WHEATON SCIENCE PRODUCTS

Transcription

1 WHEATON SCIENCE PRODUCTS MINI PILOT PLANT SYSTEM OPERATION MANUAL Wheaton Science Products 1501 North Tenth Street Millville, NJ, USA (856) FAX (856) Wheaton Science Products 1

2 GENERAL SAFETY INSTRUCTIONS NOTE: EVEN THE SAFEST EQUIPMENT CAN CAUSE INJURY IF THE USER IS CARELESS. 1. KNOW YOUR INSTRUMENT Read the operating manual carefully. Learn the equipment s applications and limitations. 2. GROUND ALL EQUIPMENT If electrical, this equipment is equipped with a grounding type plug. The green/yellow conductor in the cord wire should never be connected to a live terminal. 3. AVOID DANGEROUS ENVIRONMENT Electrical instruments designed to process liquids must be operated with extreme caution. If liquid comes in contact with internal electrical components or wire, fire or electrical shock may occur. Adequate surrounding workspace should be provided during use. Do not operate equipment in a combustible atmosphere. 4. WORK SURFACE Keep well lighted. Be certain the work surface is clean, level, and sturdy enough to support the weight of the unit, particularly if the unit is to be filled with liquid. 5. WEAR PROPER APPAREL Do not wear loose clothing, neckties or jewelry that might get caught in moving parts. Non-slip footwear is recommended. Wear protective hair covering to contain long hair. 6. WEAR SAFETY GOGGLES Wear safety goggles at all times. Everyday eyeglasses only have impact resistant lenses. They are NOT safety glasses. 7. DON T OVERREACH - Keep proper footing and balance at all times. 8. MAINTAIN INSTRUMENT WITH CARE Keep screws tight and unit clean. Check periodically for worn or damaged parts. Inspect the plug and cord before each use. Do not operate this instrument if there are signs of damage. 9. AVOID ACCIDENTAL STARTUP If electrical, always make sure the switch is in the OFF position before plugging the instrument into outlet. 10. DISCONNECT THE INSTRUMENT Always disconnect the instrument from the power source before servicing. 11. DO NOT BLOCK COOLING VENTS IF PROVIDED 12. DO NOT OPERATE THIS EQUIPMENT IN ANY MANNER NOT SPECIFIED IN THIS MANUAL. 13. KEEP THE OPERATING MANUAL FOR THE EQUIPMENT IN A SAFE PLACE NEAR THE INSTRUMENT FOR QUICK AND EASY REFERENCE 14. IT IS RECOMMENDED THAT A FIRE EXTINGUISHER ALWAYS BE LOCATED IN AREAS WHERE ELECTRICAL INSTRUMENTS ARE BEING USED. WSP- 305 Wheaton Science Products 2

3 SAFETY SYMBOLS USED IN THIS MANUAL A Warning symbol indicates attention to an operation that can cause operator injury, improper function of or damage to the equipment and possible problems with the process. A Danger symbol indicates attention to an operation that could cause electrocution or severe injury or death. Wheaton Science Products 3

4 TABLE OF CONTENTS 1.0 INTRODUCTION 2.0 INSTALLATION 2.1 Site Preparation 2.2 Delivery Day 3.0 CALIBRATION 4.0 VESSEL SIDE ARM PORT ATTACHMENTS 4.1 Probes and Pipes , 19 and 45 Liter Vessel Configurations 4.3 Cell Culture Assembly Rigs 5.0 STERILIZATION 6.0 EQUILIBRATION OF OPERATIONAL PARAMETERS 6.1 Post Autoclaving Connection 6.2 Start-Up Sequence 7.0 CELL CULTURE 7.1 Inoculating the Culture 7.2 Monitoring the Culture 7.3 Terminating the Culture 8.0 PREVENTATIVE MAINTENANCE 9.0 APPENDIX I Pipe Configurations 10.0 APPENDIX II Gas Supply Manifold Operation 11.0 APPENDIX III Sampler Operation 12.0 APPENDIX IV Troubleshooting problems 13.0 APPENDIX V System hook-up figures Wheaton Science Products 4

5 1.0 INTRODUCTION A Wheaton Science Products (WSP) Mini-Pilot Plant system consists of a one or more Control Towers, Stackable Support Units and a 10 liter 19 or 45 liter vessel with drive system. These can be operated as standalone units or connected over a network (using WSP BIOPRO software) for supervisory control and data recording. Information specific to the Control Tower and Stackable Support Unit such as calibration and operation can be found in the Control Tower Manual. Information specific to the BIOPRO software can be found in the BIOPRO software manual. This operational manual is concerned with the system set-up and operation. All the above manuals should be kept close as a ready reference. By following this manual, the user should quickly learn the basic operational procedures of the Mini Pilot Plant equipment. These basic processes will permit scale up verification and pilot scale production of products now being made at the laboratory bench scale level. Wheaton Science Products 5

