S.U.B. User s Guide. Thermo Scientific HyPerforma Single- Use Bioreactor (S.U.B.) User s Guide

Transcription

1 S.U.B. User s Guide Thermo Scientific HyPerforma Single- Use Bioreactor (S.U.B.) User s Guide Revision C DOC0014 December 2016

2 Table of Contents Warnings and Safety...4 General Safety Information...4 Protective Earthing... 5 Environmental Conditions...5 Water Jacket Vessel Information...5 Safety Interlocks...6 How to Use This Guide...7 Section 1 Section 2 Section 3 Single-Use Bioreactor (S.U.B.) Overview 1.1 Introduction Hardware Characteristics End User and Third-Party Supplied Components BioProcess Container Characteristics Additional System Components Initial Installation Preparation Installation and Setup General System Operation Information Advanced Control Information Cell Culture Operating Instructions Maintenance...53 Operating Information 2.1 Verification Procedures BPC Loading Instructions (50, 100, and 250L) BPC Loading Instructions (500 and 1,000L) BPC Loading Instructions (2,000L) Probe Assembly Sampling Pall Kleenpak Connector Instructions Troubleshooting Section 4 Specifications and Parts Information 4.1 Hardware Specifications BPC Specifications Accessories and Options Specifications Thermo Scientific 2

3 Table of Contents Section 5 General Ordering Information 5.1 Ordering Instructions Ordering/Support Contact Information Appendix A Appendix B Appendix C Appendix D Installation of Electrical Receptacle for Units with A/C Motors AC-Tech Variable Speed Drive Settings Load Cell Calibration Instructions ,000L S.U.B. Operation and Maintenance Guidelines Thermo Scientific Single-Use Bioreactor (S.U.B.) 3

4 Warnings and Safety Warning: Read and understand operator s manual before using this equipment. Thermo Scientific HyPerforma Single-Use Bioreactor (S.U.B.) is designed to be operated under traditional mammalian cell culture conditions. A general understanding of bioreactor systems and their operation is important prior to using the system for the first time. Read and understand user s manual before operating Failure to do so could result in injury Warning: Hazardous voltage inside Risk of electrical shock and injury. Disconnect power before opening. Service by trained personnel only. Consult manual. Electrical components are designed into and required for the proper function of the S.U.B. The mixer motor, motor controller, resistive heater jacket and control panel all have electrical components. Warning: Static electricity may build up in BPCs BPCs may act as insulators for electrostatic charge. If electrostatic charge is transferred to a BPC, the charge may be stored in the BPC and/or the product inside. This phenomena varies by product and use; therefore, it is the sole responsibility of the end user to ensure a hazard assessment is conducted and the risk of electrostatic shock is eliminated. Where applicable, a product contact stainless steel coupler may be grounded to the frame to dissipate electrostatic build up from the material within a BPC. It is good practice to dissipate electrostatic build up by grounding all BPCs prior to coming in contact with them. When working with BPCs, the use of nonconductive materials, such as nonconductive gloves, is recommended. Warning: Entanglement hazard Rotating and moving parts can cause injury. Keep hands away from moving parts. Do not operate this equipment without the supplied guarding in place and properly functioning. It is the responsibility of the end user to assess this equipment and ensure that equipment and safeguards are in good working condition and that all operators are trained and aware of entanglement hazards and associated protective devices, such as hazard signs and guarding. Thermo Scientific Single-Use Bioreactor (S.U.B.) 4

