ez Control for In Situ sterilizable Bioreactors

ez-Control for In Situ sterilizable Bioreactors

HARDWARE MANUAL

Project 310420; PS 70L, IST (AU)

HARDWARE MANUAL

CONTENTS ez-Control for In Situ sterilizable Bioreactors Project 310420; PS70 IST (AU)

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TABLE OF CONTENTS 1 SAFETY .....................................................................................................................................................4

1.1 Safety Symbols ..................................................................................................................................4 1.2 Safety Warnings ................................................................................................................................5 1.3 EU Declaration of Conformity ..........................................................................................................7

2 GENERAL .................................................................................................................................................8 2.1 Introduction .......................................................................................................................................8 2.2 System View ......................................................................................................................................8 2.3 Emergency Stop Button .....................................................................................................................9 2.4 Right-Side View ................................................................................................................................9 2.5 Rear View ........................................................................................................................................10 2.6 Sensor Input Specifications .............................................................................................................13 2.7 Environmental Conditions ...............................................................................................................13 2.8 Cleaning Instructions .......................................................................................................................13

3 ACTUATORS ..........................................................................................................................................14 3.1 Gas Supply Section .........................................................................................................................14

3.1.1 Mass Flow Controllers .......................................................................................................14 3.1.2 Valves in Gas Supply Lines ...............................................................................................15 3.1.3 Aearation Flow Pattern ......................................................................................................15

3.2 Actuators for Temperature Control..................................................................................................16 3.3 Pumps and Tubing ...........................................................................................................................17 3.4 How to Load the Tubing .................................................................................................................18 3.5 Actuator control ...............................................................................................................................19

3.5.1 Discrete actuators versus Continuous Actuators ................................................................19 3.5.2 Actuator for Level / Foam Control ....................................................................................19

4 AGITATION ............................................................................................................................................20 4.1 Internal Stirrer Speed Controller .....................................................................................................20 4.2 Applicable Stirrer Motors ................................................................................................................20 4.3 Stirrer Motor Specifications ............................................................................................................21

4.3.1 Motor P100, i=6 ................................................................................................................21 4.3.2 Motor P1000 ......................................................................................................................21 4.3.1 Motor ESA 6L/30 ..............................................................................................................21

4.4 Power Supplies for Stirrer Motor Assemblies .................................................................................22 4.5 Motor Cables ...................................................................................................................................22 4.6 Maintenance ....................................................................................................................................22

5 REACTOR ...............................................................................................................................................23 5.1 General Reactor Construction .........................................................................................................23 5.2 70L Reactor .....................................................................................................................................24 5.3 Head Plate Port Specifications ........................................................................................................25 5.4 Reactor Port Specifications .............................................................................................................25 5.5 Utility Connections ..........................................................................................................................26

6 AUXILIARIES .........................................................................................................................................27 6.1 Stirrer Assembly ..............................................................................................................................27

6.1.1 Bottom Drive Stirrer Assembly .........................................................................................27 6.1.2 Stirrer Motor ......................................................................................................................27 6.1.3 Turbine impellers ...............................................................................................................27 6.1.4 Baffles ...............................................................................................................................27

6.2 Optimum Impeller Configuration ....................................................................................................28 6.2.1 Power Required for Stirring ...............................................................................................29

HARDWARE MANUAL

ez-Control for In Situ sterilizable Bioreactors CONTENTS Project 310420; PS70 IST (AU)

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6.2.2 Example of Stirrer Speed Requirement Calculation ..........................................................29 6.3 Aeration ...........................................................................................................................................30

6.3.1 Auxiliaries for Aeration .....................................................................................................31 6.3.2 Off-Gas Line ......................................................................................................................32

6.4 Auxiliaries for Addition ..................................................................................................................33 6.4.1 Push Valve Assembly ........................................................................................................33 6.4.2 Rapi-Lok Sterile Connector ...............................................................................................33

6.5 Auxiliaries for Drain .......................................................................................................................34 6.6 Safety Devices .................................................................................................................................35

6.6.1 Pressure Gauge ..................................................................................................................35 6.6.2 Rupture Disk and Discharge Pipe ......................................................................................35 6.6.3 Relief Valve and De-aerator for Jacket ..............................................................................35

6.7 Blind Stoppers .................................................................................................................................36 6.8 Illumination .....................................................................................................................................36

7 SENSORS.................................................................................................................................................37 7.1 pH ....................................................................................................................................................37 7.2 Temperature ....................................................................................................................................37 7.3 DO ...................................................................................................................................................37 7.4 Level ................................................................................................................................................37

© 2015 Applikon-Biotechnology; Delft, the Netherlands. All rights reserved. This set of documents is protected by copyright law. It may not be reproduced, stored or made public in any form by any means without the prior written approval of Applikon Biotechnology B.V. Applikon Biotechnology B.V. Heertjeslaan 2 2629 JG Delft The Netherlands Tel.: +31 (0)10-2083555 Fax.: +31 (0)10-2083505 Web: http://www.applikon-biotechnology.com E-mail: [email protected]

http://www.applikon-biotechnology.com/

mailto:[email protected]

HARDWARE MANUAL

SAFETY ez-Control for In Situ sterilizable Bioreactors Project 310420; PS70 IST (AU)

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1 SAFETY

1.1 SAFETY SYMBOLS The following symbols are used on the equipment and in this manual.

WARNING Risk of personal injury or danger to life.

WARNING Risk of electrical shock hazard.

WARNING Risk of physical hand injury.

WARNING Hot surfaces. Take precautions to prevent burns!

