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Adress: zeta Bio- und Verfahrenstechnik Alte Jonastrasse 83 CH-8640 Rapperswil Switzerland

Contact: Dr. Michael Nägeli Phone: +41 55 450 60 06 Fax: +41 55 460 13 33 E-Mail: michael.naegeli@zeta.com Date: 16.11.2007

bio-t® mini – The Laboratory Bioreactor System

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Table of contents 1. Zeta Overview ............................................................................................................................. 3

1.1. Welcome to the world of Zeta................................................................................................. 3 1.2. Core competence Bio- and Process Technology................................................................... 3 1.3. Our Products .......................................................................................................................... 4 1.4. References............................................................................................................................. 5

2. Zeta bio-t® mini Laboratory Bioreactor System ........................................................................... 6 2.1. Design Overview .................................................................................................................... 6 2.2. Bioreactor ............................................................................................................................... 7 2.2.1. Glass vessel ....................................................................................................................... 7 2.2.2. Head plate .......................................................................................................................... 8 2.2.3. Agitator ............................................................................................................................... 9 2.2.4. Sampling system ................................................................................................................ 9 2.3. Control System..................................................................................................................... 10 2.3.1. Hardware.......................................................................................................................... 10 2.3.2. Technical data .................................................................................................................. 10 2.3.3. Gas mixing station............................................................................................................ 11 2.3.4. Pumps .............................................................................................................................. 11 2.3.5. Software ........................................................................................................................... 12

2.3.5.1. Touch screen............................................................................................................ 13 2.3.5.2. pH measuring and control ........................................................................................ 17 2.3.5.3. Temperature measuring and control ........................................................................ 17 2.3.5.4. Agitator speed measuring and control...................................................................... 17 2.3.5.5. Dissolved oxygen measuring and control................................................................. 17 2.3.5.6. Foam detection and control ...................................................................................... 19 2.3.5.7. External signals measurement and control .............................................................. 19 2.3.5.8. Alarm handling.......................................................................................................... 19 2.3.5.9. SCADA light.............................................................................................................. 20 2.3.5.10. SCADA full version ............................................................................................... 20

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1. Zeta Overview

1.1. Welcome to the world of Zeta The zeta Holding is a network of internationally active independent companies using the synergy of their core competences in the business areas of

• Bio- and Process Technology • Sterile Plant Construction • Automation

Spread over central Europe, more than 280 own members of staff permanently work for our customers from the various locations. The zeta group of companies is committed to continuous and sustainable development, combined with the latest technologies and active quality awareness (ISO 9001:2000, EN 729, SCC). Vitality, engagement, a visionary thought pattern as well as clear structures and perfect synergies are the attributes of which you benefit when projects are being implemented. Therefore zeta stands for “Evolution of Technology”.

1.2. Core competence Bio- and Process Technology The core competence of zeta’s bio- and process technology activities lies in the technical implementation of the concepts developed by our customers in the pharmaceutical industry. With our understanding of specific processes, design expertise, corresponding production capacities and our know how of qualified procedures, we are the perfect partner for you. In-house production of key components allows us to maintain top quality standards and to demonstrate the flexibility that is needed in implementing projects. Whether you require the whole package of engineering, production and start-up or simply one element of these services, zeta is your competent partner for process engineering – a partner that evolves dynamically to achieve your success – “Evolution of Technology”. The department Bio- and Process Technology of the Zeta affiliate in Rapperswil (Switzerland) has its core competence in engineering and planning of components and plants for the biotechnology-, the pharmaceutical- and the food industry. Based on the many years of experience of our employees, we can guarantee comprehensive and professional completion of projects from simple laboratory scale to complex production scale projects.

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1.3. Our Products Based on this long-term experience in the field of building biotechnological plants, our team of technologists and engineers have developed systems of bioreactors, which meet any specific requirement. No matter if it is bacterial, yeast or animal cell cultures – no matter what culture volume you work with - Zeta can offer a product line from small- over pilot- to large scale bioreactors to the market. All system variants – from bench-top, autoclavable laboratory bioreactor, multi-bioreactor systems as well as entire production plants are within the product portfolio of Zeta. The following figures provide you some insight in to the world of Zeta.

bio-t® mini: Autoclavable laboratory reactor bio-t® xpac: Multi-bioreactor system for screening cultivation

bio-t® pro: Sterilizable pilot scale bioreactor bio-t® pro: Production scale bioreactor

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1.4. References

Our range of products includes all types of bioreactors from small scale autoclavable laboratory bioreactors up to in-situ sterilizable production scale units with several 1000 liters working volume. The following table lists a selection of our references.