5 Warnings and Safety Warning: Hot surface. Do not touch. The heating jacket is designed to heat the outer vessel wall. Normal operating conditions generate heat and could create hot surfaces. Hot surface inside Contact with surfaces may cause burns Do not touch while in operation Warning: Burst Hazard Under normal operating conditions the S.U.B. BPC chamber is under slight pressure. Normal passive venting prevents any excess of pressure building up within the chamber. Chamber pressure and inlet line pressure should be monitored for proper settings. Contents under pressure Do not exceed 0.5psi (0.03bar) BPC pressure Do not exceed 5psi (0.34bar) inlet pressure Assure vent filter is properly positioned and working properly Protective Earthing Environmental Conditions Water Jacket Vessel Information Protective earthing must be verified prior to plugging the S.U.B. into any electrical outlet. Ensure the receptacle is properly earth grounded. Operating: 17 to 27 C; 20 to 80% relative humidity, non-condensing Storage: -25 to 65 C Installation category II (over voltage) in accordance with IEC 664 Altitude Limit: 2,000 meters S.U.B. hardware unit with water jacket has been designed to be operated with water as the heat transfer medium with temperatures not exceeding 50 C (122 F) under less than 150psig (1MPa) operating pressure. For the utmost safety it is recommended that the water jacket S.U.B. be operated at 75psig or less. NOTE: The S.U.B. BPC operating limits for temperature are 5 to 40 C. The internal pressure should not exceed 0.5psi. The water jacket is not required to be registered, inspected and stamped with the Code U symbol per section U-1(c)2(f) of the ASME Boiler and Pressure Vessel Code and/or European Pressure Equipment Directive (PED) 97/23/EC. Upon request, a Declaration of Conformity, PED Sound Engineering Practices can be made available. Thermo Scientific Single-Use Bioreactor (S.U.B.) 5

6 Warnings and Safety Use of Agitation Speed Governors and Safety Interlocks Agitation speed governors set up on the bioreactor controller are used to limit the maximum mixing speed, according to pre-defined liquid volumes. Safety interlocks, which stop agitation when the volume in a S.U.B. drops below defined limits, and speed-based governors prevent damage to the drive shaft in the bioreactor. Agitation speed governors and safety interlocks typically prevent the hazardous conditions listed below. Operating the motor at any speed while loading the drive shaft Operating the agitator above the rated speed when volumes are less than 50% of a system s working volume Operating the agitator above the rated speed at volumes between 50% and 100% of the working volume Operating above the qualified power input to volume (PIV) thresholds Operating the agitator above the rated speed during harvest at less than 50% of working volume Operating the agitator if the impeller is not fully submerged Testing has shown high system reliability when qualified interlocks and governors are in place. This eliminates the chance of human error, which could reduce system reliability. Both the amount of liquid in the vessel and the amount of power applied to the impeller have an impact on the applied deflection on the shaft. Excess deflection and/or mixer speed may damage the drive shaft. For more information about using PIV ratios and safety interlocks in 2,000L bioreactor systems, see Appendix D of this publication. Thermo Scientific Single-Use Bioreactor (S.U.B.) 6

7 How to Use this Guide How to Use This Guide Scope of this Publication Document Change Information This user s guide contains information about the standard Thermo Scientific HyPerforma Single-Use Bioreactor (S.U.B.) systems, including hardware, components, product design verification methods, installation, operation, specifications and part numbers. It is intended for use by people who may or may not have experience with Thermo Scientific systems, but who have some knowledge of bioproduction processes and large-scale bioreactors. Revision Number Date Page Number Change Made / Initial Release S. Jelus B 12/ Added How to Use This Guide section E. Hale B 12/ C 12/ C 12/ C 12/ C 12/ C 12/ C 12/ C 12/ C 12/ C 12/ C 12/ C 12/ C 12/ Fixed Electrical Power Supply Requirement in Specifications section Added infromation about safety interlocks to Warnings and Safety section Added serial number information and photo of ends of multiple-section drive shafts Added footnote to agitation rate in Table 1.4 about the need to use safety interlocks of 2000L systems Added footnote to agitation rate in Table 1.5 about the need to use safety interlocks of 2000L systems Added warning note about agitation rate and volume requirements, and the use of safety interlocks Added measurement to Table 1.7 for 2,000L drive shafts and cross-reference to Appendix D; Added Table 1.8 Added information about 2-piece drive shaft and a note anout poistion of impeller tubing inside the BPC Added serial number information and photo of ends of multiple-section drive shafts Added a note about not pushing drive shaft straight into the assembly when loading Added information and Figure to illustrate proper insertion of drive shaft Added information about 2-piece drive shaft to 2,000L specifications Added ceiling height requirements for 2-piece drive shaft and detail about mixing speed to 2,000L specifications E. Hale S. Jelus Author S. Jelus/E. Hale S. Jelus/E. Hale S. Jelus/E. Hale S. Jelus/E. Hale S. Jelus S. Jelus/E. Hale S. Jelus/E. Hale S. Jelus/E. Hale S. Jelus/E. Hale S. Jelus/E. Hale S. Jelus/E. Hale C 12/ Added drive shafts as accessories S. Jelus/E. Hale C 12/ Added Appendix D - 2,000L S.U.B. Operation and Maintenance Guidelines S. Jelus/E. Hale Thermo Scientific Single-Use Bioreactor (S.U.B.) 7

