OPERATOR MANUAL

i-CONTROLXP for 40L Pilot System GENERAL

June 2009

i-CONTROLXP

OPERATOR MANUAL

Project 48560 (40L Pilot system); UC Davis, June 2009

1. General

2. General Preparations

3. Pressure Test & Sterilization

4. Cultivation

5. Cleaning and Maintenance

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OPERATOR MANUAL
i-CONTROLXP for 40L Pilot System GENERAL
June 2009

CHAPTER 1

GENERAL
The Applikon range of controlled in situ sterilizable bioreactor systems and control equipment
is represented by the following image:

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OPERATOR MANUAL
i-CONTROLXP for 40L Pilot System GENERAL
June 2009

The bioreactor system and the described hardware (including software) can be operated as a
standalone unit.
This operator manual describes the daily operations with the i-ControlXP and the
corresponding In Situ Sterilizable Bioreactor.

Note:
In most cases Standard Operation Procedures describe the different process steps. In case this
manual contradicts with the SOP=s, the SOP=s must be followed!

Also refer to the following manuals:

S Hardware manual of the i-ControlXP,

S Software Reference manual of the i-ControlXP,
S PID-Control manual of the i-ControlXP.

In preparation of and during each bioprocess different successive steps must be performed:
- general preparation for each new process
- pressure test
- sterilization
- preparation for cultivation
- cultivation
- inactivation and draining
- cleaning

This Operator Manual refers to the hardware as indicated on the corresponding Piping &
Instrumentation Drawing (P&ID) of the i-ControlXP; e.g. V-2.06 is the steam supply valve in
the Wet Part.

Warning:
The i-ControlXP drives a stirrer motor that 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 In Situ Sterilizable Bioreactor. The
stirrer motor comes with suppliers declaration of conformity
regarding the European Community legislation on machinery! It must not be put
into service until the complete i-ControlXP with any third party auxiliaries, has been
declared to be conform with machinery directive 89/392/EEG and 91/368/EEG.

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OPERATOR MANUAL
i-CONTROLXP for 40L Pilot System GENERAL PREPARATION
June 2009

CHAPTER 2

GENERAL PREPARATION
In order to prepare for starting a new bioprocess, the following schedule has to be followed:

- Verify if the electrodes are functioning properly:

Concerning the pH electrode:

In most cases, gel-filled pH-sensors are used; those sensors do not need to be refilled!
Before mounting the sensor in the DN25 sensor holder, it must be calibrated.
Calibrate the pH-sensor, preferably with buffers pH=7 and pH=4. For information concerning
the calibration routine refer to the Software Reference manual of the controller. The
calibration data (slope and offset of the electrode) are stored in the controller memory.
The gel-filled pH-sensor is now mounted in the holder that fits in a DN25 port.

Concerning the DO electrode:

The DO electrode is calibrated after the sterilization is completed and the reactor is cooled
down to cultivation temperature. If, during the previous cultivation any malfunctioning of the
DO electrode was observed, the membrane module must be replaced. Information about
replacing this module can be found in the manual of this electrode.
Mount the DO electrode together with its electrode holder in a DN25 port at the lower reactor
wall.

Concerning other sensors (temperature, level and pressure):

These sensors do not need daily maintenance. Mark the required insertion depth of the level
sensor in the head plate with permanent marker and insert the level sensor completely so that it
will be sterilized over the total length. After sterilization the level probe can slowly be pulled
up until the mark is in sight.

- Replace all conical in- and outlets with conical steam (blind) connectors (auxiliaries with
conical connectors are: drain / sample valve and addition auxiliaries like the triple inlet and the
inoculation / addition assembly).

- Remove all push valves and autoclave them first; for specific information, refer to chapter
5.1.

- If contained assemblies are applied, the manually operated contained valve must be taken out
and be replaced by bent tubes.
The contained valves must be sterilized in an autoclave separately.

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OPERATOR MANUAL
i-CONTROLXP for 40L Pilot System GENERAL PREPARATION
June 2009

- Check to see if all connections are properly fixed. Make sure that all hose clamps are securely
tightened.
All ports in the head plate and reactor wall must be closed properly by assemblies, sensors or
blind stoppers.
Filter elements must be verified and properly fitted.

- If applied, verify if the rupture disc is free of cracks and properly fitted.

- If not in contradiction with the current S.O.P., fill the reactor with medium (in case of full
sterilization) up to working volume.

WARNING:

In case a bottom stirrer is applied, the stirrer must not be operated in an

empty reactor! Running dry will damage the slide bearing!

- Empty the jacket of the gas outlet condenser.

- Verify the steam supply pressure: 2 - 2.5 barg.

- Verify if cooling water is present.

- Verify if instrument air (for pneumatic valves) is available at required pressure (6 barg).

- Verify if gas supply for sparging/overlay is available at required pressure (2 barg).