Country Scope of supply / Plant Scale Czechia Stirrer systems, Tank and pipeline construction Production Austria Fermenter Pilot scale Austria Biotechnology Plant Production Switzerland Biotechnology Plant Pilot/Production Switzerland CIP-Plant Production Germany Fermenter Laboratory Austria Fermenter Pilot Austria CIP-plant Production Austria Multi fermenter system “Xpac” Laboratory Germany Tank and pipeline construction Production Switzerland Utilities, Tank and pipeline construction Production Switzerland Multi fermenter system “Xpac” Laboratory Germany Utilities Production Slovenia Tank and pipeline construction Production Slovenia Up-stream - Plant Production Czechia Tank and pipeline construction Production Czechia Tank and pipeline construction Production Germany Multi fermenter system “Xpac” Laboratory Germany Fermenters Production Germany Fermenters and Tank and pipeline construction Production Germany Fermenters and Tank and pipeline construction Production Germany Fermenters and Tank and pipeline construction Production Germany Mobile mixing- / sterile tanks Production Nederland Plant for Cell culture Production Switzerland Fermenter skid Production Germany Biotechnology Plant Production

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2. Zeta bio-t® mini Laboratory Bioreactor System

2.1. Design Overview As the smallest member of the zeta family, the bio-t® mini laboratory bioreactor system is designed for conventional research and development tasks, and also as a training system for universities. In addition it is used as well for Inoculum Production in GMP environments. The bio-t® mini laboratory bioreactor system is divided in two functional parts:

• Bioreactor • Control system

The bioreactor consists of an autoclavable glass vessel with double jacket and a stainless steel head plate. The head plate is equipped with a removable motor, agitation system and all necessary ports for measuring devices like pH-sensor, pO2-sensor, Temperature probe, foam, level etc. Glass vessel and head plate are sealed with an O-ring using four bolts. Three different vessel volume sizes are available. The control systems consists of a compact and easy to clean stainless steel housing, holding the 8” color touch screen as man-machine interface. The bio-t® mini’s control system is based on a PC-compatible processor system. Four free to configure feed pumps, flow meters and a heating device in conjunction with the necessary control and regulating systems allow you to cultivate the organisms of interest according to their needs. bio-t® mini: High Tech Tool for Research and Development as well as Inoculum production within GMP environment.

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2.2. Bioreactor The bio-t® mini laboratory bioreactor system is available with glass vessels with working volumes starting from 0.4 to 10 Liters. The bioreactor vessels are autoclavable and consist of the following main components:

• Graduated double jacket glass vessel (mm) manufactured from high-quality borosilicate glass • Head plate (Stainless steel 1.4435), electro polished (Ra<0.8µm) • Agitation system with removable motor and magnetic coupling or mechanical seal • Stand made from stainless steel with handles • Holder for supply bottles (optional)

2.2.1. Glass vessel Vessel type (Liter) 2L 5L 10L 2L 5L 10L Type of culture Bacteria Cell Culture Total volume (Liter) 2.9 6.7 13.2 2.9 6.7 13.2

Maximal volume1 (Liter) 22 5 10 22 5 10

Minimal volume (Liter)3 0.4 1.4 1.9 0.4 1.4 1.9 Height (mm) 220 380 465 220 380 465 Diameter (mm) 130 150 190 130 150 190 Autoclave dimensions using a fixed exhaust air cooler (mm)4

-Width -Height

350 514

350 740

380 870

350 514

350 740

380 870

Autoclave dimensions using a fold-away exhaust air cooler (mm)

