Fermentation Technology

Here you will find:

RAMOS Bioprocess Optimisation in Shaking Flasks FluidTrain Fed-batch and Sample Drawing in Shaking Flask OmniFerm - Mini-Fermenter System HiFerm-FCU Client-specific Fermenter Control Systems Gmix Precision Gas Mixing Device

AlcoTect sterilisable alcohol probe

HiSense Precision Exhaust Gas Analytics RQFeed Feeding Algorithm for optimal Productivity Packed Bed Reactor

HiTec Zang GmbH Tel: +49(0)2407/910 100 info@hitec-zang.de http://www.hitec-zang.com

Fermentation Technology
RAMOS Bioprocess Optimisation in Shaking Flasks
Fields of Application Continuous growth monitoring of prokaryotic and eukaryotic cultures including mammalian cells in shaking flasks 8-fold parallel online measurement of the respiration activity under sterile conditions Easy determination of characteristic values (OTR, CTR, RQ, max, kLa ...) Bioprocess optimisation through early identification of oxygen and substrate limitation or product inhibition Bioassays (toxicity assay, proliferation assay) Development of suitable conditions in the primary and secondary screening (culture duration, media, operating conditions) Reduction of the development times for ideal culture media and media conditions Mass balance of fermentation processes Quality control Potential Many experiments which previously could only be carried out laboriously in a fermenter with gas analytics, can now be conducted much cheaper and moreover in a fraction of the time with RAMOS. RAMOS pays off within short time due to the reduction of laboratory hours and development periods. In addition, production costs can be consistently and significantly reduced due to more efficient strains and culture procedures which can be discovered with the help of RAMOS. Up To now the development of suitable media has always been neglected due to the large connected expenditures. Exemplary for this fact is that LBmedia were used for decades as standard media for Escherichia coli bacteria. Already the initial investigation with RAMOS led to the insight that this medium does not allow an optimum growth due to its insufficient buffer effect. One can securely analyse the behaviour of microbial cultures with the help of measuring curves delivered by RAMOS, e.g.: substrate limitation oxygen limitation product inhibition diauxic growth

RAMOS (Respiration Activity MOnitoring System) is the standard measurement system online determination of respiration activities of prokaryotic and eukaryotic cultures in shaking flasks. The respiration rate is usually directly proportional to the number of cells and therefore enables continuous monitoring of the growth progression. Clear Advantages More information about your microbiological processes Distinction of process-related and biological effects. Replaces expensive experiments in the fermenter. Identical cultivation conditions as in your reference shaking flask. Easy handling Reduction of development time for bioprocess and cultivation conditions Provides ideal screening conditions. Enables rapid characterisation and targeted optimisation of media. Reduction of test period to the actually required time. Shortens the time-to-market. RAMOS was developed in cooperation with the department of biochemical engineering (Aachen University). RAMOS supplies the required information for: Process Development Process Optimisation Media Optimisation Fermentation Validation Scale-Up

30 Years of Excellence in Lab Scale Technology Over 1.000 Products around the Laboratory Reactor

Fermentation Technology
Until now, in numerous processes evaluated so far, RAMOS has contributed to reveal key insights and the decisive impulses for research that has been worked on for years. The demand for equal cultivation conditions for micro-organisms in normal shaking flasks and in RAMOS-Measuring flasks was given priority in the development of the plant. For this reason, the design of the flask was devised, in regard to the gas and hydrodynamics, so that it accords to a normal shaking flask in the bottom part containing liquid. In addition, a method was developed which enables to determine the oxygen transfer merely out of the variation of the oxygen partial pressure in the head space of the measuring flasks (no invasive electrodes). The system is patented. The RAMOS system fits on a normal benchtop. It is delivered completely installed with flasks, sensors, shaker, incubator, and control PC including an uninterrupted power supply system. For the familiarisation with the new methods, we offer one to two-day seminars and training programmes. Measurement of the Oxygen Transfer Rate The oxygen transfer rate is the most suitable and most universal variable to characterise and quantify the physiological condition and the activity of an aerobe culture because almost every metabolic activity is combined with oxygen consumption. The online measurement of the oxygen transfer rate in shaken bioreactors is state-of-the-art, however it is technically very laborious. The determination of the oxygen transfer rate in shaken bioreactors (shaking flasks) under sterile conditions has yet not been possible. RAMOS closes this gap and therefore combines the advantages of shaking flasks (little space required, cost-efficient, the possibility of parallel operation) with those of a stirred bioreactor (online-measurement). OTR- Phenomena The figure gives a schematic selection of biological phenomena and their effect upon the oxygen transfer rate (OTR). The diagrams show how the behaviour of a microbial culture can be systematically analysed with the help of the measuring curves delivered by RAMOS.

To ensure equivalent cultivation conditions, the gas composition above the culture liquid in the flasks of the measuring plant is adjusted identically to the composition in a normal flask. For this purpose, the gas flow is set client-specifically (depending on the type of sterile seals used). The system handling can be learned very easily and quickly. Within a short period of time, you will not be able to imagine to work without RAMOS

Measuring of the Carbon Dioxide Transfer Rate Next to the oxygen transfer rate, the carbon dioxide transfer rate (CTR) and the respiratory quotient (RQ) are important sources of information for microbiologists.
The RAMOS System

The temporal development of OTR, CTR and RQ can be clearly monitored online. In addition, further process parameters are calculated hereof. The evaluation can be extended according to user specifications.

HiTec Zang GmbH Tel: +49(0)2407/910 100 info@hitec-zang.de http://www.hitec-zang.com

Fermentation Technology
The development of OTR, CTR and RQ can be viewed online at your screen. The user interface presents also further computed parameters. This analysis can be adapted and extended to customer needs.

The diagramm show charts of the OTR, CTR and RQ of an yeast fermenetation with crabtree effect.

From the general overview, showing all of the 8 flask, each flasks diagram can be selected individually. Technical Data
(RAMOS installation for 8+6 shaking flasks including shaker and incubator.)

