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Manual - The Gregory P. Mullen NMR Structural Biology Facility

1. 1 NEW BRUNSWICK f O 2 S l N le o gt i VN VN ay ao p 1 Viewed from the top 3 Width 39 65 cm 15 61 in 2 Depth 40 64 cm 16 00 in 4 Height 67 56 cm 26 6 in 4 2 Environment The BioFlo CelliGen 115 fermentor operates properly under the following conditions e Ambient temperature range 10 C to 35 C e Relative humidity up to 80 non condensing BioFlo CelliGen 115 M1369 0050 Operating manual 17 4 3 Installing the Control Cabinet Position the BioFlo CelliGen 115 control station cabinet on a firm level surface in an area where utilities are readily available Connect the mains power cord to the rear of the control cabinet At a later time once the system is completely assembled and all connections have been made you will plug the mains power cord into a suitable electrical outlet Figure 2 Front View 5 E o a SS i TH 2 j O O 3 a 0 i 4 Touchscreen display see Section 6 1 Rotameters from 0 to 4 see Section 12 1 1 Pumps 3 see Section 11 ON OFF mains power switch see Section 4 9 Service connections see Figure 1d Ni a
2. pH Probe Kits 13L M1369 9970 Probe cable amp adaptor 3 0L M1369 9977 for magnetic drive and direct drive 75L M1369 9982 140L M1369 9985 pH Probes 200 mm gel filled 1 3L P0720 5582 225 mm gel filled 3 0L P0720 5584 325 mm gel filled 7 5L P0720 5580 425 mm gel filled 14 0 L P0720 5583 pH Probe Cable amp Adaptor pH probe cable all vessels P0720 2276 pH DO probe adaptor 12 mm compression M1273 5040 DO Probe Kits 13L M1369 9974 Probe cable 8 adaptor 3 0L M1369 9979 for magnetic drive and direct drive 75L M1369 9986 140L M1369 9988 DO Probes 160 mm 1 3L P0720 6580 220 mm 3 0L P0720 6282 320 mm 7 5L P0720 6283 420 mm 140L P0720 6284 DO Probe Cable 8 Adaptor DO probe cable all vessels P0720 2336 pH DO probe adaptor 12 mm compression M1273 5040 Foam Level Probe Kits 1 3L M1369 9947 Foam probe Level probe cable 8 adaptors 3 0L M1369 9951 7 5L M1369 9960 14 0L M1369 9960 Foam Level Probe Cable amp Adaptor Foam Level probe cable all vessels M1361 8014 Foam Level probe adaptor 12 mm compression M1273 5043 continued Motors Operating manual 126 Direct Drive Cell Culture All vessels M1369 3135 Direct Drive Fermentation td Eee OE M1362 2120 7 5L 14 0 L M1369 3125 Magnetic Drive All vessels M1369 3130
3. 70 6 2 8 Modifying setpoints This process is the same as entering setpoints See Section 6 2 7 above 0 Sections 6 2 9 6 2 12 will acquaint you with the primary screens accessed from the blue buttons at the bottom of each screen 6 2 9 Calibration screen This screen is used to calibrate the pH DO and level probes For details on probe calibration see Sections 7 2 pH probe 7 3 DO probe and 7 4 Level probes Figure 42 Calibration Screen Y Calibration Screen New Brunswick Cell Culture Mode 07 Dec 2008 15 33 Loops Calibrating Loop pH DO 4 Level Sens 1 Current Value Raw Value Level Sens 2 E Summary PS E Cascade 3 Pumps Setup 1 These last two loops are input from the Level probes to the Level 1 and Level 2 loops 6 2 10 Cascade screen A cascade is a control function that uses the output of one loop to influence the action and output of one or more other loop s This screen see the following page allows the user to set up cascades to view current cascade settings and to change those settings For details on setting cascades see Section 10 1 BioFlo CelliGen 115 M1369 0050 Operating manual 71 Figure 43 Cascade Screen Cascade Screen New Brunswick Cell Culture Mode 07 Dec 2008 15 34 DO Cascade Cascade Limits c None Agit Casc Low Limit lo c Agit Agit Casc High Limit GasFlo Casc Low Limit Oo
4. 1 Connect OUTPUT of first station 2 to INPUT of second station 3 If two utility stations will be installed skip to Step 4 If only one utility station will be installed connect one of the provided terminators part number M1273 8004 to the master control station s input COM port Connect another terminator to the utility station s output COM port as shown in Figure 6 Figure 6 Installation of Terminators with Master amp One Utility Station 1 Terminator on master control station 2 Terminator on utility station Operating manual 22 4 Turn on the master control station first then turn on the utility station 5 See Section 12 3 for instructions on how to add new hardware If you wish to add a second utility station continue with Steps 6 to 8 6 To add a second utility station connect the RS 495 cable provided to the first utility station s output COM port and the second utility station s input com port as shown in Figure 7 Verify that the cable is securely connected to both cabinets 7 Connect one of the provided terminators to the master control station s input COM port Connect another terminator to the 2 utility station s output COM port as shown in Figure 7 Figure 7 Installation of Terminators with Master amp Two Utility Stations p Y po x N Sa 1 Master control station note 2 1 utility station 3 2 utility stat
5. Figure 25 or Figure 26 whichever represents your sampling system 1 Check to be sure that the sample bottle is slightly loose not tight against the gasket 2 Close the valve on the sampler tube if it is open 3 Squeeze the bulb and holding it compressed tighten the sample bottle against the gasket 4 Open the valve and gradually let go of the rubber bulb to obtain the desired sample volume 5 When you have obtained the desire volume close the valve 6 Unscrew the sample bottle from the sampler Take the cap from a new bottle and place it on the sample filled bottle 7 Install the new bottle in the sampler and make sure that the sample bottle is firmly sealed against the sampler gasket Always use aseptic techniques 8 Repeat the above steps until you have the desired number of samples 13 6 Fermentation phases In a typical fermentation run you can expect to see four characteristic phases 1 the Lag phase 2 the Exponential Growth phase 3 the Steady State phase and 4 the Decline phase 13 6 1 Lag phase This initial phase is aptly named because it is the slow beginning of your fermentation run while the microbes become accustomed to their medium 13 6 2 Exponential growth phase After the initial lag a sudden spurt in growth will indicate that the environment is fully hospitable to the microbes Compared to the nearly inanimate lag phase this activity will appear to be nearly uncontrolled BioFl
6. 4 7 1 Headplate Familiarize yourself with the arrangement of the headplate ports as shown in the following diagrams before proceeding with the vessel assembly These are recommended arrangements You may find it more practical to change the arrangement the variety of ports and adapters will easily accommodate your needs 10 Figure 14 1 3 L Headplate 1 Level probe 6mm 5 Septum 12 mm 9 Exhaust condenser 12 mm 2 Tri port 12 mm 6 pH probe 12 mm 10 Tri port 12 mm 3 DO probe 12 mm 7 RTD thermowell 12 mm 4 Tri port 12 mm 8 Harvest sampler assembly 0 On the 1 3 liter headplate there is only one 6 mm port be sure to use this for the level probe 0 The RTD thermowell port should only be used for its intended purpose Operating manual 32 14 13 12 11 10 Figure 15 3 0 L Headplate 15 gt SN N A Y 1 Sparger 6 mm 9 Mounting position for sampler assembly 2 Cooling coil 6 mm 10 RTD Thermowell 12 mm 3 pH probe 12 mm 11 Sample 6 mm 4 Septum 12 mm 12 Tri port 12 mm 5 dO2 probe 12 mm 13 Exhaust 12 mm 6 Tri port 12 mm 14 Harvest Tube 6 mm 7 Level probe 6 mm 15 Cooling coil 6 mm 8 Each headplate bolt is a possible mounting position for a bottle holder dO2 and DO are abbreviations for disssolved oxygen 0 The RTD thermowell port should only be used for
7. 5 1 Certifications The BioFlo CelliGen 115 has been tested and meets the requirements of U S and Canadian electrical and safety standards And as attested in the CE Declaration of Conformity reproduced on the following page they also conform to the appropriate electrical and safety requirements set out in European Directives BioFlo CelliGen 115 M1369 0050 Operating manual 59 New Brunswick an eppendorf company CE DECLARATION OF CONFORMITY New Brunswick Scientific hereby declares that the product s listed below conform to the European Union directive and standards identified in this declaration Product s BioFlo 115 Fermentor System CelliGen 115 Bioreactor System EU Directive s Low Voltage 2006 95 EC Electromagnetic Compatibility 2004 108 EC CE Marking Directive 93 68 EEC Standard s EN61010 1 EN61000 4 3 EN55011 CLASS B EN61000 4 4 EN61000 3 2 EN61000 4 5 EN61000 3 3 EN61000 4 6 EN61000 4 2 EN61000 4 11 The conformity assessment procedures were performed at the following i Compliance Laboratory 6 Randolph Way Hillsborough NJ 08844 New Brunswick Intertek Testing Services 41 Plymouth Street Fairfield NJ 07004 Advanced Scientific 44 Talmadge RD Edison NJ 08818 The technical documentation relevant to the above equipment will be held at New Brunswick Scientific 175 Freshwater Bivd Enfield CT 06082 U S A Tel 860 253 3400 Fax 860 741 0859 ry D
8. I ALERT Risk of damage to equipment gt Never clean the vessel or its components or the control cabinet with abrasive chemicals or materials 15 1 Cleaning the vessel 0 If applicable be sure to follow the bio safety regulations regarding the release of microorganisms into the environment 1 Fill the vessel with a mild detergent and water solution Let it stand for one hour then brush it thoroughly with a soft brush Use the brush both on inside and on outside surfaces 3 Drain the vessel and rinse several times with tap water 4 Repeat rinsing with distilled water and let it dry 15 1 1 List of wetted parts For further reference in your choice of cleaning detergents Table 7 provides a list of wetted parts in the vessel assembly and the materials they are made of Table 7 Wetted Parts Wetted Parts Material Headplate O ring EPDM O ring lubricant Silicone Headplate penetration O rings EPDM Metal surfaces 316L or 316 stainless steel Vessel glass Borosilicate glass Inoculation septum Pure gum rubber color tan 15 2 Cleaning the cabinet At least once a month clean all the metal and plastic parts of your equipment Use a soft damp cloth moistened with water or mild detergent If a detergent is used remove all residue by rinsing them with clean water BioFlo CelliGen 115 M1369 0050 Operating manual 123 16 MAINTENANCE Preventive maintenance keeps your equipment in pro
9. 16 If you are using oxygen set the O2 control loop to the desired setpoint for oxygen enrichment If however you are using Air only set the O2 setpoint to 0 Zero 17 Set the O2 or Air control loop control mode to O2 Enrich 18 Enable the pumps 19 Go to the CASCADE screen and select the DO loop 20 Set up cascades as desired 0 Aeration is required whenever the agitation setpoint is greater than 750 rpm Eppendorf suggests a minimum airflow rate of 0 25 VVM when running at speeds gt 750 rpm 13 3 Inoculation Using the septum port 1 Aseptically remove the inoculum from its flask with the inoculation syringe 2 Inject the inoculum through the septum in the inoculation port If you prefer to inoculate via an addition port be sure to flame the connectors and use an inoculum flask as your addition vessel BioFlo CelliGen 115 M1369 0050 Operating manual 115 13 4 Start BioCommand if present 1 Start the New Brunswick BioCommand supervisory software on your computer reset the EFT Elapsed Fermentation Time to zero make appropriate program selections to begin logging data 2 Make sure all gas pressures are 10 PSI and the water pressure is 10 PSI 3 If your BioFlo CelliGen 115 has Rotameter air flow control adjust the airflow to the desired rate Check to see that flow is stable and that all gases are properly connected 13 5 Sampling procedure Referring to Operating manual 116
10. 4 Gently slide the top portion of the knurled port adapter part of the probe kit onto the probe Slide the two white ferrules onto the probe the narrower one on top of the deeper cup shaped one Gently slide the bottom portion of the port adapter onto the probe taking care to orient the longer threaded section toward the top of the probe Remove the two O rings installed in the dO2 port first slide the white Teflon O ring onto the probe then follow with the black 12mm port adapter O ring Do not yet close up all the elements of the port adapter Gently insert the probe into the appropriate port allowing the O rings to seat fully into the port 0 The fit may be snug Gently rotate the probe as you press it into the port 9 10 11 to avoid breakage Finger tighten the bottom portion of the port adapter into the port Adjust the probe to the desired height then nesting the ferrules close the top portion of the adapter onto the bottom portion Finger tighten the knurled adapter assembly BioFlo CelliGen 115 M1369 0050 Operating manual 81 7 3 4 DO probe polarization 0 If the probe has been disconnected from a voltage source either the system s O amplifier or a separate polarizing module for longer than 5 minutes it will need to be re polarized To re polarize Connect the probe to the operating O amplifier or polarizing module Allow SIX HOURS FOR POLARIZATION prior to calibrating t
11. A CAUTION Risk of injury to hands 5 Then as you rotate the pump s rotor by hand in a clockwise direction to clear the channel lay the tubing in the channel around the pump head 6 Fitthe other end of the tubing loop into the second spring loaded clamp making sure the tubing fits tightly around the pumphead 7 Press and hold the pump mode Prime button or change the pump mode to ON at 100 setpoint and ensure that the pump operates smoothly See Section 11 1 for details on pump assignment and Section 11 for details on pump set up and operation 9 3 Confirm pH calibration Autoclaving can alter the zero characteristics of pH probes typically by 0 1 0 3 pH To check and to compensate for any discrepancy you will need an accurate external pH meter 1 Following sterilization with the media at room temperature note the pH value on the BioFlo CelliGen 115 SUMMARY screen 2 Take asample of media and measure the pH using the external meter 3 If the two values disagree return to the pH calibration screen see Section 7 2 and Set Zero to the value reported by the external meter Do not change the Span or you will invalidate the entire calibration The pH value will now agree with the external meter s reading 9 4 Install liquid addition systems Figure 33 is a simple depiction of a typical addition system Depending on the liquids base acid nutrients media to be added your system may be slightly different 1 Asep
12. Impeller diameter in inches RPM Agitator speed in rpm 4 5x 107 Constant factor based on unaerated water at 20 C with a six bladed Rushton impeller factor based on the number of impellers used in the vessel e Use 1 for one impeller e Use 1 8 for two impellers e Use 2 4 for three impellers BioFlo CelliGen 115 M1369 0050 Operating manual 139 0 The HP requirements are substantially affected by aeration An airflow rate of one vessel volume per minute VVM may produce as much as 40 reduction in the horsepower used It is required that some air gas flow be utilized when running at speeds above 750 rpm The relationship in the reduction of horsepower when gas is added into the system is not linear A small amount of air can produce a 20 reduction in horsepower Operating manual 140 21 APPENDIX C FERMENTATION TECHNIQUES The following section outlines step by step procedures for carrying out a benchtop fermentation Provided in a question and answer format this discussion covers such topics as which media formulation tubing size and concentration of various additives should be used It also addresses the preparation autoclaving and clean up procedures for the vessel and accessories While this example refers specifically to an E coli fermentation in a BioFlo CelliGen 115 the information is generally applicable for any fermentation 21 1 Media formulation Question What kind of media should be
13. Water 10 PSIG maximum 50 pm raton SSS Gases 10 PSIG maximum _ ee Electrical requirements Control Heat With Exhaust Condenser w o Exhaust Condenser Station Blanketed Width Depth Height Width Height Utility Station cm in cm in cm in cm in cm in Dimensions 1 3 L Vessel 24 9 5 22 8 5 56 22 22 8 5 42 16 5 3 0 L Vessel 24 95 22 8 5 56 22 22 8 5 42 16 5 7 5 L Vessel 37 145 29 115 65 23 29 115 495 195 14 0 L Vessel 29 115 29 115 74 29 29 115 61 24 Water With Exhaust Condenser w o Exhaust Condenser Jacketed Width Depth Height Width Height cm In cm In cm In cm In cm in 1 3 L Vessel 29 115 29 115 52 20 5 24 9 3 41 16 3 0 L Vessel 29 115 29 115 56 22 5 24 9 3 45 18 7 5 L Vessel 29 115 29 11 5 68 26 8 29 115 52 20 5 14 0 L Vessel 29 115 29 115 80 31 5 29 11 5 67 26 5 Vessel 1 3L 3 0L TOE 14 0 L empty 9 3 kg 20 5 Ib 18 kg 39 5 Ib 19 5 kg 43 Ib Control Station Communications 29 5 kg 65 Ib USB for easy firmware upgrades Control station only BioCommand Port for communication with optional BioCommand SCADA software Ethernet for future expansion Certified to es c ETL us UL Standard UL 61010 1 CAN CSA C22 2 No 61010 1 Ambient Operating Conditions 10 35 C up to 80 relative humidity non condensing 0 Specifications are subject to change without notice
14. 100 0 Setpoint Gas Mix Selecton 100 0 EA F Air 02 CO r 02 N2 t PV Out Mieri as Sel E 100 0 100 0 100 0 4 Gas Mix Flow Cycle Times Seconds conde On Time 1120 Of Time 0 1000 100 1000 a 2222222 imits Set Low Set High 00 100 0 Decimal Places 0000 00 00 ina 00 00 00 Calibration Cascade 3 Pumps X Setup There is also a GasFlo loop when one TMFC is present settings in this loop s gauge screen turn the TMFC on and off and control the gas flow rate The GasFlo gauge screen allows you to set parameters for the TMFC that controls this gas The gauge screen for any of the gases allows you to set parameters for the TMFC that controls the gas Operating manual 108 Figure 72 GasFlo Gauge Screen Cell Culture Mode full Gauge Screen New Brunswick 07 Dec 2008 15 52 GasFlo 0 0 SLPM oma 0 0 5 oe E om ane a IA 0 0 0 0 0 0 20 0 20 0 100 0 Limits Set Low Set High 0 0 200 Totalizer Liter IS 0 0 0000 00 00 oo A A Uf Calibration TA Cascade Pumps 2 Setup 12 2 System settings Press the third tab in the SETUP screen to open the System Settings screen see below Use this feature to select the onscreen language you prefer to reset the date and or time to update the software and to calibrate the BioFlo CelliGen 115 touchscreen Figure 73 System Settings Screen Fermentation Mode Le
15. 14 Default P amp proportional amp integral values are preset at the factory We strongly recommend that you maintain the factory set parameters The DO probe is a polarographic probe Be sure to inspect the DO probe before every run changing the electrolyte solution and membrane as needed 3 8 Foam Level control Foam can be monitored during batch fermentation by a foam level probe located in the headplate The controller operates the antifoam assigned pump that adds chemical defoamer into the vessel as needed The internal level can also be controlled by using this feature Pumps can be triggered to turn on or off in response to the presence or absence of liquid 3 9 Exhaust system The exhaust gases pass into the exhaust condenser where moisture is removed then returned to the vessel The remaining gases then pass through a 0 2 um exhaust filter Be sure to inspect filters before every run replacing them as needed U WARNING Risk of explosion gt NEVER block the exhaust to pressurize the vessel 3 10 Recommended accessories amp supplies Before you begin to assemble your BioFlo CelliGen 115 it would be prudent to verify that you have all of the following accessories and supplies readily at hand e An autoclave e An inoculation syringe e Rubber gloves e Media e Silicone tubing e Antifoam agent e Atie gun e Aluminum foil e Plastic ties multiple colors can be helpful e Rubber bands e Plastic tubing
16. Heaters amp Heater Blankets Water Jacket Heaters TESOL M1362 2107 7 5L 14 0 L M1369 3108 1 3L M1369 8021 Heat Blankets 3 0 L M1369 8022 7 5L M1369 8020 14 0 L M1369 8023 Glass Vessels 1 3L M1273 9907 Heat Blanket Vessel 3 0L M1273 9909 7 5L M1273 9916 14 0L M1273 9918 1 3L M1273 9908 Water Jacketed Vessel 3 0L M1273 9915 7 5L M1273 9917 14 0L M1273 9919 Exhaust Condensers By fauct Condenser 1 3L 3 0 L 7 5L M1273 9945 14 0 L M1273 9957 Headplate Adaptors amp Plugs Tri port adaptor M1273 9961 pH DO probe adaptor 12 mm compression M1273 5040 Foam Level probe adaptor 12 mm compression M1273 5042 Septum kit M1273 3031 6 mm adaptor kit 6 mm port to 6 mm tube M1273 5054 6 mm single addition tube for 6 mm adaptor M1273 9575 Adaptor kit 12 mm port to 6 mm tube M1273 5056 Adaptor kit 12 mm port to 12 mm tube M1273 5058 6 35 mm port plug M1273 9405 12 mm port plug M1273 9406 19 mm port plug M1273 9407 Headplate port washer O ring kit M1273 9900 Thermowells 1 3L M1273 9200 Thermowell 3 0L M1273 9201 7 5L M1273 9202 14 0L M1273 9203 continued Harvest Tubes BioFlo CelliGen 115 M1369 0050 Operating manual 127 Harvest Sample tube 1 3L M1273 9260 3 0L M1273 9197 Harvest tube 7 5L M1273 9162 14 0L M1273 9194 Sparge Rings amp Cooling Coils Sparge Ring
17. Ree GasFlo Casc High Limit Agit 02 02 Mix Casc Low Limit c Agit GasFlo 02 Mix Casc High Limit GasFlo 02 c Agit GasFlo 02 E Summary Calibration Cascade Pumps 2 Setup 6 2 11 Pump screen This screen see below allows the user to access the pump gauges screens where the three standard pumps are displayed providing both current readings and the opportunity to change pump settings For details on using the PUMP screen see Section 11 1 Figure 44 Pump Screen Pump Screen New Brunswick Cell Culture Mode 07 Dec 2008 15 35 Pump1 Pump2 Pump3 Setpoint 00 Setpoint 00 Setpoint 0 0 Pv 0 0 Pv 0 0 Pv 0 0 Control Mode ____________ Control Mode Control Mode Flow Rate mL Second Flow Rate mL Second Calibrate Calibrate Calibrated Calibrated 2 000 0 000 Total Total Total 600 000 0 000 0 000 Period Sec Period Sec Period Sec E Summary Calibration 2 cnn y Setup Assignment None Flow Rate mL Second Calibrate Calibrated 0 000 Operating manual 72 6 2 12 Setup screen This master screen is actually comprised of three screens accessed by tabs which are used to set up the controller system settings and hardware for the BioFlo CelliGen 115 system This section will introduce you to those screens and their features For details on using
18. air gas supplies Never intentionally block the exhaust to raise vessel pressure Use the minimum air gas pressure that will provide adequate airflow for the application Never exceed the maximum air pressure of 10 psi This maximum pressure is necessary only to obtain the highest gas flow rates ate ALERT Risks of damage to vessel gt To protect the integrity of your glass vessel and to avoid damage familiarize yourself with these cautions Never allow hot glass to touch cold water or a cold surface Never rest the vessel on an uneven surface Never drag or roll the vessel across any surface Avoid metal to glass contact With the exception of occasional contact with baffles inside a vessel used for fermentation avoid touching the glass with any metal object Use non abrasive cleaners only and clean with soft brushes no sharp ends or bristles Any surface that comes into contact with any portion of the vessel must be clean and non abrasive Only finger tighten the knurled headplate bolts and port adapters Over tightening puts undesirable pressure on the glass Keep the glass free from contact with any diamond material diamond jewelry industrial diamonds or diamond dust from grinding wheels Operating manual 28 0 Clean the vessel thoroughly after each run with detergent otherwise debris could build up thus providing a place for bacteria to grow and produce toxins This can result in low cell viabili
19. as well as to any addition bottles or other connectors A tie gun is useful for this purpose Note that both the air sparger and the exhaust line will have a terminal filter For the BioFlo CelliGen 115 vessel the part numbers are PO200 0491 for the sparge line s small filter and PO200 0490 for the exhaust line s large filter All tubing connected to ports that have their terminus within the vessel below the liquid level i e the harvest and sparge ports Operating manual 144 must be clamped prior to autoclaving The sampler valve must be in the closed position Other hoses such as those attached to base or addition ports should be clamped to facilitate sterile hook ups Eppendorf primarily uses the following clamps a Hosecock Clamp Fisher catalog number 05 847 and a Hoffman Side Tubing Clamp Fisher catalog number 05 875B Do not rely on polymer clamps to survive autoclaving they often pop open in the autoclave If you wish to use the newer polymer clamps during the running of your fermentation then place them onto the tubing but leave them open Use easily removable metal clamps to actually close the line during autoclaving These may be removed after the vessel has been autoclaved Be sure to use the polymer clamp to close off the tubing BEFORE you remove the metal clamp Clamps can be placed at any point on tubing but be sure they don t clamp down onto a port or connector because that would interfere with proper seali
20. cccccccccccccsseeseeccccccccssssssescccccesssuenseececcesesaueaens 120 15 CLEANING AS NN 122 15 1 CLEANING THE VESSEL cccscccccccceesssssscccccccceessssescccccceceessssescccccsecueeessscecccseseeeeens 122 LIA Listof wetted DAIS DARAS A AE ile eaa ai 122 15 2 CLEANING THE CABINET irinen cues a i n ie 122 16 MAINTENANCE sisesiscscccsccescscscocscccceccoecsocccstccsccesessocoessccsecnotcsovces ccsecesdssovovssccsusvsdsses 123 16 1 PH PROBE MAINTENANCE AND STORAGE cccssssssseccccccccsssssssscecccecsausnssscescesesueenens 123 16 2 DO PROBE MAINTENANCE AND STORAGE Q cssssssseccccccccssssssssccccceseusenssscecceseeaeenens 123 16 3 VESSEL SETUBIN Gu id 124 16 4 PERIODIC INSPECTION cccccsccccccccccsssssscccccccceessssesccccceseeessssssccccceseusessssscccceseeeeeeens 124 16 5 AGITATOR BEARING HOUSING ccssssesseccccccccssessseccccceceusesescsccccseccuueaessscecceseeuuenens 124 16 5 1 Motor assembly replacement iaa a RA AA A AA RRA 124 16 6 REPLACEMENT PARTS cccccccccccccsssssssceccccccssessssceccecceseeusassceccsscessussassceccesseeuuuensncsscs 125 17 SERVICE PPP o P nn a tetas 129 Td FROUBLESHOOTING 30 SAA A A hei 129 18 DRAWIN S EEEE T R E EEEE OAE E EEE E 131 18 1 LISTOEDRAWING S ai 131 Operating manual 1822 GIST OF TABLES uta A A EA Ia ae SR 132 19 APPENDIX A SOME GENERAL CONCEPTS oocccccccccoconononononononononocononococonononos 134 19 1 WHAT IS A CONTROLLER cccccccccccccssss
21. to allow the vessel to vent more easily during autoclaving Place a clamp on the tubing Na DORA ON 0 Be sure to leave one clamp open during autoclaving to equalize pressure If you have addition foam trap or harvest bottles mounted at the base of the vessel you can autoclave them with the vessel Without detaching their tubing from the headplate 13 Remove the bottle holder s and reinstall each on one of the headplate clamping screws 14 Reinsert the bottle and turn the holder until the bottle and holder are positioned over the headplate rather than extended over the edge 15 Finger tighten the knurled nut 16 Clamp off the tubing and where appropriate remove it from the pump Probe tips must be moist during sterilization e f you will be doing batch fermentation be sure the vessel is filled with media so the media will also be sterilized Operating manual 86 e If you will be using heat liable media use at least 100 mL of a balanced salt solution such as phosphate balanced saline solution Sterilize the media separately after autoclaving the vessel 8 2 Autoclaving the vessel 1 If you have a vessel assembly that is too tall for your autoclave carefully lay the vessel still mounted in its stand if present in the optional angled autoclave rack part number XMF 8624 M1227 9231 see below Secure it in place with the strap 2 If the vessel is not water jacketed skip to Step 3 If the