-Width -Height

350 358

350 536

380 620

350 358

350 536

380 620

Surface area (mm) -Width -Height

700 600

700 600

730 600

700 600

700 600

730 600

Remarks: 1=The volume can vary due to individual measuring probe set up; Values represent Volume until the upper end of the heating jacket; 2=The maximal

working volume using magnetic coupling is 1.5 Liter; 3=All probes active

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2.2.2. Head plate The head plate made from stainless steel 1.4435, electro polished (Ra<0.8µm) is equipped with 12 connecting ports Pg13.5 (19 mm). Exceptionally the two Liter vessel has 6 x PG13.5 and 6 x 11mm connecting ports. A vessel stand is mounted on the outside of the head plate. On the inside the baffle cage is fixed to the head plate to increase turbulence wile steering (only bacteria culture vessel). Optional equipment for bubble free aeration can be fixed to the head plate. Vessel type (Liter) 2L 5L 10L 2L 5L 10L Type of culture: Bacteria Cell Culture Probes: -pH pH Electrode, Mettler Toledo -Temperature PT 100 -pO2 Oxygen Electrode, Mettler Toledo -Anti foam1 Conductivity probe Additions: -Acid X X X n/a1 n/a1 n/a1 -Base X X X X X X -Media X X X X X X -Anti foam X X X n/a1 n/a1 n/a1 Aeration: -Aeration with sparger Sparger Micro sparger -Aeration through head space n/a2 n/a2 n/a2 X X X -Exhaust air Nozzle with exhaust air cooler Unloading: -Sampling Sampling system with dip tube -Harvest/Level Height adjustable tube Remarks: 1=often not used during cell culture 2=often not used during bacterial culture

Within the scope of delivery: • 2 blind plugs • 6 sterile filters (Air inlet, Air outlet, Sampling system, 3 storage bottles) • 1 sterile coupling • 3 storage bottles

Vessel type (Liter) 2L 5L 10L 2L 5L 10L Type of culture Bacteria Cell Culture

Storage bottles 3 x 100ml 2 x 100 ml 1 x 250ml

1 x 100ml 2 x 250ml 3 x 100ml 2 x 100 ml

1 x 250ml 1 x 100ml 2 x 250ml

Baffle cage X X X n/a n/a n/a More than 12 head plate connecting ports are easily achieved using double- or triple connectors for one PG13.5 port (optional).

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2.2.3. Agitator Magnetic coupled stirrers are clearly superior to direct drive agitators concerning sterile design. Therefore zeta offers the top-mounted AMR magnetic type agitators for bench top bioreactors. Nearly every mixer element can be used in conjunction with the agitator and these elements are adjustable in height on the agitator shaft. Apart from magnetic-coupled agitators, zeta also supplies conventionally direct drive agitators with a single or double mechanical seal. These are used mainly where the risk of contamination is more of a secondary consideration. In both cases the motor is fastened directly on the head plate and can be removed for autoclaving. Different agitation speeds configurations can be chosen according to the need of the cultivation process. For microbial cultures we recommend a configuration where the speed can be controlled in the range of 100-1000 rpm (±1%) to allow a maximal gas input into the cultivation media. Using cell culture the configuration of 50-450 rpm is recommended. A baffle cage to generate a turbulent environment within the bioreactor is integrated in the “Bacteria” version. Vessel type (Liter) 2L 5L 10L 2L 5L 10L Type of culture Bacteria Cell Culture

Amount of mixer elements 21 21 31 21 21 31 Type of mixer elements 6-bladed rushton turbine 3-bladed segment stirrer Agitation speed 100-1000 rpm 50-450 rpm Magnetic coupling/Direct drive optional Remark: 1=Adjustable in height

6-bladed rushton turbine 3-bladed segment stirrer

2.2.4. Sampling system The sampling system consists of a graduated sampling container with sterile trap and sterile filter which is supported by the head plate. The sampling system is connected with a flexible hose to a dip tube going into the culture vessel. The culture broth is transported into and out of the sampling container by the use of a syringe.

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2.3. Control System The Control System consists of a compact and easy to clean stainless steel housing. On the front wall integrated is the 8“ color touch screen as man-machine interface. Furthermore four free to configure peristaltic pumps and Rotameters are installed on the front wall. Ports such as aeration in- and outlet, water for the heating circuit as well as measuring probes are installed on the side of the control system, looking towards the bioreactor. The gas mixing station with the reducing valves, electrical cable, the serial connecting ports for the external signals and the SCADA port is located on the rear side of the control system.