8 How to Use this Guide Input into Thermo Scientific Publications The accuracy of the information in this publication and your satisfaction with the scope and content of this publication are important to us. Please send any corrections or suggestions you have for this user s guide (or for any other Thermo Scientific technical publication) to TechnicalDocumentation@ThermoFisher.com. Please contact your local sales representative for information about the related publications listed below. Related Documents Please contact your local sales representative for information about the related publications listed below. Document Number -- DOC0016 Document Thermo Scientific HyPerforma Single-Use Bioreactor Data Sheets (various sizes) Thermo Scientific HyPerforma Single-Use Bioreactor Validation Guide Description Product descriptions and ordering information Information about validation procedures Thermo Scientific Single-Use Bioreactor (S.U.B.) 8

9 Section 1 - HyPerforma Single Use Bioreactor (S.U.B.) Overview This section covers the following information: 1.1 Introduction to the Single-Use Bioreactor 1.2 Hardware Characteristics 1.3 End User Supplied Components 1.4 BPC Characteristics 1.5 Additional System Components 1,6 Initial Installation Preparation 1.7 Installation and Setup 1.8 General System Operation Information 1.9 Cell Culture Operating Instructions 1.10 Maintenance Section 1.1 Overview 1.1 Introduction The Thermo Scientific HyPerforma Single-Use Bioreactor (S.U.B.) has been designed to be a single-use alternative to conventional stirred tank bioreactors currently utilized in animal cell culture. Based on years of accepted stirred tank reactor (STR) design, the S.U.B. emulates STR scalability and operating parameters, yet it has the unique advantage of being a single-use device. Ease of setup with respect to system operation and integration into existing facilities makes the S.U.B. an attractive alternative to its stainless steel counterpart. Critical design parameters such as height to diameter ratios, mixer design and location, and typical control system interfaces have been maintained. The S.U.B. BPC is supplied gamma irradiated. A key element to the single-use design is the plastic (polyethylene) impeller with a bearing/seal assembly linking to an external mixer drive. Quick setup and change over allows for faster turnover in cell culture runs over traditional reusable systems. The S.U.B. systems consist of the following primary components: 1. Outer support container with either Electric Resistive or Water Jacket heating systems 2. S.U.B. BPC and 3. Control system for units with A/C motors for agitation (Electric Resistive and Water Jacket) and temperature (resistive only) 4. Direct drive agitation mixing assembly with an A/C or D/C motor, drive shaft and impeller. Thermo Scientific Single-Use Bioreactor (S.U.B.) 9