Additional information

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ez-Control for In Situ sterilizable Bioreactors SAFETY Project 310420; PS70 IST (AU)

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1.2 SAFETY WARNINGS GENERAL

• This set of equipment has been designed in accordance with EN61010-1 "Safety Requirements for Electrical Equipment for Measurement, Control and Laboratory Use", and has been supplied in a safe condition.

• The Hardware manual contains information and warnings, which have to be followed by the user to ensure safe installation, operation and to retain the equipment in safe condition.

• Before switching on the equipment, make sure that it is set to the line voltage. • This equipment is designed for bioprocess control; it must not be used for other purposes!

CAUTION Potential hazard of machinery.

• The stirrer assembly including motor at the bioreactor is a machine with potential hazard (moving parts). The stirrer motor must not be put into service until the machinery into which it is to be incorporated has been declared to conform the machinery directive 2006/42/EEC.

WARNING Risk of electrical shock hazard.

• The cabinet that contains the stirrer speed controller must not be switched on until the motor cable and the encoder cable are properly connected to both the cabinet and the stirrer motor.

• When the cabinet that contains the stirrer speed controller is switched on, the motor cable and the encoder cable must not be disconnected. When the motor cable is disconnected, high voltage will be interrupted (risk of burns and electric shock). When the encoder cable is disconnected, the stirrer speed will increase in an incontrolled manner since no feed back is available.

CAUTION Potential hazard of machinery.

• Make sure that the stirrer motor is only operated when it is properly seated in the stirrer assembly of the bioreactor.

• When the stirrer motor has been put into operation, it will become hot. Take precautions to prevent personal injury!

WARNING Risk of physical hand injury caused by pumps.

• In most cases, one or more pumps are installed in the ez-Control. • According to the European Community legislation on machinery, a pump drive is a machine with

potential hazard. Only apply these drives with the pump heads and tubing that are described in this manual (chapter: pumps)!

• Switch off the power of the pump drive before replacing any tubes or pump heads.

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SAFETY ez-Control for In Situ sterilizable Bioreactors Project 310420; PS70 IST (AU)

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CAUTION Risk of damage to the equipment.

• Do not use the tubing pump drives for other purposes than displacement of fluids (or gas). • Advised pump tubing (silicone or norprene food) have their limitations in physical and

chemical resistance. Make sure that the pump tubing and the selected reagent type are compatible. Refer to a Chemical Compatibility Database like: http://www.coleparmer.com/Chemical-Resistance

• Application of damaged tubes may result in fluid leaking into the pump drive. • Verify the tube quality inside the pump head before every fermentation run.

Additional information

• Do not apply an Earth Leakage Circuit Breaker in the power supply of the Applikon control equipment (in an industrial environment or laboratory, an earth leakage circuit breaker is not mandatory).

• Because of the built-in main supply filters, an Earth Leakage Circuit Breaker may be activated. As a result, the power will be switched off!

• Additional electrical protection can be realized by installing a separation transformer between the mains and the control equipment.

http://www.coleparmer.com/Chemical-Resistance

HARDWARE MANUAL

ez-Control for In Situ sterilizable Bioreactors SAFETY Project 310420; PS70 IST (AU)

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1.3 EU DECLARATION OF CONFORMITY

The company Applikon Biotechnology B.V., residing in Delft, The Netherlands, hereby certifies that the instrument:

ez-Control for

In Situ sterilizable Bio Reactor Systems meets the requirements of the following Directive of the European Union: - Directive 2006/42/EC on Machinery that includes: - Directive 2014/30/EU on Electromagnetic Compatibility - Directive 2014/35/EU on Low Voltage ir A. Oudshoorn MBA January 5th, 2010 Managing Director Applikon Biotechnology B.V. Heertjeslaan 2 2629 JG Delft The Netherlands Tel.: +31 (0)10-2083555 Fax.: +31 (0)10-2083505 Web: http://www.applikon-biotechnology.com E-mail: [email protected]

http://www.applikon-biotechnology.com/

mailto:[email protected]

HARDWARE MANUAL

GENERAL ez-Control for In Situ sterilizable Bioreactors Project 310420; PS70 IST (AU)

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2 GENERAL

2.1 INTRODUCTION The Applikon In Situ sterilizable Bioreactor Systems basically consist of the following parts: • An In Situ sterilizable Bioreactor with the appropriate auxiliaries, like a stirrer assembly, baffles, sensors,

an aeration assembly, etc. • A wet part for temperature control and steam sterilization. • An ez-Control for measurement and control of process variables (pH, temperature, DO, level, pressure,

and stirrer speed) with corresponding controller outputs in order to keep process conditions on set-point. The ez-Control combines and supports actuators like pumps and valves.

2.2 SYSTEM VIEW Below the combination of the ez-Control and the Pilot System 70L is presented. The Stirrer Speed Controller (for reactor agitation) and the Power Supply for the Electrical Heater (for temperature control during cultivation) are located inside the ez-Control cabinet.

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ez-Control for In Situ sterilizable Bioreactors GENERAL Project 310420; PS70 IST (AU)

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2.3 EMERGENCY STOP BUTTON The Emergency Stop button is located at the left upper corner of the cabinet. If the Emergency Stop button is pressed, all actuators are switched off (to default position). The power supply to the sensor hardware and control device is maintained, so measuring the process parameters is continued. The calculated controller output however, is not realized until the Emergency Stop situation is reset to normal. Reset an Emergency Stop situation by turning this key clockwise until it returns to the normal position. Now log in as a System Engineer to continue normal operation.

