for Microbial Applications
Contents
1. Bio Bundles 1 15 Liter US Version for Microbial Applications a Fo i is f p Hi z Ea L Ma IA OPERATOR MANUAL April 2004 amp aoolkorrt DEPENDABLE INSTRUMENTS OPERATOR MANUAL Bio Bundles 1 15 Liter Microbial Applications US Version April 2004 Chapter 1 1 12 1 3 2 1 2 2 3 1 3 2 4 1 4 2 4 4 aa 5 2 6 1 6 2 6 3 6 4 7 1 dee 7 3 7 4 7 5 Tel TABLE OF CONTENTS Description General General Introduction Examples of Autoclavable Bio Reactor Systems Related Manuals Assembling the Head Plate General Head Plate Lay out and Assembling Instructions pH Electrode Calibration General pH Calibration Routine in the ADI 1010 Bio Controller Initializing Dose Monitors General Display Values Dose Monitor Calibration Reset Dose Monitor Values Autoclaving Preparation Steps Autoclaving Preparing Cultivation Installation Preparing for Operation Calibration of the dO Electrode Resetting Dose Monitors Cultivation Fixed PID Control versus Adaptive Control Inoculation View Process Data in a Trend Sampling Correcting the Measured pH Value Pasteurization Cleaning CONTENTS Page NND N me W U OO 10 10 10 12 13 14 15 16 17 18 19 19 21 22 22 24 24 OPERATOR MANUAL Bio Bundles 1 15 Liter Microbial Applications GENERAL INTRODUCTION US Version April 2004 CHAPTER 1 GENERAL 1 12. GENERAL INTRODUCTION The ADI autoclavable Bioreactor Systems basically consist of the following parts an autoclavable Bioreactor with the appropriate auxiliaries like a stirrer assembly baffles an aeration assembly etc _ an ADI 1010 Bio Controller together with an ADI 1010 Power Unit The Bio Controller measures process variables pH Temperature dO level and stirrer speed and calculates corresponding controller outputs in order to keep process conditions on set point The Power Unit amplifies the control signals of the Bio Controller to Power Outputs on off output signals for pumps and valves analog output signals for mass flow controllers and stirrer speed an ADI 1025 Bio Console that combines and supports actuators like pumps and valves in order to optimize the use of limited bench space Schematic overview of a Bio Bundle System lay out microbial or cell culture applications L Sensor Inputs ii Supply Acid Base Supply WE i JUU gt Actuator as gees tent VF Ant Foam Bio Controller Power Unit Bio Console Fermentor Note The ADI 1010 Power Unit is mounted inside the ADI 1025 Bio Console at the rear oopikon BIOTECHNOLOGY GENERAL INTRODUCTION OPERATOR MANUAL Bio Bundles 1 15 Liter Microbial Applications US Version April 2004 1 2
3. Version April 2004 Optimizing the I interval value of the control loop the I interval is used to correct a continuous deviation from set point in case no relevant continuous deviation from set point occurs the l interval can be kept at 0 sec First decrease the optimized P value with approx 10 relative Start with an I interval of 2700 sec 45 min Decrease the I interval until the Process Value starts oscillating around set point The I interval is now optimized Optimizing the D interval value of the control loop the D interval is used to anticipate situations like control action overshoot after set point changes in case no relevant overshoot occurs the D interval can be kept at 0 sec Start with a D interval of 600 sec 10 min Decrease the D interval until the overshoot is minimized The D interval is now optimized ADAPTIVE CONTROL In case adaptive control is used controller is set to AUTO the ADI 1010 Bio Controller will start process control with moderate control parameters and will start process identification as soon as current control is inadequate After process identification new control parameter values are calculated and applied Process identification will be repeated when the process changes its character Note Process identification is executed based on small variations in set point value to both sides the character of the process is identified through the way proces