6 2.0 INSTALLATION 2.1 Site Preparation Selection of site Cell culture production in the Mini-Pilot Plant System, as in any bioreactor, requires that ingress of media and cells and egress of samples, harvests and waste media be performed aseptically. This process may be performed in a laminar air flow (LAF) hood or by application of classic cell culture fermentation techniques on the lab bench. This manual describes the use of the Mini-pilot Plant using these techniques Production Floor Operation Convenient operation of a single vessel Mini-Pilot Plant System requires an open area of approximately 15 square feet. Services required for the area include, specialty gasses, air supply, and electrical power service. A laminar air flow (LAF) hood should be installed near the operation site such that the flexible tubing assemblies may reach the inside of the hood without resorting to physically moving the vessel to accomplish this. This manual describes the operation of the Mini-Pilot Plant as it is generally done in a non-sterile environment such as a production plant or on a laboratory table top. Classic cell culture production techniques for filling the vessel, planting cells, sampling and harvesting are described in this manual for this production style setting. These techniques have been performed successfully for many years in the cell culture industry. Successful performance requires proper connection devices and standard aseptic operator techniques. Media is put into the vessel through terminally sterilizing filters. Cells are introduced into the vessel by the use of inoculum bottles. Samples may be obtained via the optional sampling devices. Harvesting of product and final drainage to waste is performed through manifolded flexible tubing lines Laminar Air Flow [LAF] Hood Operation The rigors of aseptic culture techniques are relieved if the culture vessels with their associated tubing assemblies can be placed inside a laminar air flow hood or into a biological safety cabinet. The size of the 10, 19 and 45 liter vessels generally preclude placement of these vessels in a biological safety cabinet or vertical laminar air flow hood, however, if the vessels are placed close to the hoods, the connectors on the ends of the equipment tubing lines can reach into the hood. Consequently these production operations are often aided by the aseptic environment of an LAF hood. Many of the steps described in this manual require the utility of an LAF hood. Wheaton Science Products 6

7 2.1.2 Gas Supplies Specialty Gasses Nitrogen, oxygen and carbon dioxide in tanks may be purchased from your local gas supply outlet. Routine culture conditions generally do well with stock or standard gas qualities available [i.e. where the gas quality is assessed to be greater than 99.5%]. Gas supplies of more rigorous specifications and supplied with batch or individual tank analyses are available. These higher quality gasses may be necessary or desired in certain GMP production situations but for research work, optimization and scale-up considerations, the cost of such gasses is often prohibitive. Warning! Install all specialty gas tanks securely, chaining them to a wall or other support mechanism that will prohibit the tanks from falling and jeopardizing the safety of personnel or equipment Air Air may be supplied to the Mini Pilot Plant vessels from: 1) tanks in the same manner as the specialty gasses described above, or, 2) from an in house compressor system. Air from an in house compressor system must be oil free, having been passed through an oil coalescing filter to remove such mists from the air line. Pressure to the Stackable Support Unit must be <= 15 psi for all gasses Gas Pressure Regulators Install on each tank, gas pressure regulator equipment with a secondary pressure gauge which provides accurate control to +/-1 PSI over the range of 0 10 or 0-20 PSI. Regulators and the gauge modifications needed can be provided by the same firm which supplies the specialty gasses. If a house air supply is used, the air supply port should be equipped with a regulator having the same characteristics as those attached to the specialty gas tanks. A hydrophobic vent filter (0.2u) should be placed in-line prior to the connection to the head-space inlet. Wheaton Science Products 7

8 2.1.4 Electrical Power Service A dedicated, surge protected, circuit of 15 amps is recommended for the operation of the Mini-Pilot Plant System. A circuit of this capacity will supply the Control Tower and it s associated probes, the Stackable Support Unit, heater and a data logging computer if BIOPRO software is used. 2.1 Unpacking Unpack and verify that all items, as ordered, were shipped. NOTE: If this unit will be used for production purposes which may be subject to regulator scrutiny, the purchase orders, delivery papers, manuals etc. must all be saved for the preparation of the Installation Qualification document Assemble the Mini Pilot Plant equipment in the production area which has been prepared for this use. See the Figures at the back of this manual for suggested set-up and the proper inter-unit connections Using flexible tubing (e.g. Tygon, silicone, etc.) connect the appropriate gas tank regulator to the labeled input check valves attached to the rear of the Stackable Support Unit Adjust the regulated pressure of each gas (except air) delivered to the input ports to 8 to 10 PSI. Adjust all tanks to the same pressure. Adjust air pressure to 6 to 7 PSI. Recheck during operation Install in the nitrogen supply line a Y connection. Attach a 3 to 4 length of silicone tubing to the leg of the Y which is not connected to the Stackable Support Unit. Clamp this tubing shut. This Y connection in the nitrogen line will be used for calibrating the zero point of the DO 2 probe, pressurizing any reagent bottles used and providing a source of inert gas Plug the power lines of the Control Tower and the Stackable Support Unit into the electrical circuit. A switchable, surge protected power strip to which all of the Mini Pilot Plant components are connected is recommended. Do NOT plug the cable of the heater belt to the outlet found on the back of the pump/heater module of the Stackable Support Unit at this time. Wheaton Science Products 8

9 3.0 CALIBRATION OF PROBES AND UTILITIES Connect all probe cables to the rear of the Control Tower. Turn the power on to the Mini Pilot Plant System (see item 2.2.4). Follow the instructions in section 7.0 of the Control Tower manual and set the RATE values for all control loops to zero to stop control. Calibrate the probes in the following order. 3.1 Temperature Probe Follow the procedure in the Control Tower manual calibration section. 3.2 ph Probe If the probe tip has been covered with a silicone cup filled with storage electrolyte (or an appropriate ph buffer) proceed with the calibration process. If it is dry, soak the probe for at least one hour in storage electrolyte or buffer. Calibrate the probe according to the procedure described in the calibration section of the Control Tower manual. Make the proper selection for temperature compensation to the ph probe measurement. 3.3 DO 2 Probe Calibrate the probe according to the procedure described in the calibration section of the Control Tower manual Calibrating the zero is conveniently performed as follows. Connect a 30 ml syringe barrel to the end of the unused length of tubing connected to the nitrogen source and insert the tip of the DO 2 probe into the syringe barrel. Start nitrogen gently flowing (approximately 100 cc/min). The measured value of the dissolved Oxygen control loop, as displayed on the Control Tower, will approach zero. After setting the zero, remove the probe from the syringe barrel and clamp shut the line supplying nitrogen to the syringe. CAUTION: The tip of the DO probe can be damaged easily, be very careful when handling. Wheaton Science Products 9

