S. 1 © HiTec Zang GmbH - HRE Respiration Activity Monitoring System Bioprocessoptimisation

S. 2 © HiTec Zang GmbH - HRE Online – respiration activity measurement (OTR, CTR, RQ) in shaking flasks Respiration Activity Monitoring System The RAMOS ® System

S. 3 © HiTec Zang GmbH - HRE The Tray

S. 4 © HiTec Zang GmbH - HRE Fields of Application Online-tracing of the metabolic activity of pro- and eukaryotic cultures in shaking flasks 

S. 5 © HiTec Zang GmbH - HRE Easy Determination of parameters: - oxygen transfer rate (OTR) - carbon dioxide transfer rate (CTR) - respiration quotient (RQ) - maximum growth rate (µ max ) - volumetric oxygen transfer coefficient (k L a) …, which afford a safe Scale-Up.  Possibilities

S. 6 © HiTec Zang GmbH - HRE Unlimited growth on minimal media Oxygen limitation Product inhibition ( e.g. pH) Diauxic growth Time of fermentation Oxygen transfer rate Time of fermentation Oxygen transfer rate Time of fermentation Oxygen transfer rate Time of fermentation Oxygen transfer rate maximum oxygen transfer capacity Total oxygen consumption [mol/l] = Substrate limitation (except C-source) Time of fermentation Oxygen transfer rate Possibilities Detection of characteristic biological phenomena (OTR) 

S. 7 © HiTec Zang GmbH - HRE Possibilities Detection of characteristic biological phenomena CTR development: 

S. 8 © HiTec Zang GmbH - HRE 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

S. 9 © HiTec Zang GmbH - HRE ? online-exhaust gas analytik stirred bioreactor OTR CTR RQ online shaking bioreactor State of the Art

S. 10 © HiTec Zang GmbH - HRE 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

S. 11 © HiTec Zang GmbH - HRE 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,...)

S. 12 © HiTec Zang GmbH - HRE Unknown Fermentation Process Time culture process end of experiment A B normal shaking flask: ?

S. 13 © HiTec Zang GmbH - HRE B A Time culture process end of experiment A B Known Fermentation Process

S. 14 © HiTec Zang GmbH - HRE measures online the respiration activities (OTR, CTR, RQ) of aerobic biological systems in shaking flasks under sterile conditions Solution

S. 15 © HiTec Zang GmbH - HRE more information about microbiological processes in 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

S. 16 © HiTec Zang GmbH - HRE Graduated flask

S. 17 © HiTec Zang GmbH - HRE 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 [mol/(L·h)] Cell density [N/mL] OTR CTR cell density glutamine- and glucose consumption

S. 18 © HiTec Zang GmbH - HRE Media optimisation Example: optimum of osmolarity Growth rate µ [h -1 ] Osmolarity [osmol/kg] optimum of osmolarity at 0,318 osmol/kg Mammalian cell culture Hybridoma (50 ml liquid volume) Sample Fermentations

S. 19 © HiTec Zang GmbH - HRE Mammalian cell culture Hybridoma Comparison of RAMOS to a stirred reactor with online exhaust gas analytics OTR [mol/(L·h)] stirred tank reactor (2 litre culture volume) RAMOS (0,05 litre culture volume) Dipl.-Ing. M. Canzoneri Sample Fermentations Time of Fermentation [h]

S. 20 © HiTec Zang GmbH - HRE Bacterium Corynebacterium glutamicum Effect of different liquid volumes Sample Fermentations OTR [mol/(L · h)] 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

S. 21 © HiTec Zang GmbH - HRE Effect of different substrate concentrations Bacterium Pseudomonas fluorescens Sample Fermentations OTR [mol/(L · h)] fermentation time [h] 1x concentrated 2x concentrated 4x concentrated

S. 22 © HiTec Zang GmbH - HRE Media- and process optimisation OTR [mol/(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 Yeast Hansenula polymorpha Sample Fermentations

S. 23 © HiTec Zang GmbH - HRE Mammalian cell cultures Hybridoma Cell-growth within a RAMOS experiment Dipl.-Ing. M. Canzoneri Sample Fermentations

S. 24 © HiTec Zang GmbH - HRE Mammalian cell culture Hybridoma Time of Fermentation [h] Cell density [N/ml] time parallel measurement Dipl.-Ing. M. Canzoneri Cell proliferation within a RAMOS experiment Sample Fermentations

S. 25 © HiTec Zang GmbH - HRE 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    

S. 26 © HiTec Zang GmbH - HRE Operating Interface

S. 27 © HiTec Zang GmbH - HRE Flask Overview

S. 28 © HiTec Zang GmbH - HRE Oxygen Transfer Rate (OTR)

S. 29 © HiTec Zang GmbH - HRE Detail View for each Flask (OTR, CTR, RQ)

S. 30 © HiTec Zang GmbH - HRE O 2 -, CO 2 - Transfer Oxygen transfer (OT) Carbon dioxide transfer (CT) Balancing of the total oxygen transfer during the fermentation process 

S. 31 © HiTec Zang GmbH - HRE growth rate µ maximum Growth Rate µ maximum growth rate µ

S. 32 © HiTec Zang GmbH - HRE OTR CTR Shedding light on your process

S. 33 © HiTec Zang GmbH - HRE 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 [mol/(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

S. 34 © HiTec Zang GmbH - HRE Cell culture (Hybridoma) Dosing

S. 35 © HiTec Zang GmbH - HRE FTT ® Fluid-Train System Dosing and automated samplin

S. 36 © HiTec Zang GmbH - HRE FTT ® Fluid-Train System controlled loop dosing

S. 37 © HiTec Zang GmbH - HRE ● 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

S. 38 © HiTec Zang GmbH - HRE ● reproducable biomechanical measurement ● personalised drug and toxin research ● alternative to animal experiments ● integrated, fully automated and heat sterilisable pipetting unit ● Multiwell units with integrated sensorics CellDrum™ - Cell force measurement

S. 39 © HiTec Zang GmbH - HRE ● 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

S. 40 © HiTec Zang GmbH - HRE Cell culture (Hybridoma) Without dosing

S. 41 © HiTec Zang GmbH - HRE Cell culture (Hybridoma) Dosing according to OTR controlled loop starting at RQ<1

S. 42 © HiTec Zang GmbH - HRE Cell culture (Hybridoma) Dosing program

S. 43 © HiTec Zang GmbH - HRE Cell culture (Hybridoma) Parameterisation of taking samples

S. 44 © HiTec Zang GmbH - HRE Cooperations and Publications Cooperations: Publications: Anderlei T., Büchs J., Device for sterile online measurement of the oxygen transfer rate in shaking flasks, Biochem. Eng. J. 7(2), , 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 polymorpha Screening Cultures, J. Ind. Microbiol. Biotechnol. 30, , 2003 Anderlei T., Zang W., Büchs J., Online respiration activity measurement (OTR, CTR, RQ) in shake flasks, Biochem. Eng. J. 17(3), , 2004 Lotter St., Büchs J. Utilization of power input measurements for optimisation of culture conditions in shaking flasks, Biochem. Eng. J. 17(3), , 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) 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