10 Section 1.1 Overview Figure ,000L Single-Use BioReators The outer support container is engineered and fabricated to fully support each S.U.B. BPC and allow easy access for operation. It is a stainless steel vessel which holds and supports the BPC. The outer support container contains the mixing drive, silicone heating blanket (for units using electric resistive blankets) or water jacket, optional controllers for mixing and heating (for units using electric resistive blankets), and portable dolly. The drive shaft is detachable and reusable and is inserted into the BPC through the mixing assembly and into the bearing port. Load cells are offered standard on the 1,000 and 2,000L S.U.B.s and are optional for the smaller volume systems. The BPC includes the impeller assembly, sparger, vent filter inlet/outlet ports, probe integration ports, and filling, dispensing, and sampling ports. Each S.U.B. BPC comes fully assembled and gamma irradiated. The materials are fully qualified for biological product contact per USP Class VI plastics. Each assembly is manufactured under cgmp and is supported by qualification and validation information. No reuse cleaning is required. The BPC is provided gamma irradiated. Innovative, proprietary technology allows for the integration of the mixing shaft, ph and dissolved oxygen probes, and the resistive temperature detector (RTD). The probe and temperature interfaces are comparable to traditional systems with the design allowing for simple, aseptic connections. Integrated spargers are built into the S.U.B. BPC through universal ports. The Thermo Scientific S.U.B. utilizes an open architecture design for the control system, allowing for integration with customer systems or with third-party controllers for feed pumps, mass flow controls, and HMI Thermo Scientific Single-Use Bioreactor (S.U.B.) 10

11 (human-machine interface) screens. Controls for temperature (resistive only) and agitation are integrated into the S.U.B., with ph/dissolved oxygen (DO) probes and controls being supplied by the user or a thirdparty integrator. Water Jacket systems require a temperature control unit selected and supplied by the end user or by Thermo Scientific. This User s Guide covers the setup, operation, maintenance and troubleshooting of all S.U.B. systems in the following volumes 50, 100, 250, 500, 1,000 and 2,000 liters. Thermo Scientific Single-Use Bioreactor (S.U.B.) 11

12 Section 1.2 Hardware Characteristics 1.2 Hardware Characteristics System Features Agitation Bioreactor Control System Exhaust Vent Filter Heater Condenser System The Single-Use Bioreactor is designed for system mobility and easy integration while utilizing a straightforward operator interface. Hardware drawings and specifications tables specific to volume can be found in Section 3 of this manual. The following sections include general descriptions of the hardware components of the S.U.B. If your system uses an A/C motor and a Thermo Scientific electrical control box, the stirring speed is adjusted by using the keypad interface on the control panel. The agitation control interface utilizes a digital display to indicate stirring speed in units of revolution per minute (rpm). Power is supplied to the motor by a two position power switch. The up and down arrows on the agitation keypad adjust the stirring speed. If your system has a D/C motor and is integrated and managed by a third party controller, agitation is managed by the controller. Thermo Scientific does not provide electrical control for units with D/C motors. The S.U.B. is designed to integrate with existing bioreactor control systems in their numerous configurations. The S.U.B. control system supplied with the Thermo Scientific electrical box enclosure manages the process parameters of agitation and temperature. Parameters of ph and DO, gas management, feed addition, and base addition control must be managed by an external controller supplied by the end user or a third party integrator. An optional exhaust vent filter heater is available for increased longevity of the exhaust filter on the S.U.B. BPC. Heating the filter sufficiently to eliminate the formation of condensation is an effective means of reducing the risk of fouling of the filter membrane. The heater is factory preset to operate between C, but can easily be adjusted to the demand of the application. Temperature settings above 60 C are not recommended. The Condenser System is recommended for 2,000L S.U.B. use and is also available as optional hardware for smaller systems. It cools exhaust gases and re-circulates the condensate into the bioreactor. Thermo Scientific Single-Use Bioreactor (S.U.B.) 12