Dimensions Weight Size of Shaking Tray Measuring Flasks Electrical Connections Gas Connections Resolution Error in OTR Measurement Tempering Temperature Range Uncertainty of Temperatur Control Order Code VA-RAMOSOTR CG-RAMOSABK VA-RAMOSINKSHA VA-RAMOSRPM VA-RAMOSCTR VA-RAMOST500 VA-RAMOSTKSE VA-RAMOSTKS4 VA-RAMOSMK250 VA-RAMOSMKX VA-RAMOSFI1

width: 690 mm, height: 750 mm, depth: 490 mm approx. 66 kg (device without cooling) 420 x 420 mm 8 graduated flasks (250 ml) plus brackets for 6 auxiliary perpendicular shaking flasks (250 ml) for sampling or 8 graduated flasks (500 ml). Other sizes on request 230 V AC, 3 A, other voltages upon request none, intake of ambient air Oxygen transfer rate <= 0.001 [mol/(hxl)] (20 ml culture volume, 10 min. testing time), even better at greater volumes and longer testing periods <4% for temperature under 30 C (opt.) water cooling is required, under 20 C (opt.) a cooling device is required without cooling RT + 5C to 50 C, with cooling RT- 5C 50 C opt. 4 to 50 C 0.2 C

Description RAMOS system for OTR-measurement with 8 graduated flasks, shaker, incubator, PC, UPS UI to control a RAMOS System (details see CG-ABK2) RAMOS inkubator shaker, up to 300 rpm Increased rotational shaker speed up to 500 rpm incl. mass balancing RAMOS system upgrading CTR/RQ measuring for 8 graduated flasks Modified tray for 8 x 500 ml graduated flasks RAMOS cooling system, 4 C below ambient temperature RAMOS cooling system min. 4 C abs. RAMOS replacement flasks 250 ml (without fittings and filters) RAMOS flasks, customer specification Replacement sterile-filter, 8 pcs

For further information, please request the detailed RAMOS brochure and respective literature.

30 Years of Excellence in Lab Scale Technology Over 1.000 Products around the Laboratory Reactor

Fermentation Technology
Feeding and Sample Drawing in Shaking Flasks
Fluid-Train-Technology
Pharmaceutically relevant products, such as insulin, antibodies, and many more, are synthesised by microorganisms and cell cultures in bioreactors. With the Fed-batch regulation, the pro- or eukaryotic cells are fed on a defined and regulated basis. Up to now, the use of this procedure was restricted to large-scale production to achieve optimal biomass, respectively product yield. The innovative Fluid-Train Dosing and Sample Drawing Technology (FTT) ensures a comminglingfree dosing and sample drawing conducted through merely one tube. Consequently, it is possible to seperatly and sterilely dose two or optionally more different media into each individual shaking flask. The combination with the RAMOS measuring data enables the feeding of the cultures with exactly the required amount of substrate, which leads to ideal culture growth and ideal production, respectively.

RAMOS-Fed-batch provides fully automatic Fedbatch fermentation in shaking flasks. The parallel technology and the approved measurement procedure of the RAMOS were expanded by an automated dosing system. RAMOS-Fed-batch makes controlled feeding possible and regulates the dosing rates based on the oxygen transfer rate (OTR) or respiration quotient (RQ). The cell number-independant key parameter RQ gives information on the physiological condition and is evaluated from the oxygen and carbon dioxide transfer rate (CTR) and thus allows a metabolism-dependant regulated dosing. The targeted regulation of the ideal RQ value can therefore be utilised to achieve maximum product yield. Based on these results, a feeding algorithm was developed, which enables to to keep a physiological condition constant without drawing any samples and analytics. The dosing amount is precisely recorded by the software. Technical Data Receiver Bottle Dosing Rate Order Code VA-RAMOSFTT8 VA-RAMOSCON8

The diagram show a dosing control upon the OTR value. The starting set point of 0,004 mol/l*h is being reduced after 45 hrs. to 0,002 mol/l*h. After approx. ca. 60, 90 and 100 std. the feeding of media will be temporarily interrupted. Parallel, up to 24 samples are, either event or schedule-controlled, are drawn from the culture containers and deposited (AutoSam-s). This makes unattended operation over night or weekend possible. Rinsing and cleaning cycles warrant a contamination-free operation. The Fluid-Train Technology is applied for the fermentation of proand eukaryotic organisms.

2 Feed-Media Bottles, 1 Bottle NaOH, 1 Bottle ultra-pure Water 1-2 ml per flask and dosing cycle, cycle period Description Program-controlled dosing and sample drawing , 8 channels including 4 receivers RAMOS Dosing regulation for control variable = OTR, CTR or RQ, 8 channels

RAMOSFTT8 requires an AutoSam sampling device IL-AUTOSAM-xx and if necessary a cooling incubatro IL-AUTOSAM-KL.

HiTec Zang GmbH Tel: +49(0)2407/910 100 info@hitec-zang.de http://www.hitec-zang.com

Fermentation Technology
OmniFerm - Mini Fermentation System
closed loop control based on online analysis is possible tempering without heat carriers Options Client specific equipment Free programmable recipe control Freely configurable test report OTR- and RQ-based feed regulation for optimal product yield, e.g. within pharmaceutical protein production Gravimetric dosing You have the choice between containers with operating volumes of 150 to 2000 ml. The reactor can be removed quickly and easily for sterilisation or preparation.

The OmniFerm-Mini bioreactor system is suited for the controlled cultivation of bacteria and yeasts, as well as plant, insect and mammalian cell cultures. It features an especially small footprint so that several fermenters can be placed on a laboratory bench. This small-scale fermentation system closes the gap between shaking flasks and laboratory fermenters. Results which were achieved in shaking flask cultures can be validated parallelly with minimal substrate use and time expenditure. The modular design and the multiple equipping options allow maximum flexibility compared to conventional systems. The basic unit consists of a glass vessel with a stainless steel lid individually including the required sockets and a cooler). The pH-value is measured with an invasive electrode and can optionally be controlled by CO2/base or acid/base. The reactor is aerated via the head space, a gassing tube or a gassing ring. Heating and cooling is being managed at the vessel bottom. As cooling media tap water or a chiller can be used. The optionally integrated off-gas analytics system unit records the progression of the OTR, CTR, RQ, OT, CT, and max online. These values allow drawing conclusions in regard to the proliferation rate and the metabolic activity of the biological culture. Due to the integrated pO2-electrode, conclusions can be drawn out of comparison between dissolved oxygen and oxygen transfer rate. The fermentation control and regulation is based on a HiTec Zang MSRmanager system with a special interface module. Amongst others, it is possible to connect laboratory apparatuses like balances, special sensor systems, analysers, dosing systems, etc. Due to the proven PMC technology, simultaneous parallel operation of up to 8 reactors is possible in bus or star-shaped alignment. Highprecision dosing controllers ensure controlled feeding in semi-continuous and continuous operation mode. Characteristics full laboratory fermenter functionality high degree of automation various types of sensors 6