22. 120 Volts 50 60 Hertz 10 Amps 230 Volts 50 60 Hertz 6 Amps 0 The electrical requirements vary depending on the part number that has been ordered Model Part Number and Electrical Power Requirements for each fermentor appear on a metal label affixed to the rear of the equipment just above the connection for the mains power cord ALERT Risk of damage to equipment gt Before making electrical connections verify that the supply voltage matches the voltage and the mains power requirements marked on the electrical specification plate located on the rear panel of the cabinet and the control schematics supplied with the system 7N WARNING High voltage Risk of electrical shock gt Always make sure this equipment is properly earthed grounded Operating manual 24 4 5 2 Water and drain connections E ALERT Risk of water leaks gt Make sure all utility connections have been securely made before connecting to WATER IN and before turning on the main water supply Failure to observe these precautions will result in water leaking out of the unconnected hoses and the cabinet The water inlet and drain connections are located on the left side of the control cabinet see Figure 8 a detail from Figure 4 Water pressure should be from 5 to 10 PSIG with 50 um filtration 2 28 meter 7 5 foot lengths of tubing are supplied with an open end for water in and the drain and with quick connect fitt
23. 14 0 L fermentation vessels ONLY oooconnnoccconanicaccnononannnncnnnnnnnos 38 4 8 5 SAUS 39 4 8 6 PASIAN cooling GON ES EA IA susan ise LEAR AA REE 40 4 8 7 Install sparger 3 0 L 7 5 L amp 14 0 L vessels a ti 40 4 8 8 Install harvest MDE snose oz 4 4 8 9 Installsampler TUDELA A A aS 4 ESOO Install thermowell A A ds 4 BS LL Install Joam probe A AA 4 4 812 Install foam exhaust TUDE tia dielacnaia lanes 42 4 843 Install level DrODE S s css svccuastetivian dics os 42 4 8 14 Install addition tube S cccccccccccccccccseesecsececececssesssnsececeseceesssesssaeeesececsesessseceeesees 42 ed InstalipH RODEA oa sy a a A a Ra a 42 4S6 Install COZ A E E E AAA EE OAE EA 44 4 8 17 Install exNaUSECONAEAS CE A da A ia 46 4 646 O OE 0 ET RARA A he Tea i ee eI RAE ah ST 47 4819 Install Joamtr p A a CONCA mechan 50 4 86 20 SUG TAUSC DOTS AS AUS A E 51 4 8 21 Install 1 3 L 3 0 Lor 7 5 L fermentation vessel baffle oooonnnccnnoninicinnnono s 51 4 022 ASIAN CAG OI O AA A A a E A Aaa abs 52 YESO y Install vessel 2 ESAS AAA AAA AE TA AAA AAA 52 4 35 24 MUST motor assembly as 53 48 253 gt AMARE connections 1 A AN E e hate apa a a E da 53 4 9 ON OFFESWIICH e ACG ania aE a a aS 54 4 10 OPTIONAL BIOCOMMAND SOFTWARE sssssessesssseseestseesresstestestteseeseeserssreseserssressesets 55 3 SPECIFICATIONS wes tsdeseisshessdesocslesseinesiudscsdsdopwatedsudadasuscventscaalscstacesstsabiedsdeaveorenccstadstesons 57 5 1 CERTIFICATI
24. 19 Control Loop Definition of 134 Control Station 12 Controller Definition of 134 Cooling Coil Installation of 40 Corrosion Resistance 149 D Deadband 13 68 92 Decline Phase 117 Description of Vessel 12 DO Probe Calibration of 81 Charging of 114 Inspection of 78 Installation of 80 dO2 Probe Installation of 44 79 Double Filter System 120 Drawing Index 132 Drawings 1 3L Headplate 31 7 5L 4 14 0L Headplate 33 Non Jacketed Vessel 30 Operating manual 152 Sampler System 49 Vessel Bumper Installation 34 Drive Assembly Handling 28 E Electrical Connections 23 Electrical Requirements 23 End of Run 119 EPR General Characteristics of 150 Essential Warnings 27 Exhaust Condenser 14 53 Installation of 53 Operation Tips 120 Exhaust Filter Operation Tips 120 Exhaust System 14 Exponential Growth Phase 116 F Fed Batch Operation 117 Feed Pumps To Add Liquid 101 Fermentation Run Phases of 116 Preparing for 113 Fermentation Techniques 140 Fermentor Information Sheet 3 Filling the Water Jacket 38 Foam Control 14 113 Foam Exhaust Tube Installing the 42 Foam Level 14 Foam Probe Installing the 41 G Gas Connections 25 Gas Control 106 Gas Overlay 118 GasFlo 107 Gauge Screen 67 Glucose Feed Concentration 142 H Handling Tips 28 Harvest Tube Installation of 41 Harvesting 119 Headplate Installation of 52 Headplates 1 3L 31 3
25. 5 Attach the exhaust filter respecting the direction of flow if stamped on the filter to the condenser outlet Secure the filter with a plastic tie 6 Connect silicone tubing to the inlet port of the exhaust condenser Secure with a plastic tie Figure 23 Exhaust Condenser 1 3L 3 0 L amp 7 5 L Vessels 6 5 2 3 4 1 Sterile filter 4 Exhaust port Set screw in port adaptor 5 Water inlet Exh Cond In 3 Headplate 6_ Water outlet Exh Cond Return Be sure to see important NOTICE on the following page BioFlo CelliGen 115 M1369 0050 Operating manual 47 If the weight of the exhaust filter kinks the tubing fasten a short length of stiffening material to the tubing using rubber bands or tie wraps to support the filter Ensure that gas flow through the exhaust condenser is unobstructed during runs and during autoclaving Figure 24 Exhaust Condenser 14 0 L Vessel only 2 3 d 6 1 Sterile filter 4 Set screw in port adaptor 2 Water outlet Exh Cond Return 5 Headplate 3 Water inlet Exh Cond In 6 Exhaust port 4 8 18 Install sampler The optional BioFlo CelliGen 115 sampler system is designed to aseptically remove batch samples from the vessel The entire installation is easily autoclaved in place on the vessel If you are using the sampler install
26. 6 DO probe calibration setting Span ssccccccscssecececssecececssececessseaecesseaeeseussaeeees 82 7 4 LEVEL PROBE CALIBRATION cicirenna a a a a aa aaa 82 7 5 ABOUT PUMP CALIBRATION ccccccccccccccsssssseeccccceccusesescsceccesseaseasssscscceseseeuueasseecceseeeea 83 VESSEL STERILIZATION cisssssccscesccsssacdeisescacetacceseaiccetacecacaccesicdesacesecacesssscotacssscavecsicese 84 8 1 INITIAL PREPARATION FOR AUTOCLAVING ccccccccesssccccesssccscesssccseesssccssessceseeseccseues 85 8 2 AUTOCEAVING THE VESSEL uti iii 86 8 2 1 Sterilization time and temperalUTC ooooooconinoncncnnnonnnnnnnnnnnnnnnnnn nn nnnnnn nn nn cnn nn nn nnnnnos 87 REINSTALLING THE VESSEL ASSEMBLY cccccccononononononononononononononononononononecononos 88 9 1 REINSTALL THE VESSEL ASSEMBLY cssssssseccccceccssssssscecccscceeesssescsccesseaeuaeasececceseeaea 88 9 2 TOAD PUMP TUBING cuca a 88 9 3 CONFIRM PH CALIBRATION ccssssesseccccccccscssssssceccccccesesesscecccseeeussensscescssseeauaeneecsses 90 9 4 INSTALL LIQUID ADDITION SYSTEMS occcococononononononononononononononononononononononononononononononononos 90 9 4 1 Addition tubing ME A REA EER 91 9 5 RECONNECTGASES ii basar 92 9 6 INSTALE TEMPERATURE RTD PROBE 0d RS 92 CASCADE CONTROL iicccccscdcseccscsescccccscaiscosssocccsceccsscocssoccccsceesededesesdeesstesseccuavecseossee 93 10I CREATING A CASCADE sireh a ei 94 ABOUT PUMPS wicisissicsssccsccccscstecsuccocceckcccusesecccscesuscccsdsucsocses
27. 9953 14 0 L M1273 9956 Rotameter Kits 0 20 SLPM Rotameter with stand amp tubing M1287 3520 0 5 SLPM Rotameter with stand amp tubing M1287 3510 Spare Parts Kits Spare parts kit Heat Blanket vessel 1 3 5 3 0L M1239391 7 5L 14 0 L M1273 9992 Spare parts kit Water Jacketed vessel CIL gak MIA ADR 7 5L 14 0 L M1273 9999 Miscellaneous Start up kit All sizes M1369 0300 Autoclave rack 7 5L 14 0 L M1273 9266 Water regulator kit 4 manifolds M1273 5001 Air regulator kit 4 manifolds M1273 5002 Bearing housing cap 10 pack M1273 9936 Addition bottle kit 250 mL M1273 9989 Addition bottle kit 500 mL M1273 9990 Addition bottle holder kit M1273 9940 Silicone grease for seals amp O rings P0860 1050 Silicone tubing clamp P0160 4460 Polysufone quick connect female 6 35 mm 7 in P0240 2680 Polysufone quick connect male 6 35 mm in P0240 2670 0 2 um inlet exhaust filter P0200 0495 Allen hex key 1 98 mm 5 64 in H 960 Motor Retro Kits Retro Kit Direct Drive Cell Culture All vessels M1369 9914 Retro Kit Direct Drive Fermentation 3 QL M136 2712 7 5L 14 0 L M1369 9913 Retro Kit Magnetic Drive All vessels M1369 9911 BioFlo CelliGen 115 M1369 0050 Operating manual 129 17 SERVICE If any problems occur with your BioFlo CelliGen 115 system or its individual components do not attempt to perform any service on it Unauthorized servicing may void the warranty Please contact your local Eppendorf Servi
28. Agit Gauge Screen New Brunswick Cell Culture Mode 07 Dec 2008 15 27 Agit 134 RPM Setpoint RPM Gains Factory 3 150 Proportional 0 02 150 EN Mer 5 50 KN 00 100 0 25 200 i Set Low Set High 25 200 Decimal Places 0000 00 00 25 f Calibration T Cascade El Pumps 2 Setup 00 Pl Values Adjusting these values will determine how your system responds to changes in your culture For details see Section 19 5 If you select Manual you will control the loop by adusting the Output which offers a range of 0 100 corresponding to the loop s range For example selecting 100 for Agitation will cause the motor to run at 200 rpm the High Limit set in the Limits pane of this sample screen Step 3 Press inside the Setpoint box to open the touchpad see below Figure 41 Setpoint Touchpad full Gauge Screen New Brunswick Scientific Fermentation Mode Agit Setpoint RPM 250 1 _ MA EE EE 2 EN KH KI EE EN E EIEE h Calibration Ei Cascade ES Pumps Ke setup Step 4 Use the touchpad number keys to enter the desired setpoint Use the white Clear key at any time before Step 5 to empty the Setpoint edit box Step 5 Press the OK key to save the setpoint and to return to the GAUGE screen or press the Cancel key to return without saving the setpoint Operating manual
29. B It is unlikely that you will ever need to reset or change the P amp values Even if the mains power fails during a run the P amp values factory preset or your own are stored in memory and should still be in effect when the mains power is restored However it is recommended that you check these values at the beginning of each run 7 3 3 DO probe installation 0 Prior to installation any dissolved oxygen probe you are using should be inspected for damage and replaced if necessary 0 To avoid damage to the probes during operation be sure that there is no interference between the probes and the baffle assembly impeller blades or cooling coil 1 Wear protective gloves to protect yourself in case of accidental breakage 2 Lightly coat the dO2 probe with glycerol I ALERT Risk of damage to dissolved oxygen probe Always fit the dO2 port adaptor onto the probe first Then insert the probe with its adaptor into the headplate following Steps 3 11 shown after the drawing on the following page Never attempt to install the dO2 port adaptor in the headplate without the probe Operating manual 80 Figure 50 dO2 Probe with Port Adapter exploded 7 q i em 1 dO2 probe adapter top portion 5 Teflon O ring white 2 Bottom ferrule 6 Top ferrule 3 dO2 probe adapter bottom portion 7 Cap 4 Port O ring black With reference to the drawing above 3
30. DO NOT allow the liquid to run into the graduated cylinder yet Operating manual 100 4 Ifyou are not using a scale skip to Step 5 If you are using a scale place the graduated cylinder with the tubing on the scale and press Zero on the scale 5 In the Flow Rate pane of the PUMP screen for that pump press the Calibrate button to open the Calibration pane Figure 62 Calibrating the Pump Flow Rate Calibration Run Time Seconds 15 N 60 Cancel Amount Pumped mL 1 Step 6 Press your choice of Run Time 15 30 or 60 seconds The button you press will turn green 2 Step7 Press the Start button The button will turn green and the pump will start running 3 Step 8 When the Run Time has elapsed record the amount of liquid accumulated in the cylinder enter that number or the number registered on the scale in the Amount Pumped edit box 4 Step 9 Press the Set button to save this data to the PUMP screen 0 Calibration must be performed at operating setpoint The pump is now calibrated As the pump runs you will see that the total will increase by this calibration standard 0 Each pump and each tubing size will need its own calibration 11 5 Pump period At the bottom of each pump gauge is the Period sec pane Figure 63 Pump Period sec Period sec 5 BioFlo CelliGen 115 M1369 0050 Operating manual 101 Use the Period sec edit box and its associated t
31. Setup Screen New Brunswick Controller Setup EFG ECU Hardware Setup TouchScreen English b E 1 Date Time 7 DEC 2008 15 36 Set Software BF115 Rev 8 Update Folders 2 E Summary f Calibration E Cascade Pumps 1_ English is the default language other languages are not available at this time 2 To recalibrate the system s touchscreen press the Calib button then touch the onscreen target each time it appears You will be guided through the process 3 Here you will find a list of the current User Interface and Control Program versions To update the software see Section 16 3 2 4 To change the Date and or Time see Section 16 3 1 07 Dec 2008 15 36 3 BioFlo CelliGen 115 M1369 0050 Operating manual 109 12 2 1 Resetting date time To reset the onscreen date and or time displayed in the lower righthand corner of every screen 1 In the System Settings screen press the edit box for the numeric parameter you wish to change 2 Use the pop up touchpad to input the new number and press the OK button 3 To change the month press the down arrow and press the month you wish to select from its associated drop down menu 4 Press the Set button to save the new information You can do this after each change or after all changes have been made 12 2 2 Updating software To update the system software obtain a new version of the software as upgrades
32. Tuning is an iterative process It consists of configuring a loop with trial P and D values evaluating loop response then readjusting the constants The process is repeated until the loop responds fully and without oscillation e One usually begins with a trial P setting and D to zero After P is established a similar iterative process establishes I e Most fermentor probes respond too slowly or are too noisy to utilize the D term to advantage In most cases D should remain at zero Agitation is sometimes an exception e The magnitude of the control signal depends on the P and D constants It also depends inversely on a Normalizing Constant 19 6 What do the constants mean The control signal Sy for a loop that is N seconds into a run is expressed mathematically as Sn P en k X 1 60 e k D n en 1 K Where P I and D are respectively the proportional integral and derivative constants e is the loop setpoint minus the current value or error k is a normalizing constant for the loop The controller reevaluates Sy every second Iis divided by 60 so any value entered by the user should be in reciprocal minutes The normalizing constant k can be set to any non zero value but is usually set to the full scale reading of the loop For example if the range of expected temperatures is O to 125 setting k to 125 results in a P term value of P when the error is at a maximum e P en k P 125 125 P S
33. Vessel Size BioFlo 115 junit 1 7 5 Liter No of TMFCs 1 TMFC Range 0 20 SLPM Operatina Mode BioFlo 115 Options 1 Cell Culture Mode v pH DO F Foam Level 4 Gas Mix F Gas Mix r Gas Flow EE Summary F Calibration Cascade J Pumps 1 The new Operating Mode appears in these two places If you run the system with various vessel sizes or the size indicated is incorrect for the Unit indicated use the Vessel Size dropdown menus to change to the new vessel size see sample screen below then press the Save Changes button to allow the system to reset to new parameters Figure 68 Changing Vessel Size Le Setup Screen New Brunswick Cell Culture Mode 07 Dec 2008 15 46 Controller Setup System Settings Hardware Setup Unit Type Unit Name Vessel Size BioFlo115 funit 7 5 Liter 1 1 3 Liter No of TMFCs 1 TMFC Range 0 20 SLPM 3 0 Liter Operating Mode BioFlo 115 Options 14 Liter Cell Culture Mode v pH DO v Foam Level 4 Gas Mix v Gas Mix r Gas Flow s Summary 3 Calibration E Cascade Pumps e 1 To change the Vessel Size press the down arrow in the dropdown menu press the appropriate size It will now appear in the edit box 2 Press the Save Changes button to save the selection to memory Operating manual 106 12 1 1 Gas control Depending on your system s configurat
34. be checked M here You cannot change those selections For other options present but not checked press each as appropriate and a V will appear in the box 4 Here you can use the down arrow to change the Operating Mode 12 3 2 Removing a Utility Station If at any time you wish to remove a utility station one which has already been assigned a unit number from the system following these instructions 1 Verify that the utility station is still connected to the control station and both are turned on Press the SETUP button to open the SETUP screen then press the Hardware Setup tab 3 Press the Scan Hardware button and wait until all items are listed in the Unit panes on the right side of the screen 4 Press the lt lt lt button corresponding to the Unit you wish to remove Wait until all the hardware assigned to that utility station appear in the New Hardware box 5 Turn off the unwanted utility station and disconnect the RS 485 cable from its COM port and from the control station s COM port BioFlo CelliGen 115 M1369 0050 Operating manual 113 13 PERFORMING A RUN 13 1 Set up foam control Before you fill the vessel with medium confirm that the foam probe is working properly 1 2 our Te 8 9 10 11 12 13 14 15 16 17 18 Fill the vessel with tap water or saline solution DO NOT USE DISTILLED WATER an ionic solution is necessary for conduc
35. be present and in good condition and the rubber stoppers must be securely in place The pH probe must be properly calibrated prior to insertion in the headplate Be sure to carefully follow the manufacturer s instructions for probe calibration or the instructions in this manual It is often necessary to coat the probe with a very thin layer of glycerin or deionized water in order to avoid jamming or breaking it during insertion The pH probe must be inserted carefully using two hands with one hand holding the base of the probe near the port opening Never force the probe and never insert the pH or the DO probe until the headplate is properly secured It is absolutely critical that both the pH and DO probes have their protective caps on prior to autoclaving in fact the caps should always be on except when the probe is being hooked up to the system NEVER autoclave a pH probe or a DO probe without its protective cap BioFlo CelliGen 115 M1369 0050 Operating manual 145 Check the DO probe to be sure the required amount of electrolyte is present prior to insertion Eppendorf usually replaces electrolyte for each new run The DO probe s membrane must also be inspected prior to use The glass wool for the sampler is prepared by rolling a small quantity up and inserting it into the small tube that attaches to the bulb It may be necessary to trim any glass wool fibers that stick out Note that it is undesirable to pack glass wool too
36. box then press the gt gt gt button for the Unit name you wish to assign This name will appear at the top of the screen 4 Each system needs a unique ID number in the SCADA pane assign the correct Communication Mode and Unit ID number then press the Set button BioFlo CelliGen 115 M1369 0050 Operating manual 111 Figure 75 Adding New Hardware Le Setup Screen New Brunswick Cell Culture Mode 07 Dec 2008 15 50 Controller Setup System Settings El Fi stay 3 A Module Status Good Errors Ali Unitl Base Power Active 370198 90 Base Power Main phiDO Active 053454 1 Main phiDO al TMFCI Active 105674 0 1 TTD Tk FC1 lt lt lt Unit2 Module Good Errors Scan Hardware ES gt gt gt SCADA lt lt lt Mode INBS Modbus v gt Unit3 Module Good Errors Unit ID 2 lt lt lt Es Summary Calibration A Cascade Pumps T 1 When you press the Scan Hardware button any new hardware appears here Figure 76 New Hardware Added I gt Setup Screen New Brunswick Cell Culture Mode 07 Dec 2008 15 51 Controller Setup System Settings LEWE Sy Module Status Good Errors Base Power Active 370325 90 New Hardware Unit1 Main phiDO Active 053473 1 ct TMFCA Active 105710 0 lt lt lt Module Status Good Errors Unit2 Base Power Active 365111 74 Scan Hardware Main phiDO Active 052724 0 TMFC1 Active 104151 2 1 SCADA lt lt lt Mode
37. can be set to respond to other sensor cues such as DO level the pH reading the turbidity measurement the glucose measurement etc The pumping profile to be used must generally be determined through experimental experience BioFlo CelliGen 115 M1369 0050 Operating manual 143 21 6 Recommended process control settings Question What are the recommended process control settings i e temperature pH agitation speed DO amp gas sparge rate Answer For E coli temperature is usually set to 32 35 C and pH is set at 7 0 7 2 For yeast the values are 30 C and a pH value of 5 0 Agitation speed is usually set to a minimum of 200 300 rpm with a maximum value of 1000 rpm Dissolved oxygen or DO level is usually 30 The gas sparge rate is generally 0 5 to 1 0 VVM 21 7 Typical fermentation run Question Can you review the steps involved in set up through shutdown of a fermentation run Answer To answer properly let s break the process down as follows 21 7 1 Vessel preparation before autoclaving It is advisable to rinse the previously cleaned vessel prior to use When doing this remember that all clamps must be open and the valve for the sampling tube must be in the open position If the glass wool is going to be replaced for the run then remove it and the rubber sampler bulb prior to rinsing The protective bearing housing cap must also be in place It will be necessary to hold the protective cap in p
38. filter 8 Headplate clamping screw 3 Thumb clamp 9 Sampler bottle 4 3 16 in silicone tubing 10 Sampler bottle holder 5 Sampler port 11 Headplate clamping nut 6 Headplate 3 Connect a length of silicone tubing to the sampler tube on the headplate Secure it in place with a plastic tie 4 Slip a thumb clamp onto the tubing 5 Connect the other end of the tubing to the tall sampler inlet pipe Secure it in place with a plastic tie 6 Connect a short length of silicone tubing to the short sampler outlet pipe Secure it in place with a plastic tie 7 Connect the sterile syringe filter to the other end of the tubing taking care to respect the direction of flow if stamped on the filter Secure the tubing in place with a plastic tie 8 Insert the tip of the sampler syringe as far as it will go into the open end of the filter Although the syringe will lodge there and hang freely in place you can add a plastic tie for security 9 Close the plunger Operating manual 50 10 Remove the cap from one of the sample bottles and screw the bottle into the metal holder 11 Position the entire assembly to your satisfaction then finger tighten the clamping nut 4 8 19 Install foam trap If you are using a foam trap kit see the drawing on the following page 1 Unscrew the headplate clamping nut or base clamping nut if you prefer to mount the trap at the base of the vessel closest to the foam exhaust tu
39. fit the pH port adaptor onto the probe first Then insert the probe with its adaptor into the headplate following Steps 3 11 shown after the drawing below Never attempt to install the pH port adaptor in the headplate without the probe Figure 21 pH Probe with Port Adapter exploded 7 gt lt __ _ _ _ __ 3 R _ __ 4 6 4 5 1 Cap 5 Port O ring black 2 pH probe adapter top portion 6 Teflon O ring white 3 Bottom ferrule 7 Top ferrule 4 pH probe adapter bottom portion With reference to the drawing above 3 Gently slide the top portion of the knurled port adapter part of the probe kit onto the probe 4 Slide the two white ferrules onto the probe the narrower one on top of the deeper cup shaped one 5 Gently slide the bottom portion of the port adapter onto the probe taking care to orient the longer threaded section toward the top of the probe 6 Remove the two O rings installed in the pH port first slide the white Teflon O ring onto the probe then follow with the black 12mm port adapter O ring Operating manual 44 7 Do not yet close up all the elements of the port adapter 8 Gently insert the probe into the appropriate port allowing the O rings to seat fully into the port 0 The fit may be snug Gently rotate the probe as you press it into the port to avoid breakage 9 Finger tighten the bottom portion of the port a
40. its intended purpose BioFlo CelliGen 115 M1369 0050 Operating manual 33 Figure 16 7 5 L amp 14 0 L Headplate 1 2 19 18 17 16 6 15 7 8 A 8 l 3 9 14 AS f w sP 13 12 11 10 1_ Sparger 6mm 11 Plug 19 mm spare 2 Cooling coil 6 mm 12 Level 6 mm 3 Mounting position for sampler assembly 13 Plug 12 mm spare 4 pH probe 12 mm 14 Each headplate bolt is a possible 5 Sampler tube 6 mm mounting position for a bottle holder 6 dO2 probe 12 mm 15 Tri port 12 mm 7 Plug 6 35 mm spare 16 RTD Thermowell 12 mm 8 Lifting handle 17 Exhaust 12 mm 9 Tri port 12 mm 18 Harvest 6 mm 10 Septum 12 mm 19 Cooling coil 6 mm 0 The RTD thermowell port should only be used for its intended purpose Operating manual 34 4 7 2 Install heat blanket 1 Wrap the heat blanket as snugly as possible around the vessel taking care to leave one of the viewing windows facing forward You will probably want to orient the blanket so the mains power cord connection is out of the way 2 Secure the blanket by overlapping the Velcro strips and pressing them together A WARNING Risk of electrical shock gt NEVER cut any portion of the heat blanket gt NEVER fold the heat blanket or place any weight upon it gt For storage always lay the heat blanket flat 4 7 3 Install vessel in vessel stand 1 Place the clamping ri
41. oxygen by first increasing GasFlo to the high limit then if DO still has not reached the setpoint increasing oxygen percentage entering the system through the sparger to as much as the high limit This is not available without the Automatic GasFlo Option and Automatic Gas Mix Option Operating manual 94 e Agitation GasFlo O2 controls dissolved oxygen by first increasing Agitation to the high limit then if DO still has not reached the setpoint increasing the GasFlo entering through the sparger to as much as the high limit If the DO setpoint is still not achieved the cascade will begin to increase the O2 percentage of the gas mix This is not available without the Automatic GasFlo Option and Automatic Gas Mix Option 10 1 Creating a Cascade To create a DO cascade 1 Press the CASCADE button to open the CASCADE screen Figure 57 Cascade Screen Cascade Screen New Brunswick Cell Culture Mode 07 Dec 2008 15 34 DO Cascade Cascade Limits None Agit Casc Low Limit c Agit Agit Casc High Limit c o2 GasFlo Casc Low Limit GasFlo Casc High Limit c GasFlo 02 Mix Casc Low Limit c Agit 02 A 02 Mix Casc High Limit c Agit GasFlo c GasFlo 02 c Agit GasFlo 02 ES Summary JF Calibration ES E Pumps Setup 0 In the DO Cascade pane on the left before a selection is made the default selection is None indicated both by a dot in its option button and by the loop name in blue Any unavailab