10 3.3.3 Allow the probe to remain in room atmosphere (100% air saturation) for approximately one minute, or until a stable reading and observe that the value of the dissolved oxygen control loop displayed by the Control Tower becomes some positive value not to exceed 999. This assures the user that the probe is functional. The magnitude of the displayed value will be defined by the SPAN calibration step after the vessel and probes are autoclaved and the DO 2 probe has been fully polarized. (See the calibration section of the Control Tower manual) 3.4 Agitation Speed The 10 liter, 19 liter and the 45 liter vessels are equipped with a single 100 mm port to hold the overhead drive assembly. This port is located in the center of the vessel. Install the impeller on the drive shaft at the position desired. The impeller may be positioned at any point on the drive shaft using the two screws in the hub. Install the impeller at the desired position on the shaft (usually with the top of the impeller just breaking the media surface) and insert the headplate assembly into the center port and secure it with the large screw cap ring. Fill the vessel with water to the volume level intended for production. The agitation rate can now be set under load conditions. Set the agitation rate using the control potentiometer located on the top mounted drive. NOTE: The agitation rate (motor stirring speed) can be (as an installed option) under the Control Tower s control. NOTE: If the Overdrive head is the digital display type you may disregard the following paragraph: Count the number of revolutions per unit time at each arbitrary dial index marker and create a calibration curve relating the dial setting to the RPM of the impeller. When this is completed, drain the vessel. 3.5 Gasses Air Source House Air Supply If a house air supply is used to supply air to the control tower, the port should be equipped with a regulator of the same type as the compressed gas tanks. Adjust the regulator so the pressure is 7 PSI and feed the air through a 0.2u hydrophobic vent filter. This air supply should also be clarified as described previously. Connect the air to the input check valve labeled AIR on the rear of the Stackable Support Unit Air Pump ( fish tank type) Connect the output of a mini-air pump through a 0.2u hydrophobic vent filter then to the input check valve labeled AIR on the rear of the Stackable Support Unit. This must have a loaded pressure of 5 to 7 PSI. Wheaton Science Products 10

11 3.5.2 Calibration of Gas Flow Rates With the auxiliary air pump or the house air supply connected adjust the flow rate of air with the rotameter needle valve to provide a ml/min flow rate. Then clamp shut the line which delivers the air to the check valve on the back of the Stackable Support Unit. Manually activate the nitrogen, oxygen and carbon dioxide valves in turn [use the procedure described in the Control Tower manual] and observe the position of the indicator ball. With the gas pressures set to 10 PSI the indicator ball should show a flow rate of about ml/min when the gas valve is open. The user may need to make minor adjustments of the specialty gas pressures to obtain this gas flow The operating concept which is to be achieved is to have the specialty gasses impressed into the gas delivery system at a slightly higher pressure thus giving them a higher flow rate, than the air. The air flow serves as a carrier gas (as well as an oxygenating source) to bring the specialty gasses into the vessel headspace or to the sparger. 3.6 Pump Rate To avoid over control on the basic side of the ph set point, the pump in the stackable housing is usually operated at the lowest possible speed. Lock the Control Tower on the ph channel and depress and hold the + button. Adjust the position of pump potentiometer to the lowest possible position which still retains smooth pump operation. 3.7 Cold Finger Operation The optional cold finger is used when it is necessary to maintain a cell culture medium temperature that is close (i.e. within +5 0 C) of ambient. The cold finger is inserted into the stopper indicated in the configuration schematics of the vessels (see drawings section) Conditions for the operation of the cold finger are empirically determined by connecting the cold finger to a chiller or other water supply and adjusting the flow to the minimum needed for the heater control loop to provide a constant temperature. Typically, a flow of cc/min will allow the vessel enough cooling to keep the temperature within specifications. Wheaton Science Products 11

12 4.0 VESSEL SIDE ARM PORT ATTACHMENTS 4.1 Probes and Pipes for the Mini-Pilot Plant The 10 liter, 19 Liter vessel of the Mini-Pilot Plant is equipped with six 45 mm ports. The 45 Liter vessel is equipped with seven 45 mm ports. The vessel ordered will come equipped with the Standard Set of probes pipes and plugs shown below. Optional Equipment items listed below that have been ordered will be included but may be packed separately. Standard set of Probes, Pipes and Plugs Item Description [Type] DO 2 Probe [12 mm Mettler Toledo / Ingold] with stopper ph Probe [12 mm DPAS, 320 mm long] with stopper Thermowell with stopper Gas Vent pipe with stopper (same as innoculation pipe) Base Reagent Inlet Pipe Sparger Headspace gas inlet pipe Innoculation pipe with stopper (same as gas vent pipe) Optional Equipment Available Item Description Antifoam/Level Control (Custom Option) Sampler Sparger (extra sparger for custom configurations) Cold Finger (small for 10L, 19L large for 45L) Silicone stoppers (for 45mm ports) with the following hole configuration a. Stopper with 1 12mm hole (for 19mm D.O. probe) b. Stopper with 1 12mm hole (for ph probe or 12mm D.O. probe) c. Stopper with 1 9mm hole and 1 3mm holes (sparger, vent, base addition) d. Stopper with 1 9mm hole (thermowell or custom use) e. Stopper with 2 6mm hole (cold finger or custom use) f. Stopper with 1 9mm and 1 3mm hole for headspace gas in and media/seed g. Stopper blank for custom drilling Wheaton Science Products 12