The stirrer is powered by a rpm-regulated DC motor. High-class shaft seals ensure maximum sterility. Alternatively or complementary, a magnet stirrer is available for shear-tolerant micro-organisms. Propeller, rushton, slated blade and anchor stirrer will be used. The gas supply of the reactor can be optionally controlled by a rotary flow meter manually, or automated with high-quality mass flow controllers. It is likewise possible to control the gas mixture for the fermenter with a Gmix gas mixing station. For distinct off-gas analytics, the precise compliance of gas flow rates must be ensured. The Gmix gas mixing device meets the highest demands in this respect. The trend-setting automation software LabVision in combination with the required extension modules, like EasyBatch and HiBatch-recipe control, allows the solution of complex and automation-technically demanding problems, like individual fed-batch operation in up to 8 reactors.

30 Years of Excellence in Lab Scale Technology Over 1.000 Products around the Laboratory Reactor

Fermentation Technology
A basic project including all functionalities necessary for standard batch operation, operating displays and parameterisations is expandable almost without limit. The detectable parameters are: pH, pO2, gas supply, gas mix, fed-batch control and chemostat operation mode. There are no limits of realisation to the model driven control based upon the established self describing programming language HiText.

Technical Data Dimensions Reactor Capacity Working Capacity Bottle height (exterior depth) Length of electrode Electrical Connection Materials Sterilisation Interfaces Sensors Pumps Temperature Gassing Shaft Sealing Order Code CG-OMNIFERM-ABK VA-OMNIFERM-FE-250 VA-OMNIFERM-FE-600 VA-OMNIFERM-FE-1000 VA-OMNIFERM-FE-2000 VA-OMNIFERM-COOL VA-OMNIFERM-4RS232 IL-OXYTECT-xx IL-PHFERM-l IL-ODFERM-l VA-OMNIFERM-CHILL VA-OMNIFERM-BGR VA-OMNIFERM-AIRPUM VA-OMNIFERM-GAS1 VA-OMNIFERM-GASMF VA-OMNIFERM-GASMF2 VA-OMNIFERM-GASMF4MUX VA-OMNIFERM-REDOX VA-OMNIFERM-LEV VA-OMNIFERM-FOAM VA-OMNIFERM-FOLE VA-OMNIFERM-ETOH VA-OMNIFERM-RS232 IL-HISENSE-m-x IL-HISENSE-c-x width: 195 mm, depth: 300 mm, height: 500 mm 250 ml 600 ml 1000 ml 2000 ml 150 250 ml 300 600 ml 500 1000 ml 500 2000 ml 135mm, DN80 150mm, DN100 150Mm, DN100 300Mm, DN100 120 mm 120 mm 120 mm 225 mm 230 V AC, 3 A Glass, polished stainless steel 1.4573 Auto-clavable 2 x Pg 13.5, 7 x UNF 1/4-32, 1 x UNF 1/2-20 temperature, optional pH, dissolved O2, Redox, level, foam, alcohol 4 x rpm-regulated peristaltic pumps 0..30ml/min, opt. gravimetric controlled RT to 50 C (optional, cooling with water or cooling agent) Dip tube, sparger or head space mechanical Description Control and Operating Unit for up to 8 OmniFerm Fermentation Units OmniFerm Fermentation Unit, reactor Capacity 150 to 250 ml, with four dosing pumps OmniFerm Fermentation Unit, reactor Capacity 300 to 600 ml, with four dosing pumps OmniFerm Fermentation Unit, reactor Capacity 500 to 1000 ml, with four dosing pumps OmniFerm Fermentation Unit, reactor Capacity 500 to 2000 ml, with four dosing pumps Cooling functionality, integrate cooling coil for tapped water or thermostat, contol valve 4 RS232 connectors of e.g laboratory scales Dissolved oxygen probe, autoclavable, PG 13.5 thread pH probe pH 0..14, 0 to 130C, 0..10 bar, autoclavable, PG 13.5 thread Cell count probe (optical density), autoclavable, PG 13.5 thread Cooling thermostat 300 W at 15C (provides up to 8 fermenters) Gassing stirrer Electronic controlled gasing pump for up to 4 fermenters, max. 550 l/h Gassing control with pulsed valve Gassing control with Massflow Controller Gassings control with two Massflow Controllers, e.g. Air and CO2 Gasing control with massflow controller, tetra multiplexing, 4 gasses Redox sensor, auto-clavable, PG13.5 thread Level sensor, auto-clavable Foam sensor, auto-clavable Combination Sensor Level and Foam, auto-clavable Alcohol sensor, carrier gas supply, auto-clavable RS232 Balance Interface for gravimetric dosing Exhaust Gas Analysis System for O2, OTR, CTR and CO2 for micro biology Exhaust Gas Analysis System for O2, OTR, CTR and CO2 for cell tissue cultures

X: amount of Channels, see HiSense, further volumes upon request

Complementary products LabDos Precision Dosing Pump, AutoSam Sample Drawing System, Sterile Tube Pressure Sensor, Laboratory Scales can be found in other chapters.