42. provides proper control If the P I D constants are incorrect the control signal may be too weak for the parameter to ever reach setpoint or at the other extreme the controller may respond excessively to small errors causing the actuator to oscillate between high and low values Usable P I D constants must be determined for each P I D loop The process is largely one of calculated trial and error All loops that are configured with the P I D control mode must be tuned When delivered as part of a New Brunswick system P I D loops will have been tuned at the factory to work correctly with the New Brunswick controlled instruments For other applications the user is responsible for P I D tuning Tuning can be a complex task for those unfamiliar with the process which is why a trained engineer or technician normally performs this task A number of textbooks that explain the theory and describe the process could be useful for the mathematically inclined novice The Ziegler Nichols method described in the footnoted reference is used at our production facilities The following suggestions are intended for novices Be sure to refer to a textbook and consider utilizing the services of a technician 1 For example Chinks F G Process Control Systems Application Design and Tuning McGraw Hill 1988 New York Auckland Bogota London Toronto Sydney Tokyo Montreal Operating manual 136 e Allow sufficient time for the task
43. replaced if necessary 0 To avoid damage to the probes during operation be sure that there is no interference between the probes and the baffle assembly impeller blades or cooling coil 1 Wear protective gloves to protect yourself in case of accidental breakage 2 Lightly coat the pH probe with glycerol k ALERT Risk to pH probe gt Always fit the pH port adaptor onto the probe first gt Then insert the probe with its adaptor into the headplate following Steps 3 11 shown after the drawing on the following page gt Never attempt to install the pH port adaptor in the headplate without the probe BioFlo CelliGen 115 M1369 0050 Operating manual 77 Figure 49 pH Probe with Port Adapter exploded 7 gt ta ER 6 O 5 1 Cap 5_ Port O ring black 2 pH probe adapter top portion 6 Teflon O ring white 3 Bottom ferrule 7 Top ferrule 4 pH probe adapter bottom portion With reference to the drawing above 3 Gently slide the top portion of the knurled port adapter part of the probe kit onto the probe 4 Slide the two white ferrules onto the probe the narrower one on top of the deeper cup shaped one 5 Gently slide the bottom portion of the port adapter onto the probe taking care to orient the longer threaded section toward the top of the probe 6 Remove the two O rings installed in the pH port first slide the white Teflon O ring onto
44. the vessel eS Operating manual 82 Figure 51 Calibrating DO Calibration Screen New Brunswick Fermentation Mode 07 Dec 2008 15 40 Calibrating Loop DO Level Sens 1 Current Value Raw Value Level Sens 2 35 0 948 os ee y Summary PEE Es Cascade 3 Pumps Setup 1 Raw Value corresponds to the signal directly received from the probe before it is converted to a DO value by the controller 2 As explained in Step 4 these buttons may not be present depending on your system s configuration 7 3 6 DO probe calibration setting span 1 In the AGIT GAUGE screen set the AGIT speed to 50 rpm 2 Set the AGIT mode to AUTO 3 Vigorously sparge air into the vessel via the filter on the headplate until the display is stable for approximately 10 minutes this may take up to 30 minutes total 4 In the CALIBRATION screen select DO 5 Enter 100 in the SET SPAN edit box then press the SET SPAN button 7 4 Level probe calibration Each level sensor is connected to a conductivity probe that is sensitive to wet contact According to the use you assign to the level probe it will turn its assigned pump on or off For example if you assign the probe to be Dry you will position it in the space above the top of the media and calibrate it to be very sensitive to wetness If the wetness is expected to be the result of foam associate this level probe with the
45. tightly use the bulb and a sampling tube to see if a vacuum can be held and released properly as when a sample is normally taken Attach a sample tube prior to autoclaving This tube should be to turn loose to avoid explosion or implosion The glass wool should be covered with a piece of foil 21 7 2 Vessel sterilization When autoclaving the vessel exhausts through the exhaust filter so it is essential that the line be prevented from crimping and that the filter be good unplugged To ensure that crimping does not occur use a short piece of fairly rigid tubing If rigid tubing is not available use a small splint to support the tubing The vessel is normally sterilized for 45 minutes Note however that certain media formulations cannot be sterilized for this length of time as degradation will occur check the media manufacturer s instructions The probes must never be autoclaved dry If it becomes necessary to sterilize the vessel without media use a balanced salt phosphate buffered saline solution to cover the ends of the probes Aseptically remove the PBS prior to filling the vessel with the desired media NEVER PLACE PROBES IN DISTILLED OR DEIONIZED WATER THIS WILL CAUSE YOUR PROBE TO LOSE ELECTROLYTE The maximum fill is 70 of the vessel s maximum volume Autoclaving should be done when liquid is present in the vessel on a slow exhaust setting see autoclave manufacturer s instructions for autoclaving liquids Sterilizati
46. tube are welded into the same tri port to save space When the headplate is in place on the vessel the bottom of the harvest tube should rest at the bottom of the vessel 2 Finger tighten the knurled adapter on the harvest tube then use the Allen key provided to tighten the set screw Do not overtighten 4 8 9 Install sampler tube 1 Working from beneath the headplate install the optional sampler tube in the sample port If you are using the 1 3 L vessel the sampler tube and harvest tube are welded into the same tri port to save space 2 Finger tighten the knurled adapter on the sampler tube then use the Allen key provided to tighten the set screw 4 8 10 Install thermowell 1 Working from above the headplate insert the thermowell tube into the RTD port Be sure to use the port designated for the RTD to avoid damaging the glass I ALERT Risk of damage to equipment gt Make sure that the thermowell does not touch the cooling coil or come into contact with the glass vessel 2 Finger tighten the knurled adapter on the thermowell 4 8 11 Install foam probe If you are using a foam sensor with a foam trap kit 1 Working from above the headplate insert the foam sensor into the appropriate port 2 Finger tighten the knurled adapter Operating manual 42 4 8 12 Install foam exhaust tube If you are using a foam trap install the foam exhaust tube 1 Working from beneath the headplate insert the foa
47. vessel is water jacketed the jacket should be half full for autoclaving see Section 4 8 3 for instructions on filling the jacket Make sure that the Water In line connected to the bottom of the jacketed vessel is pinched closed to avoid water leaking from the jacket during autoclaving 3 Insert the entire vessel assembly glass jar vessel stand if present headplate and all headplate components into an autoclave and sterilize 4 When you remove the vessel from the autoclave immediately crimp the foil funnel on the addition port and close off the vent tubing to maintain sterility Figure 53 Angled Autoclave Rack Option 5 1 Exhaust condenser make sure this points upward 2 Non jacketed vessel assembly 5 Autoclave rack 3 Retention strap 6 Foam trap and or addition bottles 4 Vessel stand 7 Bearing housing cap BioFlo CelliGen 115 M1369 0050 Operating manual 87 8 2 1 Sterilization time and temperature Sterilization time varies with autoclave characteristics temperature settings vessel size and contents i e media properties As a starting point autoclave for 25 minutes after the autoclave reaches 121 C H ae ALERT Risk of vessel damage Be sure to vent the vessel at all times during autoclaving Release the autoclave pressure only when the temperature has dropped below 90 C Use slow exhaust 30 60 minutes If available put the autoclave on liquid cycle p
48. will be necessary to close the gas tank valve and its lines to the system If a recirculating chiller is in use it should be shut off when the temperature control is shut off Clamp off the feed lines from any addition bottles used prior to detaching them from the vessel The next step is to disconnect the vessel from the system Remove the temperature probe from the thermowell Remove the motor and place the protective cap over the agitation shaft bearing housing When you disconnect the water lines always disconnect the incoming lines prior to the outgoing lines Disconnect the air line from the sparger Disconnect the pH and DO probes from the system and put on their protective caps The DO probe presents an easy removal as you simply unscrew the thread and gently pull it out Immediately rinse it off then gently wipe it dry always remembering to never touch the membrane at the tip Some runs will result in an accumulation of biomaterial on the probe so and it may be necessary to wipe the probe down more vigorously nevertheless in no case should the tip be touched After cleaning the DO probe visually inspect the tip for damage If it is damaged replace the probe Store the probe in a clean area in such a way as to protect the sensitive tip Removing the pH probe is usually not so difficult inserting it because the shaft is wet and should be relatively easy to remove The danger of probe breakage is still very real however so extrem
49. wish to conduct Various formulations can be found in the handbooks and literature 21 2 Antifoam formulation Question What kind of antifoam should be used and in what concentration Answer Please visit our website at www nbsc com click on the FAQs tab then click on Fermentation and Cell Culture for recommendations on types of antifoam agents to use The initial concentration of antifoam is usually 0 1 0 5 mL L When the foam probe is used the pumping of antifoam is controlled by the system The pump should be set to add the minimum amount of antifoaming agent required to prevent foaming in your particular process That amount varies depending on the amount of protein in the media the amount of protein secreted by the microorganism agitation speed and other factors Therefore you will have to experiment to get the proper pump setting 21 3 Tubing size Question What is the correct tubing size for acid base antifoam and nutrient feed for a fed batch run Answer For vessels up to 5 liters part number TU202 This is Marprene tubing with an inside dimension ID of 1 6 mm It has an OD of 4 8mm 3 16 NOM and a wall thickness of 1 6mm Larger tubing will be required for vessels over 5 liters It may also be necessary to use a connecting fitting to allow two different tubing sizes to be used in cases when the tubing size required for the pump and the size required for the direct connection to the vessel differ Eppendorf recom
50. 0L 32 Heat Blanket Installation of 34 Horsepower Factors that Affect 138 Impellers Installation of 39 Important Warnings 27 Index of Drawings 132 Index of Tables 132 Information Sheet 3 Inoculation 114 146 Inspection of Boxes 11 Installation Gas Connections 25 Water amp Drain Connections 24 L Lag Phase 116 Level Probe s Installing the 42 Level Probes Application of 101 Liquid Addition Systems 90 Location Environment 16 Physical 16 Loop Setpoints Entering the 68 Modifying the 70 M Maintenance 123 BioFlo CelliGen 115 M1369 0050 Operating manual 153 Maintenance Inspections 124 Mass Flow Controller 13 Media Formulation 140 Microaerophilic Culture 118 Modbus Com Port Pin Designation 56 Motor Assembly Installation of 53 Motor Replacement 124 N N2 3 106 107 O O2 2 106 107 Operating Control Mode Changing the 104 Operating Controls 60 OTR Calculating an 137 Determining an 137 Factors that Affect 138 Out Mult 98 Output Multiplier 98 P Parts Lists 125 pH Control of 13 pH Probe 13 Calibration of 74 Inspection of 74 Installation of 42 76 Maintenance of 78 Storage of 78 PID Explanation of Constants 135 Explanation of Tuning 135 Preparing for a Fermentation Run 113 Preparing Vessel for Autoclaving 143 Probe Calibration Definition of 134 Probe Cleaning 147 Probe Removal 147 Probe Storage 123 147 Process Contr
51. 1 Fresh media 2 Harvested broth 13 10 Anaerobic and microaerophilic culture When growing anaerobic organisims oxygen must be excluded from the media and when growing microaerophilic organisms oxygen must be limited to a very low level in the media For anaerobes several strategies can be used to eliminate oxygen e Reducing agents can be added to the media e Vigorous agitation normally used to increase dissolved oxygen in the media is not required A low agitation rate however is required to keep the cells in suspension and to provide mixing of the liquid to maintain good temperature control An inert gas such as nitrogen can be sparged into the media to provide the necessary anaerobic conditions e Additionally a gas overlay can be installed to introduce the inert gas into the headspace The gas introduced via the gas overlay can come from splitting of the sparge gas by using aT or Y fitting For the growth of microaerophiles a premixed gas is introduced into the sparge line and overlay The gas mixture is dependent on the particular organism that you are culturing 13 11 Harvesting procedure When the vessel is set up on the control cabinet adjust the level probe s tip to the level at which you want harvesting to stop i e below the current liquid level 1 Assign a feed pump as Lvl2 Wet to pump liquid out of the vessel 2 Aseptically connect the feed pump s tubing to the harvest port BioFlo Cel
52. 1 to open the loop s GAUGE screen Figure 39 Sample GAUGE Screen pH Cell Culture Mode New Brunswick Gauge Screen 07 Dec 2008 15 30 15 93 pH E Gains pH Setpoint pH 14 00 70 00 Set PV Out Integral Deadband 2 14 00 1593 100 0 10 00 0 00 14 00 14 00 100 0 FilterBand 0 00 Filter Gain 1 00 Set Low Set High 2 00 14 00 Decimal Places 0000 00 00 000 0 0 000 2 00 2 00 100 0 Summary RP Calibration A Cascade Pumps 2 Setup 1 Step 2 Press the button that corresponds to the desired control mode 2 Deadband is a user definable pH value within which above or below the setpoint no response will be triggered 3 Step 3 To save the new control mode and return to the SUMMARY screen press the Summary button 6 2 7 Entering loop setpoints The setpoint is the value you want each loop to attain When the loop control mode is AUTO the fermentor will automatically make appropriate adjustments to maintain the value at the setpoint To enter a setpoint for any loop follow these steps Touch either the LoopName box or the Setpoint box for the desired loop on the SUMMARY screen In this example we have selected AGIT 2 The loop GAUGE screen opens see the following page 1 BioFlo CelliGen 115 M1369 0050 Operating manual 69 Figure 40 Sample GAUGE Screen
53. 54 Standard PUMP Array ceisir aaa aE AAA R KREA AAE A AS E EAA TRAAT Ait 89 Figure 55 Loading Pump Toba a SS 89 Figure 56 Typical Liquid Addition Systemic id A no 91 Fig re 7 Cascade AA A II A 94 Figure 58 Sample Cascade Screen ii dicas 95 A aaa oes aiet eases a aicstos Bogus Ta a Seas oes Oca 97 Figure 60 Pump Assignment e des os del 97 Figure 61 Setting Pump Stroll ae 98 Figure 62 Calibrating the Pump Flow Rated ii tai 100 Figure 63 Pump P riod sec Josie ed eo 100 Figure 64 Pump Assignment SCLC Si iii 101 Figure 657 Controller Setup Coti 103 Figure 66 Changing Operating Control Mode ce eeccesceseceeeereeseeeeeceseceaeceeeaeeseeeeeeeseenaes 104 Figure 67 Operating Control Mode Changed tii idas 105 Fig re 68 gt Changing Vessel oa RITO E ER ASS 105 Figure 69 Air 1 Gauge Screen with O2 Enrich oononincnncnonaconnninonaccnnnnnnnonnnnnncnn cono canonnnnos 106 Figure 70 Air 1 Gauge Screen with 3 Gas sc soci scecessesqzeacesscusteesgqsceosacsses eo cesngedeesse dadas 107 Figure 71 Air 1 Gauge Screen with 4 Gas ieiaataneaaeoae ba taeensasev ees 107 Figure 72 GasFlo Gauge Screen ii iii 108 Figure 73 Sy Stem Senasa diia 108 Figure 74 Hardware o katt alae ea ct Dale aa td ag na Aaah N ER aae 110 Figure 75 Adding New Hard Wate data tada is 111 Figure 76 New Hardware Add usina riada 111 Figure 77 Controller Settings for New Hardware Added c ooooocnocnnccnoconocconcconoconcnnnonncnnccnnccnnos 112 Figure 78 O A A
54. 7 1 pH probe inspection Inspect probe for possible shipping damage If damage is observed notify your local Eppendorf sales representative or distributor immediately Check the electrode tip for trapped air bubbles To remove any air bubbles hold the electrode upright and shake gently NEVER REST THE PROBE ON ITS TIP 7 2 pH probe calibration 0 Calibrate the pH probe before autoclaving it with the vessel 1 If you have not already done so connect the pH probe to the pH connector on the control cabinet using the appropriate cable 2 Turn the ON OFF main power switch ON 3 Press the CALIBRATION button to display the CALIBRATION screen 0 The pH probe is calibrated using two external buffer solutions of known pH usually 7 00 and 4 00 4 Rinse the pH electrode with distilled water then immerse it into pH 7 00 buffer solution and allow a few minutes for the system to equilibrate 5 Open the CALIBRATION screen see the following page Steps 6 8 are indicated as callouts around this screen BioFlo CelliGen 115 M1369 0050 Operating manual 75 Figure 48 Calibration Screen 1 Calibration een New Brunswick Fermentation Mode El 07 Dec 2008 15 39 Calibrating Loop pH Le 2 Current Value Raw Value 7 02 0 a 4 o 4 E Summary PARA E Cascade 3 Pumps Setup Step 6 Press pH in the Loops pane As a result of Step 6a pH appears in the
55. Bioprocess Equipment 6 New Brunswick New Brunswick BioFlo CelliGen 115 Benchtop Fermentor amp Bioreactor Operating Manual M1369 0050 Revision E eppendorf COPYRIGHT Copyright 2012 2013 Eppendorf AG Germany No part of this publication may be reproduced without the prior permission of the copyright owner Eppendorf reserves the right to change information in this document without notice Updates to information in this document reflect our commitment to continuing product development and improvement TRADEMARKS BioFlo CelliGen BioCommand and Eppendorf are registered trademarks and New Brunswick and the New Brunswick Logo are trademarks of Eppendorf AG Hamburg Germany Marprene is a registered trademark of Watson Marlow Limited in Falmouth Cornwall UK PharMed is a registered trademark of Saint Gobain Performance Plastics in Akron Ohio Windows is a registered trademark of Microsoft Corporation in the United States and other countries Trademarks are not marked in all cases with or in this manual Eppendorf has attempted to identify the ownership of all trademarks from public records Any omissions or errors are unintentional June 6 2012 Revision E M1369 0050 BioFlo CelliGen 115 M1369 0050 Operating manual FERMENTOR BIOREACTOR INFORMATION SHEET On this page record the information for your fermentor bioreactor and retain this for future reference M
56. Calibrating Loop box Raw Value is the signal received directly from the probe before it is filtered and convertd by the controller Step 7 Touch inside the Set Zero edit box Enter 7 0 on the popup keypad then press the OK button Step 8 When the Current Value reading stabilizes press the Set Zero button 11 12 13 Rinse the pH electrode with distilled water Immerse pH electrode into a second pH buffer solution which is several pH units above or below pH 7 00 e g pH 4 00 and allow a few minutes for the system to equilibrate Similar to step 7 above touch the SET SPAN edit box Use the touchpad that opens to enter the value of the second buffer solution e g 4 00 then press the OK button When the CURRENT VALUE reading stabilizes press the SET SPAN button To ensure accuracy repeat Steps 4 11 a few times using the same two buffer solutions 0 The pH calibration should be checked after autoclaving immediately prior to inoculation Take a sample from the vessel and compare the pH value displayed on the control cabinet screen to the pH recorded by an external pH meter Any discrepancy should be adjusted with the SET ZERO procedure Operating manual 76 7 2 1 pH probe installation WARNING Risk of broken glass gt Be sure to wear protective gloves when installing a glass electrode 0 Prior to installation any pH probe you are using should be inspected for damage and
57. Cooling Coil 13L M1273 9259 heat blanket vessels f 1 3 L jacketed M1273 9267 Sparge Ring 3 0 L M1273 9256 7 5L M1273 9246 14 0 L M1273 9251 Cooling Coil 3 0L M1273 9249 heat blanket vessels 7 5L M1273 9247 14 0 L M1273 9250 Impellers 6 Blade Rushton type 52mm ferm 1 3 L 3 0 L M1273 9291 6 Blade Rushton type 59mm ferm TOL M1273 9292 6 Blade Rushton type 74mm ferm 14 0 L M1273 9293 Pitched Blade upflow TOE MATO 2206 7 5L 14 0 L M1273 9207 Pitched Blade downflow Le ESOL Mie ie200 7 5L 14 0L M1273 9212 Marine Blade 1 3L 3 0L M1273 9901 7 5L 14 0 L M1273 9902 1 3L M1273 3201 aoe 3 0 L M1273 3202 Spin Filter suspension cells TSL M1273 3205 14 0 L M1273 3210 1 3L M1273 3211 a 3 0L M1273 3212 Spin Filter microcarriers 7 5L M1273 3215 14 0L M1273 3220 Baffles 1 3L M1273 9263 Baffle 3 0L M1273 9264 7 5L M1273 9245 14 0 L M1273 9265 continued Microspargers Sintered porous microsparger 1 3L M1273 5007 Operating manual 128 For any other spare parts please contact your local sales representative or distributor heat blanket vessel Sintered porous microsparger 13L M1273 5003 water jacketed vessel 3 0L M1273 5004 Sintered porous microsparger 75L M1273 5005 14 0L M1273 5006 Sampling Assemblies 1 3L M1273 9946 Sampling assemblies 3 0L M1273 9949 7 5L M1273
58. NBS Modbus Mi i Unit3 Module Status Good Errors gt gt gt Unit ID 2 lt lt lt sl Summary Calibration E Cascade Pumps Setup 1 When you press the gt gt gt button for the Unit involved the new hardware moves into this pane Operating manual 112 12 3 1 Identifying utility station s added Now that you have added one or more utility station s using the Hardware Setup screen return to the Controller Setup screen to name the new station s as desired and to identify the vessel size the operating mode and the options installed on the system If you make any changes be sure to press the Save Changes button to commit them to memory Figure 77 Controller Settings for New Hardware Added 1 A Setup Screen New Brunswic Fermentation Mode 22 May 2009 11 32 ENTER system Settings Hardwe Setup Unit Type Unit Name Vessel Size BioFlo 115 BioFlo 115 7 5 Liter mai _ 2 No of TMFCs 1 TMFC Range 0 20 SLPM Operating Mode BioFlo 115 Options 4 Fermentation Mode P pH DO E e 02 Enrich Direct or Cascade Driven F Foam Level F Gas Mix F Gas Flow K Pumps Save Changes a Summary Calibration Cascade 3 Pumps Ed 1_ As explained in Section 12 1 use this box to name the new system 2 Here you can use the down arrow to change the Vessel Size 3 Some of the options installed on your BioFlo 115 will be automatically detected so they will
59. O Se 16 4 2 ENVIRONMENT ali A taaboaelene 16 4 3 INSTALLING THE CONTROL CABINET cccccccccessessssececesccsceessnseaecesecceseeesssaeeeeeeceseeeens 17 44 CONNECTING UTILITY CABINETS ccsccsccsccccceceessssececesececeseessnseeeeesccsesensnseaeeeseccesenenees 20 4 5 LBRT E D l 82h bs eel E EAE a sR dns ath Se 22 4 5 1 Electrical RE QUITE CS A a INES 23 4 5 2 Water and drain connections oooooonnncncnnncnononononononononononononononononononenonononeninenenes 24 4 5 3 Gas connections 2 AAA A AA AAA AAA OI Bes 25 4 6 IMPORTANT SARE TY NOTES 5350 ti a a cd 27 4 7 VESSEL ASSEMBLY NON JACKETED cccccccccccessessssececescceceessnseseeeseceesseenssaeeeeescesenenees 29 4 7 1 Ted A A racks E A be Balad ease A Ea 31 4 7 2 Install heat blanket ooonnnnnnnnnnnnnnnnncncccnnnccononcnnonononononononono nono conoce conoce nononononononos 34 4 7 3 Install vessel in Vessel stand ooononnnnnonacnnincnnncnononononononononononononononononononininonenenes 34 4 7 4 Install baffle 14 0 L fermentation vessels ONLY ooocoonnnccccnoniccccnononcnnnnnnnnnnnoos 35 4 8 VESSEL ASSEMBLY WATER JACKETED cssssseseeeeeeesseeeeeeeseseeeseeeseseseeeeeeeeeeeeeeeeeeees 35 4 8 1 Install headplate clamping ring oeosooeeseeeennsseeeessesessssserssssesesssereesssseesssseees 37 4 8 2 Install vessel on base plate a a A A a Ai 37 BioFlo CelliGen 115 M1369 0050 Operating manual 4 8 3 Filling the water jacket AAA 38 4 8 4 Install baffle
60. O padtadetanaaeacens 117 Fig re 79 CONTA OA A a e e o do 118 18 2 List of tables Table 1i Servico Conoci 23 Table 2 Impeller Positions a A A Ad RA ne i 39 BioFlo CelliGen 115 M1369 0050 Operating manual Table 3 Table 4 Table 5 Table 6 Table 7 Modbus Com Port Pin Designation ooocnnncnincoconnconnonnnconncconocnnn cono ccon coco no naar cnnnccnnncinns 56 SUMMARY Screen ESatuTOS until tibios 64 PUmp Control Mods sectas ir A E it a a 99 PIGW Rate per Tubing Ai causes od ae a aidean aaie IES 99 O 122 Operating manual 134 19 APPENDIX A SOME GENERAL CONCEPTS 0 In this section all discussions of P I D control are to explain the theory on which it is based This product uses only P proportional integral control not D derivative 19 1 What is a controller The local process controller is a multi loop controller which means it can control several process parameters simultaneously It compares current values with setpoints and creates independent control signals for each controlled parameter The control signals are used to drive appropriate actuators that maintain the various parameters at their setpoints Using temperature as an example the controller compares the output of a temperature sensor to the user entered temperature setpoint and generates a signal to activate either a heater or a cooler to maintain vessel temperature at the temperature setpoint The controller provides the log
61. ODEL NUMBER VOLTAGE SERIAL NUMBER The above information can be found on the electrical specification plate Purchased with the following installed options Operating manual TABLE OF CONTENTS 1 USER INSTRUCCION Simi ese 9 1 1 HAZARD ICONS A AD oR DI 9 1 2 DANGER LEVEE Suenan ara A A ea 9 1 3 MANUAL CONVENTIONS r sia a a GA 10 1 4 ABBREVIATION Sita a A a ios 10 2 INSPECTION amp UNPACKING OF EQUIPMENT uuu cccccccsssssscccssssrcccsssscccesssccees 11 2 1 INSPECTION OP BOX ES isis a e a a a a aaa 11 2 2 PACKING LIST VARA CATON a a a e e A A a 11 2 3 BASIC COMPONENTS 303cdes secgeeks cats soph Sie da 11 3 INTRODUCTION OVERVIEW cssssscssssssscccssssscccssssssccsssssccssssssccssssssccsssssssees 12 3 1 SYSTEM es ia as 12 3 2 WESSELS ea AE E EAE EEEE E rine cee tel EE 12 3 3 PAVONE V E E E E EE Fees lan E E E 12 3 4 TEMPERATURE CONTROL o cccccccccscssssssssecesecccsceesensececesececeseessnseeeeeseesesensntsaeeeseceeseeeeas 13 3 5 AERATION Solari 13 3 6 PH CONTR OD neisa a SA AR E ia 13 3 7 DPDOCONIROL opn A E E EE NE 13 3 8 EOSMIEE ELN ui es 14 3 9 EXHAUST SYSTEM A A iia 14 3 10 RECOMMENDED ACCESSORIES amp SUPPLIES ccccessessscecesececessesssseceeescesceeserseaeeeseceens 14 3 11 SUPERVISORY SOFTWARE ccsccsccsccsccscsssssssccesececsessssnsececesececssssssaaeeeeescesesesenseaeesseceens 15 4 INSTALLATION ii A ds eii 16 4 1 PHYSICAL LOCATION seco o8s4 3 is A Rat sae Pees A A ids Ts Raa D
62. ONS pa o EEA E a E EE E E a To Ea EESE T 58 6 OPERATING CONTROLS essoeseessesooesoessesooesocssessosoossoessesoossocssessossocssessossocssessosssesse 60 6 1 TOUCHSCREEN zics2 st2essctatansadiaduzcteicdshoeta aau RASE Aag SEASSA ASER anA an 60 6 2 DISPLAY SCREENS a td ta 60 6 2 1 Touchscreen calibration to ii 60 6 2 2 Start Up A e E a a india Ide a site ain 61 6 2 3 Ad innit aiid ti A E T an eats 61 6 2 4 KENDO usan it dit 64 6 2 5 COUPE CTC ATA RA ASA TALA TA AAA AAA A A 66 6 2 6 Selecting loop control modes 2 67 6 2 7 Entering loop setpoints AS AA amd vue adn 68 6 2 8 MODIFYING Setpoint ee a A A a ead 70 6 2 9 Calibration OOO A AS A AA cadiz 70 LN DE rel APP II A A A 70 O24 10 PUP ECN A A ee eat 71 6 212 gt Setup NCTC AAA A A TAA 72 7 PROBE PREPARATION CALIBRATION cccsccssssscssssccsssccssssccsssssssssssssssssees 74 7 1 PH PROBE INSPECTION 0 SS ceed E cea ROE 74 Operating manual 7 2 PH PROBE CALIBRATION cccssssesceccccccccssssssceccccceceuaesssceccescesesuussssecesccsseuuuaessecceseeaea 74 7 2 1 pH probe imstall ti n A a a ai td ti 76 7 2 2 pH probe maintenance amp OTIBL ii A A a A 78 7 3 DISSOLVED OXYGEN DO PROBE PREPARATION cccccoooonccnnoonncnncnnnnnnnncnnonnnonnnnnonnnnnnnos 78 7 3 1 Inspecting the DO DOO 2 ii ASE A AAA TI 78 7 3 2 DO DEODE PEPA id iia 78 7 3 3 DO probe installati n nanen A eee aed 79 LIA DO probe POT Saad 8l 7 3 5 DO probe calibration setting ZETO iii a A dea 8l 7 3