The reactor system is now prepared for the pressure test.

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OPERATOR MANUAL
i-CONTROLXP for 40L Pilot System PRESSURE TEST AND STERILIZATION
June 2009

CHAPTER 3
PRESSURE TEST AND STERILIZATION
The pressure test and sterilization procedure are controlled by the i-Control software; also
refer to the i-Control Software Reference Manual.

BEFORE STARTING THE STERILIZATION PROCESS, MAKE

SURE THAT SAFETY MEASURES ARE TAKEN TO PREVENT
PERSONAL INJURY (TOUCHING THE REACTOR SURFACE
AND BIOWASTE LINE WILL CAUSE SEVERE BURNS!).

The pressure (hold) test and the sterilization routine is performed using the Sterilization
Settings in the software of the i-Control. Press the SIP button in the SYNOPTIC screen. The
different sterilization steps together with the current status will be presented in a list. At the
left hand bottom corner of the screen, the button SIP SETUP >> can be found. When this
button is pressed, the parameters for each of the individual sterilization phases is presented.
Edit the parameters and press the button SIP << PROCESS (right hand bottom corner) to
return to the sterilization overview screen.

At the left hand side of the sterilization overview screen, 4 Process buttons (start, stop, abort
and reset SIP) are presented. In the table below, the function of these buttons is explained:

Name Process Key: Description: Remark:

Start SIP Starts SIP-routine -

Stop SIP Stops SIP-routine Press the Reset SIP button before starting a new
SIP-routine

Abort SIP Stops SIP-routine under all Press the Reset SIP button before starting a new
circumstances SIP-routine

Reset SIP Reset phase Finalizes the SIP-routine

On next pages, the different part in the SIP-routine are described.

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OPERATOR MANUAL
i-CONTROLXP for 40L Pilot System
June 2009
Basically, the S.I.P.-routine consists of the following steps:
- Initialization Phase:
- Pressure Stabilization: before the Pressure test is started, the reactor pressure is
- Pressure Test:
- Phase 1a:
- Phase 1b:
- Pre-heat:
- Phase 2:
- Sterilization Hold:
- End Sterilization:
- Cooling 1:
- Cooling 2:
- Cooling 3:
- End SIP:
The main phases of the sterilization routine are discussed on the next pages.
PRESSURE TEST AND STERILIZATION
preparation for the Pressure test (reactor pressure
set-point of approx. 1.2 barg needs to be reached)
stabilized
the reactor pressure needs to be stable at approx. 1.15 barg;
this test is performed in order to detect leaks
transition phase to sterilization (all wet part hardware
is switched off)
the jacket of the reactor is filled with (clean) steam; the
reactor is heated up until its temperature reaches
“pre-heat” temperature
during the pre-heat interval the gas inlet filters are
heated with steam; condensate from the filters is
transferred to the drain through steam traps
clean steam is also injected into the reactor through the
gas inlet filters; this phase is completed when the
temperature set-point is reached (e.g. 122 oC)
the reactor temperature is kept at sterilization
temperature during a predefined interval (e.g. 20
minutes); when during this interval the reactor
temperature drops below set-point, the sterilization
timer is reset
steam valves are closed, gas inlet valves to sparger and
overlay are opened; preparation to the cooling phases
reactor is aerated in order to prevent a vacuum;
temperature set-point approx. 115 oC)
the jacket of the reactor is filled with cooling water; the
reactor is rapidly cooled down until the set-point for
this step is reached
the reactor is cooled down until Process temperature
(e.g. 37 oC) is reached
Sterilization routine is completed; new cultivation can
be started
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OPERATOR MANUAL
i-CONTROLXP for 40L Pilot System PRESSURE TEST AND STERILIZATION
June 2009

INITIALIZATION:

All hardware is set to a passive mode; gas overlay valve is opened. This phase is completed as
soon as the pressure set-point is reached.

Gas Supply Lines

V-10.15 Steam to gas inlet filters

Steam Supply
V-2.13 V-10.05
Exhaust
V-9.01 V-10.09

V-2.06 V-10.06
V-11.05
Water Out
V-10.07 To Bio
V-2.07 Waste
QC-5.00
Vent QC-5.01
V-10.19
V-2.08
Water In V-10.08

V-10.16

Heater
Pump
V-2.04
Drain Bioreactor To Bio
Waste
V-2.12
Steam Trap

SIP-parameters:
- SP flow air overlay
- SP reactor pressure

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OPERATOR MANUAL
i-CONTROLXP for 40L Pilot System PRESSURE TEST AND STERILIZATION
June 2009

PRESSURE HOLD TEST:

During the Pressure Hold Test, the over-pressure in the reactor must be maintained. If the
pressure hold test is successfully completed, the operator is prompted to confirm. At the end of
this phase, the manually operated valve in the off-gas line must be opened in order to release
the pressure in the reactor.