HiTec Zang GmbH Tel: +49(0)2407/910 100 info@hitec-zang.de http://www.hitec-zang.com

Fermentation Technology
HiFerm-FCU Customer-specific Fermenter Control Systems
The level of automation of common bioreactors in microbiology and cell culture technology has a time lag of twenty years in relation to the automated laboratory reactor systems for chemical laboratories. HiTec Zang makes its experience of over 25 years in laboratory automation henceforth available for the customer-specific automated bioreactor. Via serial interfaces, laboratory devices like balances, special sensor systems, analysers, dosing systems, gas mixer, etc. can be connected. Dosing controllers for pump guided or gravimetric dosing are integrated. Thus, dosing systems can be driven by online measurement and analytic values. Interfaces dissolved oxygen sensor (pO2), direct connection for pH and redox sensors, (expandable to 2), alcohol sensor, 2 special inputs with current/voltage sources e.g. for a foam sensor, 8 analogue inputs for flow rate controllers, temperature and pressure sensors etc., 8 analogue outputs for dosing pumps and stirrer control, 16 digital outputs with short-circuit-proof semiconductor switch for magnetic valves, pumps, heater, etc., 14 digital inputs for fill level, pressure monitor, etc., 8 RS232 interfaces for balances, analysers, laboratory devices, etc. 4 USB-Interfaces for keyboard, mouse, printer, memory stick, webcam, etc. With the automation software LabVision and the expansion modules, e.g. the EasyBatch or HiBatch Recipe Control, it is possible to realise complex and challenging automation tasks in an uncomplicated manner.

Example for a customised manufactured system with dosing pumps and touchscreen monitor.

The advanced fermentation control unit HiFerm FCU allows the automated operation of a fermenter with an unprecedented level of automation. It is based on the MSRmanager automation system with a special interface module for sensors, actuators, and instruments. Sensors and actuators are connected with splash waterproof IP65 plugs and bushes, so that the connection is easy to install and, if necessary, disconnected quickly. The evaluation circuitry for oxygen, pH, temperature sensors etc. is integrated and can be expanded in various ways. For control of valves and pumps, analogue and digital outputs with short-circuit-proof semiconductor switches are provided. Analogue inputs for the connection of temperature sensors, electrodes, and miscellaneous sensors are digitalised with 24 bit and yield accordingly excellent measurement accuracy. Digital inputs allow connection of limit switches or malfunction annunciators of additionally connected devices.

30 Years of Excellence in Lab Scale Technology Over 1.000 Products around the Laboratory Reactor

Fermentation Technology

An user-friendly interface and an easy handling of the system assure a short preparation time. A basic project with the essential functions, views and parameterisations can be expanded nearly arbitrarily by the user. All analogue values are displayed as curves in a chart. Limit violations will be alerted and the whole event history is saved in all details. All values, parameters, and events are stored in an adaptive database. Fully automatic Research Fermenter (. Frings Bonn) Features expandable with analysers and special sensors freely-programmable control software LabVision with numerous extension options Complex Process Control is possible touch screen for keyboard- and mouseless operation automatic managed laboratory journal optionally as complete system with pumps, sensors, etc. Integration of other manufacturers' components is possible Via the optional off-gas analysing module, the metabolic activity of biological cultures can be detected online via the respiration activity (OTR, CTR, RQ, OT, CT, max). With this knowledge you can easily draw conclusions in regard to substrate consumption and changes in metabolism or biomass. For dosing with fed-batch or continuous operation mode, up to five precision peristaltic pumps can be connected. They assure a secure and constant substrate supply for semi-continuous and continuous processes and a very accurate dosing of anti-foam, base or acid for pH control.

Cell fermentater control unit

Order Code VA-HIFERM-FCU

Description Fermenter Control Unit

HiTec Zang GmbH Tel: +49(0)2407/910 100 info@hitec-zang.de http://www.hitec-zang.com

Fermentation Technology
Gmix Gas Mixing Device
Control / Protective Devices Outlet pressures are measured and controlled for safety reasons. In case of exceedance of the adjusted outlet pressure, the gas supply will be automatically locked and an alarm signal will be activated.

The Gmix precision gas mixing device was especially developed for applications which require highest accuracy and reproducibility of gas mixtures. The gas mixing device is available in various configurations with one to four outlets. The gas mixture, consisting of up to four (opt. Six) gases, can be adjusted individually for every outlet. All inlets will be calibrated ex works upon customer request. Up to six gases for eache inlet can be calibrated. Default gases are air, N2, O2 and CO2. Units can be selected as ml/min (default: cm/min) und g/min. The Gas Mixing Device Gmix is available both as a RAMOS expansion and as independant system. Application The application areas of the precision gas mixing device are, amongst others, all biological fermentations the require a gas composition. Due to the extremely precise gas mixture, the gas mixing device can also be deployed in physical technology and trace gas analytics. One further application area is the calibration of gas sensors. Advantages Flows and percentagae will be entered into and shown in self describing dialogue mass. The actual concentrations are kept constant with high class mass flow controllers (NO time multiplexing!). Thereby, an eminently consistent and precise dosing is assured. Due to the very compact design, the device requires little space.

Operation with RAMOS During operation with RAMOS, the UI/work station of the gas mixing device is directly integrated in the RAMOS-software. The setpoint values are predetermined by the operator. The measured flow and fractions are indicated numerically and the temporal progression is displayed graphically.

Customer specific solutions e.g. for aggressive gases can be buildt upon request.

10

30 Years of Excellence in Lab Scale Technology Over 1.000 Products around the Laboratory Reactor

Fermentation Technology
Technical Data Dimensions Weight Inputs Amount Gases Inlet pressure Outlets Amount Outlet pressure Min. pressure difference Control principle Prefilter Flows Operating range
(Gas Mixing Device)

Width: 240 mm, Depth: 320 mm, Height: 150 mm with up to 4 inputs Width: 340 mm with 5 or 6 inputs approx. 6 kg with up to 4 inputs, 8 kg with 5 or 6 inputs 2 6 gases, pneumatic connection 6/4 Air, N2, Oxygen (O2), Carbon Dioxide(CO2) by default, non-condensing, other gases upon request. Up to 6 gases posisble. Distribution is freely selectable by the user. according to specifications, up to 11 bar abs, from approx. 10 slm/Input, higher pressure upon request 1, pneumatic connection according to specification, up to 10 bar abs. Approx. 1 bar, according to specifications Thermal massflow controller. Each component is controlled on each outlet, NO multiplexing! Inlet filter 20 m