63. UMMARY Screen Features Parameter Column Description LoopName Each system comes with standard factory assigned loops e g Agitation Temperature pH DO etc Loops are factory assigned according to the configuration of your system PV Process Variable here the display reflects the current value for each loop in comparison to its setpoint displayed in the next column Setpoint The current setpoint default or user set for each loop Out The current percent output for each loop This is an automatic control function to maintain current readings within the setpoint tolerance range Control Mode Depending on the loop the control mode may be Off Auto Manual On or O2 Enrich Unit of measure This is the unit of measure used for the PV and Setpoint Cascade If any cascades have been programmed they will be displayed here Navigation Buttons for screen access Summary Description This screen is command central it shows all your loops their current readings setpoints and what has been programmed for them Calibration This screen allows you to calibrate the pH DO amp Level probes and the gas flow Cascade A cascade is a control function that uses the output of one loop to influence the action and output of one or more other loop s This screen allows you to set up cascades to view current settings and to make changes to those settings Pumps Thi
64. a S En Daea a a EREE 24 Gas ONMECHONS ection asea a a E e a R E a te 26 Sparge Connection detail From Figure 4 oococonnccinoccnococonocooncconn cono nonnnoconocancconnno 26 WRONG Handling of Drive Assembly oonoociocccnocococcnoncnononcnonoconocnnonono nono nocnocnnos 28 CORRECT Handling of Drive Assembly oooooooccnnocococococccononcnnnoconocnnc nono nono ncconocnnos 28 MESAS a e ira ligue 30 1 3 Help Aa 31 JOLE Headplate ceanna este lees eiatecguetiums aad kes ten element taste 32 AOL AD E Head plate a 33 Upper Vessel Bumper Tostadas 34 Water Jacketed Vessel Assembly ic A a casts 36 Installing Headplate Clamping Ring oooonoccnocccooccconaconaconnnonononnnocono cono nonn nooo ncconocnnss 37 Water Jacket Guard Installation top VISW ooooonoocnnnonononoconoconncconoconononnncnnncconocnnos 38 pH Probe with Port Adapter exploded ooooooonoccinncinociconoconocononconncconoconnconncconnnnno 43 dO2 Probe with Port Adapter exploded ooonooonnoncnncnconccconccconaconanonnnconccconocnnos 45 Exhaust Condenser 1 3L 3 0 L amp 7 5 L Vessels ccs cccssssecssscesssecesssneesees 46 Exhaust Condenser 14 0 L Vessel only larisa incita 47 Sampler Harvest System 1 3 L Ves 48 Sampler System 3 0 L 7 5 L amp 14 0 L Vessels oooooooccnnnccocccocononnnconncconocnnncnnnnno 49 o A O O cma wishin ETE T O en 51 Vessel LOCAthOM ysis acts A dips amas 53 ON OFF Mains Power WI e rata 54 RS 232 422 Aes es seee i RO ORE ee eS e
65. aced every six months or more frequently if needed At three month intervals perform the following checks and inspections 0 Before you begin make sure that the ON OFF mains power switch is in the OFF position and that the mains power supply has been disconnected 1 Check all controls and accessible items mains power switch connectors screws nuts and bolts to make sure they are properly tightened Tighten any loose item s 2 Check that all controls and connectors are free of dust 3 Check that all O rings in the headplate and impellers are intact and in good condition Replace those that are not 16 5 Agitator bearing housing Every 3 6 months the ball bearings and the shaft seals in the bearing housing should be checked and cleaned Replace any worn out bearings and or shaft seals 16 5 1 Motor assembly replacement A WARNING Risk of electrical shock gt No one but a professional service person should touch electric or electronic parts or assemblies in the control cabinet If the motor assembly should require replacement call for an authorized Eppendorf service technician BioFlo CelliGen 115 M1369 0050 Operating manual 125 16 6 Replacement parts The following lists of replacement parts are provided for your convenience Using the part number will facilitate processing of your order by your local Eppendorf distributor
66. amp 0 Figures 1 4 represent one possible control station cabinet configuration Your control cabinet may look different depending on the particular model and options you have purchased Operating manual 18 Figure 3 Rear View 1 8 2 IL 5 E 3 1 O uji Cooling vent W Utiliity stations do not have a fan 2 Service connections see Figure 1d Label with electrical specifications 4 serial number Plug for mains power cord SCADA port see Section 4 10 Cabinet output port see Section 4 4 Cabinet input port see Section 4 4 Gas connections see Section 4 5 3 NIOO AIU 00 BioFlo CelliGen 115 M1369 0050 Operating manual 19 Figure 4 Control Station Service Connections Touchscreen gas and pump control options may or may not be present depending 1 on the configuration of your control station These connections are addressed in Section 4 5 2 I ALERT Risk of damage to equipment gt Before making electrical connections verify that the supply voltage matches the voltage and the mains power requirements marked on the electrical specification plate located on the rear panel of the cabinet and the control schematics supplied with the system Operating manua
67. and rinsed separately The glass wool can be removed at this point too Detach the sampling tube and wash it separately Open the valve on the sampling port and all clamps on all tubing connected to ports for proper washing be sure to remove the media prior to unclamping any tubing below liquid level such as a side harvest line The headplate should be detached by loosening and then removing the clamps that hold it to the rest of the vessel Those clamps may require rinsing The remaining culture broth should be sterilized or emptied into a bucket and disinfected by using bleach or other accepted disinfectant prior to disposal Note that some media may be incompatible with this procedure in which case the media can be placed into another container for sterilization prior to disposal The headplate should be washed thoroughly with warm water and then with deionized DI water It may be necessary to scrub off any accumulations of biomaterial A pad that won t scratch the steel is required for this The agitation shaft thermowell harvest and sparger tubes and the short beveled tips of the interior portion of the base type addition ports will often require special attention All tubes and shafts must be cleaned Note that there may be some residual base or acid left in those lines so extreme caution and the use of chemically resistant gloves is highly recommended for this procedure It is often necessary to hand wipe surfaces with a paper towel in or
68. ation plugs 13 2 Preparing for a fermentation run ALERT Risk of water leaks gt Before connecting or disconnecting the water hoses to from the vessel and or cabinet at any time make sure the main water supply is closed Operating manual 114 Connect water to the system and turn it on Make sure the drain line is properly connected to the system Connect the quick connect plastic water lines to the exhaust condenser Add glycerin to the thermowell and insert the temperature probe Make sure the motor is not connected Turn the mains power ON Set the TEMP setpoint to the desired working temperature Check that agitation Agit is in OFF mode Connect the motor then set agitation to the desired speed and select Auto as its control mode Remove the shorting cap from the pH probe Connect the pH cable to the pH probe Remove the protective cap from the DO probe and connect the DO cable to the DO probe 10 If you have a water jacketed vessel be sure to refill the water jacket if required nO OA eh go 0 The DO polarographic probe will need to be connected for a minimum of six hours to be properly polarized before it can be correctly calibrated 11 Calibrate the DO probe see Section 7 3 12 Set pH and DO to the desired setpoints 13 Set the pH control mode to Auto 14 Set the DO control mode to Auto 15 Open the PUMP screen and assign a pump to Acid and another pump to Base Turn the pumps ON
69. be Mount the foam trap bottle holder on the clamping screw using the hole at the end of the holder s mounting arm Secure the holder in place with the clamping nut Leave the nut loose enough to swivel the holder Firmly place the foam trap bottle 250 mL or 500 mL in the holder With the bottle cap in place aseptically install a sterile 0 2 u filter on the shorter tube that penetrates the cap Be sure to respect the proper flow direction if stamped on the filter Connect a length of silicone tubing to the longer tube in the other bottle cap penetration Secure the tubing with a plastic tie and clamp it off on the top Connect the tubing securing it with a plastic tie to the foam exhaust tube in the headplate After autoclaving you will position the bottle holder where you want it then finger tighten the clamping nut BioFlo CelliGen 115 M1369 0050 Operating manual 51 Figure 27 Foam Trap OT 2 8 7 3 i 4 5 1 Sterile filter 5 Vessel 2 Silicone tubing 6 Bottle holder mounting arm 3 Foam exhaust tube 7 Bottle holder 4 Headplate 8 Foam trap bottle 250 or 500 mL 4 8 20 Plug unused ports Close off unused ports 1 Install a blind plug without a hole in any headplate port that will not be used 2 Install sili
70. become available they are posted on the web for easy download at www nbsc com in a USB drive and plug the drive into the USB port on the control cabinet 1 In the System Settings screen press the Refresh button to update the current software status and to search for a new USB drive 2 The name of the new drive folder appears in the Update File box 3 Press the Update button to install the file The file will reboot twice this may take a little time 4 The Software pane will reflect the changes Updating software will not affect any previous user settings 12 3 Hardware Setup Ne Ae ALERT Risk of damage to equipment gt When connecting multiple utility stations be sure to connect power and configure only one at a time Any attempt to connect and power two or more utility stations simultaneously can cause communication problems between the master control and utility stations The BioFlo CelliGen 115 system you purchase is preset in the factory as Unit1 with all the accompanying hardware In the Unit1 hardware list shown in the sample Hardware Setup screen see the following page the system has the Base Power module the Main pH DO module and one TMFC This system is also set to New Brunswick Modbus communication mode see the SCADA pane and has the Unit ID number of 2 This is the system s multidrop identification number Remember when you add utility stations that no two nodes on the network can have the same
71. cabinet 10 Remove the DO shorting cap and connect the DO cable to the DO connector on the control cabinet 11 Connect the foam probe cable to the foam connector on the control cabinet 0 gt A Oo NOO 9 2 Load pump tubing If you have pumps in your configuration the three standard pumps are located on the front of the control cabinet see the following page BioFlo CelliGen 115 M1369 0050 Operating manual 89 Figure 54 Standard Pump Array Before you insert tubing into the PUMP CHANNEL verify that the PUMP is in the OFF control mode With reference to the drawing below follow these steps to properly load tubing into the PUMP HEAD Figure 55 Loading Pump Tubing 1 Upper spring loaded clamp Pump cover Pump head 3 Pump head rotor aja Pump channel Lower spring loaded clamp Operating manual 90 1 Open the PUMP cover to gain access to the interior of the pump 2 Select the desired tubing size see Table 6 in Section 11 4 for reference and cut a length sufficient to reach from the inlet source through the pump and to the outlet recipient allowing a few extra inches 3 Form a loop large enough to go around the pump head 4 Fit one side of the tubing loop into one of the spring loaded clamps pulling the clamp open with your finger gt Be careful not to pinch your fingers in the pump head levers
72. cation Verify against your Eppendorf packing list that you have received the correct materials Report any missing parts to your local Eppendorf Sales Order Department 2 3 Basic components You should have at least the following components which will be described in greater detail later in this manual e Control Cabinet with Touchscreen e Bearing Housing e Vessel e Filters amp connectors e Thermowell amp RTD e Inoculation Addition System e Baffles for fermentation only e Sampling System e Impellers e Harvesting System e Probe Kits i e pH DO Foam Level e Sparging System e Motor 0 The assembled Control Cabinet Touchscreen assembly is called a Control Station For purposes of clarity in this manual however the control cabinet which houses the controller and the touchscreen will be referred to separately by their component names Operating manual 12 3 INTRODUCTION amp OVERVIEW 3 1 System BioFlo CelliGen 115 is a versatile fermentor bioreactor that provides a fully equipped system in one compact package It can be employed for batch fed batch or continuous culture with process control for pH dissolved oxygen DO agitation temperature pump feed antifoam and foam level Systems can be configured as either control stations or utility stations Each individual stand alone system is a control station One control station can run up to two additional utility stations which are dependent on the contr
73. ce Department or your local New Brunswick distributor In any correspondence with Eppendorf please refer to the Model Number BioFlo CelliGen 115 and the Manufacturing Part Number and Serial Number of the system 17 1 Troubleshooting A WARNING Risk of electrical shock gt Always turn your BioFlo CelliGen 115 off and disconnect the mains power cord before performing maintenance As with any equipment difficulties sometimes arise If you experience a problem with the operation of your BioFlo CelliGen 115 consult the following list of symptoms You may be able to resolve the situation easily and quickly yourself If the problem is not listed below or if the suggested solutions do not work please call your Eppendorf representative to request a service technician Other than the solutions proposed below do not attempt to fix the equipment yourself Problem Possible Solution TEMPERATURE Readout is a negative value e Inspect the temperature probe for obvious damage typically 225 C replace it if necessary e Make sure the temperature probe is connected to the cabinet jack The system will not heat up e Make sure the system was primed at start up e Make sure the temperature probe is plugged into the vessel thermowell e Water pressure may be too low raise pressure within recommended range e Verify correct connection click to lock of the water inlet and outlet lines on the vessel heat exchanger e Hit r
74. cone tubing secured with a plastic tie and clamped shut on any access tube i e harvest tube that will not immediately be used 0 It is good practice to lightly lubricate the underside of the headplate with silicone before installing it on the vessel 4 8 21 Install 1 3 L 3 0 L or 7 5 L fermentation vessel baffle Hold the baffle in place with two fingers when you lift the headplate assembly Operating manual 52 1 Gently place the baffle tab facing up around all of the other instruments protruding from the headplate including the cooling coil 2 Position the tab between the two uprights of the cooling coil 4 8 22 Install headplate 1 Orient the cooling coil uprights toward the back opposite the gradations marked on the vessel glass If this isa 1 3 L 3 0 L or 7 5 L fermentation vessel squeeze and hold the baffle in place opening toward the back with thumb and forefinger You may find it convenient to squeeze the tab with your thumb 2 Carefully lower the headplate easing all of its attachments into the vessel without hitting the glass or the baffle inside if this is a 14 0 L fermentation vessel 0 If you are using a baffle after installing the headplate insert any convenient length of wood plastic or stainless steel do not use any other kind of metal through an unused port to push the baffle down as far as it will go The baffle is stainless steel repeated installations may cause it to retain a co
75. connectors e pH 4 buffer e Addition bottles e pH7 buffer e A liquid trap e Silicone O ring lubricant for e Polysulfone quick connects fermentation only User s kits and start up kits are available from Eppendorf with many of the commonly required items including a selection of tubing clamps filters connectors and addition vessels See Section 16 6 for a list of spare parts and speak to your Eppendorf sales representative for more information BioFlo CelliGen 115 M1369 0050 Operating manual 15 3 11 Supervisory software In addition to the built in software that you interface with through the touchscreen your BioFlo CelliGen 115 system can be remotely controlled from a PC via New Brunswick BioCommand optional supervisory software see Section 4 10 Consult your Eppendorf representative for details be sure to ask for ModBus protocol Operating manual 16 4 INSTALLATION 4 1 Physical location The surface on which you place the BioFlo CelliGen 115 should be smooth level and sturdy Ensure that the surface can bear the weight of the system see Section 5 Specifications for weights plus vessel contents and any applicable ancilliary equipment Also ensure that there is enough space around the back and the front of the BioFlo CelliGen 115 for proper operation and access Allow atleast 4 inches of clearance behind the equipment for heat dissipation Figure 1 Dimensions
76. d on the left side of the cabinet There are push in tube connectors for air nitrogen oxygen and carbon dioxide These connectors accept flexible 3 2 mm in ID tubing a 7 6 m 25 ft length of blue polyurethane tubing part number P0740 3113C3 is supplied with the cabinet it can be cut to the appropriate sizes to attach to the utilities Other soft flexible walled chemically inert tubing such as Marprene Pharmed etc may be used as well Gas inlets plugged with black plastic are unavailable to your configuration and must remain plugged WARNING Risk of explosion gt Use gases in this equipment only within the range between their lower explosion limit LEL and their upper explosion limit UEL gt If your process requires or produces gases be sure to verify their LEL and UEL concentration range available online or ask your gas supplier CG s WARNING ALERT Risk of explosion Risk of equipment damage gt No gas pressure should rise above 10 PSIG gt Do not use this equipment in a hazardous atmosphere or with hazardous materials for which the equipment was not designed gt All gases supplied should be medical grade All gases should be regulated using a two stage regulator The scale of the regulator gauge for gases going into the fermentor should be such that one can regulate pressure from 3 to 10 PSIG maximum Connect the barbed sparge connector part number P0242 0600 to the SPARGE outlet at
77. dapter into the port 10 Adjust the probe to the desired height then nesting the ferrules close the top portion of the adapter onto the bottom portion 11 Finger tighten the knurled adapter assembly 4 8 16 Install dO2 probe 0 Prior to installation any dissolved oxygen probe you are using should be inspected for damage and replaced if necessary 0 To avoid damage to the probes during operation be sure that there is no interference between the probes and the baffle assembly impeller blades or cooling coil 1 Wear protective gloves to protect yourself in case of accidental breakage 2 Lightly coat the dO2 probe with glycerol k ALERT Risk of damage to dissolved oxygen probe Always fit the dO2 port adaptor onto the probe first Then insert the probe with its adaptor into the headplate following Steps 3 11 shown after the drawing on the following page Never attempt to install the dO2 port adaptor in the headplate without the probe BioFlo CelliGen 115 M1369 0050 Operating manual 45 Figure 22 dO2 Probe with Port Adapter exploded 7 q i em 1 dO2 probe adapter top portion 5 Teflon O ring white 2 Bottom ferrule 6 Top ferrule 3 dO2 probe adapter bottom portion 7 Cap 4 Port O ring black With reference to the drawing above 3 4 Gently slide the top portion of the knurled port adapter part of the probe kit onto the probe S
78. der to fully remove residual traces of small particulate debris The washing of the bottom portion of the vessel requires the same procedures as the headplate Note that the sides of the vessel particularly near the baffle may require special attention The vessel can now be cleaned by washing with detergent or by using a cleaning solution If the vessel is to be sterilized all standard precautions must be taken Note that for this purpose the vessel does not need to be sealed except for those previously cited valves and tubing which run under the liquid level It will be necessary to use water in the vessel We recommend the use of DI deionized water and the fill should be at least as high as your standard level for a run Unless you have already specifically wiped the residual grease off the top of the glass cylinder there should be enough so that the headplate can be clamped to the glass vessel DO NOT tighten the headplate clamps with the same force used to install the headplate prior to a run as this could lead to vessel damage Instead the lightest possible pressure should be used The advantage to sterilization is that not only are residual viable organisms killed but also residual debris will loosen and become removable by washing after the vessel has cooled If a cleaning solution is required we recommend a 10 dilution of Micro cleaning solution International Products Corporation catalog number 6732 Alternatively if you a
79. dy calibrated the touchscreen see Section 6 2 1 This screen remains in view for a few seconds then it is replaced by the SUMMARY Screen 6 2 3 Summary screen The SUMMARY screen see below is command central it puts all the available loops at your fingertips Your BioFlo CelliGen 115 controller can run as many as three stations the dark blue Unit Tab identifies which vessel s operating parameters are being displayed in the sample screen the sytem being displayed is labeled Unit 2 if you have more than one system pressing another Unit tab will move you to the SUMMARY screen for Unit 1 or if present Unit 3 Figure 32 Sample SUMMARY Screen Fermentation with Auto Gas Mix 1 7 E Summary Screen New Brunswick Fermentation Mode 2 uma 07 Dec 2008 15 241 _ 3 LoopName Setpoint Units Casc 5 i Summary o Calibration 3 Cascade 3 Pumps Setup P Unit tabs Operating mode 0 N Current date amp time continued on the next page Operating manual 62 O The dark blue button usually represents the screen currently displayed Screen access buttons o 0a Your BioFlo CelliGen 115 comes with pre assigned loop names The available loops will change depending on your system s configuration This one is equipped with automatic Gas Mix 7 Screen name with its icon Figure 33 Sample SUMMARY Screen Fermentation with Man
80. e care must be taken while removing it Be sure to use two hands with one hand at the top of the port acting as a guide to ensure proper removal A gentle pace is required if at any point in the process the probe should jam absolutely avoid forcing It may be necessary to reinsert the probe partway and to apply a lubricant such as glycerin to the shaft and port in order to effect the removal In extreme cases it may be necessary to remove the headplate with the probe still inside so that you can approach the problem from both ends In such a case it is critical to remove the headplate very carefully We recommend that you have a spare probe available at all times in case of breakage Once the pH probe has been removed it should be immediately washed off with warm water If biomaterial has accumulated on the probe use a sponge or an equivalent that will not scratch glass with gentle pressure to clean the surface The very tip of the probe should be handled with extreme care and a Kimwipe should be used to gently dry it off after washing The probe should be stored with the tip immersed in either electrolyte or pH 7 buffer This electrolyte buffer can be reused but it should always be inspected prior to each use for precipitation or contamination Operating manual 148 Now that the vessel is detached from the system it can be cleaned Remove any remaining cotton and foil covering the ports The rubber sampler bulb should be removed
81. e ee eee 55 TOURS CI iii 60 Sample SUMMARY Screen Fermentation with Auto Gas MIX 0cooconicccocconcconon 61 Sample SUMMARY Screen Fermentation with Manual Gas MIX ooooonccniccicncn 62 Sample SUMMARY Screen Cell Culture without TMEC ooconconccocococononcnnnnnncnns 63 Sample SUMMARY Screen Cell Culture with TMFC ooconnconcccncccononononaninncnnnon 63 Alphanumeric Espada 65 N mene errada adri oi 66 Sample GAUGE Serenidad weed 67 Sample GAUGE Screen Pl A A AA ees 68 Operating manual 132 Figure 40 Sample GAUGE Screen Agit vaca ctectnccepecasartanensesunek ote eraeiaeragotdeninentnteas 69 Figure Als Setp int Touchpad rd Ad Al a e 69 Figure 42 Calibration Screen a Adee tans 70 Figure 43 Cascade E E A oo Greece ieee 71 RS 44 P mp O ed ee e oi 71 Figure 45 Controller Setup ri 72 Figure 46 System Settings Screen sy sess ccscssandsian hiatal A E ER aa eee 73 Figure 47 Hardware Setup de 73 Fig re 487 Calibration Screen e iaeia seas Gass caries teem VR CN 75 Figure 49 pH Probe with Port Adapter exploded ce ceeeseeseeseceeeceseceeeeseeeeeeeeeeaeenaeeneeees 77 Figure 50 dO2 Probe with Port Adapter exploded ooooooooccnoccnoconooccooncconanconncconoconncnnncconncnns 80 ss AAA A come ea tenement 82 Pipute 02 Calibrating Level Prope sitter ete Sate sue ects alah ca hc tata a a Satis 83 Figure 53 Angled Autoclave Rack Option oooonocnncninononnnnoconnnnnncnncnnncononnononnonnc canon cnonnannnnnos 86 Figure
82. ear C 55 This keypad is used to designate a setpoint speed for Agitation What you type on the keypad will appear here WIN When you have finished typing the entry press the OK key to save the entry and close the keypad 4 To clear the entry without saving it and to close the keypad press the Cancel key 5 To clear the entry without closing the keypad press the Clear key 6 2 5 Gauge screens Every loop has its own gauge screen To access it in the SUMMARY screen touch the screen in the appropriate blue box in the LoopName column Your touch will open that loop s GAUGE screen see the following page BioFlo CelliGen 115 M1369 0050 Operating manual 67 Figure 38 Sample GAUGE Screen Agit full Gauge 2en New Brunswi Cell Culture Mode 07 Dec 2008 15 27 Agit 134 RPM Setpoint RPM 150 0 02 3 150 134 15 7 Factory Proportional 5 50 100 0 200 200 Limits 4 Set Low Set High 25 200 Decimal Places 5 0000 00 00 25 25 0 0 E Summary i Calibration T Cascade 3 Pumps A Setup LoopName 2 Units of measure the action of this loop Agitation is measured in revolutions per minute rpm 3 Gains Proportional and integral values are what the software uses to calculate output based on differences betwee