13 4.2 Vessel Configurations The pipes, plugs and any optional equipment ordered are usually not installed during shipping. The DO 2 probe and the ph probe have been shipped in protective boxes. These probes should be inserted into their stoppers and installed into ports #1and #2 respectively. The standard configuration of pipes and probes for each vessel is shown below See Appendix 5, "System Hook-up Figures" for suggested port use for the and 45 liter vessels Wheaton Science Products 13

14 4.3 Cell Culture Assembly Rigs The standard equipment set of cell culture assembly rigs will be found in your order. They are to be attached to the pipes which are installed in the vessel side-arm ports. All optional equipment assembly rigs included must also be installed as described. Standard Equipment: Base Reagent Supply Tubing Assembly Install the base reagent supply tubing assembly on the 3.2 mm pipe. Secure the tubing to the pipe. Open the clamp installed on the line. Cover the male luer lock at the end of the tubing with foil and seal the luer lock inside an autoclave bag Headspace Gas Vent Filter Assembly Install the headspace gas vent filter assembly on the pipe installed in port #4. The silicone tubing from the port SS tubing to the filter should be short (~25mm) to keep the filter upright so condensed liquid will return to the culture and not block the gas flow. If high gas flows (>200 cc/min) are anticipated, two filters should be used on two separate ports to insure no pressurization of the vessel will take place Headspace Gas Inlet Assembly Install the headspace gas inlet assembly on the headspace gas inlet pipe installed in port #5. Secure the silicone tube to the hydrophobic vent filter Reagent Bottle A one foot (1 ) length of silicone tubing (1/6 ID x 3/16 OD) with a female luer lock fitting comes installed on the end of the dip tube of the reagent bottle, (Control Tower manual). Wrap the luer lock fitting in foil and attach a clamp to the outlet end this tubing. Clamp the tubing shut. Wheaton Science Products 14

15 Optional Equipment Available Sparger Gas Filter Assembly Option Install the sparger by inserting the pipe through the base of the silicone stopper and adjust the position until the stainless frit is close to the bottom of the vessel. Install the sparger gas filter assembly on the sparger pipe. Secure the silicone tube to the vent gas filter and to the sparger pipe with clamps Cold Finger Option Install in options port #6 or #7. Since the cold finger is a closed loop, there is no jeopardy to vessel sterility during any of the preparatory setup and sterilization by autoclaving. The cold finger is operated by flowing cold tap water or recirculating chiller water through the finger at about cc/min. The cold finger is needed as a heat sink or ballast for the culture of insect cells whose optimum growth temperature is close to ambient Sampler Option Install the Sampler in port #6 (of 10L, 19L or 45L sized vessel) adjacent to the Headspace Gas Inlet Pipe. Operation of the Sampler is described in the Appendix Gas Supply Manifold for Sparging This assembly is attached after the vessel is sterilized by autoclaving. It may added to the vessel rigging at any time during the culture process to provide smooth and efficient dissolved oxygen control. Attach the output legs of the manifold to the hydrophobic filters connected to the Sparger and to the Headspace Inlet filter. The tubing from the flow adjustment needle valve is attached to the headspace Inlet Filter. The quick disconnect end of the assembly is inserted into the gas supply support module of the support unit. The operation of this manifold is described in the Appendix. Wheaton Science Products 15

16 5.0 STERILIZATION 5.1 Vessel Autoclaving Instructions Add ml of tissue culture grade water to the reactor vessel. Orient the vessel vertically in the autoclave. Autoclave the vessel for 30 minutes at C and 15 PSI. Use a fast (or dry goods) exhaust cycle. IMPORTANT NOTE: If the vessel is oriented on its side during steam sterilization, the heavy drive shaft mechanism may crack the neck of the opening due to heat related stresses. Therefore, if the vessel must be laid on its side, remove the agitator drive shaft, and place it in a large autoclave bag or separate wrapping for independent sterilization. Cover the agitator shaft opening with autoclave wrapping and/or foil cover. After autoclaving, the drive shaft mechanism may be inserted into the vessel while protected in a laminar airflow hood. 5.2 Base Reagent Supply Bottle Autoclaving Instructions Prepare a one normal (IN) solution of NaOH and place about 800 ml of this solution into the base reagent vessel. Autoclave the base reagent solution for 20 minutes at C and 15 PSI. Allow the autoclave to come to ambient condition through a slow exhaust (or liquid) cycle. Alternatively a manual sterilizer can be turned off and allowed to come to ambient conditions overnight. 5.3 Regulatory Confirmation If validation of the sterilization process is needed, the user can insert several thermocouples through a slit in the tubing of the Headspace vent Filter Assembly (4.3.1). The thermocouples can be positioned throughout the interior of the vessel to provide a profile of the temperature rise in the vessel during sterilization. Wheaton Science Products 16