Gas Supply Lines

V-10.15 Steam to gas inlet filters

Steam Supply
V-2.13 V-10.05
Exhaust
V-9.01 V-10.09

V-2.06 V-10.06
V-11.05
Water Out
V-10.07 To Bio
V-2.07 Waste
QC-5.00
Vent QC-5.01
V-10.19
V-2.08
Water In V-10.08

V-10.16

Heater
Pump
V-2.04
Drain Bioreactor To Bio
Waste
V-2.12
Steam Trap

During the Pressure Hold Test, the over-pressure in the reactor must be maintained. In case the
leak test was completed successfully, the operator is prompted to confirm. At the end of this
phase, the manually operated valve in the off-gas line must be opened in order to release the
pressure in the reactor.

SIP-parameters:
- Max. pressure drop
- Hold time

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OPERATOR MANUAL
i-CONTROLXP for 40L Pilot System PRESSURE TEST AND STERILIZATION
June 2009

HEATING UP:

Steam flows through the jacket of the reactor; condensate is transferred to the drain. End of
this phase: the temperature set-point “Preheat Filter” in the reactor is reached.

Empty sterilization: the default “Preheat Filter” set-point = approx. 50oC (definable).
Full sterilization: the default “Preheat Filter” set-point = approx. 95oC (definable).

Gas Supply Lines

V-10.15
Steam Supply Steam to gas inlet filters

V-2.13 V-10.05
Exhaust
V-9.01
V-10.09

V-2.06 V-10.06
V-11.05
Water Out
V-10.07 To Bio
V-2.07 Waste
QC-5.00
Vent QC-5.01
V-10.19
V-2.08
V-10.08
Water In
V-10.16

Heater
Pump
V-2.04
Drain Bioreactor To Bio
Waste
V-2.12
Steam Trap

SIP-parameters:
- SP temperature

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OPERATOR MANUAL
i-CONTROLXP for 40L Pilot System PRESSURE TEST AND STERILIZATION
June 2009

PREHEAT FILTER:

Steam valve(s) to the gas inlet filter(s) and condensate valve(s) are opened; as a result, the gas
inlet filter is pre-heated with steam; condensate is transferred to the biowaste or drain. This
phase has a defined interval (e.g. 2 minutes). It automatically starts the next phase.

Gas Supply Lines

V-10.15
Steam Supply Steam to gas inlet filters

V-2.13 V-10.05
Exhaust
V-9.01
V-10.09

V-2.06 V-10.06
V-11.05
Water Out
V-10.07 To Bio
V-2.07 Waste
QC-5.00
Vent QC-5.01
V-10.19
V-2.08
V-10.08
Water In
V-10.16

Heater
Pump
V-2.04
Drain Bioreactor To Bio
Waste
V-2.12
Steam Trap

SIP-parameters:
- Hold time

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OPERATOR MANUAL
i-CONTROLXP for 40L Pilot System PRESSURE TEST AND STERILIZATION
June 2009

HEATING-UP PART 2:

Sparger and Overlay valves are opened; steam is injected in the reactor through the gas
inlet filter(s); condensate in the off-gas filter is transferred to the biowaste or drain.
This step is completed when the (definable) Sterilization temperature or pressure
set-point is reached.

Gas Supply Lines

V-10.15
Steam Supply Steam to gas inlet filters

V-2.13 V-10.05
Exhaust
V-9.01
V-10.09

V-2.06 V-10.06
V-11.05
Water Out
V-10.07 To Bio
V-2.07 Waste
QC-5.00
Vent QC-5.01
V-10.19
V-2.08
V-10.08
Water In
V-10.16

Heater
Pump
V-2.04
Drain Bioreactor To Bio
Waste
V-2.12
Steam Trap

SIP-parameters:
- SP pressure

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OPERATOR MANUAL
i-CONTROLXP for 40L Pilot System PRESSURE TEST AND STERILIZATION
June 2009

STERILIZATION (maintaining conditions from previous phase):

Sterilization temperature is maintained during the required time interval.

In case one of the measured temperature values drops below its lower limit
(see Sterilization Settings), the sterilization timer is reset until sterilization
set-point is reached again.

During this interval, the sample valve and addition valves may also be sterilized
(must be carried out manually)!
When the Sterilization interval is completed, the operator is prompted to confirm.