2.4 RIGHT-SIDE VIEW Sensor and I/O connections The following four sensor connections can be found at the right-side panel: • BNC connector for the pH sensor • BNC connector for the DO sensor • 3-pins connector for the Temperature sensor • 2-pins connector for the Level sensor Below the sensor connections, two I/O connections are located for the stirrer motor: • 5-pins connector for the Stirrer motor • 8-pins connector for the Stirrer encoder (feedback of actual stirrer speed)

Sensor and I/OConnections

Bottle Rack(optional)

Wet PartConnections

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Wet part connections At the bottom of the side panel, the following connections can be found: • LL-0.03: Connection for illumination in the reactor • HE-2.51: Connection for the filter heater • P-2.41: Connection for the wet part circulation pump • HE-2.71: Connection for the wet part heating element

2.5 REAR VIEW Communication Ports • RJ-45 Ethernet connection: - Supervisory Control And Data Acquisition (SCADA)

program on a Host - Access for remote control • Mini USB port • Two USB ports • 9-pin sub-D connector Pin layout of the 9-pins sub-D connector

Pin Function

1 System Alarm (normally closed) 2 System Alarm (normally open) 3 System Alarm (Common) 6 Process Alarm (NC) 7 Process Alarm (NO) 8 Process Alarm (Common)

4, 5, 9 Reserved for future use System Alarms will interrupt operation of the ez-Control (e.g. Emergency Stop). Process Alarms are caused by Process Parameter values (pH, Temp., DO, etc.) that are outside the Alarm Limits. The alarm pins of the 9-pins sub-D connector are potential-free and can be connected to a 24 VDC power supply with maximum current of 2 A.

In case additional I/O (inputs and/or outputs) are required, the Extended I/O Module will be mounted at the rear of the ez-Control. For more information, refer to the Extended I/O Manual.

The SIP Control Connections are connected to the SIP Actuator Module that is located below the air in- and outlets (see the next page).

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Power Section • Mains Cable Entry • Power Switch • Fuse holders; fuses must comply with IEC 127:

- 230 VAC: 4 x T 5A (5 x 20 mm) - 115 VAC: 2 x T 8A (electronics) (5 x 20 mm)

2 x T 6.3A (heater) (5 x 20 mm) Air in- and outlets The air in- and outlets are Quick Connectors for tubing OD = 10 mm. Required inlet pressure for the gasses: 2 barg. Bottle rack The ez-Control Bottle Rack for Reagent Bottles is optional. It is available in two versions: Z310113010 Bottle Rack 3 x 0.5 liter bottles Z310113020 Bottle Rack 1 x 0.5 liter and 2 x 1 liter bottles SIP Control Connections The SIP Control Connections are externally connected with the SIP Actuator Module (at the bottom part of the rear panel) through a power cable and a cable for serial communication. The SIP Actuator Module contains connectors for I/O (actuator) hardware for aeration, temperature control, etc.. Image the SIP Actuator Module (electric hardware connection): The I/O connections of the SIP Actuator Module are listed on the next page.

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The table below lists the actuator connectors and their functions. The valve notation like V(Y)-2.04 means that it may represent a valve that is pneumatically (V-2.04) or electrically (VY-2.04) driven. Connection Description POWER Power Cable connection from Control Part SERIAL Serial Cable connection from Control Part V(Y)-2.04 Circulation Valve 1 V(Y)-2.06 (Clean) Steam Inlet Valve V(Y)-2.08 Circulation Valve 2 V(Y)-2.09 Water Return Valve V(Y)-2.12 Drain Valve V(Y)-2.13 Main (Clean) Steam Inlet Valve V(Y)-10.15 Steam to Inlet Filter Valve V(Y)-10.16 Condensate Valve (Inlet Filter to Biowaste) V(Y)-10.19 Gas Inlet Valve TSH-2.01 Temperature Switch (Heater Tank)

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2.6 SENSOR INPUT SPECIFICATIONS The applied sensor input amplifiers have the following specifications. pH amplifier: Range: 0 pH to 14 pH Accuracy: ±0.01 pH Rin: > 1015 Ω Resolution: 0.01 Temperature amplifier: Type: Pt-100 (3-wire type) Range: 0 oC to 150 oC Accuracy: ±0.1 oC Resolution: 0.1 oC DO amplifier: Type: Polarographic Range: 0 % to 500 % (air) 0 % to 100 % (oxygen) Accuracy: ±0.1 % Resolution: 0.1 % Level amplifier: Type: On/Off signal Sensitivity: Firmware-selectable: High: Conductivity ≥ 26 µS equals “Contact” Low: Conductivity ≥ 200 µS equals “Contact” (see ez-Control Software Reference Manual, section “Additional Foam / Level Controller)

2.7 ENVIRONMENTAL CONDITIONS The ez-Control for In Situ sterilizable Bioreactor Systems may be used at locations with the following environmental conditions: • Use indoor • Altitude: up to 2000 m • Temperature: 4 °C to 45 °C • Relative humidity: maximum 80 % for temperatures up to 31 °C,

decreasing linearly to 50 % relative humidity at 45 °C • Mains supply voltage: 115/230 VAC (+15%/-20%), 50/60 Hz • Transient over voltages according to Installation category II • Pollution: Pollution degree 2 in accordance with IEC 664 • Power consumption: P-max = 1300 VA

2.8 CLEANING INSTRUCTIONS The ez-Control for In Situ sterilizable Bioreactor Systems may be cleaned with a moist tissue in combination with a non-abrasive cleaner.

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ACTUATORS ez-Control for In Situ sterilizable Bioreactors Project 310420; PS70 IST (AU)

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3 ACTUATORS The Actuator control part of the ez-Control supports the following actuators: • pH control: acid / base addition or aeration with carbon dioxide • DO control: aeration with air, nitrogen or oxygen, stirrer speed control • Temperature control: the wet part contains a heater tank for temperature control during cultivation and

steam valves for In Situ sterilization • Level control: Level / Foam pump This chapter describes the applicable hardware.