17 6.0 EQUILIBRATION OF OPERATING PARAMETERS 6.1 Post Autoclaving Connections After autoclaving and as soon as the vessel can be conveniently handled, clamp all lines except the following: the headspace Inlet filter, the headspace vent filter and the sparger vent filter. Insure that the manifolds of the drain/harvest line and the media Inlet/Inoculation assembly are all completely clamped shut. Remove the vessel from the autoclave and place it on the site selected. If the drive mechanism was autoclaved separately from the vessel allow both to cool and place each in an LAF hood. Unwrap and insert the drive into the center port of the vessel. Secure the drive in place with the large coupling ring. Then install the vessel in the intended operation site. IMPORTANT NOTE: If the Rate value for the temperature, ph and DO 2 control loops has not previously been set to zero (0) do it NOW! If this is not done, the temperature control loop will cause the heating belt to overheat Attach the heating belt securely around the vessel and secure it with the velcro strip connectors. Plug the heating belt power cord into the heater connector of the Stackable Support Unit Attach the Gas Supply Manifold to the sparger gas filter Assembly, the headspace gas inlet assembly and the Stackable Support Unit Attach the cables to the DO 2 and ph probes. Add about ml of water, ethylene glycol or glycerol to the thermowell and slip the RTD probe into the thermowell. Connect all cables to the Control Tower Apply power to the Control Tower Start the air supply and adjust the rate of air flowing into the vessel to ml/min Allow the vessel to cool. No further activity should occur with the vessel until the RTD probe indicates that the temperature in the vessel is <50 0 C. Wheaton Science Products 17

18 6.1.8 Connect the NaOH or reagent vessel 6.2 Start-Up Sequence Prepare the volume of medium that will be put into the vessel. When the vessel temperature is <50 0 C, pump this medium into the vessel through the media inlet/inoculation assembly. After addition of the medium to the vessel is complete, clamp the input line shut Set the agitation speed. This is manually done by counting the revolutions with the help of a timer or using a digital display (if supplied) Set the Temperature control loop operating constants as follows: Kp = 45.00, Ki = 0.03, Kd = 5.00, RATE = 20, IB = 0.5, DB = Allow the media to warm to it s set point temperature Let the system equilibrate and the DO 2 probe polarize for 6-8 hours with only air flowing into the headspace portion of the vessel Set the DO 2 probe span value to 100% as specified in the Control Tower Manual Initiate control of ph and DO 2 control loop by entering a Kp of and Rate of 5 for DO 2 and a Kp of 80 and a Rate of 10 for ph control. NOTE: These values may need to be turned for your particular system, however, they are appropriate starting values. See the Control Tower Manual PID section Allow the Control Tower to bring the operating parameters of ph, DO 2 and Temperature into full equilibrium with the set points before inoculating (or seeding) with cells. NOTE: THE HEATING BELT MAY LOOSEN WHEN IT INITIALLY WARMS AND COULD SLIP FROM POSITION, IF SO, PULL IT TIGHT USING THE VELCRO AREA (WHICH IS UNHEATED) AFTER THIS TIGHTENING WHILE WARM THE BELT WILL REMAIN IN PLACE. 7.0 CELL CULTURE Wheaton Science Products 18

19 7.1 Initiate the Culture Harvest the inoculum or seed cells from T-Flasks, roller bottles or smaller bioreactor vessels Add to a sterile reagent bottle, a sufficient number of the harvested cells to provide the appropriate seeding density for your particular application Place the reagent bottle containing the harvested cells in an LAF hood and bring the inoculation line into the hood Connect the reagent bottle containing cells, media and microcarriers to the inoculation line Using gravity feed or by use of a peristaltic pump move the cell slurry into the vessel If the culture is composed of attachment dependent cells, microcarriers must be added to the culture. This may be done separately by following the steps outlined above or the microcarriers may be added simultaneously with the cells Rinse the reagent bottle and line by adding a small aliquot of complete media and pumping this into the vessel. After the cells (and microcarriers) are in the vessel and the line is rinsed, clamp the line shut If the system is being used for attachment dependent cell culture, adjust the speed for the optimum conditions for attachment of cells to the microcarriers. After attachment has occurred adjust the speed to the desired culture conditions. 7.2 Monitor the Cell Culture Samples may be removed from the vessel by following the directions in the Appendix. Samples removed may be used for determining cell density, measure metabolites, determine product formation, check ph drift Preserve appropriate samples for other analyses as needed. 7.3 Terminating the Culture Wheaton Science Products 19

20 7.3.1 Turn off all control loops by setting each loop s Rate value to zero Drain the vessel to a suitable harvest level by use of the drain line. Temporarily close the headspace gas vent by placing a finger over the exit port of the vent filter and allowing the pressure to increase in the vessel. Unclamp the drain line. The culture fluids will begin to flow out the drain line into the harvest vessel. WARNING! GLASS VESSELS MAY CRACK OR SHATTER IF PRESSURIZED. BE EXTREMELY CAREFUL TO SHIELD FOR GLASS OR BIOHAZARDS Rinse the vessel by adding tap water, sterile media or balanced salt solution with subsequent drainage. The rinse solution may be introduced through the drain line or the inoculation port and thereafter, drained to a suitable waste container. Decontaminate the rinse fluid as specified in the users operating SOP s Autoclave the vessel if required for decontamination. Wheaton Science Products 20