Gas Supply Lines

V-10.15
Steam Supply Steam to gas inlet filters

V-2.13 V-10.05
Exhaust
V-9.01
V-10.09

V-2.06 V-10.06
V-11.05
Water Out
V-10.07 To Bio
V-2.07 Waste
QC-5.00
Vent QC-5.01
V-10.19
V-2.08
V-10.08
Water In
V-10.16

Heater
Pump
V-2.04
Drain Bioreactor To Bio
Waste
V-2.12
Steam Trap

SIP-parameters:
- SP temperature
- Min. temperature
- Drop time SP
- Hold time
- Overage time
- Reset time SP

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OPERATOR MANUAL
i-CONTROLXP for 40L Pilot System PRESSURE TEST AND STERILIZATION
June 2009

COOLING 1:

The first cooling step is designed to replace steam by air (preventing vacuum in the
reactor). Steam inlet is closed and air inlet is opened. Pressure set-point is maintained
(value = user-definable). This phase is completed when the temperature in the reactor
has dropped to approx. 115oC (definable). The next phase will automatically be
started.

Gas Supply Lines

V-10.15
Steam Supply Steam to gas inlet filters

V-2.13 V-10.05
Exhaust
V-9.01
V-10.09

V-2.06 V-10.06
V-11.05
Water Out
V-10.07 To Bio
V-2.07 Waste
QC-5.00
Vent QC-5.01
V-10.19
V-2.08
V-10.08
Water In
V-10.16

Heater
Pump
V-2.04
Drain Bioreactor To Bio
Waste
V-2.12
Steam Trap

SIP-parameters:
- SP temperature
- SP air flow sparger
- SP air flow overlay
- SP pressure

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OPERATOR MANUAL
i-CONTROLXP for 40L Pilot System PRESSURE TEST AND STERILIZATION
June 2009

FURTHER COOLING STEPS:

Cooling 1: Air inlet is open. Wet part is in cooling mode (cooling water enters the
jacket of the reactor). Phase is completed as soon as the temperature drops below the
set-point of this phase. The next phase is started automatically.

Cooling 2: Continuation of Cooling 2 with a different temperature set-point. Wet part

is in cooling mode (cooling water enters the jacket of the reactor). Phase is completed
as soon as the temperature reaches the process temperature set-point (e.g. 37oC).

Gas Supply Lines

V-10.15
Steam to gas inlet filters
Steam Supply
V-2.13 V-10.05
Exhaust
V-9.01
V-10.09

V-2.06 V-10.06
V-11.05
Water Out
V-10.07 To Bio
V-2.07 Waste
QC-5.00
Vent QC-5.01
V-10.19
V-2.08
V-10.08
Water In
V-10.16

Heater
Pump
V-2.04
Drain Bioreactor To Bio
Waste
V-2.12
Steam Trap

SIP-parameters:
- SP temperature

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OPERATOR MANUAL
i-CONTROLXP for 40L Pilot System PRESSURE TEST AND STERILIZATION
June 2009

END:

Pressure is controlled at set-point (e.g. 0.2 barg). Temperature control is turned off.
Reactor in circulation mode.

Gas Supply Lines

V-10.15
Steam Supply Steam to gas inlet filters

V-2.13 V-10.05
Exhaust
V-9.01
V-10.09

V-2.06 V-10.06
V-11.02
Water Out
V-10.08 To Bio
V-2.07 Waste
QC-5.00
Vent QC-5.01
V-10.19
V-2.08
V-10.07
Water In
V-10.16

Heater
Pump
V-2.04
Drain Bioreactor To Bio
Waste
V-2.12
Steam Trap

SIP-parameters:
- SP pressure

The sterilization procedure is now completed.

On next pages, the Actuator Activity matrix is presented in tables (one table for
automatic actuators and one for manually operated valves)!

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OPERATOR MANUAL
i-CONTROLXP for 40L Pilot System PRESSURE TEST AND STERILIZATION
June 2009

EMPTY STERILIZATION, AUTOMATIC VALVES:

Init. Pressure Preheat Sterilizati Natural
Tag Description (default text) Phase Hold Test Phase 1 filter Phase 2 on Phase Cooling Cooling 1 Cooling 2 End
V-2.04 Circulation valve #1 CLOSED CLOSED CLOSED CLOSED CLOSED CLOSED CLOSED OPEN OPEN OPEN

V-2.06 Plant steam supply valve CLOSED CLOSED PID PID PID PID CLOSED CLOSED CLOSED CLOSED

V-2.08 Circulation valve #2 CLOSED CLOSED CLOSED CLOSED CLOSED CLOSED CLOSED OPEN OPEN OPEN

V-2.09 Cooling valve CLOSED CLOSED CLOSED CLOSED CLOSED CLOSED CLOSED PID PID CLOSED

V-2.12 Drain valve CLOSED CLOSED OPEN OPEN OPEN OPEN CLOSED CLOSED CLOSED CLOSED

V-2.13 Clean steam supply valve CLOSED CLOSED OPEN OPEN OPEN OPEN OPEN OPEN OPEN OPEN

P-2.41 Circulation pump OFF OFF OFF OFF OFF OFF OFF ON ON OFF

HE-2.71 Heating element OFF OFF OFF OFF OFF OFF OFF PID PID OFF

VY-1.31 Solenoid O2 Valve sparger CLOSED CLOSED CLOSED CLOSED CLOSED CLOSED CLOSED CLOSED CLOSED CLOSED