3.1 GAS SUPPLY SECTION The gas supply section supports (a combination of) up to four rotameters (at the outside of the front panel) and/or four mass flow controllers (inside the cabinet).

In this project, no rotameters are used.

3.1.1 MASS FLOW CONTROLLERS The MFC comes with a mounting set (mounting material, connectors and tubing). It also contains a non-return valve at the gas outlet that prevents pressure differences to cause backflow through the MFC. The Controller Output is communicated digitally to the MFC using a RS485 protocol. V0R2551100 Mass Flow Controller 0 – 100 L/min

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ez-Control for In Situ sterilizable Bioreactors ACTUATORS Project 310420; PS70 IST (AU)

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3.1.2 VALVES IN GAS SUPPLY LINES At the inner rear side of the gas supply drawer, per gas a solenoid valve is mounted before the mass flow controller. Specifications for the gas inlet lines: • Gas inlet connection: bulkhead union 10 mm • Gas outlet connection: bulkhead union 10 mm • Required inlet pressure: 2 barg V0R5029312 Solenoid Valve Assembly 24 V G¼” 0 – 100 L/min

3.1.3 AEARATION FLOW PATTERN Below, the schematic aeration flow pattern is presented: The mixture of air and oxygen is directed to the gas inlet filter. Behind the filter, the gas flow is split-up into an overlay and a sparger flow with separate block valves (V-10.07 and V-10.08).

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3.2 ACTUATORS FOR TEMPERATURE CONTROL The wet part of the system contains actuators for temperature control during cultivation and sterilization. During cultivation, water is circulated through the jacket of the reactor. The temperature in the reactor is controlled by either electrical heating or introducing cold water into the jacket of the reactor. During sterilization, steam is introduced into the jacket of the reactor and in a later phase also through the overlay and sparger into the reactor.

Schematic layout of the wet part and gas in and outlet lines Tag # Description Tag # Description HE-2.71 Heating Element V-10.07 Overlay Block Valve P-2.41 Circulation Pump V-10.08 Sparger Block Valve V-2.04 Circulation Valve 1 V-10.10 Gas Inlet Filter Condensate Valve V-2.06 Steam Valve to Jacket V-10.11 Gas Inlet Filter Vent Valve V-2.08 Circulation Valve 2 V-10.15 Steam to Filter Valve V-2.09 Water Return Valve V-10.19 Gas Inlet Valve V-2.12 Drain Valve V-11.02 Off-gas Valve V-2.13 Steam Valve to Auxiliaries V-11.10 Off-gas Filter Condensate Valve V-11.11 Off-gas Filter Vent Valve

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3.3 PUMPS AND TUBING At the Pump Section up to three tubing pump drives with pump heads may be installed. The figure shows the default pump configuration.

WARNING

• According to the European Community legislation on machinery, a pump drive is a machine with potential hazard. Only apply these drives with the pump heads and tubing that are described in this chapter.

• Do not use the tubing pump drives for other purposes than displacement of fluids (or gas). • Advised pump tubing (silicone or norprene food) have their limitations in physical and

chemical resistance. Make sure that the pump tubing and the selected reagent type are compatible. Refer to a Chemical Compatibility Database like: http://www.coleparmer.com/Chemical-Resistance

• Switch off the power of the pump drive before replacing any tubes or pump heads. • Application of damaged tubes may result in fluid leaking into the pump drive. • Verify the tube quality inside the pump head before every fermentation run. Fixed-speed pump drives for ez-Control: Z310116060 Pump drive 20 rpm with Pump head package 102R (standard) Z310116050 Pump drive 20 rpm with Pump head package 313D (advanced) AVAILABLE TUBING: Pump Head 102R (20 rpm) Pump Head 313D (20rpm) Silicone Tubing Type L

(m) ID

(mm) Flow

(ml/rev) Flow

(ml/min) Flow

(ml/rev) Flow

(ml/min) Z364111300 13 7.5 0.8 0.05 1 0.06 1.2 Z364111400 14 7.5 1.6 0.22 4.4 0.26 5.2 Z364111600 16 7.5 3.1 0.81 16.2 1 20 Z364112500 25 7.5 4.8 1.66 33.2 2.2 44 Z364111800 18 7.5 7.9 N.A. N.A. 5 100 Norprene Food Tubing Type L

(m) ID

(mm) Flow

(ml/rev) Flow

(ml/min) Flow

(ml/rev) Flow

(ml/min) Z364021300 13 15 0.8 0.05 1 0.06 1.2 Z364021400 14 15 1.6 0.22 4.4 0.26 5.2 Z364021600 16 15 3.1 0.81 16.2 1 20 Z364022500 25 15 4.8 1.66 33.2 2.2 44 Z364021800 18 15 7.9 N.A. N.A. 5 100

http://www.coleparmer.com/Chemical-Resistance

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Mounting the Pump Tubing:

Front View:

Top View:

Tubing instraight line

Lift Lever andinsert tubing

Close pumphead

Tubing bentbackwards

Tubing bentforwards

Tubing forcedupwards

Tubing forceddownwards

Tubing mountedcorrectly

Tubing mountedcorrectly

3.4 HOW TO LOAD THE TUBING Pump head type 102R

WARNING Risk of physical injury Pump head 102R must be started manually. Carefully load the tubing in the pump head: watch your fingers!

Procedure: 1. Open the cover 2. Place the tube in lower clip 3. The pump with 102R head must be started manually from ez-Control.