4. at 37 C DO CALIBRATION CHOOSE CALIBRATION MODE TEMPERATURE COMPENSATION CALIBRATION RESET TO DEFAULT CALIBRATION VALUES THEN PRESS Calib PRESS Setp TO EXIT TO MAIN MENU 6 98pH 32 6 C 48 3 NO CONT 1112 rpm Use Dial to toggle calibration mode Press Calib key to proceed DO CALIBRATION CONDITION TO CORRECT ENVIRONMENT ENTER VALUE THEN PRESS CALB PRESS Setp TO STEP BACK VALUE 0 0 32 6C 48 3 NOCONT 1112rpm Use Dial to generate the dO value 100 Press Calibrate key to proceed A100 Iko7 BIOTECHNOLOGY 17 OPERATOR MANUAL PREPARING CULTIVATION Bio Bundles 1 15 liter Microbial Applications US Version April 2004 It is now advised to measure the electrode signal at 0 dO in order to verify the electrode performance Use nitrogen to drive all oxygen out of the medium The time that is required to saturate the medium with nitrogen is equal to the time that was required to saturate the medium with oxygen Monitor the dO signal In case the dO value does not drop below 0 5 dissolved oxygen after 10 15 minutes the sensor requires maintenance refer to the user manual of the dO sensor After finalizing the dO electrode calibration the default aeration configuration must be restored constant supply of air with additional controlled supply of oxygen Edit the set point of the dO control loop and start this control loop in
5. mm ports baffles are inserted at the bottom side and secured by a nut at the top side Mount the auxiliaries in the 10 and 12 mm ports auxiliaries are inserted at the bottom side and secured by a nut at the top side Mount the auxiliaries in the M18 x 1 5 ports the threaded auxiliaries are mounted from the top side of the head plate Mount the dO sensor holder in the G3 4 port Mount the dO sensor in its electrode holder do not mount the pH sensor in its holder yet needs to be calibrated first Mount the head plate on top of the reactor and affix the 6 mill nuts OTe eelea BIOTECHNOLOGY OPERATOR MANUAL ASSEMBLING THE HEAD PLATE Bio Bundles 1 15 liter Microbial Application US Version April 2004 THE 7 LITER REACTOR DISHED BOTTOM M30 x 1 central port M18 x 1 5 Medium inlet triple 10 mm Baffle assembly 10 mm Sparger 10 mm Sample pipe assembly 10 mm Thermometer pocket 10 mm 2 x Heat exchanger 10 mm Spare blind stopper 12 mm Spare blind stopper 12 mm Spare blind stopper Assembling instructions Loosen the six mill nuts that affix the head plate on the reactor remove the head plate First mount the stirrer assembly without impellers Mount the impeller s at the shaft of the stirrer assembly Mount the baffle assemblies in 3 exterior 10 mm ports baffles are inserted at the bottom side and secured by a nut at the top side Mount the other a
6. reactor remove the head plate First mount the stirrer assembly without impellers Mount the impeller s at the shaft of the stirrer assembly Mount the head plate on top of the reactor Mount the auxiliaries in the M18 x 1 5 ports the threaded auxiliaries are mounted from the top side of the head plate Mount the dO sensor holder in the G3 4 port Mount the dO sensor in its electrode holder do not mount the pH sensor in its holder yet needs to be calibrated first Affix the 6 mill nuts OPERATOR MANUAL Bio Bundles 1 15 liter Microbial Application ASSEMBLING THE HEAD PLATE US Version April 2004 THE 3 LITER REACTOR DISHED BOTTOM Port Head Plate Auxiliary M30 x 1 central port Top Stirrer Assembly G3 4 dO electrode holder M18 x 1 5 pH electrode holder M18 x 1 5 Condenser M18 x 1 5 Septum Holder M18 x 1 5 Medium inlet triple M18 x 1 5 Heat exchanger 6mm Baffle assembly 6mm Baffle assembly 6mm Baffle assembly 10 mm Sample pipe assembly 10 mm Foam Level detector 10 mm Thermometer pocket Spare blind stopper Spare blind stopper Spare blind stopper Spare blind stopper Assembling instructions Loosen the six mill nuts that affix the head plate on the reactor remove the head plate First mount the stirrer assembly without impellers Mount the impeller s at the shaft of the stirrer assembly Mount the baffle assemblies in the 6
7. 0 Bio Controller is switched on The dO measurement is based on the polarographic principle Clark cell Therefore the electrode must be polarized for at least 6 hours before it is calibrated Connect the heat exchanger tubing to the cold water valve Hook up the flow console outlet of the ADI 1025 to the air inlet filter Connect cooling water to the condenser Mount the stirrer motor on top of the stirrer assembly make sure that the stirrer motor is not running Connect all sterile fluids acid base etc that must be added to the medium aseptically to the inlet auxiliaries Insert the Pt 100 sensor into the thermometer pocket Note To improve heat transfer between the thermometer pocket and the Pt100 sensor fill the thermometer pocket with water or silicone oil This will decrease the dead time of the sensor and will make temperature control more accurate When operating at higher temperature silicone oil has the advantage of a lower vapour pressure less evaporation Connect the level control cable with one jack to the level sensor and with the other to the hole in the head plate of the reactor Pull up the level sensor to such an extent that it will make contact with the medium surface at the required level Do not push the level sensor back into the reactor since this may cause contamination oopikon BIOTECHNOLOGY 15 OPERATOR MANUAL PREPARING CULTIVATION Bio Bundles 1 15 lite