13 Section 1.2 Hardware Characteristics Temperature Heating Performance External Control Load Cells The S.U.B. can be operated within the temperature range from ambient to 40 C. For systems with resistive heaters and Thermo Scientific electrical control boxes, temperature setpoints can be adjusted via the temperature controller located on the front panel of the S.U.B. electrical control box. This controller is pre-programmed to avoid overshoot during heat up and to maintain a target temperature of +/-0.5 C based on the set value display. The process temperature is measured by means of a supplied resistive temperature detector (RTD) (pt-100) that is inserted into the thermo-well of the S.U.B. BPC. Water Jacket system temperature control is maintained through the temperature control unit (TCU). Heating times for the S.U.B. systems vary based upon operating liquid volume and temperature, ambient or heating fluid temperature, sparge rate, and mixing rate. Users should adjust process liquid staging and seeding strategies to the unique aspects of the S.U.B. Process controllers and heaters in resistive heating systems are designed to provide optimum heat transfer and to minimize heat up times, while maintaining the material integrity of the polymer film construction of the S.U.B. BPC. Refer to Section 1.8 for expected heating times. Users can choose to bypass the temperature control, mixing speed control and display pressure systems provided with the S.U.B. and utilize existing bioreactor controllers. Refer to Section 1.8 for more details. Load cells, which are used to determine the weight of the contents of a S.U.B., are standard on the 1,000 and 2,000L S.U.B. hardware and optional for the smaller S.U.B. volumes. Load cell kits can be installed at the factory or can be added later by a certified service technician. The load cell kit comes with three load cells, summing block, wiring and display with a choice of several data interfaces. Load cells arrive uncalibrated; it is recommended that the load cell manufacturer or a qualified technician calibrate these systems on site. Thermo Scientific Single-Use Bioreactor (S.U.B.) 13

14 Section 1.3 End User and Third-Part Supplied Components 1.3 End User and Third-Party Supplied Components ph and Dissolved Oxygen (DO) Probes The following table shows the length and diameter requirements for traditional sensors (probes) that can be integrated into the S.U.B. These requirements are based on the necessary insertion depth of the probe when used with the probe ports. NOTE: The presence of a properly positioned O-ring on the probe is critical for use with the S.U.B. Recommended ph/do Probes for use with the S.U.B. Probe Lengths (from O-ring to tip) Must Not Exceed 235 mm Probe Part Number Diameter Thread Type O-ring to probe tip Print/Lit. Actual Length Length AppliSens DO Z mm 13.5 PG 235mm 235mm AppliSens ph Z mm 13.5 PG 235mm 235mm Mettler Toledo DO Mettler Toledo ph InPRO 6800/12/220, PN DPAS-SC-K8S/225, PN IG 12mm 13.5 PG 215mm 215mm 12mm 13.5 PG 195mm 219mm Broadley-James DO D140-B220-PT-D9 12mm 13.5 PG 215mm 214mm Broadley-James ph F-635-B225-DH 12mm 13.5 PG 225mm 219mm Finesse DO DOS-OFF-VP mm 13.5 PG 225mm 220mm Finesse ph PHS-EFP-K mm 13.5 PG 225mm 220mm NOTE: Consult probe manufacturer s Web site for appropriate probe cable connection and part number. Table 1.1 Manufacturers and models of S.U.B. compatible ph/do probes. Controllers Thermo Scientific operates with an open architecture approach to the integration of controls. Our industry-leading S.U.B. has been integrated with most controllers on the market, allowing our customers to choose the control system they want, or to reduce expense by integrating with a controller that is already onsite. In order to facilitate integration, electrical schematics are provided in the Equipment Turn Over package supplied with the HyPerforma S.U.B. Companies that offer control solutions in either cgmp or non-cgmp format for Thermo Scientific S.U.B. units are listed below. Thermo Scientific Single-Use Bioreactor (S.U.B.) 14

15 Section 1.3 End User and Third-Part Supplied Components ABEC Bellco Broadley-James Dasgip Emerson Honeywell New Brunswick Scientific Pendotech Sartorius Stedim Biotech The HyPerforma S.U.B is also available as a complete turnkey system through Thermo Scientific. These S.U.B. units may be provided with integrated controls, pump towers, a control monitor, and advanced features such as data logging, multiple S.U.B. connections and optional 21CFR part 11 compliance for cgmp manufacturing. A variety of single use sensors are available for ph, DO and pressure control. Thermo Scientific can provide complete, integrated solutions using the manufacturers listed below. Applikon PLC ez-controller Finesse PC controller Emerson Delta V Allen Bradley Siemens Contact your local sales representative for more information. NOTE: The S.U.B. will work well with any of the various control system platforms, such as PLC, PC, DCS or proprietary operating system based controllers. Thermo Scientific Single-Use Bioreactor (S.U.B.) 15