VY-3.31 Solenoid N2 Valve sparger CLOSED CLOSED CLOSED CLOSED CLOSED CLOSED CLOSED CLOSED CLOSED CLOSED

VY-3.32 Solenoid Air Valve sparger OPEN CLOSED CLOSED CLOSED CLOSED CLOSED OPEN OPEN OPEN OPEN

VY-3.33 Solenoid O2 Valve sparger CLOSED CLOSED CLOSED CLOSED CLOSED CLOSED CLOSED CLOSED CLOSED CLOSED

V-9.11 Back pressure control valve PID CLOSED OPEN OPEN PID PID PID PID PID PID

V-10.15 Valve Clean steam sparger filter CLOSED CLOSED CLOSED OPEN OPEN OPEN CLOSED CLOSED CLOSED CLOSED

V-10.16 Valve Biowaste sparger filter CLOSED CLOSED CLOSED OPEN OPEN OPEN CLOSED CLOSED CLOSED CLOSED

V-10.19 Gas supply valve sparger OPEN CLOSED CLOSED CLOSED CLOSED CLOSED OPEN OPEN OPEN OPEN

V-11.01 Valve biowaste offgas filter CLOSED CLOSED CLOSED CLOSED OPEN OPEN CLOSED CLOSED CLOSED CLOSED

EMPTY STERILIZATION, MANUALLY OPERATED VALVES:

Init. Pressure Preheat Sterilizati Natural
Tag Description (Manual Valves) Phase Hold Test Phase 1 filter Phase 2 on Phase Cooling Cooling 1 Cooling 2 End
V-10.07 Block valve overlay OPEN OPEN CLOSED CLOSED OPEN OPEN OPEN OPEN OPEN OPEN

V-10.08 Block valve sparger CLOSED OPEN CLOSED CLOSED OPEN OPEN CLOSED CLOSED CLOSED CLOSED

V-11.02 Offgas block valve OPEN CLOSED OPEN OPEN OPEN OPEN OPEN OPEN OPEN OPEN

V-11.03 Steam to off gas heat exchanger CLOSED CLOSED OPEN OPEN OPEN OPEN OPEN OPEN OPEN OPEN

BV-12.01 Bottom valve OPEN OPEN OPEN OPEN OPEN OPEN OPEN OPEN OPEN CLOSED

V-12.02 Bottom valve to biowaste CLOSED CLOSED CLOSED CLOSED OPEN OPEN CLOSED CLOSED CLOSED CLOSED

V-14.02 Clean steam supply cont. add. 1 CLOSED CLOSED CLOSED CLOSED CLOSED CLOSED CLOSED CLOSED CLOSED CLOSED

V-14.03 Inlet valve cont. add. 1 OPEN OPEN OPEN OPEN OPEN OPEN OPEN OPEN OPEN OPEN

V-14.04 Biowaste valve cont. add. 1 CLOSED CLOSED CLOSED CLOSED OPEN OPEN CLOSED CLOSED CLOSED CLOSED

V-14.12 Clean steam supply cont. add. 2 CLOSED CLOSED CLOSED CLOSED CLOSED CLOSED CLOSED CLOSED CLOSED CLOSED

V-14.13 Inlet valve cont. add. 2 OPEN OPEN OPEN OPEN OPEN OPEN OPEN OPEN OPEN OPEN

V-14.14 Biowaste valve cont. add. 2 CLOSED CLOSED CLOSED CLOSED OPEN OPEN CLOSED CLOSED CLOSED CLOSED

V-17.02 Sample to biowaste valve CLOSED CLOSED CLOSED CLOSED OPEN OPEN CLOSED CLOSED CLOSED CLOSED

V-20.02 Clean steam to sample valve CLOSED CLOSED CLOSED CLOSED OPEN OPEN CLOSED CLOSED CLOSED CLOSED

V-20.03 Clean steam to bottom valve CLOSED CLOSED CLOSED CLOSED CLOSED CLOSED CLOSED CLOSED CLOSED CLOSED

For valve numbering, refer to the Piping & Installation Drawing (P&ID).