8. 4 PREPARING CULTIVATION 6 3 CALIBRATING THE dO ELECTRODE After autoclaving the reactor the dO electrode must be calibrated autoclaving may affect the characteristics of the gas permeable membrane of the electrode First the medium in the reactor must be stable at process temperature e g 37 C next the medium must be saturated with air Open the main air supply valve is not included in the Bio Bundle Tune the rotameter needle valve to such an extent that the bioreactor is aerated properly Make sure that the dO control loop is switched off The medium is now saturated with air Record the measured dO signal until it reaches a stable value 10 15 minutes saturation interval is normally sufficient During calibration sparging the medium with must be continued Now start the calibration by pressing the dO key followed by the Calib key The following options are presented View current calibration values Enable disable temp correction Execution of dO calibration Resetting the calibration parameters to default Use the dial to select the option Calibration and press the Calib key again Use the dial to generate the value 100 and press the Calib key to proceed The stabilized value is used to calculate the slope of the electrode Press the Setp key to finalize the calibration routine Expected value for the Slope of the electrode 8 15 at 25 C 10 20
9. E MONITOR LEVEL PUMP Note DOSE MONITOR N2 VALVE DOSE MONITOR AIR VALVE The configured actuators are DOCE MON ORKO VAINE marked with a DOSE MONITOR OUTPUT 1 N2 DOSE MONITOR OUTPUT 2 02 DOSE MONITOR OUTPUT 3 AIR Press the Setp key to return to the DOSE MONITOR OUTPUT 4 CO2 previous screen PRESS Setp TO STEP BACK 6 98pH 32 6 C 48 3 NO CONT 1112rpm Press Set Point key to return 4 3 DOSE MONITOR CALIBRATION The configured actuators pumps valves or mass flow controllers that are used for dose monitoring need to be calibrated in order to convert on time or controller output to volume Make sure that the corresponding control loops are switched off green pilot at the parameter key is off CHOOSE DEVICE TO CALIBRATE When the Calibrate option for the THEN PRESS Menu Dose Monitors is selected the DOSE MONITOR BASE PUMP follow d DOSE MONITOR ACID PUMP or CO2 VALVE ollowing screen 1s presented DOSE MONITOR LEVEL PUMP DOSE MONITOR N2 VALVE DOSE MONITOR AIR VALVE DOSE MONITOR O2 VALVE DOSE MONITOR OUTPUT 1 DOSE MONITOR OUTPUT 2 PRESS Setp TO STEP BACK 6 98pH 32 6 C 48 3 NO CONT 1112rpm Use Dial to select Dose Monitor Use the dial to select a dose monitor function and press the Menu key to proceed Press Menu key to proceed 100 IKOT BIOTECHNOLOGY 11 OPERATOR MANUAL INITIALIZING DOSE MONITORS Bio Bundles 1 15 liter Microbial Appli
10. EXAMPLES OF AUTOCLAVABLE BIO REACTOR SYSTEMS Below two examples of autoclavable bioreactor systems are presented BIO REACTOR SYSTEM WI TH INTERNAL STIRRER CONTROLLER Bio Controller ADI 1010 gt Stirrer Motor P100 Bio Console Stirrer ADI 1025 Assembly 3L Bioreactor 3L Jie Bio Console ADI 1025 d3 Bio Controller es Motor ADI 1010 q Stirrer Controller 2 ADI 1032 Stirrer Assembly 15L Bioreactor 15L 1 3 RELATED MANUALS Hardware and Installation manual Bio Bundles 1 15 liter Firmware Reference Manual ADI 1010 Bio Controller autoclavable applications PID Control Manual Autoclavable Bio Reactor Systems QOOIKON BIOTECHNOLOGY 2 OPERATOR MANUAL Bio Bundles 1 15 liter Microbial Application ASSEMBLING THE HEAD PLATE US Version April 2004 CHAPTER 2 ASSEMBLING THE HEAD PLATE 2 1 GENERAL When unpacking the equipment verify if there 1s any transport damage and if the reactor is complete the way you ordered it Clean all parts with 70 ethanol to remove dust or dirt from shipping When assembling the reactor make sure not to damage the threaded ports always screw in the auxiliaries straight and preferably by hand Note In case too