Default: 0,05 slm 50 slm set value, Dynamic 1:30 optionally smaller or greater, dynamics 1:100 Uncertainty Default: < 1,5 % of set value, opt. +/- 0,3% of set value, +0,5% of measured value Reproducibility <0,1 % of set point Long time stability <1 % of set point Set up uncertainty flow according to range, components 0,01 % Rise time < 1 s for 2 % of set point Heating period 10 s Temperature coefficient 0.025 %/C Pressure coefficient -0.2 %/bar Materials Material in contact with Standard: aluminium, brass (part. nickel-plated), FPM (viton), stainless steel, media: PUR, Si, Si3N4, SiOx, Epoxid. optionally metals and PUR can be replaced by stainless steel. Monitoring Outlet pressure Limit values for alarming and shut-down for each outlet channel adjustable, accuracy 100 mbar relatively. Flow Current value interface with alarming at under- and over-usage Operating PC Operation with provided software with graphical interface. Data export to Excel Interfaces Optional: Remote Control via serial COM interface or the Windows-Pipe mentioned above, Remote Control by other programs is possible via instruction set according to NAMUR General Operating voltage 110 230 V, 50 60 Hz Current consumption 0,34 A Temperature range 0 C to 40 C Industrial protection IP 20
* mLn, Ln = Norm(milli)liter (1013 mbar, 0 C)

HiTec Zang GmbH Tel: +49(0)2407/910 100 info@hitec-zang.de http://www.hitec-zang.com

11

Fermentation Technology
Order Code IL-GMIX21 Description Gas mixing device (2 Inlets, 1 Outlets) for non-condensing gases (Air, O2, N2, CO2) Inlet 0,05 to 5 slm, uncertainty 1,5 % of span, Dynamic 1:30 IL-GMIX31 Gas mixing device (3 Inlets, 1 Outlets) for non-condensing gases (Air, O2, N2, CO2) Inlet 0,05 to 5 slm, uncertainty 1,5 % of span, Dynamic 1:30 IL-GMIX41 Gas mixing device (4 Inlets, 1 Outlets) for non-condensing gases (Air, O2, N2, CO2) Inlet 0,05 to 5 slm, uncertainty 1,5 % of span, Dynamic 1:30 IL-GMIX21-X Gas mixing device with increased accuracy (2 Inlets, 1 Outlets) for non-condensing/corrosive gases (Air, O2, N2, CO2). Inlet 0,05 to 5 sLM, uncertainty (0,3 % of span + 0,5 % of setpoint) Dynamic 1:100 IL-GMIX31-X Gas mixing device with increased accuracy (3 Inlets, 1 Outlets) for non-condensing/corrosive gases (Air, O2, N2, CO2). Inlet 0,05 to 5 sLM, uncertainty (0,3 % of span + 0,5 % of setpoint) Dynamic 1:100 IL-GMIX41-X Gas mixing device with increased accuracy (4 Inlets, 1 Outlets) for non-condensing/ corrosive gases (Air, O2, N2, CO2). Inlet 0,05 to 5 sLM, uncertainty (0,3 % of span + 0,5 % of setpoint) Dynamic 1:100 IL-GMIX21-XET Gas mixing device with increased accuracy , stainless steel model, dead volume optimised (2 Inlets, 1 Outlets) for non-condensing/ corrosive gases (Air, O2, N2, CO2). Inlet 0,025 to 0,5 sLM, uncertainty (0,3 % of span + 0,5 % of setpoint) Dynamic 1:100 IL-GMIX31-XET Gas mixing device with increased accuracy , stainless steel model, dead volume optimised (3 Inlets, 1 Outlets) for non-condensing/ corrosive gases (Air, O2, N2, CO2). Inlet 0,025 to 0,5 sLM, uncertainty (0,3 % of span + 0,5 % of setpoint) Dynamic 1:100 IL-GMIX41-XET Gas mixing device with increased accuracy , stainless steel model, dead volume optimised (4 Inlets, 1 Outlets) for non-condensing/ corrosive gases (Air, O2, N2, CO2). Inlet 0,025 to 0,5 sLM, uncertainty (0,3 % of span + 0,5 % of setpoint) Dynamic 1:100 IL-Gmix-S-RAMP Program Control for User-defined Profiles Systems with more outlets can be accomplished by cascading multiple devices or can be manufactured upon request. Upon request we can equip all devices with further options such as: System adjustments to special gases, aggressive gases, multiple gas datasets per inlet, analogue inputs 4...20 mA and controller for dissolved O2 and pH, digital input for externally control (standby, ramp start and stop).

We are also able to construct a gas mixing device exactly according to your individual requirements and specifications!

AlcoTect Alcohol Probe

High Durability Short Response Time High Precision Technical Data Probe Diameter Probe Length Process Connection Measuring Range Measurement Uncertainty Temperature Range Pressure Range Response Time 95% Materials Carrier Gas Output Signal 19mm and 25mm 105, 120, 220 or 320mm 13 mm, opt. Ingold Flange 0..15% +/- 0,1% 10 C to 143 C 0...2bar < 5 min 316 Ti SS / Silicon Membrane Synthetic Air, 20ml/min 4..20mA or 0..10V

The AlcoTectTM Alcohol Probe, amongst other application options, is suited for the monitoring of fermentation processes, the monitoring of beer production, the destillation process and the dealcoholisation process. Product Features: Exact online determination of ethanol and methanol Sterilisable in-situ (SIP and CIP) Order Code IL-ALCOTECT-d-l IL-ALCOTECT-AIRS

Description Alcohol Probe with Evaluation Unit for ethanol and methanol Gas Supply synthetic air 20ml/min for alcohol probe, without gas bottle

d: 19, 25 = Diameter 19 or 25 mm, l: 105, 120, 220, 320 = 105, 120, 220 or 320mm 12
30 Years of Excellence in Lab Scale Technology Over 1.000 Products around the Laboratory Reactor

Fermentation Technology
OxyTect Optical Dissolved Oxygen Probe
High Precision Short Response Time Exchangeable Sensor Cap Technical Data The OxyTect, optical dissolved oxygen probe amongst other application options, is suited for the monitoring of fermentation processes. The disadvantages connected with the conventional Clark probes are no longer existent. Product Features: Exact online determination of the dissolved oxygen concentration Compatible with the connections and signals of the conventional Clark probe Sterilisable in-situ (SIP and CIP) Auto-diagnosis Immediately ready for measurement High Durability Order Code IL-OXYTECT-xx Probe Diameter Probe Length Process Connection Measuring Range Detection Limit Temperature Range Pressure Range Response Time 98% Materials Output Signal Power Supply 12mm 120, 160, 225, 325 o. 425mm PG-13,5 Thread 4 ppb/0,05%...300% O2 Air Saturation 0,01 Vol% -10 C to 80 (130) C -1...12bar < 30s High Grade Steel, Silicone 4..20mA, Clark-Signal, RS485 ( MODBus RTU Fieldbus), VP 8.0 Jack 7..30V DC, 0,6W