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OPERATOR MANUAL
i-CONTROLXP for 40L Pilot System PRESSURE TEST AND STERILIZATION
June 2009

FULL STERILIZATION, AUTOMATIC VALVES:

Init. Pressure Preheat Sterilizati
Tag Description (default text) Phase Hold Test Phase 1 filter Phase 2 on Phase Cooling 1 Cooling 2 End
V-2.04 Circulation valve #1 CLOSED CLOSED CLOSED CLOSED CLOSED CLOSED OPEN OPEN OPEN

V-2.06 Plant steam supply valve CLOSED CLOSED PID PID PID PID CLOSED CLOSED CLOSED

V-2.08 Circulation valve #2 CLOSED CLOSED CLOSED CLOSED CLOSED CLOSED OPEN OPEN OPEN

V-2.09 Cooling valve CLOSED CLOSED CLOSED CLOSED CLOSED CLOSED PID PID PID

V-2.12 Drain valve CLOSED CLOSED OPEN OPEN OPEN OPEN CLOSED CLOSED CLOSED

V-2.13 Clean steam supply valve CLOSED CLOSED OPEN OPEN OPEN OPEN OPEN OPEN OPEN

P-2.41 Circulation pump OFF OFF OFF OFF OFF OFF ON ON ON

HE-2.71 Heating element OFF OFF OFF OFF OFF OFF PID PID PID

VY-1,31 Solenoid CO2 Valve CLOSED CLOSED CLOSED CLOSED CLOSED CLOSED CLOSED CLOSED CLOSED

VY-3.31 Solenoid N2 Valve CLOSED CLOSED CLOSED CLOSED CLOSED CLOSED CLOSED CLOSED CLOSED

VY-3.32 Solenoid Air Valve OPEN CLOSED CLOSED CLOSED CLOSED CLOSED OPEN OPEN OPEN

VY-3.33 Solenoid O2 Valve CLOSED CLOSED CLOSED CLOSED CLOSED CLOSED CLOSED CLOSED CLOSED

V-9.11 Back pressure control valve PID CLOSED OPEN OPEN PID PID PID PID PID

V-10.15 Valve Clean steam sparger filter CLOSED CLOSED CLOSED OPEN OPEN OPEN CLOSED CLOSED CLOSED

V-10.16 Valve Biowaste sparger filter CLOSED CLOSED CLOSED OPEN OPEN OPEN CLOSED CLOSED CLOSED

V-10.19 Gas supply valve sparger OPEN CLOSED CLOSED CLOSED CLOSED CLOSED OPEN OPEN OPEN

V-11.01 Valve biowaste offgas filter CLOSED CLOSED CLOSED CLOSED OPEN OPEN CLOSED CLOSED CLOSED

FULL STERILIZATION, MANUALLY OPERATED VALVES:

Init. Pressure Preheat Sterilizati
Tag Description (default text) Phase Hold Test Phase 1 filter Phase 2 on Phase Cooling 1 Cooling 2 End
V-10.07 Block valve overlay OPEN OPEN CLOSED CLOSED OPEN OPEN OPEN OPEN OPEN

V-10.08 Block valve sparger CLOSED CLOSED CLOSED CLOSED OPEN OPEN CLOSED CLOSED CLOSED

V-11.02 Offgas block valve OPEN CLOSED OPEN OPEN OPEN OPEN OPEN OPEN OPEN

V-11.03 Steam to off gas heat exchanger CLOSED CLOSED OPEN OPEN OPEN OPEN OPEN OPEN OPEN

BV-12.01 Bottom valve CLOSED CLOSED CLOSED CLOSED CLOSED CLOSED CLOSED CLOSED CLOSED

V-12.02 Bottom valve to biowaste CLOSED CLOSED CLOSED CLOSED CLOSED CLOSED CLOSED CLOSED CLOSED

V-14.02 Clean steam supply cont. add. 1 CLOSED CLOSED CLOSED CLOSED CLOSED CLOSED CLOSED CLOSED CLOSED

V-14.03 Inlet valve cont. add. 1 CLOSED OPEN OPEN OPEN OPEN OPEN OPEN OPEN OPEN

V-14.04 Biowaste valve cont. add. 1 CLOSED CLOSED CLOSED CLOSED OPEN OPEN CLOSED CLOSED CLOSED

V-14.12 Clean steam supply cont. add. 2 CLOSED CLOSED CLOSED CLOSED CLOSED CLOSED CLOSED CLOSED CLOSED

V-14.13 Inlet valve cont. add. 2 CLOSED OPEN OPEN OPEN OPEN OPEN OPEN OPEN OPEN

V-14.14 Biowaste valve cont. add. 2 CLOSED CLOSED CLOSED CLOSED OPEN OPEN CLOSED CLOSED CLOSED

V-17.02 Sample to biowaste valve CLOSED CLOSED CLOSED CLOSED OPEN OPEN CLOSED CLOSED CLOSED

V-20.02 Clean steam to sample valve CLOSED CLOSED CLOSED CLOSED OPEN OPEN CLOSED CLOSED CLOSED

V-20.03 Clean steam to bottom valve CLOSED CLOSED CLOSED CLOSED OPEN OPEN CLOSED CLOSED CLOSED

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OPERATOR MANUAL
i-CONTROLXP for 40L Pilot System CULTIVATION
June 2009

CHAPTER 4

CULTIVATION
After finishing the sterilization routine, the bioreactor can be prepared for cultivation. Since
the contents of the Bioreactor must be kept sterile, all actions must be carried out aseptically!