83. ecccccesssusesceccscsessuueaeasscescssssuaenens 143 21 7 1 Vessel preparation before autoclaving comoooconcnnnocnnccnnnnnnnnnnnnnnnnnnnno nn ncnnnnnnnos 143 21 7 2 Vessel Sterilization cccsscccccnseecccccssecccccsseececsseececcuseececsenecceccsecccaneseeceeauaeeess 145 21 7 3 Post sterilization vessel SC UD Gi iaa estes 145 2 Vessel operation Ad at 146 21 7 5 Vessel shutdown amp CIC A EE io si 147 22 APPENDIX D CORROSION RESISTANCE ocomcncnmommssss 149 23 APPENDIX E GENERAL CHARACTERISTICS OF EPR 0 00000000000000000 150 23 1 SIDENTIEVYINGEP Ri A A a eel cokes 150 23 2 GENERAL CHARACTERISTICS ccsssssseccccccccsesssssscecccccesseusaeseeccscscssueeaessecseceseeanenens 150 24 INDEX sesisasdesscciccsesccestscseccsesocccdsdosecccocseaccdseocsdcvescnacsdsdasecccoesesccdsesesecvcscoscedsdassdevocsosvcdce 151 BioFlo CelliGen 115 M1369 0050 Operating manual A bu 1 USER INSTRUCTIONS CAUTION Risk of damage to personnel and or equipment This equipment must be operated as described in this manual Please read the entire Operating manual before attempting to use this equipment If operational guidelines are not followed equipment damage and personal injury can occur Do not use this equipment in a hazardous atmosphere or with hazardous materials for which the equipment was not designed Eppendorf is not responsible for any damage to this equipment that may result from the use of an accessory not
84. ectable e None which means that dissolved oxygen will not be controlled by means of a cascade e Agitation controls dissolved oxygen through automatically controlled agitation speed When the actual DO2 value drops below the setpoint the system will increase the agitation speed up to as much as the high limit to meet the culture demands Once the DO setpoint is reached or exceeded the agitation will fall back down to the low limit e Oxygen controls dissolved oxygen by automatically adjusting the mix of air and oxygen This is not available without the Automatic Gas Mix Option When the actual DO2 value drops below the setpoint the system will increases the percentage of O2 to as much as the high limit to meet the culture demands e Agitation Oxygen controls dissolved oxygen by first increasing Agitation to the high limit then if DO still has not reached the setpoint increasing the oxygen percentage being entered through the sparger to as much as the high limit This cascade is most frequently used in fermentation This is not available without the Automatic Gas Mix Option e Agitation GasFlo controls dissolved oxygen by first increasing Agitation to the high limit then if DO still has not reached the setpoint increasing the GasFlo entering through the sparger to as much as the high limit This cascade is most frequently used in fermentation This is not available without the Automatic GasFlo Option e GasFlo O2 controls dissolved
85. edle will corrode but it saves the fermentor vessel Insert the needle though a septum port so that the drip point is away from stainless steel components and fairly close to the liquid level You may also use a more diluted solution of the acid or base However take note that this may cause the complication of adding a larger volume of liquid to the vessel Also it is not a good idea to add acid and base through a single double or triple port adapter The combined effects of both causes rapid corrosion of the adapter The pump setting is usually 20 0 25 0 under acid or base mode For these concentrations of acid and base Marprene tubing should be used To avoid damage to the stainless steel headplate use a septum port for introduction of these strong solutions into the vessel If you are using silicon tubing reduce the concentration of H2SO to less than 8 about 5 and use a 20 solution of NaOH When selecting an acid for use in fermentation select the lowest possible concentration that allows for pH control 21 5 Glucose feed Question What is the proper concentration of glucose feed Answer The glucose is 50 concentration The feed rate is not usually a constant value as this will differ not only from run to run but it will vary greatly over the course of a run depending upon the organism s growth This operation can be controlled automatically by BioCommand New Brunswick proprietary Windows based software Glucose feeding
86. ements 70 C max temperature 65 C max temperature for 14 0 L Control PID for heating and cooling Heat blanketed Vessels External heating blanket and immersed stainless steel coiling coil Water jacketed Vessels Water jacket heater and circulation loop Sensor Platinum RTD probe Pt 100 Agitation Drive Magnetic Drive or Direct Drive Indication Digital display in 1 rpm increments Direct Drive 50 1200 rpm for fermentation 25 400 for cell culture Magnetic Drive 25 200 rpm Rushton style standard with fermentation system Pitched blade standard with cell culture Optional Spin filter Aeration Gas Flow 0 4 Rotameters options e 0 150 mLpm e 250 2500 mLpm e 1 5Lpm e 1 20 Lpm 1 Thermal Mass flow Controller TMFC e 0 04 20 00 SLPM Automatic 4 Gas Mixing via 4 solenoids options Manual Gas mixing via 4 gas manifold Sparger Standard O sparger Peer Spiorat vierosparger o Inlet Filter___ 0 2um interchangeable cartridge N Gas For calibration of DO probe Indication Digital display in 0 01 pH increments 2 14 pH PID link to pumps or gases adjustable deadband Sensor pHprobe pH probe continued Operating manual 58 BioFlo CelliGen 115 System Indication __ Digital display in 0 1 increments Range 0200 2 Sensor Polargraphic DO probe Filter 0 2um interchangeable cartridge Condenser Stainless steel water cooled in headplate 7 Pumps Control m SSS
87. ercentage of DO See Section 10 for further information on setting up cascades Default P amp proportional amp integral values are preset at the factory We strongly recommend that you maintain the factory set parameters See Sections 19 4 19 6 for more information on P amp values BioFlo CelliGen 115 M1369 0050 Operating manual 13 3 4 Temperature control The culture temperature setpoint may be selected within the range from 20 C above coolant temperature to 70 C for 1 3 to 7 5 liter vessels and from 20 C above coolant temperature to 65 C for 14 0 liter vessels It is controlled by the process control software which then sends information to either a heater blanket and cooling coil or to a water jacket The media temperature is sensed by a Resistance Temperature Detector RTD submerged in the thermowell Default P amp proportional amp integral values are preset at the factory We strongly recommend that you maintain the factory set parameters 3 5 Aeration Up to four gases including air nitrogen carbon dioxide and oxygen can be introduced into the media through the ring sparger or optional microsparger The flow rate is controlled manually by one two three or four Rotameter s or automatically by thermal mass flow controller TMFC according to the definition of your system The TMFC is regulated automatically according to values set via the control station touchscreen The gas mix can ei
88. es It has exceptionally good weather aging and ozone resistance excellent water and chemical resistance excellent resistance to gas permeability and excellent temperature usage range up to 350 F 177 C Ethylene Propylene is a polymer where oil and solvent resistance is poor however it is fairly good in ketones and alcohols It is not recommended for exposure to aromatic hydrocarbons BioFlo CelliGen 115 M1369 0050 Operating manual 151 A Acid Concentration 142 Acid Type 142 Adding a Utility Station 110 Adding New Hardware 110 Addition Tubing Size of 91 Aeration 13 Agitation System 12 Air 1 106 107 Airflow Control Automatic 13 Manual 13 Anaerobic Culture 118 Antifoam Formulation 141 Antifoam Probe 14 Autoclaving 143 Preparing for 85 Autoclaving the Vessel 86 145 Baffle Installation of 35 38 51 Base Concentration 142 Base Plate Installing Vessel on 37 Base Type 142 Batch Operation 117 Bearing Housing Maintenance of 124 BioCommand 15 55 56 115 BioFlo 115 Options Setting the 112 Bottom Clamping Ring Installing the 37 C Cabinet Cleaning of 122 24 INDEX Calibration of Touchscreen 108 Calibration Screen 70 Cascade Creating a 94 Cascade Screen 70 94 Certifications 58 59 Cleaning 122 147 CO2 4 106 107 Connecting Cabinets 20 Connecting Stations 20 Continuous Operation 117 Control Cabinet Installing the 17 Control Cabinet Connections
89. eset button on hot plate if appropriate The system is leaking water e Inlet water pressure may be too high lower pressure within the recommended range e Check for any loose connection of inlet hoses tighten if necessary continued Operating manual 130 Problem Possible Solution AGITATION Agitator does not turn or turns e The motor drive coupling may not be installed only slowly properly read the motor adaptation instructions in this manual then check the coupling e Remove replace the O ring e Make sure the motor is plugged into the cabinet receptacle TURN OFF MAINS POWER SWITCH BEFORE CONNECTING THE MAINS POWER PLUG DO and pH PROBES DO probe readings are erratic e Recalibrate the probe carefully following instructions in this manual e Recharge the probe carefully following instructions in this manual e Probe may need a new membrane and a refill of electrolyte e Check for a secure connection e Replace probe cable or DO probe pH probe readings are erratic e Recalibrate the probe carefully following instructions in this manual Check for a secure connection Gel filled probe may need replacement Liquid filled probe may need a refill of electrolyte Probe cable may need replacement Probe does not hold calibration e Probe may be defective replace it e _pH DO board may be defective call for service GASFLOW There is insufficient gas
90. flow e Inlet or exhaust sterile air filter may be wet or clogged replace it e Check that the air pressure is within the specified range e Make sure the control mode for DO and for pH is set to AUTO not OFF e Make sure that the GasFlo loop is ON e Make sure that the Air loop is in O2 Enrichment mode e Make sure that the DO cascades are Enabled GENERAL Touchscreen is not responding e Calibrate touchscreen BioFlo CelliGen 115 M1369 0050 Operating manual 131 18 1 Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Figure 10 Figure 11 Figure 12 Figure 13 Figure 14 Figure 15 Figure 16 Figure 17 Figure 18 Figure 19 Figure 20 Figure 21 Figure 22 Figure 23 Figure 24 Figure 25 Figure 26 Figure 27 Figure 28 Figure 29 Figure 30 Figure 31 Figure 32 Figure 33 Figure 34 Figure 35 Figure 36 Figure 37 Figure 38 Figure 39 18 DRAWINGS List of drawings MEST STON eas a ae a re aS ttc aa 16 A each tea Pode Baten odecel econ hla cine duos aiii 17 Rear VI ia 18 Control Station Service Connections ecceecceesseceseceseeeeeeeececeaeceeeeeeeeeeseecsseeeeaeens 19 Connecting AI TIES eases did arisia 21 Installation of Terminators with Master amp One Utility Station eee 21 Installation of Terminators with Master amp Two Utility Stations ee 22 Water Connection Skaara area aan e ban a anaa Ear ea
91. fore proceeding consult the dimensions of your vessel assemblies to be sure your autoclave is large enough to accommodate the vessel with its various components WARNING Risk of explosion gt During autoclaving the vessel exhaust filter must be vented to avoid explosion A WARNING Risk of burns gt Use protective gloves when handling hot components H ae ALERT Risk of water leaks gt Make sure the main water supply is closed before connecting or disconnecting the water hoses to from the vessel and or cabinet at any time k ALERT Risk of equipment damage from steam gt Install the bearing housing cap on the fermentation vessel bearing housing before sterilization to keep steam from damaging the internal bearings E ALERT Risk of damage to tubing gt Never autoclave PVC tubing clear with white braiding There are four objectives to preparing a vessel for sterilization A To minimize pressure differences throughout the sterilization process by ensuring that the air can transfer freely between the inside and the outside of the vessel BioFlo CelliGen 115 M1369 0050 Operating manual 85 B To ensure that minor pressure differences do not expel liquid from the vessel by clamping off all penetrations that go below liquid level C To protect hydrophobic filters from blockage which would occur if condensation were allowed to wet and block the filter surface D To protect susceptible vessel as
92. g half so that the plate extends beyond the ring Figure 19 Installing Headplate Clamping Ring 8 Q O O 2 In the same manner install the second mounting plate on the other end of the ring half 3 Bring the two halves of the headplate clamping ring together under the vessel flange with the mounting plates on the bottom for easy access from below 4 Align the mounting plates with their corresponding holes on the other ring half and drop in the remaining Phillips head screws Tighten the screws to fasten the ring in place 4 8 2 Install vessel on base plate 1 Place the base plate on a level surface 2 Lightly lubricate the base plate O ring and seat it securely in its groove 3 Fit the one piece water jacket guard rubber gasket around the outside of the bottom vessel flange against the water jacket see the following page Operating manual 38 4 With the clamping screws in place on the ring fit the bottom clamping ring onto the base plate Figure 20 Water Jacket Guard Installation top view 1 Rubber gasket part number M1155 9902 1 3 L amp 3 0 L or M1227 9903 7 5L 8 14 0 L 2 Bottom vessel flange 5 With the gradations marked on the glass facing front toward the user slide the vessel into the bottom clamping ring until it rests securely against the base plate Make sure the water inlet tube stands free not kinked inside the water jacket 6 Finger tighten the si
93. hScreen English Date Time 4 7 dec 2008 15 36 Software Fermentation Mode 07 Dec 2008 15 36 Maintenance BF115 Rev 8 Update Folders g Summary Calibration Cascade 3 Pumps Ad 1 English is the default language No other choice is currently available When other choices Francais Deutsch Espa ol become available the user will select the language here using the VW dropdown menu 2 Use this pane to calibrate the touchscreen see Section 12 2 for details 3 Use this pane to view the Software Firmware version installed and to update Software via the USB port see Section 12 2 2 for details 4 Use this pane to change Date and Time see Section 12 2 1 for details Figure 47 Hardware Setup Screen Ke Setup Screen New Brunswick Fermentation Mode 07 Dec 2008 15 38 Controller Setup System Settings 1 New Hardware Unit1 EA E Base Rower Acive 304980 74 PENN TMFC1 Active 104113 1 Mode INBS Modbus zl Unit3 Module Status Good Errors 2 ee ES Unit ID 2 Es Summary t Calibration TA Cascade Pumps ey Setup 1 Use this screen to view and add hardware for as many as 3 Units installed in the system and to set Unit IDs for software see Section 12 3 for details 2 Use the SCADA pane to choose software connections as explained in Section 12 3 Operating manual 74 T PROBE PREPARATION amp CALIBRATION
94. he probe 7 3 5 DO probe calibration setting zero 0 The DO probe is calibrated AFTER sterilization There are two methods to obtain zero for calibrating the DO probe Review both methods and use the one you prefer Method 1 1 Remove the DO cable from the DO electrode 2 Go to the CALIBRATION screen and select DO 3 Enter 0 in the SET ZERO edit box see the following page then press SET ZERO 4 Reconnect the DO cable to the DO electrode 0 If you use Method 1 make sure the probe is not disconnected for more than one minute Method 2 0 Nitrogen is needed for Method 2 There is an No gas inlet on the control cabinet for this purpose make sure that your nitrogen source is connected to this inlet Connect the DO cable to the DO electrode and the control cabinet Go to the CALIBRATION screen and select DO Press the N2 3 ON button Depending on your system s configuration however this button may not be present In this case manually turn the N2 loop on from the SUMMARY screen or manually turn on the Rotameter and set it to 1 20 SLPM depending on vessel size and flow controller 4 In approximately 10 30 minutes the current value reading will stabilize 5 Press the SET ZERO edit box see the following page use the touchpad to enter 0 press the OK button then press the SET ZERO button 6 Press N2 3 OFF or if in Step 3 you manually turned the Nz loop on now manually shut off the nitrogen flow to
95. hould also be checked to ensure that its rubber stoppers have not been displaced Note the time that the DO probe is connected since the probe requires a minimum of 6 hours for polarization Remove the bearing housing cap and attach the motor Open the SUMMARY screen and set the air from OFF to O2 Enrichment Return to the SUMMARY screen and make sure that GasFlo is in ON mode Connect the air line from the system to the sparger s terminal filter as aseptically as possible although the filter will prevent external contamination good technique is always a good idea Open the clamp on the sparger line and visually observe the vessel to ensure that air is flowing properly Then set the agitation to the minimum desired value After set up the system should be carefully observed to ensure that there are no problems especially no water line leaks 21 7 4 Vessel operation The vessel must have any and all necessary addition bottles connected prior to use If another bottle such as the glucose feed is not initially required it can be hooked up later The pH will probably need to be adjusted This is done by setting the pH control to Auto Note that due to the system s tendency to overshoot the target pH during this initial adjustment it is desirable to set the initial pH setpoint a little conservatively For example post sterilization pH reading is 6 8 and desired setpoint is 7 2 Set the system to setpoint 7 0 when conducting the ini
96. ic that generates appropriate drive signals to various actuators so that process parameters remain at their setpoints 19 2 What is a control loop A control loop is the basic element of automatic process control Three components comprise one control loop a sensor a controller and an actuator Based on information from a sensor the controller generates an actuator control signal that maintains a parameter at its setpoint Control will fail if any element in the control loop fails 19 3 What is probe calibration In bioprocess control calibration generally refers to establishing a correspondence between a probe s output and the actual value of whatever that probe senses For example pH probes are often calibrated with pH 7 0 and pH 4 0 buffers to establish a Zero pH 7 0 and a span pH 4 0 Other buffers can be used but the principle is always the same For any probe calibration two values a zero and a span are required for the controller to correctly translate inputs from that probe DO and pH probes are routinely calibrated before each use Most other probes need be calibrated only infrequently 19 4 What are P I D constants BioFlo CelliGen 115 M1369 0050 Operating manual 135 The mathematics of P I D control is familiar to most control and process engineers In P I D mode the controller creates a control signal that is based upon the deviation between the setpoint and input from a sensor The magn
97. imilarly with a full scale error the I term after 1 minute and the D term will be and D respectively BioFlo CelliGen 115 M1369 0050 Operating manual 137 20 APPENDIX B OTR 20 1 Determining an oxygen transfer rate The oxygen transfer rate OTR of all New Brunswick fermentors is determined by a standard sulfite oxidation test The standard operating conditions for determining OTR are Temperature 30 C Agitation 1000 rpm Aeration 1 VVM 20 1 1 OTR calculations OTR can be estimated by titrating a fixed amount of sodium sulfite Na2SO3 with air CU 2 2503 02 gt 2S0 The Procedure Calibrate the DO electrode e Setzero on DO Fully oxygenate the fermentor with agitation and airflow e Setspan to 100 Introduce a known amount of Na2SOs into the fermentor when fully oxygenated e OTR 30 000 n mM O L hr VAT number of moles of sodium sulfite vessel volume in liters time taken from DO curve at two points of 50 DO min n V AT Operating manual 138 20 2 Some factors that affect OTR and horsepower Many factors influence OTR not the least of which are type size and placement of impellers in the reactor Factors which effect OTR are vessel dimensions impeller diameter type of impeller i e turbine marine pitched blade etc Eppendorf selects and recommends the placement of impellers in the vessel to attain a minimum of 350 mM O L hr of OTR The BioFlo CelliGen 115 fer
98. ings to attach to the cabinet The tubing part number P0740 1631 has an inner diameter of 6 35 mm 1 4 in and an outer diameter of 11 1 mm 7 16 in Figure 8 Water Connections 1 For the EXHAUST CONDENSER IN amp RETURN connections 0 9 m 3 ft lengths of 4 76 mm 3 16 in ID silicone tubing part number P0740 2505 are pre assembled They have a quick connect on one end to be connected to the cabinet They are open at the other end to connect to the exhaust condenser s inlet and outlet The connection points should be secured with cable ties 2 For the COOLING LOOP IN amp RETURN connections 0 9 m 3 ft lengths of 4 76 mm 3 16 in ID silicone tubing part number PO740 2505 are pre assembled They have a quick connect on one end to be connected to the cabinet The other end is to be connected to 1 the cooling coil s inlet and outlet on the headplate of heater blanket vessels or 2 to the water inlet and outlet lines coming from water jacketed vessels The connection points on the open ends should be secured with cable ties BioFlo CelliGen 115 M1369 0050 Operating manual 25 ALERT Risk of water leaks gt Before connecting or disconnecting the water hoses to from the vessel and or the cabinet at any time make sure the main water supply is closed 4 5 3 Gas connections Gas inlets are located on the rear panel of the control cabinet The sparge outlet is locate
99. inserted k ALERT Risk of damage to pH probe gt Never let a pH probe rest on its tip and never leave a pH probe in DI water 7 3 Dissolved oxygen DO probe preparation 7 3 1 Inspecting the DO probe Inspect the probe for possible shipping damage Immediately report any damage you may observe to your local Eppendorf sales representative or distributor Remove the protective cap from the electrode end The membrane is delicate and care must be exercised to prevent accidental damage NEVER REST THE PROBE ON ITS MEMBRANE 7 3 2 DO probe preparation To ensure stable output the probe should be sent through two or three sterilization autoclaving cycles prior to use The probe will be operable after the second cycle but it will be more stable with additional sterilizations The shorting plug should be installed on the probe during autoclaving or sterilization BioFlo CelliGen 115 M1369 0050 Operating manual 79 Default P amp proportional amp integral gains are preset at the factory They are different for each operating mode fermentation and cell culture to ensure proper DO control It is recommended that you use the factory set P I values Do not attempt to change the settings unless you are experienced with P amp control If you choose to make changes P amp gains for the DO loop can be modified by using the touchpad on the front of the control cabinet For details see Section 20 Appendix
100. ion note terminator on top terminator on bottom 8 Follow the instructions in Section 12 3 again to complete the utility station installation and identification so the control station and the utility stations can work together 4 5 Utilities E ALERT Risk of damage to equipment Do not use this equipment in a hazardous atmosphere or with hazardous materials for which the equipment was not designed All control and utility stations must be properly connected to gases water supply vessel water electrical mains power and an open drain The gas connections are located on the rear panel of the cabinet All other service connections are on the lefthand side of the cabinet BioFlo CelliGen 115 M1369 0050 Operating manual 23 Using standard plant practices and respecting all applicable codes connect services to the appropriate connections as recapped in Table 1 and explained in greater detail in Sections 4 5 1 4 5 3 Table 1 Service Connections Service Utilit Requirement Connection 120 VAC 50 60 Hz Single Phase 10 120 VAC 1 phase Amp fluctuations not to exceed 10 field wired to 15 Amp disconnect in panel 230 VAC 50 60 Hz Single Phase 6 230 VAC 1 phase Amp fluctuations not to exceed 10 field wired to 15 Amp disconnect in panel ong 60S ES Nitrogen 3 10PSIG_ Pushintue Exhaust 1 2 PSIG maximum backpressure 4 5 1 Electrical Requirements
101. ion you may have the following possibilities for gas control 1 4 Rotameters with manual gas mixing 1 Rotameter with automatic gas mixing 1 TMFC with manual gas mixing or 1 TMFC with automatic gas mixing If your system is equipped with no TMFC or one TMFC the system will be preconfigured to one Control Mode in the Controller Setup screen O2 Enrich Direct or Cascade Driven for Fermentation or 4 Gas Mix for Cell Culture Your system has 4 gas solenoid valves No TMFC means that all gas flow is manually controlled using one or more Rotameter s When you have Fermentation as the Operating Mode and O2 Enrich as the Control Mode the gas process loops you will find in the SUMMARY screen are labeled Air 1 as shown in Figure 42 and O2 2 Figure 69 Air 1 Gauge Screen with O2 Enrich ful Gauge Screen New Brunswick Fermentation Mode 07 Dec 2008 15 48 Air 1 100 0 Setpoint 100 0 Set PAM Out 100 0 1000 100 0 Limits Set Low Set High 100 0 100 0 0 0 100 0 100 0 000 0 0 000 0 0 0 0 0 0 Decimal Places 0000 00 00 CS fi Calibration Cascade 3 Pumps Setup 1 This gas loop is currently set to O Enrich which is why there is an O2Enrh button When you have Cell Culture as the Operating Mode and 3 Gas mix as the Control Mode the process loops are labeled Air 1 O2 2 and N2 3 or Air 1 O2 2 and CO2 4 The loops n
102. irector of QA RA a Operating manual 60 6 OPERATING CONTROLS 6 1 Touchscreen Your primary interface with the BioFlo CelliGen 115 is the touchscreen on the control cabinet Figure 31 Touchscreen 1 Touchscreen display only the control station is equipped with a touchscreen Utility stations do not have one 2 Rotameters you may have from 0 to 4 Rotameters The quantity of and flow rates for the Rotameters is determined by your system specification Pumps AJ ON OFF mains power switch 5 Control cabinet 6 2 Display screens 6 2 1 Touchscreen calibration The first time you turn the system on you may be prompted to calibrate the screen to your touch 0 For optimal results be sure to stand or sit in the position from which you are most likely to work Height and angle of reach will affect calibration Follow the onscreen instructions to touch and hold the target each time it appears Usually you will be prompted to touch the four corners of the screen twice in succession BioFlo CelliGen 115 M1369 0050 Operating manual 61 6 2 2 Start Up screen The Start Up screen which tells you which operating software version is installed in your BioFlo CelliGen 115 is the first screen you see each time you turn on the system if you have alrea