21 8.0 PREVENTATIVE MAINTENANCE 8.1 DO 2 Probe Follow the manufacturer s directions for the maintenance of the polarographic DO 2 probe Visually inspect the membrane for damage prior to each calibration. If the membrane is dirty, clean it with a moist, soft cloth The electrolyte will have to be changed periodically; it should not be used longer than six months Replace the membrane cartridge itself when the probe response time becomes long, the measured value drifts or is noisy, calibration is not possible or mechanical damage to the membrane is observed. 8.2 Gel Filled ph Probe The gel filled ph probe is essentially considered a disposable item. As such, there are no recommended preventative maintenance procedures. However, there are signs that can be considered by the user in making decisions about the utility of the probe. They are: The response of the probe will become sluggish and erratic at the end of its useful life When the sintered glass diaphragm (frit) at the base of the probe becomes dark brown or gray, this is an indication that the life span of the probe has been reached. Mettler-Toledo / Ingold recommends that the user attempt to clean this using their diaphragm cleaner; however, they also have determined that due to the construction of the probe (i.e. gel verses liquid interior) the cleaning process may not work in the same manner as it does for the liquid probes The user may, with experience, determine a finite number of autoclave cycles that the probe will be subjected to. After this limit is reached, the probe will be replaced. Depending on the conditions of use and sterilization, this number of cycles may be in the range of 10 to 50. Wheaton Science Products 21

22 8.3 Vessel Dissemble the vessel completely and wash all cell culture residue from the interior surface of the vessel When the vessel is cleaned, the agitator should be completely dissembled and each part washed separately The silicone plugs in the headplate should be removed and the interior of the headplate ports cleaned carefully when the vessel is dissembled following a culture study. Wheaton Science Products 22

23 APPENDIX I 9.0 PIPE SUPPLY SET 9.1 Thermowell, 8 mm pipe sealed at one end. 9.2 Off Gas Vent Pipe, 8 mm diameter pipe, 10 cm long. 9.3 Sparger, 3.2 mm OD, pipe with sintered 2 um pore size sparger attached. 316L stainless steel. 9.4 Reagent Pipe: 3.2 mm OD, 20 cm long. 9.5 Headspace Gas Inlet / innoculation Tube. 9.5 mm diameter pipe 20 cm long 9.6 Sampler Devices Optional These samplers are self supporting and can be used on 10L, 19L and 45L. vessels Sampler without plug valve The sampler is constructed of a single ¼ OD 316l SS tube with connects the 15 ml sample tube holder to the vessel contents. Samples are taken by evacuating the sample tube through the vent filter and removing the sample by aspiration Sampler with plug valve The sampler is constructed from two ¼ OD 316L SS tubes connected by a plug valve. The tube/plug assembly connects the 15ml sample tube holder to the vessel contents. The sample is taken by pressurizing the vessel which pushes the culture media into the sample tube. Alternatively, the sample may be taken by aspiration as in the sampler without the plug valve. Wheaton Science Products 23

24 APPENDIX II 10.0 STREAM SPLITTER ACCESSORY OPERATION The following procedure is to be performed after the Mini-Pilot Plant vessel has been assembled, sterilized and filled with media for a cell culture study. The sparger and the headspace gas supply ports will have been installed in the vessel side arms, (see section 4.2) Connect the Stream Splitter Accessory to the sparger and the headspace supply lines as shown in the Figure Fully open the accessory needle valve. The gas flow will preferentially go the head space as the path of least resistance Clamp shut the nitrogen, oxygen and carbon dioxide gas supply lines Turn on the air and adjust the rotameter mounted on the Stackable Support Unit to provide an air flow of 125 ml/min Clamp the air supply line shut and turn off the air pump Use the Control Tower to manually open, one by one, each gas solenoid. Refer to the Control Tower manual, section 8.2 for this procedure Without changing the position of the Support Unit rotameter (4.0 above), adjust the pressure on each gas tank regulator gauge so that the gas will flow at 150 ml/min. through the rotameter as each solenoid is opened Open the air supply line Begin to close the needle valve. The valve furnished with the Stream Splitter Accessory is a ten turn needle valve, i.e. it requires approximately ten turns to close the valve from fully open to fully closed. The closure of this needle valve will restrict the gas flow to the headspace and ultimately begin distributing the gas to the sparger. This distribution, however, will not occur until the needle valve is almost fully closed (between 10.0 and 10.5 turns). Wheaton Science Products 24

25 10.10 Close the needle valve until the first evidence of bubbles begin to come from the sparger. Then carefully back open the valve until this effusion of bubbles ceases The total air flow through the Stackable Support Unit rotameter will have diminished to approximately 50 ml/min. during the closure of the needle valve. Readjust the needle valve of the rotameter installed on the Gas Control Unit to provide 100 ml/min. of air flow Increasing the flow in step may cause air to bubble through the sparger. If this occurs, open the stream splitter needle valve very slightly until the effusion of bubbles ceases Lock the Control Tower on the DO 2 control loop with the arrow keys Depress the + button opening the oxygen solenoid. The sparger should begin to gently bubble. The needle valve is now adjusted to provide a gentle sparging when the DO 2 solenoid is opened and no sparging when the DO 2 solenoid is closed (i.e. the + button is released) When an acceptable split is obtained (this will be determined by your experience) press the Clear/Scan button on the Control Tower and installed the appropriate PID values for the control of D.O. on the DO 2 loop When the measured value of dissolved oxygen is below set point and the Control Tower begins to open and shut the oxygen solenoid, the rotameter falling ball indicator will alternately rise and fall between 150 ml/min. and 100 ml/min. When the oxygen solenoid opens, observe the fritted sparger for the evolution of gas bubbles. Let several cycles of the solenoid opening and closing occur Minor adjustments in the needle valve may need to be made until gas evolves from the sparger only when the oxygen solenoid opens. BE PATIENT! Observe the stabilization of the measured value of DO 2. The above procedure will generally provide accurate control of this parameter to within 2-5% of set point. APPENDIX III Wheaton Science Products 25