16 Section 1.4 BPC Characteristics 1.4 BPC Characteristics Single-Use Bioreactor BPC Features Operating Pressure Working Volume Draining and Harvest The cell culture itself is contained inside the S.U.B. BPC. The chamber is manufactured from film which is a co-extruded structure specifically designed for biopharmaceutical process usage. All materials are qualified for a broad range of physical, mechanical, biological and chemical compatibility requirements. (Refer to data in our BPC Catalog. Contact your sales representative for a copy). The bioreactor BPC is supplied gamma irradiated. The S.U.B. BPC does not operate as a closed system, as it has both inlet and exhaust filters that are utilized to maintain an environment for cells to grow without concern for contamination. However, conditions can be encountered when gas inlet flow rate may exceed exhaust flow rate. This may be encountered in the unlikely event of a pressure regulator failure on a gas feed, or when excessive foaming creates conditions of vent blockage. The S.U.B. BPC is not rated as a pressure vessel (gas pressure should not exceed 0.5 psi within the BPC). Custom BPCs can be ordered with an optional single-use pressure transducer for monitoring the pressure within the S.U.B. (supplied standard with 1,000 and 2,000L systems). Each S.U.B. is designed for a working volume range. The minimum working volume and the rated working volume are listed in the specification tables provided in Section 4 of this manual. The total volume listed includes the headspace needed for proper aeration and gas management. Actual working volumes should not exceed the indicated rated working volumes. In addition, working volumes less than the minimums listed can result in damage to the BPC and S.U.B. hardware malfunction. The S.U.B. is equipped with a bottom drain line that allows for liquid harvest by means of peristaltic pump. Connection of the bottom drain line can be accomplished by use of a tubing welder or the quick connect or fitting that is provided. Manipulation of the BPC as the last few liters of media is removed can minimize liquid hold-up within the S.U.B. Gas to liquid mass transfer in cell culture bioreactors is controlled by the solubility of the gas in the liquid, its distribution, and the temperature and pressure. Direct air sparging is a way of providing for the oxygen requirements of mammalian cell cultures. The standard S.U.B. BPC incorporates a unique single-use dual sparging design that allows for optimal aeration of the culture process and effective CO 2 stripping. Multiple aseptic connection options exist for S.U.B. users. The standard Thermo Scientific Single-Use Bioreactor (S.U.B.) 16

17 Section 1.4 BPC Characteristics Aeration Connections Sampling BPCs include tubing welder sections, quick connects and Pall Kleenpak connections. The S.U.B. BPC is designed with various lengths and dimensions of thermoplastic tubing for the purpose of addition to and dispensing from the S.U.B. BPC. Refer to Ordering Information in Section 3 for custom end treatment options. The S.U.B. is equipped with a small volume sample port that is adjacent to the BPC thermo-well. This small diameter silicone dip tube of 152.4mm length (6 ) allows low void volume samples to be taken for cell viability and density, as well as analyte analysis. This dip tube is supplied with an luer lock connector (SmartSite) that allows for direct sampling or attachment of various sampling manifolds by use of standard luer lock connection. Alternatively, manifolds can be welded onto the C-Flex sample line by tubing welder. BPC drawings and standard configuration tables specific to volume can be found in Section 4 of this manual. Thermo Scientific Single-Use Bioreactor (S.U.B.) 17