The Prime function can be used (Menu->Tubing pump control->Prime) 4. Carefully load the tubing in the pump head: watch your fingers! 5. Stop the pump by releasing the Prime button 6. Place the tube in higher clip 7. Close the cover of the pump head Pump head type 313D (advanced)

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3.5 ACTUATOR CONTROL

3.5.1 DISCRETE ACTUATORS VERSUS CONTINUOUS ACTUATORS Discrete (digital) actuators like valves and pumps are controlled in a “Pulse-Width Modulated” manner (PWM): • within a predefined “cycle time”, the output (Actuator) is

switched on during the “On-time” and off during the rest of the cycle.

Example 1 If Cycle time = 20 sec. and Actuator Output = 30 % then On/Off Output will be repeatedly switched: on during 6 sec (30 % of 20 seconds). off during 14 sec. Continuous (analog) actuators like mass flow controllers and stirrers do not have a “cycle time” or an “on-time”. Deviation from set-point results in a controller output that relates to the actuator output in a linear way. Example 2 If Controller Output = 30 % Then Continuous output has an Actuator Output = 30 %.

3.5.2 ACTUATOR FOR LEVEL / FOAM CONTROL The actuator for the level / anti-foam controller is either off (AO = 0 %) or on (AO = 100 %). In most cases the desired situation is “No Contact”. When the status “No Contact” changes into “Contact”, first a “Dead Time” elapses after which the actuator is switched on during the “Pulse Time” interval. This sequence is repeated until the status changes into “No Contact” again.

Values for Dead Time and Pulse Time and the function of the level sensor (discrimination between “no contact” and “contact”) can be edited by using the Parameter option Controller setup. Refer to the related Software Reference Manual.

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AGITATION ez-Control for In Situ sterilizable Bioreactors Project 310420; PS70 IST (AU)

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4 AGITATION

4.1 INTERNAL STIRRER SPEED CONTROLLER The internal stirrer speed controller of the ez-Control is used to drive the stirrer motor on top of the bioreactor. Depending on the reactor size and required stirring power, different stirrer motors can be used.

REMARK Current limitation of the stirrer speed controller

• Depending on the number of impellers and the viscosity of the medium, the required power (torque) of the stirrer motor may reach its maximum. If this is the case, the stirrer speed set point cannot be reached!

• In case the maximum torque of the stirrer is reached, decrease the stirrer set point or decrease the number of impellers.

4.2 APPLICABLE STIRRER MOTORS This section describes the DC permanent magnet stirrer motors that can be used in combination with the Microbial and Cell Culture bioreactors. An encoder, present in the motor, is used for stirrer speed feedback to the motor controller. The stirrer motor is delivered with four motor studs to fit in the stirrer assembly and a covered (flexible) coupling fork to avoid noise and vibrations during operation. No tools are required to mount the stirrer motor on top of the stirrer assembly.

WARNING

• The stirrer motor is a machine with potential hazard (moving parts). • In order to avoid dangerous situations, make sure that the stirrer motor is only operated when it is

properly seated in the stirrer assembly of the bioreactor. • A running motor will be hot. Take precautions to prevent personal injury. • The stirrer motor must only be used as part of a Bioprocess (bioreactor with control system). The table below lists the applicable stirrer motors with the corresponding torque and stirrer speed range. Motor Type Maximum Torque

(Nm) Stirrer Speed Range

(rpm) Stirrer Power Supply in

ez-Control P100 i=6* 1.0 0 … 200 48V/200W P1000 3.0 0 … 1000 60V600W ESA6L30 8.0 0 … 3000 60V600W

*The P100 i=6 stirrer motor is equipped with a planetary gear box that reduces the stirrer speed (6 times).

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4.3 STIRRER MOTOR SPECIFICATIONS

4.3.1 MOTOR P100, I=6 Standard Top Stirrer motor for cell culture bioreactors with a standard stirrer speed range of 0 to 200 rpm. • Maximum torque: 1.0 Nm. • Weight: 2.4 kg. • Includes: motor studs and coupling. • Gear box Stirrer speed is reduced 6.25 times by planetary gearbox. Z510000011 Stirrer Motor Assembly P100, i=6

4.3.2 MOTOR P1000 Standard Top Stirrer motor for bacterial culture bioreactors with a stirrer speed range of 0 to 1000 rpm. • Maximum torque: 3.0 Nm. • Weight: 8.1 kg. • Includes: motor studs and coupling. • Gear box none Z510000040 Stirrer Motor Assembly P1000

4.3.1 MOTOR ESA 6L/30 Special Stirrer motor for the In Situ sterilizable bioreactors with a stirrer speed range of 0 to 3000 rpm. The stirrer motor can be used in combination with a top or bottom stirrer assembly. • Maximum torque: 8 Nm. • Weight: 17 kg. V0R2001180 Stirrer Motor Assembly ESA6L30

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4.4 POWER SUPPLIES FOR STIRRER MOTOR ASSEMBLIES Z310110130 Power Module SIP Standard for P100 Z310110140 Extended Power Module SIP Extended 230 VAC for P1000 Z310110150 Extended Power Module SIP Extended 115 VAC for P1000

4.5 MOTOR CABLES Z510121011 Motor Cable for Stirrer P100 Z510121014 Motor Cable for Stirrer P1000 (L = 4m) Z510121022 Encoder Cable (L = 4m)

4.6 MAINTENANCE The stirrer motors are virtually maintenance-free. The only parts that require regular maintenance are: • Carbon brushes: life span depends on operating conditions (normally approx. 10,000 operating hours). • Ball bearings.

CAUTION • When carbon brushes are replaced, it is strongly advised to replace the ball bearings of the

motor as well. • Operating the stirrer motor with worn-out carbon brushes will cause irreversible damage!