2.3.1. Hardware The bio-t® minis Hardware comprise the following Hardware:

• Compact and easy to clean stainless steel housing • bio-t® mini control system with 8” color touch screen • CPU: Intel PXA255 200MHz Processor • Heating circuit with circulation pump and heating device (1000W) • Power controller for temperature and agitation • Pressure reducing valves with gas mixing station (Standard, Pulse Flow Control, Continuous

Flow Control • Integrated controller for pO2, pH • 4 free to configure peristaltic pumps • Serial port (RS232) or USB for data transfer to PC • Ports for two external signals (e.g. pumps) • Rotameter for aeration using a dip tube • Rotameter for aeration using head space • SCADA light for data export

2.3.2. Technical data Control system (L x W x H): 550 x 250 x 520 mm Color touch screen (L x W): 231 mm x 176 mm Power cord: Place of delivery standard Weight (kg): 29 kg Electrical service: 100 -120 V 50/60 Hz or 200 -230 V 50/60 Hz

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2.3.3. Gas mixing station The following three gas mixing stations are available: Gas mixing station

Standard

Pulse Flow Control

Continuous Flow

Control

Function

Mixing of one

additional gas to air

Mixing of 3 additional

gases to air

Mixing of 3 different

gases to air (incl. four Mass Flow

Controllers) Type of culture: (B)acteria; (C)ell culture

B C B C B C

Aeration using (S)parger; (H)ead space

S (vvm)

S (vvh)

S (vvm)

S (vvh)

H (vvh)

S (vvm)

S (vvh)

H (vvh)

Air 2 1 2 1 n/a 2 1 n/a

O2 0.83 1 0.83 1 n/a 0.83 1 n/a

CO2 n/a n/a n/a n/a 0.2 n/a n/a 0.2

N2 n/a n/a 0.83 1 0.5 0.83 1 0.5

2.3.4. Pumps Four free to configure peristaltic pumps with Watson Marlow pump heads (Type OEM 102) are within the standard set-up of the bio-t mini control system. All pumps can be operated also in a manual mode. The pumps are preconfigured for the addition of acid, base, anti-foam and substrate. The residual pump, if control strategies with CO2 are used, can be configured as a harvest pump. The software allows changing this configuration. In total 6 pumps can be connected to the bio-t® mini control system. The maximal dosing rate for each pump can be calibrated with the hose empirically.

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2.3.5. Software The bio-t® mini software is a user friendly program to automate and configure your cultivation process. The menu-drive software allows the several control strategies. Data supervision, trending and alarming are further features within the package.

• Simple menu-driven data entry • Measuring and control of up to 11 parameters • Ramp functions • Control of two external signals • Alarming • Access control with password • Data exporting function to PC (SCADA light)

Beside the data exporting function of the SCADA light, a full version is available too, which allows to control the bioreactor from a PC. The following functions are supported by the software Evolution mini for the three Gas mixing stations: Standard Pulse Flow Continuous

Flow Temperature control √ √ √ pH Control (Acid/Base) √ √ √ Anti-foam control √ √ √ Level control √ √ √ Agitation control √ √ √ pO2 control as a function of the agitator, one gas or dosing of substrate √ √ √

Automatic switch between Sparger and Head space aeration. √ √

pO2 control as a function of the agitator, four gases and dosing of substrate √ √

pH Control (CO2/Base) √ √ Automatic control of gas flux √ Data export function (SCADA light) √ √ √ SCADA full version optional optional optional Weight control optional optional optional Pressure control optional optional optional

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2.3.5.1. Touch screen

The user-friendly menu-driven software is operated via a color TFT touch screen. All menu selections and data entries are made by pressing the keys on the screen.

After bio-t® mini control system has been started the following menu arrives:

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With the menu “WATCH” that is shown in the picture below the actual status of the fermentation can easily be followed:

In the following picture the trend visualization can be seen as it appears on the touch screen:

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The following picture shows the alarm list as it appear on the touch screen.