103. itude of the control signal is determined by a mathematical formula that can include proportional P integral I and derivative D terms The P and D constants are three numbers that determine the relative sizes of the proportional integral and derivative terms respectively To use a temporal analogy the P or proportional part of the control signal reflects present deviations between setpoint and current value The I or integral component reflects past deviations and the D or derivative term anticipates future values of the error Generally with noisy or slow responding sensors such as dissolved oxygen and pH probes the D constant should be set to zero If the constants for a loop are too large that loop will oscillate displaying extreme swings in actuator output If for example agitation changes suddenly and frequently between minimum and maximum rpm one should suspect incorrect P I and D values for the agitation control loop This condition can easily be mistaken for a defective component when it actually results from incorrect settings If the constants are too small control response will be slow and setpoints may never be reached Again this can be mistaken for defective components P I D constants are usually established by methodical trial and error 19 5 What is P I D tuning Tuning consists of establishing controller settings the proportional integral and derivative constants such that the controller
104. l 20 4 4 Connecting utility cabinets ALERT Risk of damage to equipment function gt When connecting multiple utility stations be sure to connect power and configure only one at a time Any attempt to connect and power two or more utility stations simultaneously can cause communication problems between the master control and utility stations ALERT Risk of damage to equipment function gt If only one utility station will be installed connect the provided terminators to the master control station s input COM port and to the utility station s output COM port If a second utility station will be installed connect the provided terminators to the master control station s input COM port and to the 2 utility station s output COM port 0 The terminators are provided in your BioFlo CelliGen 115 shipping kit If you have a control station and one or two utility stations use the bus cable s and terminators provided in the following way 1 Verify that the first utility station is not yet connected to the control station and that both are turned off 2 Connect the RS 495 cable provided to the control station s output COM port and to the utility station s input COM port as shown in Figure 5 Verify that the cable is securely connected to both cabinets BioFlo CelliGen 115 M1369 0050 Operating manual 21 Figure 5 Connecting Cabinets AN wh
105. lace if you plan to invert the headplate while rinsing it In this case it is usually advisable to also remove the clamps that hold the headplate onto the rest of the vessel as failure to do so will result in their falling out during inversion The pH and DO probes should not be in the headplate while you rinse it All gas filters must be removed prior to rinsing The sparger must in particular be checked to ensure that it isn t clogged The headplate must be oriented in combination with the vessel and the internal baffle so as to allow for the exhaust condenser lines to be connected Also the baffle must be positioned so that it does not interfere with the insertion of the pH and DO probes into their ports Do not place the sample port to the rear of the vessel and position it so that ample room is available to take a sample It is advantageous to have the addition ports for acid base etc on the same side as or at least not opposite the pumps The old grease on the top of the glass cylinder should be wiped clean Reapply grease Dow Corning silicone grease prior to installing the headplate smear a very thin layer around the top of the cylinder with your fingertip Take care to ensure that no residual grease remains on your hands when you touch other parts of the vessel When the headplate is in place be sure to properly tighten the headplate clamps All tubing connected to the headplate should be secured at the headplate connection point
106. large viewing windows so the culture remains visible for inspection DM WARNING Risk of electrical shock gt NEVER cut any portion of the heat blanket gt NEVER fold the heat blanket or place any weight upon it gt For storage always lay the heat blanket flat The drawing on the following page shows a typical installation of the vessel in its vessel stand with the most commonly used accessory equipment To provide a full view of how the internal components are arranged the heat blanket is not shown in the larger vessel view Operating manual 30 Figure 13 Vessel Assembly Z o eee i 12 1 11 l N E dea bo 9 J J lt gt 8 i J 1 Heat blanket 7 Cooling coil hides sparger 2 Lifting handle 8 Baffle 3 Cooling coil 9 Clamping ring 4 Sparger 10 Headplate 5 Vessel stand 11 Exhaust 6 Thermowell 12 Agitation motor coupled to bearing housing BioFlo CelliGen 115 M1369 0050 Operating manual 31
107. lculated through PID It is common when a batch is running to see that pH remains steady at the setpoint yet the acid and or base pumps are continually alternating in making additions This is an indication that the controller is overcompensating for minor fluctuations in pH Output Multiplier is a feature that attenuates controller output to the acid and base pumps and the CO gas line providing more nuanced control of additions to maintain pH Contrary to the number shown in the Figure 35 Pump 1 Out Mult edit box we recommend that you begin by implementing a multiplier of 25 This means that if the controller s output to the base pump for example is 100 then the 25 multiplier will reduce pump output to 25 If the controller s output to the pump is 50 the 25 multiplier factor will reduce pump output to 12 5 If after applying an Output Multiplier of 25 you find the results are attenuated but the controller seems unable to maintain the setpoint increase the Multiplier by small increments until the controller is able to maintain setpoint BioFlo CelliGen 115 M1369 0050 Operating manual 99 11 3 Pump control mode There are three available control modes for each pump as explained in Table 5 Table 5 Pump Control Modes Control Mode Description Off The pump will receive no input and will not operate On The pump will operate according to the parameters you have set Prime This button toggles the pump on
108. le cascade will be greyed out not selectable 2 Select the Cascade To loop or series of loops in the DO Cascade pane Your selection s will now have a dot in the option button and will have changed from black to blue see the sample on the following page 3 In the Cascade Limits pane enter the desired Low and High limits for the Cascade To loop s in their associated edit boxes BioFlo CelliGen 115 M1369 0050 Operating manual 95 Figure 58 Sample Cascade Screen Cascade Screen New Brunswick Cell Culture Mode 07 Dec 2008 15 52 DO Cascade Cascade Limits None Agit Casc Low Limit 25 c Agit Agit Casc High Limit 200 02 GasFlo Casc Low Limit 0 GasFlo Casc High Limit 20 GasFlo 02 Mix Casc Low Limit Agit 02 02 Mix Casc High Limit Agit GasFlo GasFlo 02 Agit GasFlo 02 ffi Summary Calibration Cascade x Pumps Setup In this sample cascade as the system demands an increase of DO agitation will increase from 25 to 200 If there is still a need for more DO the GasFlo loop will kick in until the need is satisfied Operating manual 96 11 ABOUT PUMPS After assigning the pumps see Section 11 1 you will need to select a setpoint and a control mode for each calibrate their flow rates and select their pulse periods This section will walk you through those operations There are three standard 12 rom pumps on the front right of your cont
109. liGen 115 M1369 0050 Operating manual 119 3 Turn the pump ON Since the liquid is in contact with the probe the circuit will close and the pump will begin pumping liquid out of the vessel 4 When the liquid drops below the probe tip the pump will stop See also Section 11 1 Pump Assignment If you assign the pump to None instead of Lvl2 Wet it will harvest as much as possible 13 12 Shutdown procedure At the end of a run to shut down the system follow these steps Set GasFlo to OFF Set Agit and Temp to OFF Set all other control loops to OFF Turn off the mains power If the system is not to be used for several days disconnect the mains power plug Remove drain and clean the vessel as outlined in Section 15 ON ics See also Section 21 7 5 for shutdown and cleaning tips 0 Never wash the filters or get them wet Operating manual 120 14 ESSENTIAL OPERATING TIPS 14 1 Precautions for glass vessel assembly There are certain precautions you should take to avoid cracking or breaking the glass vessel during assembly and autoclaving e Glass can crack or break during assembly if the clamping screws are overtightened As a precaution tighten the screws only finger tight prior to autoclaving You should be able to insert a business card between the glass and the metal e If the vessel is not sufficiently vented during autoclaving it can crack or break As a precaution make certain that the exhaus
110. lide the two white ferrules onto the probe the narrower one on top of the deeper cup shaped one Gently slide the bottom portion of the port adapter onto the probe taking care to orient the longer threaded section toward the top of the probe Remove the two O rings installed in the dO2 port first slide the white Teflon O ring onto the probe then follow with the black 12mm port adapter O ring Do not yet close up all the elements of the port adapter Gently insert the probe into the appropriate port allowing the O rings to seat fully into the port 0 The fit may be snug Gently rotate the probe as you press it into the port to avoid breakage 9 Finger tighten the bottom portion of the port adapter into the port 10 Adjust the probe to the desired height then nesting the ferrules close the top portion of the adapter onto the bottom portion 11 Finger tighten the knurled adapter assembly Operating manual 46 4 8 17 Install exhaust condenser WARNING Risk of explosion gt Never intentionally block the exhaust to raise vessel pressure If you are using the optional exhaust condenser 1 Unscrew the spare exhaust port plug from the headplate saving it for reuse 2 Place the 12mm exhaust condenser adapter into the port 3 Place the exhaust condenser inlet See drawings below into the port and finger tighten the knurled adapter 4 Tighten it with the Allen key provided until it is secure
111. lines may be established by pre calibrating the pumps using the PUMP screen This screen controls all pump parameters for the three standard fixed speed pumps supplied with each control cabinet and for any additional pumps added through the available analog input and output connections Using the PUMP screen you can view total pump flow rate in mL second and set the pump s cycle time and assign each pump to one of eight functions None Acid Base Foam Lvl1 Lvl2Wet Lvl2Dry bearing in mind that the level dry function turns the pump on when the probe is not in contact with liquid see Section 11 1 for details BioFlo CelliGen 115 M1369 0050 Operating manual 103 12 USING THE SETUP SCREEN The SETUP screen is used to change Controller Setup see Section 12 1 to adjust System Settings select onscreen language when available change date amp time update software and calibrate the touchscreen see Section 12 2 and to check or change the Hardware Setup see Section 12 3 Additionally this screen provides the status of installed modules and the firmware version which you will need to know if you speak with a Customer Service representative about your equipment 12 1 Controller Setup When you open the SETUP screen normally the Controller Setup screen shown below will display first If you find any other Setup screen in the display press the Controller Setup tab to open this screen Figure 65 Controlle
112. m exhaust tube into the appropriate port close to a headplate clamping nut where you will later mount the foam trap 2 Finger tighten the knurled adapter If you need to raise or lower the tube at any time use the Allen key provided to adjust the adapter s set screw 4 8 13 Install level probe s If you are using a level probe as part of the antifoam system and or a level probe to detect media level one at a time 1 Working from above the headplate insert the level probe into the appropriate port 2 Finger tighten the knurled adapter 4 8 14 Install addition tube s Insert addition tubes and or tri ports in the appropriate ports for any or all of the following additions media nutrients acid base antifoam For each insertion 1 Finger tighten the knurled addition or tri port adapter 2 Working from above the headplate insert the addition tube or tri port into the appropriate port 4 8 15 Install pH probe 0 Prior to installation any pH probe you are using should be inspected for damage and replaced if necessary 0 To avoid damage to the probes during operation be sure that there is no interference between the probes and the baffle assembly impeller blades or cooling coil 1 Wear protective gloves to protect yourself in case of accidental breakage 2 Lightly coat the pH probe with glycerol BioFlo CelliGen 115 M1369 0050 Operating manual 43 I ALERT Risk of damage to pH probe Always
113. manufactured by Eppendorf 1 1 Hazard Icons General hazard Risk of burns gt gt Electrical shock hazard AN Risk of material damage Explosion hazard 1 2 Danger levels The following danger levels are used in safety messages throughout this manual DANGER Will lead to severe injuries or death WARNING May lead to severe injuries or death CAUTION May lead to light or moderate injuries ALERT May lead to material damage Operating manual 10 1 3 Manual conventions Depiction Meaning gt This prompts you to complete an action 1 Perform these actions in the sequence described 2 n List O NOTICE References useful information 14 Abbreviations d02 amp DO Dissolved Oxygen EPR Ethylene Propylene ID Inner Diameter LEL Lower Explosion Limit OD Outer Diameter OTR Oxygen Transfer Rate rpm Revolutions per minute RTD Resistance Temperature Detector UEL Upper Explosion Limit BioFlo CelliGen 115 M1369 0050 Operating manual 11 2 INSPECTION amp UNPACKING OF EQUIPMENT 2 1 Inspection of box es When you have received your order from Eppendorf carefully inspect all parts of the shipment for damage that may have occurred during shipping Report any damage immediately to the carrier and to your local Eppendorf Sales Order Department 2 2 Packing list verifi
114. mbly Da Aly AA la mios a 15 8 La 14 13 12 11 10 E l i i 2 A z a 3 E i 5 6 7 See legend on the following page BioFlo CelliGen 115 M1369 0050 Operating manual 37 1 Shown installed on the jacket water heater 9 Cooling water inlet cool loop in 2 Agitation motor 10 Base plate 3 Bearing housing 11 Thermowell 4 Headplate 12 Water jacket 5 Cooling water outlet tube 13 Baffle 6 Sparger 14 Top clamping plate 7 Bottom clamping ring 15 Lifting handle 8 Cooling water outlet cool loop return tubing connected inside the jacket 4 8 1 Install headplate clamping ring The clamping ring that secures the headplate to the vessel is split in half to facilitate installation under the vessel flange They are joined with two rectangular mounting plates 1 As shown below install one mounting plate with two Phillips head screws provided on the end of one rin
115. mends silicon tubing for use with the pump heads provided as standard on BioFlo fermentors However Marprene tubing may be used as well as long as the tubing size does not exceed 3 16 bore x 1 16 wall Marprene tubing of this size or smaller can be used with Watson Marlow 101 pump heads under low pressure and with clockwise rotation Operating manual 142 Take note that silicon tubing should not be used with hydrochloric acid HCL sulfuric acid H2504 or sodium hydroxide solutions since this material deteriorates rapidly when in contact with such solutions Another reason for avoiding HCL is that HCL and to a lesser extent H2SO causes corrosion of stainless steel NaOH solutions equal to or less than 20 can be used in silicon tubing at temperatures less than 120 F without destroying the tubing Solutions of sulfuric acid less than 10 can cause moderate damage to silicon tubing 21 4 Acid amp base Question What concentration and type of acid and base should be used Answer The acid solution is 2 3N H2804 The base solution is either 5N NaOH or NH4OH 29 which is the standard commercially available concentration Note that these are fairly concentrated The acid can affect the stainless steel parts of the fermentor vessel To avoid damage to the entry ports it is a good idea to use a sterile disposable needle at the end of the addition tubing and to add the acid or base through the disposable needle The ne
116. mentor is supplied with two properly sized Rushton Impellers Placement of the impellers should be as indicated in Section 4 8 5 In some processes users may wish to use a third impeller Should this be the case however a smaller impeller diameter is required since the systems are specifically designed such that the vessel diameter motor impellers to produce a specific OTR When any of the factors is changed other features may also change For example the standard impeller used on the 10 liter BioFlo CelliGen 115 has a 3 24 inch 0 015 diameter If three impellers are to be used 3 06 diameter impellers are required This size impeller is normally used in a 7 5 L BioFlo CelliGen 115 vessel These impellers should be placed such that the bottom impeller is placed one impeller diameter from the bottom of the vessel The second impeller should be placed one impeller diameter above the bottom impeller and the third impeller should be placed one impeller diameter above the second To determine the horsepower utilized by a given number of impellers the following formula can be used The impeller diameter varies to the 5 power with respect to horsepower A very slight change in the diameter of an impeller can make a great deal of difference in the HP required to drive that impeller The approximate horsepower utilized to drive a given set of impellers is determined as follows HP D x RPM x 4 5 x 109 x I Where HP Horsepower D
117. mpressed position Expand it before you squeeze it for installation so it will spring back against the vessel walls 3 Align the headplate holes with the vessel stand pillars then slide it down until it rests securely against the vessel flange 4 Finger tighten each clamping nut a little at a time to secure the headplate on the vessel stand working diagonally from one to another rather than working around the circle to apply equal pressure I ALERT Risk of damage to vessel gt To avoid vessel stress cracks especially during autoclaving never overtighten vessel clamping nuts 4 8 23 Install vessel Position the vessel next to the control cabinet in the rounded cut out designed for vessel placement between pumps and connectors Be sure to keep the water line quick connects to the left BioFlo CelliGen 115 M1369 0050 Operating manual 53 Figure 28 Vessel Location 1 Place vessel here 4 8 24 Install motor assembly 1 Position the motor assembly on top of the bearing housing using the locating pin or locating slot if applicable to orient it properly 2 Connect the motor cable to its receptacle on the control cabinet 4 8 25 Make all connections 1 Connect cables from all probes to their respective sockets on the face of the control cabinet 2 Connect the exhaust condenser to the exhaust condenser port 3 Using flexible tubing connect the exhaust fil
118. multidrop identification number Operating manual 110 Figure 74 Hardware Setup Screen te Setup Screen New Brunswick Fermentation Mode 1 HE 07 Dec 2008 15 38 Controller Setup System Settings sete Et sr Oe P Module Status Good Errors Sial Unit1 Base Power Active 367683 87 Main phiDO Active 053090 1 aa TMFC1 Active 104957 0 lt lt lt S Hard Unit2 Module Status Good Errors Base Power Active 364980 74 Con het gt gt gt Main phiDO Active 052704 0 TMFC1 Active 104113 1 SCADA lt lt lt Mode NBS Modbus z Unit3 Module Status Good Errors gt gt gt Unit ID 2 2 lt lt lt ES Summary Calibration Cascade 3 Pumps e 1 The dark blue Unit Name tab indicates that the hardware shown in this screen belongs to Unit 1 2 This the Unit ID is this system s multidrop number To add new hardware such as a new utility station remember to do this one at a time after you connect the module to the system 1 Press the Scan Hardware button in this screen All new hardware scanned will appear in the New Hardware box see the first screen on the following page 2 Press the gt gt gt button for the Unit name you wish to assign Unit2 for example and the new hardware list will move into that system s Module box see the second screen on the following page 3 Toreassign a Unit name press the lt lt lt button next to the original system s Module
119. n setpoint and PV process variable O Changing these may seriously affect your system s performance If you think you may have accidentally changed the P amp l values press the Factory Default button to return to the original settings See Section 19 4 for more information on P amp I Gains 4 Limits Here you adjust the high and low settings for this specific loop When adjusted the scaling for the gauge will also be adjusted to reflect the high and low limits selected 5 Decimal Places Press the appropriate button to display values with 0 1 2 or 3 decimal places 6 2 6 Selecting loop control modes A control mode is the logic by which a controller generates the desired control signal The operator has a choice of control modes the most common of which are ON OFF AUTO and MANUAL Other available control modes in certain cases are O2ENRICH 2 GAS 3 GAS or 4 GAS In cascaded control one sensor influences an actuator that is normally associated with a different sensor The onscreen control mode choice will be the name of the loop chosen to have influence on the actuator See Section 10 for details Control modes vary according to the loop and process mode There are also modes for all of the pumps see Section 11 3 for details Operating manual 68 To change control modes for any of the displayed loops Press either the LoopName or the Control Mode box in the row for the appropriate loop
120. n the proper operation of a P amp I pH control system If the tubing is too large excessive doses will be added The result is that the system will overcontrol alternating in close succession between adding one liquid then the other providing little or no change in pH reading A user selected deadband value is an aid to control pH within the user assigned range no acid or base will be added when the pH value falls within the deadband tolerance above or below the setpoint 5 normal solutions make a good trade off between moderate addition volume and good control characteristics The correct tubing diameter varies a little with process but inside diameters as small as 0 2 mm sometimes eliminate overcontrol while supplying sufficient liquid during high demand culture phases 0 Whatever the tubing ID the tubing wall thickness must be 1 16 inch 1 6 mm Eppendorf suggests that you begin with the supplied tubing which is correct for most applications If the system oscillates reduce the tubing ID where it passes through the pump Use commonly available step up step down adapters and narrower bore tubing to make the tubing modifications if required Consult Table 6 the flow rate tubing size chart for further information 9 5 Reconnect gases Ensure that all gas lines air oxygen etc are routed to the appropriate ports and secured at both ends with plastic ties 9 6 Install temperature RTD probe Turn the ON OFF mains power s
121. ng The open end of the tubing should be covered with cotton then with aluminum foil The clamp on the tubing be below the foil amp cotton The sparger filter should also be covered but not quite as tightly The exhaust filter is usually not covered All tubing should be inspected both prior to and after autoclaving to insure integrity The above description also applies to any side harvest ports in use Note that this type of port is often below the media fill line It is also possible to use a hose that has been tied off and crimped at one end to provide a cap for the base port amp addition port as well as other ports These caps must fit very securely over the port in order to avoid loss of sterility due to displacement while autoclaving All O rings should be checked for damage prior to autoclaving All fittings must be checked for tightness A loose fitting is often an indication that the small O ring in the fitting assembly requires replacement Verify that the bottom of the glass cylinder is properly secured to its base The agitation shaft must have its protective cap on prior to autoclaving It is advisable to check that the connectors from the system to the vessel exhaust gas condenser are compatible This is a good time to check that the air and water lines to the system are open and that if required an oxygen source is available and correctly connected The pH probe must be inspected prior to insertion enough electrolyte must
122. ng on the vessel stand align the clamping ring holes with the vessel stand pillars then slide it into place It will come solidly to rest on the shoulder of each pillar 2 Place sections of U shaped rubber bumper equidistantly around the inside of the clamping ring there are three pieces for 1 3 L amp 3 0 L vessels and two larger pieces for 7 5 Land 14 0 L vessels Press each section securely against the inner edge of the ring Figure 17 Upper Vessel Bumper Installation 1 Vessel clamping ring 2 Section of rubber bumper your vessel may have as few as two BioFlo CelliGen 115 M1369 0050 Operating manual 35 3 Gently lower the glass vessel through the center of the clamping ring until the vessel flange rests snugly against the rubber bumpers 4 Orient the vessel so the gradations on the glass are clearly visible at the front facing the user and situated between two vessel stand pillars 4 7 4 Install baffle 14 0 L fermentation vessels ONLY For installation of the 1 3 L 3 0 L and 7 5 L vessel baffle see Section 4 8 21 If you are using a 14 0 L vessel install the baffle assembly inside the glass vessel 1 Gently compress the baffle ring at its ends to avoid scratching the vessel walls You may find it convenient to squeeze the tab with your thumb 2 Slide the assembly inside with the tab facing up until it comes to rest at the bottom of the vessel 3 Orient the baffle s
123. nnnncncncnnononnnnnnocononnononnnnnnioniccccnnononos 113 13232 INOCULATION EE E E AAIE AAEE E A 114 13 4 START BIOCOMMAND IF PRESENT usais tinta ironia 115 13 5 GSAMPLING PROCEDURE cui tea 115 13 6 FERMENTATION PHASES ccccccccccccssssssesccccccceusesssssccccceseuuessssececcceseuuuussececcesesaeenens 116 AS DS O COP RR CRS E A e SPIO IT Re Nr MRP Terre en 116 13 6 2 Exponential growth PASO anta A a A A EE AS 116 SA II AAA O AN 117 136 4 Decline phase cucd auiiiieatncaw ac inne a 117 13 7 BATCH OPERATION cccccccccccccccccssssscscccccccsesssssescccccessesssssesccsscesseussscsccesseeseassssesces 117 13 8 FED BATCH OPERATION ccccccccccccccsssssssccccccccusssssesccccceseususssescecccescaueussscescsseeaaseens 117 13 9 CONTINUOUS OPERATION csi ai blind 117 13 10 ANAEROBIC AND MICROAEROPHILIC CULTURE csssssseccccccccesssssscecccccessenseseceses 118 13 11 HARVESTING PROCEDURE cssssssceccccccccsssssscecccccccseusnsscscceseessuuessscscceseseeausnsssses 118 13 12 SHUTDOWN PROCEDURE cscsceccccccccesesssesceccccccessussescecccsceesuusaesesccesseaeuaeaescecceseeaes 119 14 ESSENTIAL OPERATING TIPS cccccscscscssssssssscssscssscscscccccccccccscscscscscscsesesssess 120 14 1 PRECAUTIONS FOR GLASS VESSEL ASSEMBLY cccccccccccccssssssssccccccccssesssscecccseeueenens 120 14 2 EXHAUST CONDENSER amp EXHAUST FILTERS cccccceccccccccssssssssccccceccussaesssceccesesaeenens 120 14 3 INSTALL A DOUBLE FILTER SYSTEM