Description Dissolved Oxygen Probe with Evaluation Electronics 4..20mA, Clark Signal, RS485 ( MODBus RTU Fieldbus), VP 8.0 Jack, autoclavable, PG 13.5 Thread

L: 120, 160, 225, 325, 425mm = 120, 160, 225, 325 or 425mm

CellTect Probe for optical density

High Durability High Precision Short Response Time Technical Data The CellTect probe is suitable for transimisive measurements of optical densities within a fermenter. Optical density correlates with the cell density. The raw signal (AU) can analysed as optical density (OD) and cell density (cells/L) or dry matter (g/L) with the aid of calibration spread sheets or the curve adjusting programm CellFit. Product Features: Online evaluation of cell density In Situ sterilisable (SIP and CIP) Immediately ready for measurement Bestellcode IL-CELLTECT-xx-ww IL-CELLFIT Probe Diameter Probe Length Process Connection Optical path length Detection limit Uncertainty Reproducibility Temperature Range Pressure Range Response Time 98% Materials Output Signal Power Supply 12mm 120, 225, 325 o. 425mm PG-13,5 Thread 5 oder 10 mm (cell culture) 0,1mg/ml (probe depending) +/- 0.005 AU at 25C +/- 0.001 AU at 25C 15 to 45 (134) C -1...2bar < 30s High Grade Steel, PFA 4..20mA 24V DC, 2,5 W

Beschreibung Probe for optical density with evaluation electr. 4..20mA, autoclavable, PG 13.5 Thread Programm module for calculation of optical density, cell density and dry matter

xx: 120, 225, 325, 425 = 120, 225, 325 or 425mm ww: optical path length 05, 10 = 5, 10 mm
HiTec Zang GmbH Tel: +49(0)2407/910 100 info@hitec-zang.de http://www.hitec-zang.com

13

Fermentation Technology
Autoclavable pH-Probe
Extended application area in medias with extreme ion concentration, strongly oxidising characteristics, high alkali or acid concentrations or solvents Integrated Pt100 Technical Data Probe Diameter Probe Length 12mm 120, 225, 325, 360 and 425mm Process Connection PG-13,5 Thread Measurement Range 0 14 pH Measurement +- 0,1% Uncertainty Temperature Range 0 to 130 C Pressure Range 0...10bar Connection SMEK- or VP- Plug-In Head Protection Class IP 68

This autoclavable ph-probe is in particular suited for the monitoring and controlling of fermentation processes. It is distinguished by especialially high durability and robustness. Product Features: Low-Maintenance 2 Pinhole Diaphragms Duralid Electrolyte with high KCl-Content High Durability Fast and consistent Measurement Values Proven H-Membrane Low Alkaline Error

Order Code IL-PHFERM-l

Description pH Probe pH 0..14, 0 to 130C, 0..10bar, autoclavable PG 13.5 Thread

l: 20, 225, 325, 360, 425 = 120, 225, 325, 360 and 425mm

Autoclavable Conductance Probe

Pt100 integrated Technical Data The autoclavable conductance probe is suitable for monitoring and control of fermentation processes. It is characterised as longevity and robustness. Product Features: Maintenance free Long-life cycle Fas and stable measeurement values Platinum electrodes Probe Diameter Probe Length 12mm 120, 225, 325, 360 und 425mm Process Connection PG-13,5 Thread Cell constant 1,0 cm-1 Measurement Range Bis 200 mS/cm Uncertainty +- 0,1% Temperature Range -30...+135 Pressure Range 0...10bar Connection SMEK- oder VP- Steckkopf Protection Class IP 68

Bestellcode IL-LFFERM-xx

Beschreibung Conducttance probe max. 200 mS/cm, -30 to 135C, 0..10bar, autoclavable, PG 13.5

xx: 120, 225, 325, 360, 425 = 120, 225, 325, 360 and 425mm Delivery without connecion Kabel (please send special request).

14

30 Years of Excellence in Lab Scale Technology Over 1.000 Products around the Laboratory Reactor

Fermentation Technology
HiSense Emission Analysis System
Precision Gas Analysis System for Biotechnology
due to the displacement of the of the HCO3-/-CO2 balance by metabolites (e.g. lactate, NH3), make a useable measurement impossible. This is the only method that enables the determination of the actual RQ. The high measurement resolution enables reliable measurements under the usual conditions also at the low respiratory rates of animal cell cultures and within a very broad flow range (1.8 to 100.000 Ln/h, depending on version). For these measurements, hardly any expenditure for calibration is required. The software conducts all calculations, including all corrections/compensations, online. All measurement values are stored in a data base and can be displayed as current values or in the x-t-recorder. The export is conducted as data file (csv, txt and other formats) or via RS232/Windows-Pipe. The HiSense Gas Analysis systems produced by HiTec, in contrary to conventional waste gas analysis technology, are also able to deliver the biological variables OTR, CTR and RQ. The devices are suited for R&D and laboratory applications within production and quality assurance. Measurement Technology: For the oxygen analysis, sensor are deployed which distinguish themselves by low interference, lownoise measurement results and long term stability. For the CO2- measurement, NDIR photometers and flow sensors with minimal zero point and inclination faults are deployed. Due to a novel special measurement method, the precise measurement of very minute activities both at small differences and minute measurement gas flows is possible. At the cell culture version, the deployment of additional measurement technology and the offsetting of unavoidable influences, e.g. total pressure, humidity etc., enables highly precise compensation. At conventional waste gas analysis methods and the low metabolic rates and long culture periods of cell cultures, the influence can be very significant and impede or even prevent a comparison of cultivations and make the deployment of a waste gas analysis system for quality assurance impossible. Usually, without the compensation of these factors, it is not possible to gain any useful measurement values at all. As the HiSense-c Precision Waste Gas Analysis System for cell culture and fermentation applications in addition, in most cases, is also able to perform the online determination of the true CO2 production (CER) at the usual carbonate buffer systems (optional pH input is required) usual in cell cultures. For this purpose, the faults are compensated which, Extension Options: The provision of further inputs/outputs, e.g. for pumps, valves and the arrangement of regulator circuits is possible on an optional basis. Additional inputs, e.g. for pH, temperature etc., are optionally provided for. Due to the free programmability in HiText Syntax (optional), additional algorithms, such as control and regulatory functions, can be integrated into the HiSenseTM Waste Gas Analysis Station. This also includes the RQFeed process for the optimisation of the productivity of cell cultures (if required, please request the respective special documentation). . A coupling with other systems, e.g. for control/regulation purposes is possible via network, RS232, RS485 and analogue interfaces (optional). Advantages: 1 to 8(5) Measurement Channels for 1 to 4 Fermenters, optional 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 15