4.1 PREPARING FOR CULTIVATION:

The following preparation steps must be followed:

- Height adjustment of the level sensor

Carefully pull out the level sensor until the mark becomes visible.

- Calibration of the DO sensor

When the reactor contents (medium) are at process temperature, the DO electrode can be
calibrated. Aerate the reactor for such a period (approx. 15 minutes) that the signal of the DO
electrode is stable. Carry out a one point calibration at e.g. 100% DO. For more information
concerning this calibration procedure, refer to the electrode manual and Software Reference
manual of the controller!
After calibrating the DO electrode, it is strongly advised to verify the quality of the electrode;
this is achieved by driving all oxygen out of the reactor by purging it with nitrogen! Close the
air valve and open the nitrogen valve. Purge the reactor long enough to drive all oxygen out of
the medium (approx. 15 min.).
Verify the measuring value of the DO electrode; in case the reading is > 0.5 % of air saturation,
the membrane module needs to be replaced before the next sterilization is performed (it is a
good custom to record the residual signal of the DO electrode, so that can be predicted when
electrode maintenance will be required).

- Connection of addition fluids like acid and base

Addition of fluids can be realized through the following options:

S push valves in upper side wall or head plate of the reactor
S contained addition assemblies in upper side wall of the reactor
S triple inlet with conical connectors in the head plate of the reactor

-Push Valves

During operation a push valve is mounted in an upper side wall pocket (16 or 27 mm) or head
plate (16 mm). The hose barb is connected by a hose (through a pump) to the liquid addition
bottle.

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Example of a push valve ID 3 for a 27 mm port in open position:

At the end of the cultivation, the push valve can be closed by pushing the spring knob and
pulling the hose barb outwards (see example below):

The push valve can be removed from the reactor wall or head plate by turning the Lock Nut
(M16 or M27) counter clockwise and pulling the valve out of its cylinder.

Before the next sterilization is started, the push valve (together with the hose and liquid
addition bottle) must be autoclaved in order to make it sterile.

After autoclaving the push valve, it can be re-inserted in its cylinder in the reactor (do not yet
open the valve!). Turn the Lock Nut clockwise until the valve is secured.

During sterilization of the reactor, the valve remains closed.

Just before starting a new process, the valve is re-opened (push in the spring knob and push the
valve to its open position).

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-Contained addition assembly

Contained addition assembly supports Aaerosol free@ addition.

It is In Situ sterilizable and is mounted in a ι 27 mm port at the upper side wall of the reactor.
It contains four (manually operated) membrane valves and is connected to the drain through a
steam trap (for drain purposes during sterilization (outlet C)).

During sterilization, the inlet part AA@ is replaced by the replacement tube AB@.

The inlet part AA@ (together with hose and liquid addition bottle) is separately sterilized in an
autoclave.

After sterilizing the reactor (including the contained addition assembly where a replacement
tube is placed), the inlet part together with addition bottle and tubing is put in place of the
replacement tube. The connector part of the inlet assembly must now be sterilized for at least
20 minutes (open the steam valve V-14.*2 and the valve to the drain V-14.*4). A steam flow is
now realized through the connections of the contained addition inlet assembly.
After completing the sterilization interval, close the steam valve V-14.*2 and drain valve
V-14.*4. Insert the tubing in the corresponding tubing pump and open addition valve V-14.*1.

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- Start the cultivation

- Before starting cultivation, make sure the cooling water flow to condenser
jacket (if previously disconnected for during sterilization purposes) is
reconnected. During sterilization, cooling water flow to condenser is stopped by
closing the manual valve V-5.01 and at the same time, QC-5.02 is disconnected
from chilled water return and connected to QC-5.03 in order to empty/drain the
water from the condenser jacket (to the drain). In order to restart cooling water
flow to condenser, reconnect QC-5.02 from chilled water return and open the
manual valve V-5.01. This valve is opened halfway or tuned to obtain sufficient
cooling water flow to condenser jacket.
- Also, make sure that steam to off gas heater is turned on by opening the manual
valve V-11.03 in case it is closed.

From the overview screen, the operator is able to navigate to the process cultivation screen
shown below:

The cultivation process is activated by pressing the START button, after which, the unit will
transition into inoculation mode and the control loops for temperature and stirrer will be
placed in auto mode, while all other control loops will be placed in idle mode. The operator
can proceed to inoculate the bioreactor phase by pressing the inoculate button. The operator
can also choose to stop or reset cultivation.

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4.2 ACTIONS DURING CULTIVATION:

The actions during cultivation depend on the type of process. Refer to the Standard Operation
Procedures.

-Taking culture samples

During cultivation, the bioreactor contents can be sampled via the contained sample system or
for fairly large amounts of sample, via the contained drain valve. The contained Sample and
Drain Valves support Aaerosol free@ operation.