The ESA6L30 stirrer motor is a brushless motor (does not contain carbon brushes). As a result, only the ball bearings need to be replaced after 10,000 operating hours.

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5 REACTOR

5.1 GENERAL REACTOR CONSTRUCTION The main components (vessel, jacket, tubing, frame) of the reactor are made of SS 316L (1.4404) stainless steel. The sight glass is made of borosilicate, and the gaskets and O-rings are made of silicone, EPDM or PTFE. The rectangular sight glass in the vessel sidewall is mounted at working volume level. The minimum working volume of a reactor is determined by the location of the sensor ports. Design code and parameters The reactor is constructed in accordance with the codes: • The PED Code (harmonized European standard) Finishing The internal reactor surface and the auxiliaries that are mounted inside the reactor are electrolytically polished to achieve a surface roughness of Ra ≤ 0.4 µm. The inner surfaces are free of crevices to facilitate cleaning and to avoid contamination. The external reactor surface is mirror polished. The reactor is equipped with an insulation layer.

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5.2 70L REACTOR The insulated Bioreactor 70L is designed and built according to the following specifications: Part Number V6L1130711

Total Volume 73 L

Working Volume 50 L

Min. Mixing Volume 13 L

Jacket Volume 10 L

Insulation Yes (50 mm)

H/D – ratio Total vol. 3.0

H/D – ratio Work. vol. 2.1

Design Pressure Reactor -1 .. 3.1 barg

Design Pressure Jacket -1 .. 6.2 barg

Test Pressure Reactor -1 .. 4.0 barg

Test Pressure Jacket -1 .. 9.4

Working Temp Reactor 10 .. 150 oC

Heat Exchange Surface 6100 cm2

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ez-Control for In Situ sterilizable Bioreactors REACTOR Project 310420; PS70 IST (AU)

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5.3 HEAD PLATE PORT SPECIFICATIONS Port Number Port Size Description N1 69 mm Blind Plate (spare) N2 16 mm Level / Foam Sensor N3 16 mm Blind Stopper (spare) N4 16 mm Blind Stopper (spare) N5 27 mm Illumination N6 27 mm Push Valve (antifoam) N7 1.5”TC Condenser and Off-gas Line N8 1.5”TC Blind Stopper (spare) N9 2”TC Blind Stopper (spare) N10 2”TC Blind Stopper (spare)

5.4 REACTOR PORT SPECIFICATIONS Port Number Port Size Description N20 27 mm Head Space Aeration (overlay line) N21 27 mm Push Valve Addition (acid) N22 27 mm Push Valve Addition (alkali) N23 27 mm Contained Addition Valve (other addition) N24 27 mm Blind Stopper (spare) N25 1.5”TC Rupture Disc and Discharge Pipe N26 1.5”TC Pressure Gauge N30 1.5”TC Medium Aeration (sparger line) N31 1.5”TC Blind Stopper (spare) N32 DN25 Blind Stopper (spare) N33 DN25 Sensor Temp (Pt-100) N34 DN25 Sensor DO N35 DN25 Sensor pH N40 58 mm Bottom Stirrer N41 76 mm Welded Bottom Drain Valve N50 1/2”TC Wet Part Connection Jacket N51 1/2”TC Wet Part Connection Jacket N52 G1/2” Pressure Gauge Jacket

HARDWARE MANUAL

REACTORS ez-Control for In Situ sterilizable Bioreactors Project 310420; PS70 IST (AU)

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5.5 UTILITY CONNECTIONS The Wet Part of the Bioreactor System has utility connections for water, steam, biowaste and drain. In the image and table below, the different connections are described (rear view of wet part).

Connection # Connection Type Description A (1/2”TC) Industrial Steam Supply B (1/2”TC) Water Supply C (1/2”TC) Water Return D (1/2”TC) Drain E (1/2”TC) Clean Steam Supply F (1”TC) Clean Steam Condensate (Biowaste)

HARDWARE MANUAL

ez-Control for In Situ sterilizable Bioreactors AUXILIARIES Project 310420; PS70 IST (AU)

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6 AUXILIARIES This chapter describes the reactor auxiliaries, i.e. devices that can be mounted in the head plate, sidewall or bottom.

6.1 STIRRER ASSEMBLY The bioreactor is equipped with: • A top or bottom driven stirrer assembly that is magnetically coupled and is designed for long periods of

operation with minimum maintenance. • Impellers that are installed on the stirrer shaft. The impeller diameter is related to the reactor diameter. • Baffles that may be used to increase the mixing efficiency (preventing a laminar flow pattern).

6.1.1 BOTTOM DRIVE STIRRER ASSEMBLY Magnetically Coupled Bottom Drive Stirrer Assembly for 70L reactor: V6LYK66001 Double Magnetic Bottom Drive 70 bioreactor Max. Torque = 7 Nm

6.1.2 STIRRER MOTOR V0R2001180 DC Magnet Motor with Encoder 8.0 Nm, 0 … 3000 rpm (ESA6L30) Also refer to Chapter 4 of this Hardware Manual.

6.1.3 TURBINE IMPELLERS Z82313R642 Turbine impeller, diameter = 100 mm (15 mm shaft) Z82313R643 Turbine impeller, diameter = 100 mm (38 mm shaft) The impeller for the 38 mm shaft fits on the lower part of the stirrer shaft.