26 11.0 SAMPLER OPERATION Procedures for using the Sampling Device Refer to Appendix I, item and for a description of the Sampler(s) available Aspiration Method. This method can be used on either type of sampling device Attach a ml syringe to the vent filter with a short length of silicone tubing Evacuate the air from the sample tube by withdrawing the syringe barrel The culture fluid will from the vessel into the sample tube Fill the sample tube to the desired level Depress the syringe barrel to restore pressure to atmospheric and stop the flow Remove the sample tube and replace with a new sample tube. Note: The threads on the sampler head are Falcon (brand) polypropylene 15 ml centrifuge tubes fit the sampler head and are autoclavable. Wheaton Science Products 26

27 WARNING! GLASS VESSELS MAY CRACK OR SHATTER IF PRESSURIZED BE EXTREMELY CAREFUL TO SHIELD FOR GLASS OR BIO-HAZARDS Pressure Method. This method can only be used with a sampling device with a plug valve Clamp the headspace Off-Gas vent Close the plug valve The incoming headspace air will build up pressure in the headspace Open the plug valve and allow the desired volume of medium to collect in the sample tube Close the plug valve and remove the sample tube Replace the sample tube with a new tube Multiple samples can be taken while the vessel is under pressure. WARNING! After the desired number of samples are taken, UNCLAMP THE HEADSPACE OFF GAS VENT and relieve the vessel pressure. Wheaton Science Products 27

28 APPENDIX IV TROUBLESHOOTING PROBLEMS NOTE: section Also see the Control Tower Manual troubleshooting Temperature control is erratic Be sure the temperature probe is in the thermowell and the thermowell is in the media. Also, check that the thermowell has at least a 1 fill of water or glycerin for proper heat transfer. Be sure the heating pad is positioned with the heating side towards the vessel. Be sure the heater is plugged into the correct outlet. Be sure the correct PID values are used in the temperature channel (see the Control Tower manual) If ambient temperature is within about 5 0 C of the setpoint, the controller will have a difficult time controlling smoothly. The cure for this is to use the optional cold finger and run cool water (at least 15 0 C below the setpoint) through it at about cc/min. ph Control is Erratic Check probe calibration, remember, offset must be set to zero during probe calibration (see the calibration section of the Control Tower manual) Check to be sure the base reagent is correct. Check that the base pump speed is sufficient and that the speed control has not been turned off. Check to be sure the CO 2 gas is connected to the correct gas input. If you are using ATC, be sure the channel is correct. (see the calibration section of the Control Tower manual). Wheaton Science Products 28

29 Check your probe. Long cell culture runs can sometimes cause problems of clogging with the probe reference frit. This is only cured by proper maintenance and discarding probes after a recommended number of autoclavings. (see manufacturers instructions). Be sure the PID values are set correctly for the ph channel of your unit. (see the calibration section of the Control Tower manual). D.O. Control is Erratic Check probe calibration. (see the calibration section of the Control Tower manual). Check that the O 2 and air are connected to the correct input ports on the gas module. Be sure to use the correct scheme of D.O. control (i.e. headspace or sparge/mixture) as described above. Failure to do this will result in very erratic control. Check your probe, a membrane leak will cause a very high D.O. reading. This is only cured by proper maintenance and replacing membranes after the recommended number of autoclavings (see manufacture s instructions). If gas pressures are too low (<3 psi) the check valves at the rear of the gas module will not allow gas flow and control will not be possible. Typical gas pressures should be approximately 7 psi for air and approximately 10 psi for the other gasses. Be sure the PID values are set correctly for the D.O. channel of your unit. (see the PID section of the Control Tower manual). Allow sufficient time (>6 hours) to allow the D.O. probe to polarize before calibration. Failure to do this will result in drifting of the reading (see manufacturer s instructions). Wheaton Science Products 29

30 APPENDIX V SYSTEM HOOK-UP FIGURE Wheaton Science Products 30

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38 FIGURE A - MINI PILOT PLANT SYSTEM PARTS LISTS BASIC 4 CHANNEL SYSTEM ITEM NO. QTY WHEATON NO. DESCRIPTION 1 1 WL VAC Control Tower-General 1 1 WL VAC Control Tower- General 2 1 W Top motor drive w/display 120 VAC 2 1 W VAC top motor drive w/display 3 1 WL VAC Stackable Service Unit- BASIC 3 1 WL VAC Stackable Service Unit-Basic 4 1 I Assy, 1L Reagent BTL, Fermentation 5 2 I Filter, 0.2 micron 6 1 WL L flask w/six sidearm L flask w/six sidearms 6 1 W L flask w/seven sidearms 7 1 W Probe, DO, Ingold 8 1 WI Probe, temperature w/cable 9 1 WI MM OD PROBE, ph 10 1 WI V heater, silicone w/plug 10L 10 1 WI V heater wrap, silicone w/plug, 10L 10 1 I V Heater, silicone w/plug, 6x40 19L 10 1 I Assy, heater wrap w/plug 230v, 19L 10 1 I V 6X54 heater, silicone /rub wplug 45L 10 1 I V Assy, heater wrap, 45 L 11 1 I Cable, tower to stack unit 8 CH 12 1 WI Probe cable, ph, proteus Assy, cable, D. O. pin Control Tower 14 6 I MM pbt wht 1-1/4 ctr hole (10l, 19l) 14 7 I MM pbt wht 1-1/4 ctr hole (45l) Luer, male, to 1/8 tube barb Luer, female, to 1/8 tube barb 17 5FT I Tubing, silicone, 6MM ID 18 12FT I MM ID silicone tubing 19 2 I Tube, SS w/45 DEG bend, 9 OAL 20 2 I Tube, SS 3/8 DIA. 3 OAL 21 1 I Tube, 316 SS, thermowell, long 22 1 WI Assy, impeller/cap RDSN 10l, 19l overdrive Wheaton Science Products 38