11. OSE MONITORS 4 1 GENERAL The amount of liquid and gasses that is supplied to the reactor by controlled actuators can be monitored as a Dose Monitor function This Dose Monitor function needs calibration in order to convert on time into volume This procedure needs to be carried out once after changing tube diameters 4 2 DISPLAY VALUES Press Menu key to obtain list The function key Menu opens the list with corresponding options en Use the dial to scroll through the list and select the ECON LEVEL CONTROL TIMING menu option Dose Monitor DEVICE INFORMATION INITIALIZE CONTROLLERS CONTROLLERS SETTINGS CHOOSE OPTION THEN PRESS Menu Press the menu key again to continue PRESS Setp TO CANCEL OPERATION Use Dial to select option Press Menu key to start The Dose Monitor options are now presented Display Value RESET ALL Calibrate pumps valves or MFC s Reset All dose monitors Use the Dial to select the option DISPLAY CHOOSE OPTION THEN PRESS Menu VALUE and press the Menu key in order to PRESS Setp TO CANCEL OPERATION proceed Use Dial to select option Press Menu key to proceed OTe cela BIOTECHNOLOGY 10 OPERATOR MANUAL Bio Bundles 1 15 liter Microbial Applications INITIALIZING DOSE MONITORS US Version April 2004 The integrated values of dosed volume are now presented DOSE MONITOR BASE PUMP DOSE MONITOR ACID PUMP or CO2 VALVE DOS
12. UE THEN PRESS Calib key again PRESS Setp TO CANCEL OPERATION After having reached a stable signal the first calibration point is obtained Insert the sensor in the second buffer BUFFER VALUE 7 00 pH A eee eet proce with the dual point calibration procedure Use Dial to set buffer value or press the pH Parameter key in order to ES Press Calibrate key to apply the one point calibration procedure proceed only offset correction Perform one or two point calibration Finally the calibration data are displayed Expected values Slope 0 95 1 02 Offset lt 0 3 TWO POINTS CALIBRATION Press the Setp key to return to the main SLOPE 1 00 OFFSET 0 02 screen Mount the electrode in the pH nipple in the head plate of the reactor or store it in 3M KCI PRESS Setp TO CONTINUE Note _400PH_ 326C 48 3 NOCONT The body of the pH nipple is already mounted in the head plate Slide the nut metal flange and rubber clamping ring Press Set Point key to continue onto the pH electrode ring may be wetted Insert the electrode in the pH nipple and fasten the nut slightly when too much force is used the clamping ring will stick to the electrode The pH electrode is now ready for operation A100 Iko7 BIOTECHNOLOGY OPERATOR MANUAL INITIALIZING DOSE MONITORS Bio Bundles 1 15 liter Microbial Applications US Version April 2004 CHAPTER 4 INITIALIZING D
13. al is used to scroll through the list select the calibration option and press the Calib key in order to proceed Press Parameter key cab Press Calibration key PH CALIBRATION CHOOSE CALIBRATION MODE CALIBRATION SAMPLE CORRECTION RESET TO DEFAULT CALIBRATION VALUES THEN PRESS Calib PRESS Setp TO CANCEL OPERATION 32 6 C 48 3 NOCONT 1112rpm Use Dial to toggle calibration mode Press Calib key to proceed ODDIIKON BIOTECHNOLOGY OPERATOR MANUAL Bio Bundles 1 15 liter Microbial Applications pH ELECTRODE CALIBRATION US Version April 2004 The calibration routine 1s now started ENTER BUFFER TEMPERATURE OR PRESS First the buffer temperature must be Ste GOPI TO START MERDURE GYGEE 1 1 PRESS Calib TO CONTINUE entered Use the dial in order to generate Sees S ANE OPERATION the numeric buffer temperature value or press the Start Stop key in order to start measuring the temperature of the Pt 100 sensor both the pH en Temp sensor BUFFER TEMPERATURE 20 0 must be inserted in the buffer solution Press the Calib key again to proceed 6 98pH 32 6 C 48 3 NO CONT 1112 rpm Use Dial to set buffer temperature or use Pt 100 sensor Press Calibrate key to proceed Now the pH value of the first buffer solution is required Use the dial to SE OEE REFER ENTER generate this value and press the Calib FIRST BUFFER VAL
14. cations US Version April 2004 Press the Start Stop key to Start the actuator the timer starts CALIBRATION DOSE MONITOR counting Press the Start Stop ACTUATOR ON TIME VOLUME again to stop the actuator as soon as enough liquid or gas is dosed See nee E Measure the amount of dosed PRESS Start Stop TO TOGGLE ACTUATOR liquid gas AFTER ACTUATOR IS STOPPED ENTER DOSED VOLUME THEN PRESS Menu Note PRESS Setp TO STEP BACK When calibrating dose monitors of analog outputs like MFC s 1112rpm during the calibration interval the actuator output 50 Press the Start Stop key to toggle the _ actuator action On Off Use the dial to generate the numeric value of the dosed Use the dial to generate the dosed volume value volume Press the Menu key to finish the Gen perese MEnURKGY 10 procaea calibration of this dose monitor Repeat this procedure in order to calibrate all other dose monitors When all Dose Monitors are calibrated press the Setp key until the Set Point screen is presented 4 4 RESET DOSE MONITOR VALUES When the option Reset All is selected and the Menu key is pressed the integrated Dose Monitor values are reset to 0 ml OTe eeleg BIOTECHNOLOGY 12 OPERATOR MANUAL Bio Bundles 1 15 liter Microbial Applications AUTOCLAVING US Version April 2004 CHAPTER 5 AUTOCLAVING The assembled Bioreactor is autoclaved before cultivation in order t