6.1.4 BAFFLES Baffles are used to increase the mixing efficiency. Without baffles, the medium flow may become laminar, causing poor mixing efficiency and mass transfer. The baffle assembly consists of one baffle and mounting material. Z82327KS70 Baffle Assembly 70 liter Pilot System bioreactor Applicable number of baffles: 1 – 4

Stirrer Motor

RotorwithoutShaft

BottomBioreactor

StirrerAssembly

HARDWARE MANUAL

AUXILIARIES ez-Control for In Situ sterilizable Bioreactors Project 310420; PS70 IST (AU)

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6.2 OPTIMUM IMPELLER CONFIGURATION The following images show the advised impeller configuration (position and diameter) for stirred tank bioreactors (microbial and cell culture):

Microbial Culture H/D ~ 1.5 …. 2.5 Dr = 3 x Di Di = H1 H2 ~ 0.6 Dr H3 = height Working Volume

Cell Culture H/D ~ 1.0 Dr = 2.5 x Di Di = H1 H3 = height Working Volume

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6.2.1 POWER REQUIRED FOR STIRRING The power (Watt) of the stirrer motor that is required depends on the number, type and diameter of the impellers, density of the medium and the stirrer speed. The required power per impeller of a stirrer motor in non-aerated media is given by the equation:

P = ρ x (N/60)3 x D5 x Np where:

P Required Power of the Stirrer Motor (W)

ρ Density of the Medium (kg/m3) N Stirrer Speed (rpm) D Diameter of the Impeller (m) Np Power Number of the Impeller*)

*) Rushton turbine impellers: Np = 6 Applikon marine impellers: Np = 1.5 When mounted according to the given configuration, a second or third impeller on a shaft requires only 90 % of the power of the first impeller. This means that the equation above is multiplied with the factor 1.9 for two impellers and with a factor 2.8 for three impellers. Required Torque The required torque of the stirrer motor is related to its power according to the following equation:

M = P x 60 /(2 π N) where:

M Required Torque of the Stirrer Motor (Nm) P Required Power of the Stirrer Motor (W) N Stirrer Speed (rpm)

Impeller Tip Speed The Tip Speed (m/s) of an impeller at a certain stirrer speed is given by the equation:

Tip speed = (N / 60) x π x D Where:

N Stirrer Speed (rpm) D Diameter of the Impeller (m)

6.2.2 EXAMPLE OF STIRRER SPEED REQUIREMENT CALCULATION Required stirrer power, torque and corresponding tip speed for non-aerated media with a density of 1100 kg/m3 are calculated as follows. 70 liter reactor with three turbine impellers of 100 mm, at stirrer speed of 600 rpm:

required power: P = 1100 * (600/60)3 * (0.100)5 * 6 * 2.8 ≈ 185 W required torque: M = 185 * 60 / (2 * π * 600) ≈ 2.2 Nm tip speed: T = (600 / 60) * π * 0.100 ≈ 3.1 m/s

In bacterial cultures with aeration of approximately 2 VVM (2 gas volumes per reactor (working) volume per minute), the required motor power and torque will show a substantial decrease in relation to the calculated value.

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6.3 AERATION This section describes the reactor auxiliaries for gas supply (inlet and outlet). For information concerning rotameters, mass flow controllers, valves, etc., refer to Section 3.1. Since the amount of dissolved oxygen (DO) normally affects the character of the Bioprocess, this parameter is usually strictly controlled. The table below lists the “Rules of Thumb” for gas flow rates for Sparger and Overlay.

Cell culture Microbial culture

Gas Sparging Overlay Sparging Overlay

Air approximately 0.1 vvm 0.1 vvm 1 - 2 vvm 10 % of “air to sparger”

O2 10 % of air flow NA 30 % of air flow NA

CO2 30 % of air flow 10 % of air flow to sparger NA NA

N2 10 % of air flow NA 50 % of air flow NA

Where: vvm = gas volume per reactor (working) volume per minute. This paragraph is continued on next page.

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6.3.1 AUXILIARIES FOR AERATION The bioreactor is aerated through a sparger- and overlay inlet (ports in the side wall of the bioreactor). See schematic presentation below:

Overlay Sparger Part # Description Part # Description V7KAP31001 PG-10.00 Process Gas PS70 V6K0330701 C-Sparger Tube V6KWV33111 Inlet 27 mm V6LXS30381 1.5”TC Mounting Set Silicone V6LXS30191 27 mm Mounting Set Silicone

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6.3.2 OFF-GAS LINE The off-gas leaves the reactor through the condenser, the off-gas filter and the exhaust valve. See schematic image below: Due to the Electric Heater between the Condenser and the Off-gas Filter, condensate formation on the filter surface is prevented. The off-gas that leaves the condenser has been cooled down and will therefore be saturated with water. If the off-gas meets the hydrophobic filter membrane right away, it will be clogged with tiny water droplets. By heating the off-gas before in enters the filter housing, clogging will be prevented.

Off-gas Line Part # Description Z82308C030 Spiral Condenser 1.5”TC V6KAP31101 PG-11.00 Process Gas Outlet PS70

The PG-11.00 Process Gas Outlet includes the following parts: V0M2028048 Novasip Filter 1.5”TC V1KH126161 Heating Element 130W, 230V (2x 1”TC)

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6.4 AUXILIARIES FOR ADDITION Additions can be performed through the use of push valves and contained addition assemblies. In this project, only Push Valves are used.