18 Section 1.4 BPC Characteristics BPC Features Figure 1.2 Typical 50L, 100L and 250L BPC schematic Figure 1.3 Typical 500L and 1,000L BPC schematic Figure 1.4 Typical 2,000L BPC schematic 9 Part Description 1. Exhaust Vent Filter Single-use capsule filter for exhaust gas exchange 2. Gas Overlay Port Protected by gas filter 3. Ports For addition of media and other liquids 4. Seal/Bearing Assembly Links with mixer motor and allows impeller to turn while retaining integrity of the S.U.B. BPC 5. Impeller Injection molded plastic; Linked to seal/bearing assembly by C-Flex tubing contact material of the shaft 6. Ports with Kleenpak Connectors For integration of standard 12mm monitoring ph and DO probes 7. Temperature RTD Port For integration of temperature probe while retaining integrity of the S.U.B. BPC 8. Sampling Port For needleless sampling or connection to sampling manifold 9. Drain Port Used when draining the S.U.B. 10. Gas Sparge Lines 11. Condenser System Bag Table 1.2. Key to figures 1.2, 1.3 and 1.4 Sparger integrated into the chamber and protected by gas filters; Dual micro sparger (porous frit) and macro sparger (with open pipe or drilled hole) options are available Integrates with chill plate to remove condensate from exhaust; Supports effective use of 2000L systems and can also be used with other sizes of S.U.B.s that require custom gassing strategies and demand higher exhaust rates and longer durations Thermo Scientific Single-Use Bioreactor (S.U.B.) 18

19 Section 1.5 Additional System Components 1.5 Additional System Components Probe Integration The probe assembly is an innovative design to package user supplied ph and DO probes for sterilization and to aseptically connect them to the S.U.B. BPC. The probe assembly (Figure 1.5) includes the following components: 1. Molded bellows cover 2. Threaded probe adaptor 3. Pall Kleenpak Connector (KPCHT Series - high temperature) 4. Cable ties Figure 1.5. Probe assembly Required and Optional Accessories To assist in the operation of the Single-Use Bioreactor the following additional accessories are available (see Section 3 for ordering information): Heavy duty tubing clamps - sold separately Required (typically four or five) Sampling manifold with luer lock Optional S.U.B. temperature sample port (for resistive temperature dectector calibration/validation) Optional Vent Filter Heater System Optional - For users who require additional protection for the exhaust vent filter on the standard S.U.B. BPC. Includes filter heater, programmable controller and power cord. Condenser System Recommended for 2,000L S.U.B. Optional on smaller systems. Load cells Standard on 1,000 and 2,000L S.U.B.s. Optional on smaller sizes. Load cell packages includes three load cells, summing block, wiring and an optional display screen with your choice of several data interfaces. Thermo Scientific Single-Use Bioreactor (S.U.B.) 19

20 Section 1.6 Initial Installation Preparation 1.6 Initial Installation Preparation Hardware Shipment and Setup Hardware Uncrating The Single-Use Bioreactor hardware will arrive crated. For unpacking instructions and detailed contents of the crate, please refer to Packaging and Assembly Instructions, which are included in the shipping crate and in the Integrator s Reference Guide. Be sure to follow the unpacking instructions provided and retain all packaging materials. The S.U.B. hardware will arrive with the following items: Outer support container (platform, tank and control panel) Drive shaft, resistive temperature detector (RTD), probe brackets (4) and standard tool set (spanner wrench and torque wrench) Electronic turnover package (ETP) provided on a USB drive (shipped separately) Detailed instructions for crating, uncrating and assembly of 50 to 1,000L S.U.B. units are included in the Packaging and Assembly Instructions document. Uncrating, crating, and assembly of 2,000L S.U.B. units are found in the Integrator s Reference Guide. After uncrating, contact your Sales Representative immediately if any damage has occurred. Thermo Scientific Single-Use Bioreactor (S.U.B.) 20