15. ce of proteins and or peptides cleaning the poral sparger may require a special procedure remove the poral sparger from the air inlet pipe soak it overnight in a solution of pepsin 10 mg ml HC M 0 01 use ultrasonic cleaning with the following solvents d i water 70 ethanol store the poral sparger dry until next run oopikon BIOTECHNOLOGY 24
16. ectrode may result in sudden boiling of the electrolyte Note In case of a jacketed reactor the sterilization interval might need to be increased since the empty jacket has a poor heat transfer capacity If the medium cannot stand a longer sterilization interval you can fill the jacket with water to improve the heat transfer connect and close the lower tube fitting of the jacket do not close the upper one Step 6 Remove the bioreactor from the autoclave and install it at its proper location Connect the dO sensor to the ADI 1010 Bio Controller Note The dO sensor is a polarographic Clark cell This type of sensor needs to be polarized for approx 6 hours before calibration is started As soon as the sensor is connected to the ADI 1010 Bio Controller power must be switched on polarization of the sensor is started More information concerning this topic can be found in the User Manual of the sensor A100 Iko7 BIOTECHNOLOGY 14 OPERATOR MANUAL Bio Bundles 1 15 liter Microbial Applications PREPARING CULTIVATION US Version April 2004 CHAPTER 6 PREPARING CULTIVATION After autoclaving the Bioreactor liquid addition bottles etc it must be reassembled 6 1 INSTALLATION Put the reactor alongside the control equipment the Bio Controller and Bio Console and connect both pH and dO sensor cables to the corresponding electrodes Note After connecting the dO sensor cable make sure that the ADI 101
17. ial to scale the time axis possible resolution last 1 3 6 12 24 48 72 hours press another parameter key to switch to the trend display of the other parameter press the Setp key to return to the Main screen The Process Data are also displayed in a trend by first pressing the View key followed by the corresponding Parameter key Press Parameter key Press View key Use Dial to change Time Axis Measured process values of the last 72 hours are stored in memory Older process data can not be recalled displayed Note After power down the volatile memory is initialized so all collected data will be erased Teco eg BIOTECHNOLOGY 21 CULTIVATION OPERATOR MANUAL Bio Bundles 1 15 liter Microbial Applications US Version April 2004 7 4 SAMPLING To sample a fermentation broth a qyginc sample pipe is needed to which a sample system is attached Connect a syringe to the tubing that is connected to the out barb of the sample system In this tube there should i DISPOSABLE be a filter or cotton AIR FILTER Pull the piston of the syringe to create a SYRINGE vacuum in the sample bottle As a result the fermentation broth is pulled into the sample bottle Replace the sample bottle aseptically with a sterilized empty one SAMPLE SYSTEM SAMPLE PIPE 7 5 CORRECTING THE MEASURED pH VALUE In order to verify the correctness of the measured pH a sample of the culture ca
18. ls are used to assemble the head plate auxiliaries like baffles and blind stoppers etc make sure not to apply force gt 4 Nm Make sure that an O ring is present between the auxiliary and the head plate in order to ensure sterility 2 2 HEAD PLATE LAY OUT AND ASSEMBLING INSTRUCTIONS The head plate of the reactor contains a number of ports in order to accommodate auxiliaries for agitation aeration etc The exact head plate lay out depends on the reactor type volume The next pages present examples of head plate configurations The available head plate auxiliaries are described in the Hardware amp Installation Manual chapter 5 Reactors amp Auxiliaries OTe eelea BIOTECHNOLOGY OPERATOR MANUAL ASSEMBLING THE HEAD PLATE Bio Bundles 1 15 liter Microbial Application US Version April 2004 THE 1 LITER REACTOR DISHED BOTTOM The head plate of the liter reactor comes with six dedicated auxiliaries Inoculation pipe Air outlet pipe Heat exchanger 2 conn 7 Sample PIPS Inoculation Pipe Thermometer pocket An Air Overlay Air inlet pipe i lt N M18 x 1 5 Port 4x The port usage is listed in the table below M30 x 1 central port Top Stirrer Assembly dO electrode holder os a f Heat Exchanger Sample Pipe Thermo Pocket Air Inlet Pipe Foam Level Connection Assembling instructions Loosen the six mill nuts that affix the head plate on the