6.4.1 PUSH VALVE ASSEMBLY The push valve assembly is designed for easy, aseptical addition through 16 and 27 mm ports in the head plate or upper sidewall section of the bioreactor. Device for 16 mm port

Description Device for 27 mm port

Description

Z82324MT26 Push Valve ID=3 / OD = 6 mm Z82324MT27 Push Valve ID=3 / OD = 6 mm Z82324MT29 Push Valve ID=6 / OD = 9.5 mm Z82322BP33 Blind Stopper Push Valve Z82322BP34 Blind Stopper Push Valve Z82324MT32 Adapter for Push Valve Z82324MT33 Adapter for Push Valve

When a push valve is inactive (or autoclaved), it may be protected with a tube (valve must be open): Z81324MT35 Protection tube push valve 27 mm port

6.4.2 RAPI-LOK STERILE CONNECTOR The autoclavable Rapi-Loks are a fast and reliable way to make or break tubing connections during fermentation or cell culture process. Materials: Body and blind caps: SS 316L O-rings: silicone Part # Description Z81324MT50 Rapi-Lok Coupling Male; bore size (ID) = 2 mm Z81324MT51 Rapi-Lok Coupling Female; bore size (ID) = 2 mm Z81324MT52 Rapi-Lok Coupling Male; bore size (ID) = 4 mm Z81324MT53 Rapi-Lok Coupling Female; bore size (ID) = 4 mm Z81324MT54 Rapi-Lok Coupling Male; bore size (ID) = 6 mm Z81324MT55 Rapi-Lok Coupling Female; bore size (ID) = 6 mm Z81324MT56 Rapi-Lok Blind Cap for Male Coupling Z81324MT57 Rapi-Lok Blind Cap for Female Coupling

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6.5 AUXILIARIES FOR DRAIN The bottom drain is used for taking relatively large samples from the culture and for harvesting / draining the system after finishing the process. The bottom drain consists of: • a bottom drain valve BV-12.01. • a steam entry membrane valve V-20.03. • a hose that connects the drain valve with the Steam Trap (during sterilization) or with the Drain / Harvest

line during (or after) cultivation. Screw Clamp: During cultivation, the drain valve can be sterilized by connecting it to the steam trap (biowaste line) and by opening steam supply valve V-20.03. Maintain the steam flow through the sample valve during approx. 20 min.

In case the Bottom Drain valve is used for taking large samples, after sterilizing the valve a screw clamp must be applied on the drain tube to keep the drain valve sterile.

Connect the drain tubing (in a sanitary manner) to the drain / harvest line in order to drain / harvest the bioreactor.

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6.6 SAFETY DEVICES

6.6.1 PRESSURE GAUGE A pressure gauge is required in a bioreactor and in the jacket in order to monitor the pressure in the system. Upper Side Wall Bioreactor: V3MJ050030 Pressure gauge -1 to 4 barg 1.5" TC V6LXS30381 Mounting Set Silicone 1.5”TC Jacket Bioreactor: V0P5326027 Vacuum / Pressure Gauge -1 to 9 barg G1/2 port

6.6.2 RUPTURE DISK AND DISCHARGE PIPE The rupture disc will burst at a defined pressure. The rupture disc (together with the discharge pipe) fits on a 1.5" TC port in the head plate or at the upper side wall of the reactor. V0M20A0018 Rupture disc 1.5" TC 2.27 – 2.59 barg ASME Z811302085 Discharge Pipe Standard 1.5" TC

6.6.3 RELIEF VALVE AND DE-AERATOR FOR JACKET In the Jacket-Return Line, a Pressure Safety Device with De-Aerator is mounted. V6K0720501 Pressure Safety Device with Air-eliminator for Jacket 1/2”TC, CE

Discharge Pipe

Tri Clamp

Rupture Disc

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6.7 BLIND STOPPERS Blind stoppers are used to blind unused ports in the head plate and in the upper and lower sidewall. Blind stoppers for 10, 16 and 27 mm ports: Z82322BP06 Blind Stopper 16 mm Z82322BP10 Blind Stopper 27 mm Blind stopper for DN 25 port: Z82322BP20 Blind Stopper DN 25 port V1M1320077 Puller for Blind Stopper DN 25

A puller is used to remove the Blind Stopper DN 25 from the reactor: it is screwed into the blind stopper after which the blind stopper can be removed.

Blind Cap for the 1.5" TC port: Z82322BP30 Blind Cap 1.5" TC Blind Cap for central bottom and head plate entry: Z82322BP21 Blind Cap Stirrer Port Top Plate

6.8 ILLUMINATION The illumination assembly is used to illuminate the inside of the reactor. The contents can be viewed through the sight glass at the front side of the bioreactor. The illumination assembly contains a 12 V / 10 Watt halogen bulb and an illumination holder, which is mounted at the head plate. For information on usage of this illumination assembly, refer to the ez-Control Software Reference manual. Z821320090 Illumination ass. 27 mm port

HARDWARE MANUAL

ez-Control for In Situ sterilizable Bioreactors SENSORS Project 310420; PS70 IST (AU)

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7 SENSORS Several sensors can be used to measure process parameters in the bioreactor. Dependent on the sensor, it can be mounted in a head plate port or in a lower sidewall port.

7.1 pH The gel-filled sensor hardly needs maintenance (except proper cleaning after cultivation). It fits directly into a sensor holder for a lower DN 25 port. Z001012051 Sensor pH+ Z100500010 Cable pH sensor L = 5 m Z823002510 Sensor Holder EASYFIX DN 25 port

7.2 TEMPERATURE Z722200002 Sensor Pt-100 Temperature DN 25 port V1LR082161 Cable Temp. sensor L = 5 m

7.3 DO This dissolved oxygen sensor operates on a polarographic principle. This sensor fits directly into a lower DN 25 port. For specific information concerning the sensor, (including information regarding maintenance and the detachable sensor cable), refer to the sensor’s user manual (supplied with the sensor). Z010007025 Sensor, In Situ DO L = 70 mm DN 25 port Z100500012 Cable DO sensor L = 5 m

7.4 LEVEL Z72205AF20 Sensor Level 16 mm port head plate Z710402140 Cable Level sensor

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ez Control for In Situ sterilizable Bioreactors

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