HiTec Zang GmbH Tel: +49(0)2407/910 100 info@hitec-zang.de http://www.hitec-zang.com

Fermentation Technology
Technical Data (HiSense-m for microbiology) General Dimensions Operating Voltage Current Consumption Temperature Range Protection Category Measurement Channels Supply air/ Exhaust gas Operating Range of Flow Pressure Loss Input Pressure O2 Measurement Principle Measurement Range Measurement Uncertainty Resolution Long-term Drift Relevant Resolution CO2-Measurement Principle Measurement Range Measurement Uncertainty Resolution Zeropoint Drift Automatic Compensation Relevant Resolution Mass Flow Measurement Principle Standard Optional Measurement uncert. Reproducibility Prefilter Measurement Section Sample Gases OTR-CTR Measurement Values Sensitivity OTR** Sensitivity CTR** Heating Period Further Measurement Variables Operation PC Order Code IL-HISENSE-m-1 IL-HISENSE-m-2 IL-HISENSE-m-3 IL-HISENSE-m-5 IL-HISENSE-m-5 Width: 458 mm, Depth: 370 mm, Height: 140 mm at small flows 110 230 V, 50 60 Hz 3A 0 C up to 30 C IP 20 1 5 (optionally) channels for measurment in supply or exhaust gas 18 - 180 Ln/h (l/h at 0 C / 1013 mbar), optionally from 6 to 300 Ln/h < 1000 Pa atmospheric, 3 bar absolute Solid Electrolyte System 0 60 Vol. % +/- 1 % v. Measurement Range 0.02 Vol. % at 20 % O2, better upon request < 0.2 Vol. % Difference, humidity, and pressure-corrected, resolution approx. 0.02 % NDIR 0 20 Vol.%, others upon request +/- 1 % v. measurement range 0.02 Vol.% at 5 % CO2 < +/- 0.2 % Humidity, Absolute Pressure Difference, humidity, and pressure-corrected, resolution <= 0.02 % Thermal Mass Flow Measurement CO2-corrected All Channels Air supply channels, air supply regulation (mass flow regulator) Better than 2 % of setpoint 0.2 % of the setpoint None, sterile filtration at the fermenter At non-condensing ambient temperature, exhaust gas cooling required 1-10 minutes depending on hardware/software configuration approx. 1 x 10-3 Mol/l at 1 l culture volume, 5 vvh (V gas / V liquid / h ) gassing rate approx. 1 x 10-3 Mol/l at 1 l culture volume, 5 vvh gassing rate 60 min pH, OTR, CTR, OT, CT, RQ, growth rate and many peripheral values like solvant O2, CO2... Operation via software included in scope of delivery with graphic user interface. Data export to Excel or as text data file. Description Gas Analysis System for the Determination of Gas Analysis System for the Determination of Gas Analysis System for the Determination of Gas Analysis System for the Determination of Gas Analysis System for the Determination of O2 and CO2, OTR, CTR and RQ, 1 channel O2 and CO2, OTR, CTR and RQ, 2 channels O2 and CO2, OTR, CTR and RQ, 3 channels O2 and CO2, OTR, CTR and RQ, 4 channels O2 and CO2, OTR, CTR and RQ, 5 channels

Additional in- and outlets and data interfaces are realised with the components contained in the chapter Laboratory Automation, Hardware. For free programmability, the software module HiText contained in the chapter Laboratory Automation, Software is required.

16

30 Years of Excellence in Lab Scale Technology Over 1.000 Products around the Laboratory Reactor

Fermentation Technology
Technical Data (HiSense-c for cell cultures) General Dimensions Operating Voltage Current Consumption Temperature Range Protection Category Measurement Channels Supply air/ Exhaust gas Operating Range of Flow Pressure Loss Input Pressure O2 Measurement Principle Measurement Range Measurement Uncertainty Resolution Long-term Drift Relevant Resolution CO2-Measurement Principle Measurement Range Measurement Uncertainty Resolution Zeropoint Drift Automatic Compensation Relevant Resolution Mass flow measurement Principle Standard Measurement uncertainty Reproducibility Prefilter Measurement Sample Gases OTR-CTR Measurement Values Sensitivity OTR** Sensitivity CTR** Heating Period Further Measurement Variables Operation PC Order Code IL-HISENSE-c-2 IL-HISENSE-c-4 IL-HISENSE-c-8 Width: 458 mm, Depth: 320 mm, Height: 280 mm at small flows 110 230 V, 50 60 Hz 3A 0 C up to 30 C IP 20 2..8 (optionally) ch. for measurment in supply or exhaust gas of 1..4 fermenters 1,8 - 20 Ln/h (l/h at 0 C / 1013 mbar), optionally higher < 1000 Pa atmospheric, 3 bar absolute solid electrolyte system, optionally paramagnetic 0..25 vol. %, paramagnetic 0..60 vol. % +/- 1 % v. Measurement Range 0.02 Vol. % at 20 % O2, better upon request < 0.2 Vol. % Difference, humidity, and pressure-corrected, resolution approx. 0.02 % NDIR 0..20 Vol.%, others upon request +/- 0.5 % v. measurement range 0.01 vol.% at 5 % CO2 < +/- 0.05 % humidity, absolute pressure difference, humidity, and pressure-corrected, resolution approx. 0.005 % thermal mass flow measurement, CO2-corrected all channels better than 2 % of measurement range 0,2 % of measurement range None, sterile filtration at the fermenter At non-condensing ambient temperature, exhaust gas cooling required approx. 10 min, with 2 fermenters approx. 15 min approx. 1 x 10-5 Mol/l at 1 l culture volume, 5 vvh (V gas / V liquid / h ) gassing rate approx. 1 x 10-5 Mol/l at 1 l culture volume, 5 vvh gassing rate 120 min pH, OTR, CTR, OT, CT, TQ, CER, CE, RQ (corrected TQ), growth rate and many peripheral values like solvant O2, CO2... Operation via software included in scope of delivery with graphic user interface. Data export to Excel or as text data file. Description Gas Analysis System for the Determination of O2 and CO2, OTR, CER and RQ, 2 channels Gas Analysis System for the Determination of O2 and CO2, OTR, CER and RQ, 4 channels Gas Analysis System for the Determination of O2 and CO2, OTR, CER and RQ, 8 channels