19. mm ports the auxiliaries are inserted at the bottom side and secured by a nut at the top side mounted from the top side of the head plate Mount the dO sensor in its electrode holder do not mount the pH sensor in its holder yet needs to be calibrated first Mount the head plate on top of the reactor and affix the 6 mill nuts oopikon BIOTECHNOLOGY pH ELECTRODE CALIBRATION OPERATOR MANUAL Bio Bundles 1 15 liter Microbial Applications US Version April 2004 CHAPTER 3 pH ELECTRODE CALIBRATION 3 1 GENERAL Before the reactor is autoclaved the pH electrode needs to be calibrated in order to obtain accurate measurement values during cultivation Prepare the electrode in the following way Remove the Watering Cap from the electrode Rinse the electrode with d i water and dip it dry with a tissue Connect the pH electrode to the rear side of the ADI 1010 Bio Controller 3 2 pH CALIBRATION ROUTINE IN THE ADI 1010 BIO CONTROLLER Switch on the ADI 1010 Bio Controller and make sure that the control loop is switched off The pH sensor calibration routine is started by pressing the pH key followed by the Calib key Four options are now presented View the current Calibration values Calibration executed pre ceding autoclaving Sample Correction offset correction during the process based on offline samples Reset to Default Calib ration values slope 1 00 and offset 0 00 pH The di
20. n be measured off line Note Off line measurement of the acidity of a sample of the culture must be carried out under strict conditions pH measurement in the sample must be carried out directly after sampling any CO or NH that is stripped off the sample will change the current pH value off line pH measurement must be carried out at process temperature changes in temperature of the sample will lead to measuring errors change in temperature will cause a shift in the equilibrium between H O and its salt form This procedure is continued on next page 22 OTe eel eg BIOTECHNOLOGY OPERATOR MANUAL Bio Bundles 1 15 liter Microbial Applications US Version April 2004 CULTIVATION The offset of the pH electrode can be corrected according to the following procedure Press Parameter key Press Calibration key Take a sample from the broth and PH CALIBRATION measure the pH off line CHOOSE CALIBRATION MODE VIEW CALIBRATION VALUES Start the Sample Correction Poroa procedure SAMPLE CORRECTION RESET TO DEFAULT CALIBRATION VALUES THEN PRESS Calib PRESS Setp TO CANCEL OPERATION 6 98pH 32 6 C 48 3 NO CONT 1112 rpm Use Dial to toggle calibration mode Press Calibration key to proceed Use the dial to generate the pH value SH a that was measured off line and press PRESS Setp TO CANCEL OPERATION the Calib key The offset of the pH electrode calibratio
21. n is now corrected igen BBBNpN NO CONT 1112 rpm Press Calibrate key to proceed Teco eg BIOTECHNOLOGY 23 OPERATOR MANUAL CULTIVATION Bio Bundles 1 15 liter Microbial Applications US Version April 2004 7 6 PASTEURIZATION After completing the fermentation the broth can be pasteurized to kill the organisms To do this follow the sterilization procedure however the operating temperature of the autoclave should now be approx 80 85 C After pasteurization the head plate of the reactor can be removed and the fermentation broth can be collected 7 CLEANING After finishing the fermentation process the glass and stainless steel parts should be cleaned thoroughly Use hot water 70 ethanol or other suitable cleaners to clean all parts Never use abrasive materials to clean the metal parts After cleaning dry the parts and reassemble the reactor Take care not to damage or forget any O rings since this will cause contamination during the next run When the reactor is used frequently it is advised to replace the O rings of the auxiliaries twice a year Note Make sure that the pH and dO electrode are cleaned thoroughly Refer to the cleaning instructions that are mentioned in the user manual of these electrodes Store the pH electrode in ADILYTE or another 3M KCI solution The dO electrode may be stored dry Depending on the type of medium that is used presen