124. o CelliGen 115 M1369 0050 Operating manual 117 13 6 3 Steady state phase Most of your run will be the desired steady state of growth As long as the temperature pH DO and other essential parameters are stable and you feed your batch appropriately this phase can last for a standard E coli fermentation for example approximately 2 3 hours Eventually however you must expect your batch to decline 13 6 4 Decline phase This final phase is marked by a slow dying off which is of course inevitable 13 7 Batch operation A batch operation is a closed growth environment in the sense that it contains a finite amount of media The inoculum grows through the various phases of fermentation until it begins to decline and you harvest the desired product It is easy to run and yields results quickly 13 8 Fed batch operation A fed batch operation includes the addition of media to feed the batch fresh nutrient and to dilute any build up of toxic by products in the broth thereby extending the life and growth of the desired product Figure 78 Fed Batch Operation 1 Fresh media 13 9 Continuous operation A continuous operation is exactly as its name suggests as broth is harvested fresh medium is added to make more The fermentation vessel contains at all times the optimum amount of media with an established thriving culture Operating manual 118 Figure 79 Continuous Operation
125. o the opening is opposite the gradations on the vessel and the tab is aligned with the back vessel stand pillar 4 8 Vessel assembly water jacketed Water jacketed vessels need no stand the water jacket which is part of the vessel is flared and flat at the bottom to provide secure stable support At the bottom is a metal base plate to provide additional security against breakage In operation the jacked vessel sits on the Jacket Water Heater The jacket water heater is designed so that the vessel water inlet and outlet fit in a notch at the rear and the vessel feet fit into the four holes at the perimeter of the heater plate Figure 8 on the following page shows a typical installation of the double walled water jacketed vessel with the most commonly used accessory equipment k ALERT Risk of damage to equipment The Jacket Water Heater base see the following page includes a magnetic stir bar and plate For stability during shipping the stir bar is tied to the inner cage by cable gt Do not fill the water jacket or operate the vessel until you have cut the cable ties and released the stir bar Familiarize yourself with the arrangement of the headplate ports as shown in Section 4 7 1 before proceeding with the vessel assembly You may find it more practical to change the arrangement the variety of ports and adapters will easily accommodate your needs Operating manual 36 Figure 18 Water Jacketed Vessel Asse
126. ol Settings Recommendations for 143 Pump Array Standard 96 Pump Assignment 96 Pump Assignment Screen 101 Pump Calibration 99 102 Pump Control Modes 99 Pump Flow Rate 99 Pump Period sec 100 Pump Screen 71 Pump Setpoints 97 R Regulatory Compliance 58 Removing a Utility Station 112 Renaming Control Loops 66 Replacement Parts 125 Rotameter 13 RTD 41 RTD Probe Installation of 92 S Sampler Installation of 47 Sampler Tube Installation of 41 Sampling 115 Save Changes Button 104 Saving a Process Configuration 134 135 Service 129 Service Connections 19 23 Service Utility Electrical 23 Setting Up the Vessel 145 Setup Screen 72 103 Shutdown 119 147 Spare Parts 125 Sparger Installation of 40 Start Up Screen 61 Steady State Phase 117 Sterilization Preparing for 85 Sterilization Temperature 87 Sterilization Time 87 Operating manual 154 Sterilizing the Vessel 145 Summary Screen 61 Summary Screen Features 61 Supervisory Software 15 T Table Index 132 Table of Contents 8 Temperature Control 13 RTD 13 Setpoint 13 Temperature Probe Installation of 92 Terminators Installation of 21 Thermowell Installation of 41 Touchscreen Calibrating the 108 Troubleshooting 129 Tubing Recommendations 141 Tubing Size 141 U Unused Ports 51 Utilities 22 Utility Station 12 Adding a 110 Removing a 112 V Vessel Description of 12 Installati
127. ol station 3 2 Vessels One of the most versatile features of the BioFlo CelliGen 115 is the wide variety of glass vessels available There are two types of vessels non jacketed heat blanketed and water jacketed Each type of vessel is available in four sizes 1 3 liters 3 0 liters 7 5 liters and 14 0 liters Ports in the headplate are provided for but not limited to the following purposes inoculation base and acid addition a thermowell for a resistance temperature detector RTD a foam probe a sparger a harvest tube a sampling tube an exhaust condenser and dissolved oxygen DO and pH electrodes The drive bearing housing is also located on the headplate 3 3 Agitation system A removable agitation motor located on top of the bearing housing on the headplate is connected to the agitation shaft with a direct drive coupling or a magnetic coupling The motor can be easily disconnected before autoclaving the vessel and easily replaced after sterilization The motor will provide a speed range from 50 to 1200 rpm for fermentation with direct drive from 25 to 400 rpm for cell culture with direct drive or from 25 to 200 rpm for cell culture with magnetic drive The process control software ensures agitation speed control throughout the speed range It is possible to cascade Dissolved Oxygen DO to Agitation AGIT so the agitation speed will vary between the user specified minimum and maximum setpoints in order to maintain the set p
128. on is at 121 C When sterilization is complete check the exhaust line to verify that it didn t crimp and check the vessel s integrity 21 7 3 Post sterilization vessel set up The vessel must be handled gently when removed from the autoclave to prevent the media from boiling up Confirm that any unprotected vented lines are clamped off upon removing the vessel from the autoclave Check the vessel s integrity again then transport it to the bench system Place the vessel next to the control cabinet The orientation must allow for proper hook up to the the exhaust gas condenser lines Connect the water lines connecting the outgoing return lines before the incoming delivery lines and ensuring that the delivery and return lines are not inverted Insert the temperature probe into the thermowell Check that the water lines to the system are open Set the temperature value below ambient temperature and set the control to Auto After 2 5 minutes the system can be switched to the desired temperature setting This can be checked by making sure that water is truly leaving the system observe the water drained through the Drain or Water Out port Operating manual 146 Remove the protective caps from the pH and DO probes and connect the probes to the system Be careful with the pH probe do not twist the probe into its connection to the system as this can compromise sterility The connection must be screwed onto the probe The pH probe s
129. on of 52 Vessel Assembly Non Jacketed 29 Vessel Assembly Precautions 120 Vessel Bumpers 34 38 Vessel Cleaning 122 147 Vessel Operation 146 Vessel Preparation for Autoclaving 143 Vessel Pressurization 25 Vessel Set Up 145 Vessel Shutdown 147 Vessel Size Changing the 105 Vessel Stand 34 Vessel Sterilization 145 W Water amp Drain Connections 24 Water Jacket Filling the 38 Wetted Parts 122 BioFlo CelliGen 115 M1369 0050 Operating manual Evaluate your operating manual www eppendorf com manualfeedback eppendorf In touch with life Your local distributor for New Brunswick products www nbsc com ContactUs New Brunswick Scientific 175 Freshwater Boulevard Enfield CT 06082 4444 USA Eppendorf AG 22331 Hamburg Germany Tel 49 40 538 01 0 Fax 49 40 538 01 556 E mail eppendorf eppendorf com New Brunswick Scientific Europe B V Nijmegen The Netherlands Tel 31 0 24 3717 600 E mail europe nbsbv nl Eppendorf North America Inc Hauppauge NY USA Tel 1 516 334 7500 1 800 645 3050 E mail info eppendorf com Application Support Europe International Tel 49 1803 666 789 E mail support eppendorf com North America Tel 1 800 645 3050 menu option 2 E mail techserv eppendorf com Asia Pacific Tel 603 8023 6869 E mail support_asiapacific eppendorf com
130. or off manually as long as you press the button the pump will run continuously When you release the button the pump will stop running 0 If pumps are linked to a cascade this may affect the ability to manually change setpoints and control modes To select a Control Mode for any pump press the appropriate button in the Control Mode pane of the PUMPS gauge screen 11 4 Pump flow rate amp calibration methods The pump will always run at the same speed but its flow rate depends on the diameter of tubing you use Table 6 provides the pump flow rates according to various tubing diameters Table 6 Flow Rate per Tubing Size Tubing Wall Thickness 1 16 inch 1 6mm Inside Diameter inch mm 1 50 0 5 1 32 0 8 1 16 1 6 1 8 3 2 3 16 4 8 12 rpm Flow mL minute 50 Hz 0 25 0 60 255 9 44 190 12 rpm Flow mL minute 60 Hz Pump speed will vary slightly depending on frequency To calibrate any pump with the tubing you have selected 1 Load approximately three feet of the tubing into the pump head 2 Set up a reservoir with water at the input end of the tubing and an empty graduated cylinder capable of measuring small quantities at the output end of the tubing 3 Read this step completely before you do it with the input end of the tubing in the water reservoir prime the tubing line by pressing the pump s Prime button but allow it to run only until liquid starts to flow into the tubing
131. ouchpad to enter a pump cycle time in seconds For example if the pump setpoint is 30 setting a period of 5 seconds as illustrated will cause the pump to run 1 5 seconds stop for 3 5 seconds then cycle back on again 0 Running at a very low percentage renders the totalizer s results inaccurate We recommend the use of smaller tubing to avoid choosing a very low percentage for the pump setpoint 11 6 Using level probes to program feed pumps 11 6 1 Setting a feed pump to add liquid A feed pump can be set to add liquid whenever the associated level probe installed in the vessel informs the pump that an addition is needed to maintain level Prior to autoclaving the vessel make sure that the level probe that you wish to use is fully inserted into the vessel When the vessel is set up at the control cabinet raise the probe to the level at which you want addition to begin Never lower a probe after autoclaving 1 Open the PUMP screen 2 Select the feed pump you wish to pump liquid into the vessel and press that pump s ASSIGNMENT button to open the PUMP ASSIGNMENT screen Figure 64 Pump Assignment Screen Assignment Foam Lvlit Acid Lvi2 Wet Base Lvi2 Dry 3 Press the Lvl2 Dry button which corresponds to the probe s connection on the control cabinet 4 Press the Summary navigation button to save the pump assignment and to return to the SUMMARY screen In DRY control mode e when the liquid is not in contac
132. per working condition When performed routinely maintenance results in longer life for your equipment It also reduces time lost due to equipment failure LN WARNING Risk of electrical shock gt Always turn your BioFlo CelliGen 115 off and disconnect the mains power cord before performing maintenance 16 1 PH probe maintenance and storage The pH probe should be stored standing upright with the electrode tip immersed in a solution of 3 molar KCI or a buffer solution between pH 4 00 and pH 7 00 k ALERT Risk of damage to pH probe gt Never let a pH probe rest on its tip gt Never leave a pH probe in DI water 16 2 DO probe maintenance and storage Use soft facial tissue to clean the DO probe Check the probe s Teflon membrane to be sure there are no punctures puckers or wrinkles If there are the probe should be replaced When it is not in use in the vessel the DO probe should be stored standing upright with the shorting cap in place and the membrane isolated from the air environment At no time should the probe be allowed to rest on its membrane ALERT Risk of damage to DO probe gt Never let a DO probe rest on its tip Operating manual 124 16 3 Vessel amp tubing After each and every run clean the vessel and the headplate with its associated parts All tubing and filters should be replaced 16 4 Periodic inspection 0 To maintain the best possible seal O rings should be repl
133. pump assignment s and to return to the SUMMARY screen 0 For details on the choice of Level Wet and Level Dry see Section 11 6 1 11 2 Pump setpoint To enter a setpoint for a pump Operating manual 98 1 Open the PUMP screen Gauges for Pumps 1 3 are displayed in this screen Figure 61 Setting Pump Setpoint Pump Screen New Brunswick Fermentation Mode 07 Dec 2008 15 43 Pumpi ii Pump2 Wi Pump3 Out Mult 100 0 Setpoint 50 0 Setpoint 0 0 PV 0 0 Pv 50 0 Pv 0 0 Control Mode Control Mode Control Mode Assignment Acid Assignment None Assignment None ow Rate mL Second ow Rate mL Second ow Rate mL Second Calibrate Calibrate Calibrate q OB q OB HEE Calibrated Calibrated Calibrated 2 000 0 000 0 000 Total Total Total 600 000 0 000 0 000 Period Sec 10 Period Sec 10 Period Sec 10 Es Summary Calibration a Cascade ES A Setup 2 Press the Setpoint edit box for the pump Pump1 Pump2 or Pump3 3 Use the touchpad that opens to enter the desired setpoint then press the OK button to save it and return to the PUMP screen or press the Cancel button to return to the PUMP screen without saving a setpoint This sample PUMP screen shows Pump 1 assigned to Acid see Section 11 1 for details on assigning a pump Instead of a Setpoint edit box there is an Out Mult Output Multiplier edit box The output for this pump is ca
134. pump you assign to add antifoam see Section 11 1 for details about pump assignment When the foam is gone the probe sensing that it is no longer wet will shut off the antifoam addition pump If on the other hand you assign the probe to be Wet you will position it within the media and calibrate it to be sensitive to dryness Associate this level probe with the pump you BioFlo CelliGen 115 M1369 0050 Operating manual 83 assign to add media so that if the probe becomes dry it will turn the pump on until the probe is wet again Figure 52 Calibrating Level Probes Calibration Screen New Brunswick Fermentation Mode 07 Dec 2008 15 41 oops Calibrating Loop 50 Level Sens 1 Sensitivity Raw Value Level Sens 2 50 0 Dry Es Summary Calibration TA Cascade 3 Pumps X Setup To calibrate the level sensor as Dry expose the dry probe to foam or media depending on the element you wish to control until the Raw Value changes to Wet To calibrate the level sensor as Wet immerse it in media to show Wet as the Raw Value then remove it from the media until the Raw Value changes to Dry Sensitivity is the level at which the probe will turn its associated pump on or off 7 5 About pump calibration To assure the most accurate flow rate calibrate the pump each time you change tubing See Section 11 6 4 for details Operating manual 84 8 VESSEL STERILIZATION 0 Be
135. r Setup Screen Le Setup Screen New Brunswick Fermentation Mode 6 07 Dec 2008 15 36 Controller Setup System Settings Hardware Setup Unit Type Unit Name Vessel Size SS s gt mIHOIHINNvAppA junit 1 7 5 Liter Ea 7 1 No of TMFCs 1 TMFC Range 0 20 SLPM Operatina Mode BioFlo 115 Options 4 Fermentation Mode y pH DO ___________________ 9 e O2 Enrich Direct or r Foam Level Cascade Driven Y Gas Mix r Gas Flow El Summary i Calibration a Cascade 3 Pumps Setup See Figure 41 to select another Vessel Size 2 This pane indicates the options installed on your system See Section 12 3 1 for details After making selections press the Save Changes button to save any new selections AJO The Operating Mode is factory set to Fermentation See Figure 39 to change this 5 Press here the name you enter into this box using the touchpad will appear on a dark blue button tab on the top menu line see item 6 6 The new Unit Name button tab appears here Operating manual 104 If you have more than one station the dark blue Unit Name button tab is the one actively represented in the screen To move to another station s setup parameters press the light blue button When that button changes to dark blue its parameters will be actively represented in the screen and you can make changes See Section 12 1 1 for details on gas control through the Con
136. r installation and start up instructions in addition to the general instructions provided below A 25 pin RS 232 422 D connector com port labeled SCADA is provided on the rear panel of the control cabinet see below to connect the BioFlo CelliGen 115 to a supervisory host computer Figure 30 RS 232 422 Interface 1 SCADA connector Operating manual 56 Communication to BioCommand software is assured via an optional RS 232 interface cable 1 Connect the 25 pin end of the RS 232 cable to the SCADA port and make sure that the connection is secure 2 Hand tighten the thumbscrews 3 Refer to the BioCommand operating manual for instructions on connecting the RS 232 interface cable to the supervisory host computer A New Brunswick BioCommand advanced supervisory software program is available which will enable the operator to interface with a computer that has a Windows 2000 or higher operating system With this software you will be able to establish or change the setpoints for temperature pH DO agitation speed and pump flow rate You will also be able to read and log the current values of any parameters temp pH DO air flow pump flow rate levels and agitation that are monitored The data can also be stored plotted and afterwards transferred to other commonly available programs to be manipulated and analyzed in various ways Table 3 iden
137. re Slide the impeller onto the agitation drive shaft from the bearing housing Position the impeller at least 10 mm above the sparger Clamp it down in place 0 It is normal for the agitation impeller shaft to be very resistant to turning by hand The shaft seal resistance ensures sterile operation B For Fermentation Slide one impeller onto the agitation drive shaft from the bearing housing Position this lower impeller according to the table below Clamp it down in place Then install the second upper impeller in the same manner Table 2 Impeller Positions Distance from Bottom of Headplate to Top of Impeller Blade 14 0L Lower Impeller 105 mm 170 mm 225 mm 305 mm 4 1 8 in 6 11 16 in 8 7 8 in 12 in Upper Impeller 67 mm 102 mm 165 mm 235 mm 2 5 8 in 4 in 6 in 9 in 0 The distances indicated above provide a recommended starting point As working volumes and agitation rates change you may wish to adjust the impeller location s 0 It is good practice to lightly lubricate all O rings port threads and adapter threads with silicone grease part number P0860 1050 before you install equipment in the headplate Also inspect the headplate O ring to be sure it is securely seated in its groove For cell culture you may want to use IPA or glycerol instead of silicone Operating manual 40 4 8 6 Install cooling coil 1 3 L Vessel Cooling Coil Sparger Assembly The cooling coil and sparger connection
138. re using the vessel for consecutive runs with the same media rinsing it with warm tap water and with DI water may suffice Note that if water will run over a vessel surface that is greased the grease should be removed wipe it off with a wet paper towel In cases where the vessel must be decontaminated prior to cleaning add water so that the liquid level reaches the maximum working volume of the vessel This will help prevent biological materials from adhering BioFlo CelliGen 115 M1369 0050 Operating manual 149 22 APPENDIX D CORROSION RESISTANCE Websites such as www outokumpu com provide up to date information about the 316 type stainless steel used in your BioFlo CelliGen 115 vessels Operating manual 150 23 1 23 APPENDIX E GENERAL CHARACTERISTICS OF EPR Identifying EPR EPR EPT EPDM Trade Names Resist O NordleR Compound No AX 60660 OCA SS AA ASTM D 2000Classification CA Military MIL STD 417 Chemical Definition Ethylene Propylene 23 2 General Characteristics Durometer Range Shore A 30 90 Eppendorf uses 80 for most O rings Tensile Range P S l Elongation Max 800 ooo Compression Set Resilience Rebound Abrasion Resistance Tear Resistance Solvent resistance Oil resistance Low Temperature Usage High Temperature Usage Aging Weather Sunlight FairtoGood S Adhesion to Metals Fair to Good Ethylene Propylene is a polymer with outstanding properti
139. ressure release 0 Filter manufacturers generally advise limiting filter sterilization to 30 minutes but the longer time required for slow exhaust is essential to protecting the vessel integrity New Brunswick s long experience has shown no adverse effects at all on filters exposed to longer autoclaving times Adjust the time and temperature as needed If after autoclaving most of the liquid has left the vessel the autoclave is exhausting too quickly Adjust the autoclave to exhaust more slowly Operating manual 88 9 REINSTALLING THE VESSEL ASSEMBLY 9 1 Reinstall the vessel assembly I ALERT Risk of damage to vessel gt Cold water and hot glass is a potentially dangerous mix Be sure to let the vessel cool for a few minutes before reconnecting the water line 1 Position the vessel next to the BioFlo CelliGen 115 control cabinet Connect the water lines to the heat exchanger and the exhaust condenser see Vessel Assembly section Connect the drain line Connect the Cooling Loop In and Cooling Loop Return between the cabinet and the vessel Connect Exhaust In and Return between the cabinet and the exhaust condenser if present Secure all connections Connect the Water In to your water supply Turn your water utility on to 10 PSIG Carefully place the motor on the bearing housing on top of the vessel assembly Remove the pH shorting cap and connect the pH cable to the pH connector on the control
140. rol cabinet As shown below they are labeled from top to bottom Pump 1 Pump 2 and Pump 3 Figure 31 Standard Pump Array smm oc y 11 1 Pump assignment If there are pumps in your configuration the user has the ability to assign each pump present in the system To assign a pump 1 From any screen press the PUMPS button at the bottom to open the PUMP screen see the following page BioFlo CelliGen 115 M1369 0050 Operating manual 97 Figure 59 Pump Screen Pump Screen New Brunswick Cell Culture Mode 07 Dec 2008 15 35 Pump1 Setpoint 0 0 Setpoint 0 0 Setpoint 0 0 Pv 0 0 PV 0 0 PV 0 0 Control Mode Control Mode Control Mode MA A O E Flow Rate mL Second Flow Rate mL Second 7 Flow Rate mL Second a Calibrate Calibrate Calibrate Calibrated Calibrated Calibrated 2 000 0 000 0 000 Total Total Total 600 000 0 000 Reset 0 000 Reset Period Sec ii Period Sec 10 Period Sec 10 ES Summary Calibration T Cascade PTH Setup 1 Step 2 Press the Pump 1 Assignment button The Pump Assignment screen will open Figure 60 Pump Assignment Screen Assignment Acid LvIi2 Wet 1 Step 3 Press the button that corresponds to your choice of assignment for Pump 1 It will turn green 4 Repeat Steps 2 amp 3 for the other pumps you wish to assign 5 Press SUMMARY to save the
141. s are welded into one special 12mm tri port assembly 1 From beneath the headplate insert the assembly into the appropriate port s 2 From above the headplate lock the assembly in place with a knurled 12mm to 12mm adapter Finger tighten 3 There are three set screws in the adapter If you need to raise or lower the adapter tri port assembly use the Allen key provided to adjust the set screw that is easiest to access You only need to adjust one 3 0 L 7 5 L amp 14 0 L Vessel Cooling Coil 1 From beneath the headplate insert both ends of the coil into the Cooling Coil In port and the Cooling Coil Out port 2 From above the headplate finger tighten the knurled adapter on each side of the cooling coil 4 8 7 Install sparger 3 0 L 7 5 L amp 14 0 L vessels 1 From beneath the headplate insert the sparger tube into the sparger port 2 Finger tighten the knurled adapter on the sparger then use the Allen key provided to tighten the set screw Do not overtighten I ALERT Risk of damage to ferrule gt Only finger tighten any adapter that has a white Teflon ferrule tapered cone shaped insert under the Teflon washer The ferrule can deform under too much pressure BioFlo CelliGen 115 M1369 0050 Operating manual 41 4 8 8 Install harvest tube 1 Working from beneath the headplate install the harvest tube in the harvest port If you are using the 1 3 L vessel the harvest tube and sampler
142. s screen gives you access to the Pump Gauges screen where the three pump gauges are displayed providing both current readings and the opportunity to change pump settings Setup This screen allows you to make changes to your system settings hardware setup amp controller setup 6 2 4 Keypads When an alphanumerical or a numerical keypad is needed for you to put information into edit boxes clicking in the edit box will open the required keypad see the following pages BioFlo CelliGen 115 M1369 0050 Operating manual 65 Figure 36 Alphanumeric Keypad 1 I Setup Screen New Brunswick Scientif Fermentation Mode Unit Name Unit 1 E Caps Lock Clear BackSp TEE Cancel 3 o This keypad is used to designate a Unit Name What you type on the keypad appears here AIOIN gt Pressing the Cancel key clears the entry and closes the keypad When you have finished typing the entry press the OK key to save the entry and close the keypad Pressing the BackSp backspace key clears the entry one character at a time each time you press the key without closing the keypad Pressing the Clear key clears the entry without closing the keypad Operating manual 66 Figure 37 Numeric Keypad 1 ful Gauge Screen New Brun ick Scientific Fermentation Mode Setpoint RPM Agit 250 Cancel A A Cl
143. sembly components from steam damage The first objective is met by leaving at least one vessel port open the second by clamping shut flexible tubing attached to immersed penetrations and the third by wrapping filters with a protective cap of aluminum foil Use protective caps on probes and bearings to meet the fourth objective 8 1 Initial preparation for autoclaving To prepare the vessel for sterilization Remove the motor from the top of the vessel and carefully put it aside Lubricate the vinyl bearing housing cap with silicone grease to facilitate sliding the cap securely onto the housing Place the bearing housing cap on the top of the bearing housing Disconnect the air and or gas lines from the inlet filter on the sparger Disconnect the water lines Remove all PVC tubing Clamp off the harvest tube the sample tube and all other penetrations that are immersed in the media Remove the RTD from the thermowell Disconnect all probes and sensors and remove their cables If you are using pH and DO probes install each probe s shorting cap provided in the probe kit 10 Before placing the vessel into the autoclave loosen the glass sample bottle by turn 11 Wrap all filters with aluminum foil to protect them from steam 12 Attach a piece of tubing wrapped with some non absorbent material such as glass wool or non absorbent cotton to each of the addition ports Wrap foil around the end of the tubing shaped like a funnel