Additional in- and outlets and data interfaces are realised with the components contained in the chapter Laboratory Automation, Hardware. For free programmability, the software module HiText contained in the chapter Laboratory Automation, Software is required.

HiTec Zang GmbH Tel: +49(0)2407/910 100 info@hitec-zang.de http://www.hitec-zang.com

17

Fermentation Technology
RQFeed Feeding Algorithm
The development of highly productive scalable processes has significantly gained significance for industrial production of recombinant proteins. Since Fed-batch fermentations are comparatively reliable in operation and relatively easy to handle, they are widely distributed. Exactly tuned feeding of substrates can significantly increase the productivity. However, the control or even the regulation of the substrate feeding in a bioreactor is a great challenge. The compliance with ideal, as constant as possible culture parameters is of particular importance for cell cultures. Inappropriate culture conditions do not only lower the productivity of the cells, but also affect the chemical structure (e.g. of side chains) of the secreting protein (in particular the glycosylation) which causes an undesired heterogeneity of the active substance often combined with a significant influence upon the pharmacological effectiveness. Traditional feeding models are based on the parameters oxygen uptake rate (OUR) as well as offline measured cell density substrate concentration. Samples must be drawn during the process at regular intervals and subsequently analysed using external devices and rapid tests, respectively. Based on the result of these, the feeding rate is re-evaluated and adjusted. The regulation of the feeding rate based merely on these offline parameters often leads to suboptimal results. The HiTec Zang GmbH has developed the novel regulation method RQFeed for cell cultures which is based on the respiration activity of the cells. The aerobic degradation of the substrate is accompanied by oxygen consumption (respiratory chain) which in ultimately influences the oxygen uptake OUR. This parameter thus allows to draw conclusions about the actual condition of the culture. Essential for the new Fed-batch technology is the RQFeed algorithm for the specific cultivation and production process. This algorithm is the result of intensive research into the cultivation progression as well as the deduction of according procedures and operational instructions. Organisms in aerobic fermentation consume oxygen and produce carbon dioxide. The cell-specific uptake rate of the essential substrates, e.g. glutamine and glucose, is closely connected with the oxygen uptake rate (OUR) and the carbon dioxide emission rate (CER). These two values enable the Order code SP-RQFEED-AC The diagram shows fermentation at two different RQ levels. At the RQ level 2, the productivity is not satisfactory, while Level 2 displays an ideal productivity. The RQFeed regulation algorithm is both suited for laboratory reactors as well as fermenters in pilot plants and in production. An exact determination of the RQ requires a controlled gas feed and an exhaust analysis system for the measurement of the CER and OUR. HiTec Zang offers the RQFeed regulation algorithm together with its OmniFerm laboratory fermenters in connection with a gas supply and analysis plant as an upgrading module for existing fermenters, but also on license basis for own use. In addition, training courses and instructions for the deployment of the algorithm in R&D applications and within large-scale production are also offered. determination of the respiratory quotient (RQ), a key parameter that is independent of the number of cells. It reveals the different physiological states of the cell during batch cultivation and changes dependant on concentration of substrates and products so that alterations in the physiological condition can be detected and examined on a differentiated basis. Based on these findings, a feeding algorithm was developed which is able to maintain a desired physiological condition without the necessity of having to conduct offline measurements. This leads to a significant shortening of the fermentation periods, respectively to a significant increase in production rates.

Description Feed Regulator based on RQ for Animal Cell Cultures

Information to feed regulators for other cultures is available upon request.

18

30 Years of Excellence in Lab Scale Technology Over 1.000 Products around the Laboratory Reactor

Fermentation Technology
Packed Bed Reactor
One alternative to stirred tank reactors are packed bed reactors with macroporous carriers for the cultivation of adherent animal cells which facilitates a continuous cultivation at high volume-oriented cell density and low shear stress. One significant advantage of this system is the opportunity to specially adjust the retention times of viruses and monoclonal antibodies under process conditions. When scaling up packed bed reactors, in particular the oxygen supply is problematic, as the height of the bed is restricted due to the oxygen depletion. A packed bed can, at minor overall lengths, be aerated up to 10 cm axially without occurring oxygen limitations in the upper sector of the packed bed. A further scale-up requires a radial aeration. By selecting a suited carrier, it is also possible to cultivate challenging cell types. In comparison to a stirred tank reactor, packed bed reactors possess a higher performance capacity in regard to the reachable volume-specific cell density and productivity. Due to the cell retention in the packed bed, integrated separation procedures using ceramic membranes are easy to realise. Additionally, the operation of a packed bed is very uncomplicated and cost-effective. The packed bed system has been used in the biochemical laboratory for the largescale production within the scope of the following projects: Production of retroviral particles for genetherapeutic applications with the Paul-Ehrlich Institute and the Technical University Hamburg Harburg. Production of monoclonal antibodies for in vitro diagnostics on novel ceramic carriers in cooperation with the company Therapharm Deutschland GmbH. Order Code VA-FESTBETT Description Packed Bed Reactor with microporous Carriers for the Cultivation of adherent Cell Cultures

One of our main strengths is the construction of Special Custom Designs! Do not hesitate to contact us with any problem, we will help you to find a solution.

HiTec Zang GmbH Tel: +49(0)2407/910 100 info@hitec-zang.de http://www.hitec-zang.com

19