22. o create a sterile environment inside the reactor 5 1 PREPARATION STEPS 1 Empty the applied heat exchanger use compressed air to drive the water out 2 Fill the bioreactor with medium do not exceed the working volume of the reactor refer to the Hardware and Installation Manual Be sure to leave enough space for additions after sterilization e g inoculum sterilized nutrients etc 3 Fasten the six mill nuts of the head plate crosswise by hand 4 Insert the Level probe as far as possible into the vessel and fasten it after autoclaving the position of this probe can be adjusted upward to the desired height 5 Disconnect the cables of both the pH and dO electrodes cover the electrode connectors with the corresponding screw cap Make sure that the rubber gasket is in place 6 Verify the mounting of all nipples and other auxiliaries Make connections for liquid additions air in and air out using silicone tubing or other suitable autoclavable material 7 Use appropriate filters hydrophobic material pore size 0 2 micron for both air in and outlet 8 To avoid wetting of the inlet filter during autoclaving use a hose clamp to close the tubing between the filter and head plate 9 Close all connections except the air outlet air tight with a hose and a hose clamp Close all open tubing ends with cotton and cover the ends with autoclavable foil or paper 10 Make sure that the air outlet of the bioreactor is
23. open pressure differences during autoclaving may damage the reactor or the sensors The Bioreactor is now ready for autoclaving Note Liquid addition bottles with the appropriate liquids extra medium acid base anti foam agent must also be autoclaved Teco ea BIOTECHNOLOGY 13 OPERATOR MANUAL AUTOCLAVING Bio Bundles 1 15 liter Microbial Applications US Version April 2004 9 2 AUTOCLAVING Step 1 The Bioreactor with all accessories except the stirrer motor 1s placed in an autoclave The space needed in the autoclave is given in the Hardware and Installation Manual chapter 5 Reactors amp Auxiliaries Step 2 Adjust the temperature set point of the autoclave to approx 125 C and switch it on Step 3 After the temperature set point of the autoclave is reached it should stay ata temperature gt 121 C for at least 20 minutes in order to kill all organisms and thermo resistant spores Step 4 After completing the sterilization interval let the autoclave cool down without opening it until the temperature has dropped below 90 C Step 5 When the temperature in the autoclave has dropped below 90 C it can be opened to allow it to cool down to environmental temperature Note This cooling procedure should be performed to avoid low pressure in any part of the reactor system When the tube is connected to a peripheral device low pressure in tubing might result in contamination Low pressure in a pH el
24. or automatic adaptive control refer to the Firmware Reference manual chapter 2 7 For information concerning preset parameter values refer to the PID Control manual PRESET PARAMETER VALUES In case preset PID parameter values are used the default settings may have to be optimized In case the controller behaviour for one or more control loops is not satisfactory proceed according to the following list of actions Prepare the bio process according to operating conditions Switch on the relevant control loops and wait until the environment in the reactor is OK acidity temperature and dissolved oxygen Optimizing the P value of the control loop an optimized P value is of major importance Set the I value and D value of the control loop of the concerned control loop to O P value at default or at any other predetermined value Inoculate the process and wait for substantial process activity In case the Process Value measured value for the parameter that is being optimized is oscillating around set point the P value must be reduced to such an extent that the Process Value becomes stable In case the Process Value is stable from the beginning the P value can be increased until the Process Value starts oscillating around set point _ The P value is now optimized Teco eg BIOTECHNOLOGY 19 CULTIVATION OPERATOR MANUAL CULTIVATION Bio Bundles 1 15 liter Microbial Applications US
25. order to create optimum process conditions When all control loops are at set point the reactor system is prepared for cultivation 6 4 RESETTING DOSE MONITOR VALUES Press the Menu key The sixth option in the Menu List is Dose Monitor After selecting this option use the dial and pressing the eel Menu key the following three options are presented Display Values Z Calibrate CHOOSE OPTION THEN PRESS Menu PRESS Setp TO CANCEL OPERATION Reset All dose monitors Use the Dial to select the option Reset All Use Dial to select option and press the Menu key in order to proceed enu Press Menu key to proceed All Dose Monitor value are now reset to 0 ml A100 Iko7 BIOTECHNOLOGY 18 T OPERATOR MANUAL Bio Bundles 1 15 liter Microbial Applications US Version April 2004 h CHAPTER 7 CULTIVATION After proper preparations the reactor is ready to for cultivation The first step is to create an optimum environment in the reactor This is realized by activating the control loops of the ADI 1010 Bio Controller press a parameter key followed by the Start Stop key repeat this action for all other control loops FIXED PID CONTROL VERSUS ADAPTIVE CONTROL The pH Temp and dO control loops of the ADI 1010 Bio Controller can be set to control with preset PID parameters or to adaptive control For information concerning the controller settings preset PID parameters