Within the menu „SETTINGS“ the different parameters can be calibrated and preconfigured according to the process. The following screen shows up:

2 2006/04/12 13:28 O2 High 1 2006/04/12 12:10 12:15 T Low 0 2006/04/12 11:00 11:10 pH High

Date

Alarm Number Time of appearance

Time of acknowledgement

Cause

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Beside the set points and dead zones, different ramps can be preconfigured. As an example a process can run at an early stage at low temperature and after the preset time has elapsed the temperature decreases or increases to a second preset temperature level. These so-called „ramp “functions can be preset for the parameters temperature, oxygen, agitator speed, medium addition, level and weight.

A correct password has to be entered as a prerequisite to adjust parameters:

The following three access levels arise when entering the menu “ACCESS CONTROL”:

1st level: general rights (the password can be changed) 2nd level: administrator rights (the password can be changed) and you have more

access rights, e.g. to change PID parameters 3rd level service level (this password cannot be changed) the service engineer has

to become access even when the password went loss.

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2.3.5.2. pH measuring and control The pH of the culture media is measured with a Mettler Toledo pH electrode. The software allows a temperature compensated 2 point calibration before autoclaving. The set point of the pH and the corresponding range can be preset. The culture media’s pH is controlled by either the addition of acid or base or CO2 and base. If the measured pH value is lower or higher than the set point, the controller activates the peristaltic pump to either add acid or base into the bioreactor to adjust pH. Type of sensor: pH electrode, Mettler Toledo Calibration: Via software Control range: 2-12 pH Units Control accuracy: ± 0.1 pH Units Control mode: PI regulator with puls modulated outputs Control characteristics: logarithmic with dead band Temperature compensation: Via software

2.3.5.3. Temperature measuring and control Temperature within the culture media is measured by a temperature probe installed in the bioreactor. The software allows you to configure the set point of the temperature as well as the minimum and maximum temperature. The culture media’s temperature is controlled by circulating thermostatic water through the vessel jacket. If the culture media temperature is lower than the desired set point, the thermostatic water is heated by the electric water heater within the loop. An open loop with the heater off and cooling water entering is employed for cooling the vessel jacket. Type of sensor: PT 100 Ohm RTD, vessel Control range: 20-80°C, depending on supply water temperature Control mode: PID Control accuracy: ± 0.1°C

2.3.5.4. Agitator speed measuring and control Agitator speed is measured with a Hall sensor near the motor. The software allows you to configure the set point of the agitator speed. The speed can be configured as explained in chapter 2.2.3.

2.3.5.5. Dissolved oxygen measuring and control The aeration system provides the necessary gas for your organism to grow. The inlet of the external gas supply (regular air, oxygen, nitrogen, carbon dioxide) is located on the rear of the control system. Within the control system the gas blending unit with the flow meter is providing the gas concentration and mass flow according to the set point. The gas is then transported from the control system to the bioreactor were a sparger disperse the gas into the culture medium. In addition the agitator in the bioreactor is dispersing the gas bubbles within the culture medium. The gas dissolve and can be taken up by the organism of interest. The bioreactor can either be operated by head space aeration or submerse.

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2.3.5.5.1. Dissolved oxygen measurement (DO) Dissolved oxygen measurement within the culture media is performed by a Mettler Toledo Oxygen electrode. The software allows a 2 point calibration (0% and 100%). The set point of the dissolved oxygen concentration can be preset. Type of sensor: Mettler Toledo Dissolved Oxygen electrode Calibration: via Software Control range: 0 – 100% dissolved Oxygen Control mode: PID Control characteristics: Temperature compensation: yes The culture media’s dissolved oxygen can be controlled by the strategies given in the following text.