39 FIGURE B WHEATON IMPELLER DRIVE ASSEMBLY PARTS LIST ITEM NO. QTY WHEATON NO. DESCRIPTION 1 1 I CAP, OPEN TOP, WHT PLUG, DRIVE 3 1 WL RING, SILICONE SIZE I BEARING, BALL, SS 5 1 WL HEADPLATE AND HOUSING 6 1 WL O-RING SILICONE SIZE I GASKET, HEADPLATE 8 1 WL MAIN SHAFT ASSEMBLY 9 2 WL COLLAR, SHAFT CLAMP 10 4 I SCW,, X 5/8, 316 SS 11 1 WL SEAL, X.150,.48 OD 12 1 WL HOUSING, CAP 13A 1 I SHAFT, SS (323.85MM) 10L & 19L) 13B 1 WL SHAFT, 16.0 (406.40MM) ADJUSTABLE, 316 SS (45L) 14 2 I PADDLE, TEFLON 3-5/16 WIDE 15 2 I COLLAR, PADDLE CLAMP KEY, HEX, LONG ARM 5/ KEY, HEX, LONG ARM ¼ Wheaton Science Products 39

40 FIGURE C - MINI PILOT PLANT SYSTEM PARTS LISTS BASIC 8 CHANNEL SYSTEM ITEM NO. QTY WHEATON NO. DESCRIPTION 1 1 WI V General Control Tower-Twin 1 1 WI V general Control Tower- Twin 2 2 W Top motor drive w/display 120 VAC 2 2 W VAC top motor drive w/display 3 2 WL VAC Stackable Service Unit- Basic 3 2 WL VAC Stackable Service Unit-Basic 4 2 I Assy, 1 L Reagent BTL, Fermentation 5 4 I Filter, 0.2 micron 6 2 WL L flask w/six sidearms L flask w/six sidearms 6 2 W L flask w/seven sidearms 7 2 W Probe, DO Ingold 8 2 WI Probe, temperature w/cable 9 2 WI MM OD PROBE, ph 10 2 WI V heater, silicone w/plug, 10 L 10 2 WI V heater wrap, silicone w/plug, 10L 10 2 I Heater, silicone w/plug, 6x40 (19 L) 10 2 I Assy, heater wrap, 230V, 19L 10 2 I X54 heater, silicone/rub w/plug (45 L) 10 2 I V Assy, heater wrap, 45 L 11 2 I Cable, tower to stack unit 8 CH 12 2 WI Probe cable, ph, proteus Assy, cable, D. O. pin Control Tower I MM pbt wht 1-1/4 ctr hole (10l, 19l) I MM pbt wht 1-1/4 ctr hole (45l) Luer, male, to 1/8 tube barb Luer, female, to 1/8 tube barb 17 10FT I Tubing, silicone, 6MM ID 18 24FT I MM ID silicone tubing 19 4 I Tube, SS w/45 DEG bend, 9 OAL 20 4 I Tube, SS 3/8 DIA. 3 OAL 21 2 I Tube, 316 SS, thermowell, long 22 2 WI Assy,impeller/cap RDSN 10l, 19l overdrive Wheaton Science Products 40

41 FIGURE D - PLUG/STOPPER PARTS LISTS 10 L & 19 L ITEM NO. QTY WHEATON NO. DESCRIPTION SIDE ARM PLUG, 12MM HOLE 2 1 I STOPPER. W/375 HOLE, 45 MM 3 2 I PLUG, SILICONE, 45 MM, 2 HOLES BLANK NO HOLES (OPTIONAL) Wheaton Science Products 41

42 FIGURE E - PLUG/STOPPER PARTS LISTS 45L ITEM NO. QTY WHEATON NO. DESCRIPTION SIDE ARM PLUG, 12MM HOLE 2 1 I STOPPER. W/375 HOLE, 45 MM 3 2 I PLUG, SILICONE, 45 MM, 2 HOLES BLANK NO HOLES (OPTIONAL) Wheaton Science Products 42

43 FIGURE F SPARAGER ASSEMBLY PARTS LIST ITEM NO. QTY WHEATON NO. DESCRIPTION 1 10 FT I MM ID TUBING, SILICONE /16 O. D. CONN, T, PLASTIC 3 2 I CONNECTOR, HOSE 4 1 I MICRONS FILTER /8 NPT VALVE, NEEDLE 6 1 I SPARAGER ASSEMBLY, S) Wheaton Science Products 43

44 FIGURE G - ANTI FOAM PROBE CONNECTION PARTS LIST (OPTIONAL) ITEM NO. QTY WHEATON NO. DESCRIPTION 1 1 I Assy cable, Antifoam 2 1 I Assy Probe, Antifoam Wheaton Science Products 44