26. r Microbial Applications US Version April 2004 6 2 PREPARING FOR OPERATION After connecting all cables and tubing adjust the set points of the pH Temp and stirrer speed controllers to the desired value Press Parameter key press a parameter key _ J Press Set Point key press the Setp key use the dial to edit the Set Point inverse value after a short interval the edit mode is completed return to Main Screen Note Set Points can also be edited by first pressing the Setp key followed by a Parameter key 6 98pH 34 6 C 48 3 NOCONT 1112rpm Use Dial to change Set Point Make sure that the supply of tap water air and oxygen is available Switch on the stirrer speed control loop Switch on the control loops for pH and Temperature Control loops can be started by pressing buttons in the following sequence 9 press a parameter key a Press Parameter key press the Start Stop key 6l Press Start Stop The control loop is now started green pilot LED lights Note Control Loops can also be started stopped by first pressing the Start Stop key followed by a Parameter key When temperature and pH control are at set point and are stabilized the dO electrode can be calibrated after polarizing the electrode for at least 6 hours oooikon BIOTECHNOLOGY OPERATOR MANUAL Bio Bundles 1 15 liter Microbial Applications US Version April 200
27. s control reacts to these minor disturbances in set point value Finally Internal Model Control converts the identified process character into new PID control parameters The adaptive control algorithm will characterize the process based on changes in the process parameters If necessary the Judge of the Bio Controller may decide that a new process identification must be carried out oopikon BIOTECHNOLOGY 20 OPERATOR MANUAL Bio Bundles 1 15 liter Microbial Applications US Version April 2004 7 2 INOCULATION CULTIVATION Inoculation can be performed in several ways Two commonly used methods are described below l Fill a sterile flask to which a sterile hose is connected aseptically with inoculum The other end of the hose should be connected to a sterile needle Turn off aeration pierce the needle through the septum and transfer the inoculum to the reactor by gravity feed or by using a pump A sterile syringe is filled with inoculum The needle is pierced through the septum and the inoculum is injected into the reactor this method is suitable for inoculum volumes smaller than 100 ml Other methods of inoculation can be used as long as they are performed aseptically 7 3 VIEW PROCESS DATA IN A TREND Measured Process Data sensor signals and stirrer speed can be displayed in a trend by pressing buttons in the following sequence Note press a parameter key press the View key use the d
28. uxiliaries in the 10 and 12 mm ports auxiliaries are inserted at the bottom side and secured by a nut at the top side Mount the auxiliaries in the M18 x 1 5 ports the threaded auxiliaries are mounted from the top side of the head plate Mount the dO sensor holder in the G3 4 port Mount the dO sensor in its electrode holder do not mount the pH sensor in its holder yet needs to be calibrated first Mount the head plate on top of the reactor and affix the 6 mill nuts OPERATOR MANUAL Bio Bundles 1 15 liter Microbial Application ASSEMBLING THE HEAD PLATE US Version April 2004 THE 15 LITER REACTOR DISHED BOTTOM 69 mm central port 10 mm Baffle assembly Sample pipe assembly Foam Level sensor 10 mm Thermometer pocket 10 mm 2 x Heat exchanger 10 mm Spare blind stopper 12 mm Spare blind stopper 12 mm Spare blind stopper Assembling instructions Loosen the six mill nuts that affix the head plate on the reactor remove the head plate Mount the stirrer assembly without impellers Mount the impeller s at the shaft of the stirrer assembly Mount the baffles in 3 exterior 10 mm ports baffles are inserted at the bottom side and secured by a nut at the top side Mount the other auxiliaries in the 10 and 12 mm ports auxiliaries are inserted at the bottom side and secured by a nut at the top side Mount the auxiliaries in the 27
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