2.3.5.5.2. Dissolved oxygen control strategies Agitator speed: By increasing the agitator speed the exposure time of the gas within the surrounding medium is enhanced. Therefore more gas can be dissolved in the medium. The agitator speed can be preconfigured with a minimum and a maximum value. If the measured dissolved oxygen concentration falls below the set point, the agitator speed gradually increases so that more gas can dissolve in the culture medium. The agitator speed is decreased if the value is higher than the set point. Gas mass flow: By increasing the gas mass flow into the bioreactor more gas can dissolve in the culture medium. The gas flow can be preconfigured with a minimum and a maximum value. If the measured dissolved oxygen value falls below the set point, the gas flow gradually increases so that more gas is delivered to the culture medium. The gas flow is decrease if the value is higher than the set point. For this option a mass flow controller can be installed in the biot-t® mini control system. Gas blending: Alternatively the gas concentration within the bioreactor can be enhanced by the use of concentrated oxygen or nitrogen. The integrated gas blending station is mixing the desired concentration with the feedback information (DO) form the bioreactor. A constant gas flow rate is manually set with a rotameter. The oxygen concentration of the incoming gas is limited between preset limit. If aeration with regular air is not sufficient to reach the lower set point, pure oxygen gas will be added to the air stream. Substrate Addition: The bio-t® mini control system can be configured to enhance the gas concentration within the bioreactor by adding gas enriched culture media by using the substrate pump. Is the gas concentration within the culture media decreasing the controller activates the substrate pump to add gas enriched substrate.

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Combined control strategies: Any of the before mentioned control strategies can be combined. Moreover the bio-t® mini control system can be configured to run different oxygen concentration over time.

2.3.5.6. Foam detection and control A conductivity probe placed in the head space of the bioreactor is detecting foam. Foam is controlled by dosing antifoam agent into the bioreactor with a peristaltic pump. The quantity of the antifoam dose, delay between doses and the detection sensitivity of the probe can be set so that minimal quantities of antifoam are added. Type of sensor: Conductivity probe, PTFE coated Control range: 50 -1000 kOhm Type of controller: Impulse pause algorithm

2.3.5.7. External signals measurement and control An upgrade board is installed in the biot-t mini control system which allows up to two 4-20 mA galvanically isolate input signals from external devices (i.e. optical density meter, glucose analyzer, pumps, etc.) to be measure and controlled. The inputs signals are calibrated using the software and expressed as percentage (i.e. 0 – 100 %). Input signal from sensor: 4 – 20 mA, galvanically isolated Control range: 0 – 100%, full scale Type of controller: proportional regulator with dead band

2.3.5.8. Alarm handling Limits can be configured for each cultivation parameter that is measured and controlled. Deviations to the set limits are activating an alarm. Alarms are then recorded and displayed on the touch screen. In case of a sensor failure, an additional Fail Low/High function ensures that the particular control function is switched off.

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2.3.5.9. SCADA light Included within the the bio-t® mini standard configuration is a data monitoring and export function (SCADA light). The system allows transferring data from the control system to the customers PC. In this way data can be exported to Excel. Data is exported using a serial interface (RS 232) or USB.

2.3.5.10. SCADA full version A complete SCADA system can be offered as well. The difference to the SCADA light system is that the full version, besides following the process, can be used to control a current process by the use of a PC. Visualizing trends and recipe handling are further feature of the system. Using the internet allows you to follow your process from your office or home. These functions are password protected of course. The menu will be provided in English with the following features:

• Archiving of alarms and other status signals • Trending • User administration • Control functions • Audit trail according 21 CFR Part 11 • Archiving of batch data • Visualization and operation with touch screen und keyboard • Optional interface to existing plant control system

• Easily and clearly arranged graphic user interface

- Main menu - Process overview - Object pictures with hand operation - Controller pictures - Calibration menus - Recipe editor - Recipe controlled batch process operation

Please refer to the example pictures in the following text.

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2.3.5.10.1. Start picture with user administration

The start picture will be divided into three sections. In the upper section navigation buttons for operation pictures will be located. Furthermore the picture name and the last alarm signal /acknowledge will be displayed.

On the middle part the actual pictures will be displayed.

In the lower section the navigation will be started and shortcut buttons and the actual user will be displayed.

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2.3.5.10.2. Main menu The main menu on the left side will be used to navigate between the operation pictures. The navigation rail will be used to choose the several pictures, for example:

• Alarm logging • Historical trends • Audit trail • Recipe editor • User administration

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2.3.5.10.3. Overview picture The overview picture will be used to display the main parameter for supervision of the process

• Media feed • Acid, base, anti foam • Aeration • Temperatures • Step sequences • Parameter:

o pO2 o pH-value o temperature o agitation speed o Level o Gas flow

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2.3.5.10.4. Valve overview picture In the valve overview picture the status of all valves, pumps, agitators and instrumentation will be displayed.

The authorized user will be able to operate the active components via hand mode.