-Operation procedure for the Contained Sample Valve

Schematic overview is presented below.

SV-17.01 = Sample Valve

V-20.02 = Steam Inlet Valve
V-17.01 = Valve to Sample Bottle
V-17.02 = Valve to Bio Waste
T-17.01= Steam Trap to Bio Waste

V-20.02

SV-17. 01 Sample Bottle

V-17.01

V-17.02

Replacement
T-17.01 For V-17.01

During sterilization of the bioreactor, V-17.01 is replaced by the piece of replacement tube
(replacement for V-17.01). The assembly of V-17.01, a hose and a sample bottle are
autoclaved separately and remounted after the bioreactor sterilization is completed.
After mounting the sampling assembly, both valves V-20.02 and V-17.02 must be opened for
at least 20 minutes in order to sterilize the connection tubing before sampling. Sampling is
performed by opening SV-17.01 and V-17.01 at the same time. After sampling, first the tubing
must be resterilized for at least 20 minutes by opening valves V-20.02 and V-17.02. Next the
sample bottle assembly (including V-17.01)
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may be removed.

-Operation procedure for the Contained Drain Valve

Schematic overview is presented below:

BV-12.01 = Bottom Drain Valve

V-20.03 = Steam Inlet Valve
V-12.01 = Valve to Drain / Sample Bottle
V-12.02 = Valve to Bio Waste
T-12.01= Steam Trap to Bio Waste

V-12.01 V-20.03
BV-12. 01
Harvest

V-12.02
Replacement
T-12.01 For V-12.01

During sterilization of the reactor, V-12.01 is replaced by the piece of replacement tube
(replacement for V-12.01). When the process is started, the harvest assembly consisting of
V-12.01 and a hose to the harvest vessel can be mounted (this assembly must be autoclaved
separately). After mounting the harvest assembly, both valves V-20.03 and V-12.02 must be
opened for at least 20 minutes in order to sterilize the tubing. Harvesting the bioreactor
contents is performed by opening BV-12.01 and V-12.01 at the same time. After harvest, first
the tubing must be resterilized for at least 20 minutes by opening valves V-20.03 and V-12.02.
Next the drain / sample bottle assembly (including V-12.01) may be removed.

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CHAPTER 5

CLEANING AND MAINTENANCE

Proper cleaning of the bioreactor is important to maintain the sterility of the system during
cultivation. Also, cleaning before and after a cultivation process presents the opportunity for
general inspection of the bioreactor vessel and associated parts.

The i-Control can support automatic Clean In Place routine. However, this requires specific
hardware (CIP-On Frame system), which is not included in this project. For information, you
are advised to contact your local dealer.

5.1 CLEANING:

After ending the cultivation (the reactor content may be inactivated) the reactor is drained.
Remove the pH and DO electrodes and clean them thoroughly. Make sure that no broth
remains on the electrode surface! Store the pH electrode in a KCl solution (c = 3M)). The DO
electrode may be stored dry. For more instructions on handling pH and DO electrodes refer to
the specific user manuals that are delivered in the electrode box.

Blind the electrode ports with DN25 blind stoppers.

Normally the cleaning procedures are defined in Standard Operation Procedures. An example
of such a procedure may be:

Cleaning the bioreactor

- For a first cleaning step, fill the reactor with a sodium hydroxide solution ( c NaOH = 0.1M )

- Heat up the liquid to approx. 80 oC. This may be done by entering a temperature set-point of 80
o
C.

The temperature set-point of 80 oC is just an example! Please use temperature set point
relevant to your process. Refer to your SOP!

- Set the stirrer speed to its maximum. All surfaces must be wetted and heated for at least 45
minutes.
- Drain the reactor.
- Now repeat the cleaning step that is described above with a solution of 1 % Na3PO4.12H2O /
0.1 % Clymax Alcasol (or any other FDA approved detergent).
- Clean the external surface by sponging it with the described solution.
- Drain the reactor.
- Rinse the in- and outside of the bioreactor three times with tap water.
- Rinse with de-ionized water.

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Cleaning auxiliaries and tubing

- Switch off the power of the i-Control.

- Disassemble the bioreactor system and remove all tubing and auxiliaries that are inserted in
the bioreactor via the side wall and head plate. These assemblies must be cleaned off place
with the same cleaning solution as used for the bio reactor.
- Rinse thoroughly with tap water (3 times) and next with de-ionized water.
- During reassembling check the quality of O-rings and gaskets. Replace them if necessary.

The Stirrer Assembly contains moving seals! In order to prevent unnecessary wear, it
is essential to keep all parts of a dismantled stirrer assembly together in order to be able
to re-assemble it correctly.

5.2 MAINTENANCE

Maintenance instructions can be found in the Maintenance Data Sheets that are available at the
Applikon Biotechnology Service Department.

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