HRE (OTR, CTR, RQ)
O 2 -, CO 2 - Transfer Oxygen transfer (OT) Carbon dioxide transfer (CT) © Hi. Tec Zang Gmb. H - HRE 4 Balancing of the total oxygen transfer during the fermentation process
maximum Growth Rate µ growth rate µ © Hi. Tec Zang Gmb. H - HRE maximum growth rate µ
Shedding light on your process 32 CTR © Hi. Tec Zang Gmb. H - HRE OTR
OTR [mol/(L·h)] Economic efficiency consideration Media with 100% comp. 1, 30 ml liquid Media with 200% comp. 1, 20 ml liquid 4 The variation of the media concentration led to an reduction of the time of fermentation of ca. 37 % 4 Time of amortisation: ca. 6 months © Hi. Tec Zang Gmb. H - HRE Time of Fermentation [h]
Cell culture (Hybridoma) © Hi. Tec Zang Gmb. H - HRE 34 • Dosing
FTT® Fluid-Train System © Hi. Tec Zang Gmb. H - HRE 35 • Dosing and automated samplin
FTT® Fluid-Train System © Hi. Tec Zang Gmb. H - HRE 36 • controlled loop dosing
RQFeed™ - Feeding algorithm 37 determination of RQ by OUR, CER online measurement exact feeding of cultures significant increase in production rates shortening of the fermentation periods © Hi. Tec Zang Gmb. H - HRE ● ●
Cell. Drum™ - 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 © Hi. Tec Zang Gmb. H - HRE 38 ● ●
Hi. Sense™ - Precision Gas Analysis 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 © Hi. Tec Zang Gmb. H - HRE ● ● ●
Cell culture (Hybridoma) © Hi. Tec Zang Gmb. H - HRE 40 • Without dosing
Cell culture (Hybridoma) © Hi. Tec Zang Gmb. H - HRE 41 • Dosing according to OTR controlled loop starting at RQ<1
Cell culture (Hybridoma) © Hi. Tec Zang Gmb. H - HRE 42 • Dosing program
Cell culture (Hybridoma) © Hi. Tec Zang Gmb. H - HRE 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 Publications: 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. , Büchs. J. , Effect of oxygen-limitation and medium composition on Escherichia coli in small-scale cultures, Biotechnol. Progress. (accepted) © Hi. Tec Zang Gmb. H - 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