144. ssceccccccccssssssssccccceseuueasssececcsecsuuenssscecceseeauenens 134 19 2 WHAT IS A CONTROL LOOP ccseseececcccccccssssescecccccsscsusesscecceccessuusassceccesseeseuensscsses 134 19 3 WHAT IS PROBE CALIBRATION cccccccccccccceccccccccceccecceeeeeececeeeeeceeecseececeeecececesevevecs 134 19 4 WHATARE P I D CONSTANTS satis lianas idas 134 19 5 WHATISP ED TUNING ER AAA AAA ARA 135 19 6 WHAT DO THE CONSTANTS MEAN ccccccccccccccssssssseccccccccssssessecccccsccauessssceeccsesauenens 136 20 APPENDIX B OT R ieiccictelededeicccesedcstedscslevcdeckcstededslevccevdceestacatevscdistivatsvatavacdedcivscseate 137 20 1 DETERMINING AN OXYGEN TRANSFER RATE ooococcccconononnnanicicncnnonononanirncccnconononananioss 137 20 1 1 OTR calculator 137 20 2 SOME FACTORS THAT AFFECT OTR AND HORSEPOWER coooccccconononnnnnicocncnnnnononanininos 138 21 APPENDIX C FERMENTATION TECHNIQUES u scccsssssscccsssrcccssssecees 140 21 1 MEDIA FORMULATION cccccccccccccccsssssesccccccccsssssssscsccceccsseesssesccscsesseaeessssccscceseuaeeens 140 21 2 ANTIFOAM FORMULATION cccccccccsssssssscccccccceesssssscecccccesseueaesceccscccssuuesesscecccessuaenens 141 2413 TUBING ZE DL Sab II E iE LAOS e dido 141 DNA ACID SN BASE ii A EE ATA AD es 142 Qed GLUCOSE FEED a tcs 142 21 6 RECOMMENDED PROCESS CONTROL SETTINGS 0cooooocccncnnononnnnnnoncnnncnnononnnnnacncncncnnononos 143 21 7 TYPICAL FERMENTATION RUN cssssssceccccccccssssssssc
145. t filter s is are not wet or clogged Also loosen the inoculation diaphragm cap for additional venting e After autoclaving tighten the inoculation cap When the vessel is installed on the control cabinet and air is freely flowing through it you may retighten all nuts and screws again taking care not to overtighten 0 To maintain the best possible seal O rings should be replaced every six months or more frequently if needed 14 2 Exhaust condenser amp exhaust filters The inner assembly of the exhaust condenser can be removed for cleaning 1 Pass warm water and detergent through the top of the condenser but not through the quick connects Do this twice 2 Run clear water through once 3 Blow out with air 4 Autoclave Clean the exhaust condenser after each run This is most critical when operating as a chemostat for protracted fermentation times 14 3 Install a double filter system Double exhaust and double inlet filters are recommended To install them 1 Attach a Y fitting to the top of the condenser with a piece of tubing Be sure to secure the tubing with a tie at each end BioFlo CelliGen 115 M1369 0050 Operating manual 121 2 Attach an exhaust filter to each branch of the Y This allows you the flexibility to exchange sterilized filters during a run should one filter become clogged all you have to do is pinch off the unused line with a clamp Operating manual 122 15 CLEANING
146. t with the probe the feed pump is turned on so that more liquid will be added e when the liquid is in contact with the probe the pump is turned off Operating manual 102 11 6 2 Setting a feed pump to harvest A level probe can also be used to set up a feed pump to harvest Prior to autoclaving the vessel make sure that the level probe that you wish to use is fully inserted into the vessel When the vessel is set up at the control cabinet raise the probe to the level at which you want harvesting to begin i e above the current liquid level Never lower a probe after autoclaving Open the PUMP screen Select the feed pump you wish to pump liquid out of the vessel and press that pump s ASSIGNMENT button to open the PUMP ASSIGNMENT screen see Figure 34 repeated above Select the Lvl2 Wet button which corresponds to the probe s connection on the control cabinet In WET mode e when the liquid is not in contact with the probe the pump is turned off e when the liquid is in contact with the probe the pump is turned on 11 6 3 Level control off When OFF is selected from any level Foam HiFoam Lvl2 Wet Lvl 2 Dry Acid or Base control mode menu the pump is off 11 6 4 Pump calibration 0 To assure the most accurate flow rate calibrate the pump see Section 11 4 each time you change tubing Pump flow rates are provided in Table 6 Section 11 4 However more accurate flow rates through the various
147. ter to the top of the condenser Secure it with tubing ties 4 Insert the RTD into the thermowell 5 If you have not already done so connect the sparge line silicone tubing to the inlet filter WARNING Risk of explosion gt Never block the exhaust to pressurize the vessel see Section 4 6 Operating manual 54 4 9 ON OFF switch The ON OFF mains power switch is located on the righthand side of the control cabinet as you face the touchscreen see the drawing below Be sure to read the safety note on the following page before you turn the system on Figure 29 ON OFF Mains Power Switch 6 Yr 4 o gt omens Elo ot ETHERNET port not in use at this time USB port ON OFF mains power switch WIN BioFlo CelliGen 115 M1369 0050 Operating manual 55 k ALERT Risk of damage to equipment gt Before turning on the ON OFF mains power switch make sure that 1 The input water hose is connected the drain line is connected and the water supply is turned on 2 The vessel is in place and the quick connect water lines are connected to the vessel s heat exchanger 3 The mains power cord is properly connected to the control cabinet and plugged into a suitable mains power outlet 4 10 Optional BioCommand software If you are using New Brunswick supervisory software be sure to consult your BioCommand user s manual fo
148. the SETUP screen see Section 12 When you press the SETUP button the screen that opens is actually the first tab the CONTROLLER SETUP screen Figure 45 Controller Setup Screen 1 2 Setup Screen New inswick Fermentation Mode MI 07 Dec 2008 15 36 Comtroller Setup System Settings lI rdware Setup Unit Type Unit Name Vessel Size BioFlo 115 unit 1 7 5 Liter M 3 5 X No ofTMFCs 1 TMFC Range 0 20 SLPM Operatina Mode BioFlo 115 Options 4 gt Fermentation Mode v 7 pH DO e O2 Enrich Direct or 7 Foam Level Cascade Driven 7 Gas Mix r Gas Flow E Summary Calibration A Cascade El Pumps gt Setup 1 Controller Setup tab 2 The Unit Name is user selected Press inside this box then use the pop up keypad to type in the desired name 3 The Vessel Size is user selected press the W to access the dropdown menu then press the appropriate vessel size Choosing the correct vessel size here assures the application of accurate PID values 4 The default Operating Mode is Fermentation To select Cell Culture press the V then select Cell Culture from the dropdown menu 5 The number of TMFCs 0 means manual gas flow usually by Rotameter and the TMFC Range are factory set BioFlo CelliGen 115 M1369 0050 Operating manual 73 Figure 46 System Settings Screen 1 Py Setup Screen New Brunswick Controller Setup BESET Hardware Setup Touc
149. the kit as follows using the following drawings for reference 1 Remove a headplate clamping nut adjacent to the location of the sampler tube 2 Mount the metal sampler bottle holder arm on the clamping screw and secure it in place with the clamping nut Operating manual 48 Figure 25 Sampler Harvest System 1 3 L Vessel Mame 4 Cy if I I I I I g A o f 2 ii I fra 6 7 14 9 10 0 11 13 12 1 Syringe 8 Headplate 2 Syringe filter 9 Spare tube 3 Thumb clamp 10 Sample tube 4 Reducing elbow to 4 76 mm 3 16 in OD 11 Harvest tube tubing on sampler holder 5 3 2 mm 1 8 in ID silicone tubing 12 Headplate clamping screw 6 Headplate clamping nut 13 Sampler bottle 7 Sampler harvest port 14 Sampler bottle holder part number P0740 2396 BioFlo CelliGen 115 M1369 0050 Operating manual 49 Figure 26 Sampler System 3 0 L 7 5 L amp 14 0 L Vessels 1 2 4 LK P P l a ani l ae 11 i AT 5 Ga A aT 6 10 1 Syringe 7 Sample tube 2 Syringe
150. the probe then follow with the black 12mm port adapter O ring 7 Do not yet close up all the elements of the port adapter 8 Gently insert the probe into the appropriate port allowing the O rings to seat fully into the port 0 The fit may be snug Gently rotate the probe as you press it into the port to avoid breakage 9 Finger tighten the bottom portion of the port adapter into the port 10 Adjust the probe to the desired height then nesting the ferrules close the top portion of the adapter onto the bottom portion 11 Finger tighten the knurled adapter assembly Operating manual 78 I ALERT Risk of damage to equipment gt We recommend that you avoid the use of hydrochloric acid HCI with the BioFlo CelliGen 115 for pH control or any other purpose because HCI corrodes stainless steel Over time it will severely damage the headplate a costly component to replace and other stainless steel components Phosphoric and sulfuric 10 maximum concentration acids are acceptable and are commonly used for pH control 7 2 2 pH probe maintenance amp storage Check for any trapped air bubbles in the electrode s tip to remove bubbles hold electrode upright and shake electrode gently The probe should be stored standing upright and the electrode tip should be immersed in the solution of 3 molar KCI or a buffer solution between pH 4 00 and pH 7 00 If the probe is so equipped the two rubber T stoppers should be
151. ther be controlled manually by adjusting the flow of gases through their Rotameters or automatically if 4 gas mixing was purchased as an option For further information on cascading see Section 10 4 gas mixing allows the system to automatically calculate the gas mix in response to culture needs Default P amp proportional amp integral values are preset at the factory We strongly recommend that you maintain the factory set parameters 3 6 PH control pH is controlled in the range of 2 00 14 00 The pH is sensed by a gel filled pH probe Control is maintained by a P amp proportional amp integral controller which operates peristaltic pumps assigned to perform acid or base addition or which controls the use of gas es for this purpose The user can also select a deadband value to control pH within the user assigned range no acid or base will be added when the pH value falls within the deadband tolerance above or below the setpoint Default P amp proportional amp integral values are preset at the factory We strongly recommend that you maintain the factory set parameters 3 7 DO control Dissolved oxygen DO is controlled in the range of 0 200 It is sensed by the DO electrode and control is maintained by the P amp controller by changing the speed of agitation the thermal mass flow controller regulated flow rate if your system is so equipped and or the percentage of oxygen in aeration Operating manual
152. tial adjustment Note that the pH reading must be taken from a vessel that has already cooled down Additional media components that are not autoclaved can be added once the vessel has cooled sufficiently The protocol for this is the same as for inoculation as described below Inoculation can be performed by aseptically pouring liquids into the vessel through the inoculation port although Eppendorf normally uses the harvest port to inoculate A peristaltic pump or gravity is used to introduce the inoculum The shake flask is connected to the port terminus using aseptic techniques and then the clamps are opened to allow for addition Once the material is all in except for any residual inoculum which must be retained for testing secure the clamps and disconnect the shake flask At this point the harvest port terminus must be covered up again using asceptic techniques with sterile cotton and foil To harvest from the vessel attach a line to the harvest port and use a peristaltic pump to pump the culture broth out BioFlo CelliGen 115 M1369 0050 Operating manual 147 21 7 5 Vessel shutdown amp cleaning When the fermentation run is complete it is necessary to carefully shut the process down First all operating parameters agitation temperature DO level pH and gas feed must be set from their current control modes such as Auto Manual or ON to the OFF mode Additionally if a supplemental oxygen feed was used it
153. tically install if applicable a sterile 0 2 um filter in one of the two penetrations on the addition bottle cap 2 Aseptically connect the tubing securing it with a plastic tie to the harvest tube in the addition bottle Clamp it off at the top 3 If you have not already done so thread the tubing through the selected feed pump BioFlo CelliGen 115 M1369 0050 Operating manual 91 4 Connect the tubing securing it with a plastic tie to the appropriate addition port on the headplate 5 Remove the clamp E ALERT Risk of incorrect pH control gt Be aware that proper pH control is critically dependent on tubing size which should be as small as possible gt Consult Table 6 the flow rate tubing size chart for guidance 9 4 1 Addition tubing size pH can be controlled by automatic additions of liquid acid and base Additions are triggered by the BioFlo CelliGen 115 controller which is constantly comparing current pH value with the pH setpoint and making adjustments as necessary i Figure 56 Typical Liquid Addition System 4 3 4 5 1 Peristaltic pump 4 Plastic ties 2 Tubing 5 Addition bottle 3 Breathing port with sterile filter 0 2 u 6 Access to addition port Operating manual 92 The concentrations of acid and base and the inner diameter of the acid and base addition tubing where they pass through the peristaltic pumps are critical parameters i
154. tifies the pin designations for this 25 pin RS 232 422 connector Table 3 Modbus Com Port Pin Designation Pin Number Signal Comments 1 4 6 8 11 14 20 22 23 NC not assigned 2 TXD RS 232 Data Output from fermentor 3 RXD RS 232 Data Input to fermentor 7 GND Earth Ground reference for all signals 12 IRXD a 24 IRXD RS 422 paired data input to fermentor 13 ITXD i 25 ITXD RS 422 paired data output from fermentor 9 IOS Open selects RS 232 Earthed Grounded selects RS 422 Unless otherwise requested the baud rate is factory selected at 19200 and the connector is configured as an RS 232 port i e no jumper between pin 7 and pin 21 The factory set address for the machine is 8 BioFlo CelliGen 115 M1369 0050 Operating manual 57 5 SPECIFICATIONS All systems may differ in configuration please refer to your sales representative for details BioFlo CelliGen 115 System Vessels Volume Pete Ai vesser are presse glass autoclavable vith cishec bottom All vessels are borosilicate glass autoclavable with dished bottom Station of supporting 2 additional utility stations and vessels Utility Station Display 21 3 cm 8 4 in industrial color touchscreen display i is standard with control la but not included with for 2 or 3 utility station Function Fermentation and cell culture monitoring and control Temperature Indication Digital display in 0 1 C incr
155. tivity Fill an addition bottle with the antifoam you will use Attach small bore tubing to the bottle Plug the end with cotton and wrap the cotton with aluminum foil Autoclave the bottle and tubing Thread the tubing through the pump then aseptically connect the tubing to the headplate antifoam addition port Turn the pump on to prime the line Install the foam probe in its headplate port Connect the foam probe cable to Lvl 1 on the control cabinet then attach the cable to the foam probe Open the PUMP screen Select the feed pump you are using by assigning Foam to that pump Enter the pump setpoint and press the ON button Remove the water saline solution from the vessel Add medium to the vessel Ensure that all appropriate sensors and feed harvest tubes including the foam probe and antifoam addition system are properly inserted and secure Make sure the DO probe and the pH probe are capped Ensure that the temperature probe is not in the thermowell it cannot be autoclaved Close off all connectors with cotton and aluminum foil clamp off all tubing and autoclave the entire assembly After the vessel has cooled connect all probes to the control cabinet and all addition tubes to the appropriate pumps Make sure that all harvest and sample tubes are at the right level Make sure the impeller shaft is correctly and completely seated into the bearing housing Make sure that any unused ports are plugged with the supplied penetr
156. top left side of the cabinet see the following page connect the silicone tube attached to the sparge connector to the inlet filter on the vessel headplate The sparge connector tubing assembly is found in the tubing kit provided with your system Operating manual 26 Figure 9 Gas Connections 1 Insert the tubing into the connection simply by pushing it in Check to be sure the connection is secure by pulling gently on the tubing 2 If the controller is equipped with an automatic gas mixing module set up your gas supply this way Gas 1 Air Gas 2 O Gas 3 No and Gas 4 CO Figure 10 Sparge Connection detail From Figure 4 1 Connect the barbed sparge connection here BioFlo CelliGen 115 M1369 0050 Operating manual 27 4 6 Important safety notes Before you begin to assemble or operate your vessel be sure to read this section for it contains essential information to protect your safety and the safety of your equipment A WARNING Risk of explosion q NEVER PRESSURIZE A GLASS VESSEL Always use eye protection and exercise caution in the vicinity of glass If the vessel exhaust becomes blocked pressure can build up possibly shattering the vessel and endangering personnel As soon as you open the airflow valve s verify by feel that air is flowing freely from the exhaust If not immediately close the valve s or turn off the
157. troller Setup screen and the gas process loop gauge screens The Save Changes button saves your new selections and reconfigures all control loops accordingly Although you can save each change one at a time in this screen by pressing it you can also wait until all changes have been selected If you leave this screen however and wish to save your changes be sure to press the Save Changes button before you move to another screen Figure 66 Changing Operating Control Mode Le Setup Screen New Brunswick Fermentation Mode 07 Dec 2008 15 45 Controller Setup System Settings Hardware Setup Unit Type Unit Name Vessel Size BioFlo 115 junit 1 7 5 Liter y No of TMFCs 1 TMFC Range 0 20 SLPM Operatina Mode BioFlo 115 Options Cell Culture Mode EE 1 Fermentation Mode Cell Culture Mode 7 Foam Level A Gas Mix Y Gas Mix r Gas Flow Es Summary ti Calibration 2 Cascade Pumps 1 To change the Operating Mode press the down arrow then press the desired mode in the dropdown list When you change Operating Mode it will also change in the upper righthand corner of the SETUP screen after you press the Save Changes button BioFlo CelliGen 115 M1369 0050 Operating manual 105 Figure 67 Operating Control Mode Changed Setup Screen New Brunswick Cell Culture Mode 1 07 Dec 2008 15 46 ENTERO system Settings Hardware Setup Unit Type Unit Name
158. ty Whenever you assemble or disassemble the vessel components if you need to lay the drive assembly aside while it is still attached to the headplate and the agitation impeller shaft note that there is a correct and an incorrect way to position the assembly on a flat surface The wrong way which is resting the headplate and impeller shaft on a surface see illustrations below puts the impeller shaft at risk for damage Figure 11 WRONG Handling of Drive Assembly 1 Headplate 2 Impeller shaft 3 Drive assembly The correct way which is resting the drive assembly and headplate on the surface see below protects the impeller shaft from bearing weight Naturally you will have to take care not to hit the shaft as you work around it Figure 12 CORRECT Handling of Drive Assembly 1 Headplate 2 Impeller shaft 3 Drive assembly BioFlo CelliGen 115 M1369 0050 Operating manual 29 4 7 Vessel assembly non jacketed The vessels are available in four sizes 1 3 liters 3 0 liters 7 5 liters and 14 0 liters total volume for more detail see Specifications Every single walled non jacketed vessel comes with a stainless steel stand from which the vessel is suspended The stand has four rubber feet to provide stability An electric heat blanket provides temperature control for the contents of the vessel The blanket shown in the smaller vessel views on the following page has two
159. ual Gas Mix ES Summary Screen New Brunswick Fermentation Mode 07 Dec 2008 15 21 Setpoint Units Casc TE fil Calibration Cascade 3 Pumps XA Setup This is essentially the same as the previous sample screen except that this system is configured with Manual Gas Mix BioFlo CelliGen 115 M1369 0050 Operating manual 63 Figure 34 Sample SUMMARY Screen Cell Culture without TMFC ES Summary Screen New Brunswick Cell Culture Mode 07 Dec 2008 15 32 LoopName i Casc FR Calibration FE Cascade Pumps 7 Setup This is essentially the same as the previous sample screens except that this systen is configured with Gas Mix without thermal mass flow controller TMFC and is in Cell Culture operating mode The screen below is essentially the same as the one above in Cell Culture mode and with Gas Mix but this system is configured with at least one TMFC Figure 35 Sample SUMMARY Screen Cell Culture with TMFC Cell Culture Mode New Brunswick Summary Screen 07 Dec 2008 15 19 LoopName PV Setpoi Out Mod i Casc GasFlo 0 0 0 0 0 0 Off FR Calibration Cascade 3 Pumps Setup 1 A GasFlo loop indicates that the system is configured with at least one thermal mass flow controller TMFC Operating manual 64 Table 4 identifies the other interactive features of the SUMMARY screen Table 4 S
160. uccacesnsecose se sses toesas cecsuaces 96 11 1 PUMP ASSIGNMENT csssessecccccccccsssssssceccccccesssesssescccccessuuesssceccccssesuuusensncescssseuaeuens 96 E2 SPUMP SETPOINT 2 cbs aettte dees ccc botanic aged eerie be dios 97 11 3 PUMP CONTROL MODES ti ii a at 99 11 4 PUMP FLOW RATE amp CALIBRATION METHODS ccccccccccccccssesesceccccceccusesessececccsseauenens 99 LS PUMPE PERIOD a AS ANA e 100 11 6 USING LEVEL PROBES TO PROGRAM FEED PUMPS ooocccccononnnnnnnoccnncnnonononanicicicinonononos 101 11 6 1 Setting a feed pump to add liquid lt tds 101 11 6 2 Setting a feed IN RA A A 102 TEO Levelcontrol off EAT IA AA E A e A ARAGAO 102 EEO SPUD CODA ri dt tad 102 USING THE SETUP SCREEN cocccococonononononononononononononononoconococononococonococonoconocecononos 103 12 1 CCEONTROLEER SETUP A A A ia 103 12 1 1 Gas controla dad dd td Nett Sale e a do ad a dah SES 106 122 SY STEM SETTINOS ii iaa 108 12 21 Resetting date time scine nna bea aise la ees 109 BioFlo CelliGen 115 M1369 0050 Operating manual 12 22 Updating SO MOTE di 109 1237 HARDWARE SETUP a e A A A Mice teh tae ek tee lace T 109 12 3 1 identifying utility station s A aie eaaaataeass 112 12 3 2 ise AA AA A E IA E IAN 112 13 PERFORMING A RUN dicccsscccccssescscccecsescescssasccseccdcscscsscbscceseccbaccestecsecssdasaccsacccescsace 113 1340 SET UPFOAM CONTROL A a a r aR 113 13 2 PREPARING FOR A FERMENTATION RUN ooccccconononn
161. umbers 1 2 3 amp 4 correspond to the gas connections on the cabinet BioFlo CelliGen 115 M1369 0050 Operating manual 107 Figure 70 Air 1 Gauge Screen with 3 Gas ful Gauge Screen New Brunswick Cell Culture Mode 07 Dec 2008 15 49 Air 1 100 0 Setpoint Gas Mix Selection 1 ii L 100 0 Oow T Air 02 CO 02 N2 Y Air O2 N2 4 Gas Set PV Out E E 100 0 and Mix Flow Cycle Times Seconds On Time 120 Off Time O 100 0 100 0 100 0 MES Limits Set Low Set High 0 0 100 0 Decimal Places 0000 00 00 000 0 0 000 00 00 0 0 ili Summary fj Calibration Cascade Pumps 2 Setup 1 Available Cell Culture Gas Mix selections choosing O2 N2 gives you a 2 Gas button choosing Air O2 CO2 or Air O2 N2 as shown above gives you a 3 Gas button choosing 4 Gas gives you a 4 Gas button as shown in the sample screen below 2 Selecting Manual in this or any other gas gauge screen allows you to adjust the percentage of that gas air always makes up the remainder if any of 100 When you have Cell Culture as the Control Mode and 4 Gas mix as the Operating Mode the process loops are labeled Air 1 02 2 N2 3 and CO2 4 The loops numbers 1 2 3 amp 4 correspond to the gas connections on the cabinet Figure 71 Air 1 Gauge Screen with 4 Gas Gauge Screen Now Brunswick Coll Culture Mode 07 Dec 2008 15 47 Air 1
162. used and does it differ from media used in shake flasks Answer The media used in shake flasks does differ from the standard media used in a fermentation vessel Shake flask media is generally of a much simpler composition LB Broth and Tryptic Soy Broth are standard shake flask media Here is an example of a more complex media used in a recombinant E coli fermentation Chemical g L KH2PO4 3 5 K2HPO4 5 0 NH4 2HPO4 3 5 MgS0O4 7H20 0 5 Glucose 5 0 for fed batch 30 0 for batch Yeast Extract 5 0 Trace Metals 1 0 mL L Antifoam 0 5 mL L Trace metals formulation FeCl3 1 6 CoCl26H20 0 2 CuCl2 0 1 ZnCl24H20 0 2 NaMoO4 0 2 H3BO4 0 05 Hcl 10 mL H20 to 1000 mL For fermentation the glucose solution is usually sterilized in a separate flask It is then added aseptically to the other heat labile components that cannot be subjected to BioFlo CelliGen 115 M1369 0050 Operating manual 141 autoclaving such as Ampicillin and the trace metal solution These are prepared in advance by sterile filtration so that they are available as stock solutions The magnesium sulfate is sometimes sterilized separately Most materials are available from a variety of vendors Note that Sigma and Difco are often the best sources for the more unusual biological and chemical materials The exact formulations of the trace metals solution and the fermentation media for the fermentors will depend on the precise fermentation you
163. witch ON Add 1 2 mL of glycerin to the thermowell and insert the RTD temperature probe Attach the RTD cable to the RTD connector on the control cabinet Set agitation AGIT to the desired speed and then set its control mode to AUTO Set TEMP to the desired working temperature and set its control mode to AUTO OF ON BioFlo CelliGen 115 M1369 0050 Operating manual 93 10 CASCADE CONTROL A cascade is a control scheme in which the output of one control loop influences the setpoint of one or more other loops In other words it uses one or more parameter s to influence others For example if the DO control loop is cascaded to Agitation whenever the DO process variable drops below its setpoint causing an increase in DO control loop output the agitation setpoint will increase This is effective because agitation strongly influences DO With this type of cascade errors in DO are corrected by changes in agitation rpm The BioFlo CelliGen 115 controller allows cascading from the DO loop to as many as three other loops usually agitation gas flow and oxygen each complete loops with their own probes and actuators When more than one loop is configured as the recipient of a cascaded loop they respond sequentially as one maxes out the next begins to ramp up Depending on the options installed in your system you will have the ability to select one of the following cascades Those unavailable will be greyed out and not sel
164. x knurled thumb screws to securely attach the clamping ring to the base plate This seals the water jacket 4 8 3 Filling the water jacket To fill the water jacket 1 After the tubing and water supply are connected make sure the solenoid valve cable and the RTD cable are plugged into the Power Controller Set the temperature control mode to Off Check that the temperature reading is higher than 5 C Allow water to enter the piping system it will stop at the solenoid valve Set the temperature loop control mode to Auto Enter a temperature setpoint SP that is at least 12 C below the current value CV The controller will respond to the call for cooling by opening the solenoid valve filling the jacket with water Oo 4 8 4 Install baffle 14 0 L fermentation vessels ONLY For installation of the 1 3 L 3 0 L amp 7 5 L vessel baffle see Section 4 8 21 BioFlo CelliGen 115 M1369 0050 Operating manual 39 If you are using a 14 0 L vessel install the baffle assembly inside the glass vessel 1 Gently compress the baffle ring at its ends to avoid scratching the vessel walls You may find it convenient to squeeze the tab with your thumb 2 Slide the assembly inside with the tab facing up until it comes to rest at the bottom of the vessel 3 Orient the baffle so the opening is opposite the gradations on the vessel 4 8 5 Install impeller s Install the impeller s as follows A For Cell Cultu

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