21 Section 1.6 Initial Installation Preparation Site Preparation Electrical Connections for Units with A/C Motor and Electrical Control Box Electrical Connections for Units with D/C Motors Outer Support Container Preparation S.U.B. hardware using A/C motors cannot be used on circuits equipped with GFCI circuit protection because of the potential for nuisance tripping. The electrical plug on the S.U.B. is a connector that offers a secure ground. These connectors meet the electrical safety codes for portable equipment and are International Electrical Code (IEC) rated (meet IEC standard 60309). This plug serves to provide electrical ground prior to power connection. The supplied electrical receptacle should be hardwired into the facility by a qualified electrical technician; for US installations the receptacle will require the use of an adapter mounting plate (supplied) which will fit into a two gang box. For additional information on the adapter mounting plate, please see the ETP. Alternatively, the system can be hardwired directly into the facility. NOTE: The yellow plug and receptacle are for 120VAC and the blue are for 240VAC S.U.B.s. S.U.B. units using D/C motors are not supplied with electrical control boxes. When using a D/C motor, electrical connections must be supplied by a third party integrator. Each outer support container is shipped directly from the manufacturer and arrives with various safety mechanisms in place. Please follow the guidelines below to setup the Single-Use Bioreactor upon arrival. Warning: Any procedures that require the control box to be open should be performed with the main electrical disconnect in the locked out position and all power sources removed from the control box. For operator safety, secure the location of the S.U.B. outer support container by disabling the swivel casters before servicing. Electrical Preparation for 50L, 100L, 250L and 500L with Electrical Control Box and Electric Resistive Heating Systems 1. S.U.B. units are shipped with the electrical breakers in the OFF position to prevent accidental activation of the heaters during hardware installation. NOTE: The heater should only be enabled when a liquid filled BPC is present. Using a flat head screwdriver, open the control box and locate the breakers for the PID temperature controller (middle of top row) and heater (located in middle of lower row) (Figure 1.6). These breakers should be in the upper ON position during operation. For electrical schematics, please refer to the Equipment Turnover Package (ETP) which is provided on a USB drive. 2. Verify that the three-way motor controller switch is in the middle position. For reference, the middle position enables the speed control Thermo Scientific Single-Use Bioreactor (S.U.B.) 21

22 Section 1.6 Initial Installation Preparation keypad, the top position is for 0-10V controllers and the bottom is for 4-20mA controllers. Verify that the position of the two-position temperature control toggle switch is in the up position (for reference, the up position enables the PID temperature controller). Temperature controller breaker Heater breaker wired in 50, 100 and 500L only Heater breaker wired in 250 and 500L only Motor controller switch (3-way) Temperature controller switch (2-way) Figure 1.6 Control box for 50, 100, 250 and 500L electric resistive heater S.U.B. units 3. Close the control box and lock the panel using a screwdriver before continuing. 4. For S.U.B. hardware units purchased with factory installed load cells, the load cells are shipped in the locked position (threaded up) for equipment protection. Refer to Load Cell Preparation instructions in this section of this manual. Electrical Preparation For 50L, 100L, 250L and 500L Water Jacket Systems with A/C Motors and Electrical Control Boxes 1. Before beginning, refer to electrical schematics included with the Equipment Turnover Package (ETP) which is shipped on a USB drive. 2. Using a flat headed screwdriver, open the control box. Verify that the three-way motor controller switch is in the middle position (Figure 1.7). Thermo Scientific Single-Use Bioreactor (S.U.B.) 22

23 Section 1.6 Initial Installation Preparation Motor Controller Switch (3 way) 1,000L Electrical Preparation for Units with A/C Motors and Electrical Control Boxes Figure 1.7 Control Box for 50, 100, 250 and 500L Water Jacket S.U.B. 3. Close the control box and lock the panel using a screwdriver before continuing. 4. For S.U.B. hardware units purchased with factory installed load cells, the load cells are shipped in the locked position (threaded up) for equipment protection. Refer to Load Cell Preparation instructions in this section of this manual. 1. For resistive systems, S.U.B. units are shipped with the electrical breakers in the OFF position to prevent accidental activation of the heaters during hardware installation. NOTE: The heater should only be enabled when a liquid-filled BPC is present. Using a flat head screwdriver, open the control box and locate the breakers for the Proportional Integral Derivative (PID) temperature controller, heaters, pressure sensor and load cells (middle of top row) (Figure 1.8). These breakers should be in the upper ON position during operation. For electrical schematics, please refer to the Equipment Turnover Package (ETP) which is provided on a USB drive. NOTE: The heater and PID breakers are present in the water-jacketed versions, but they are nonoperational. 2. Verify that the three-way motor controller switch is in the middle position (for reference, the middle position enables the speed control keypad). 3. For resistive systems, verify that the two-position temperature control toggle switch is in the up position (for reference, the up position enables the PID temperature controller). NOTE: This switch is present but not operational in water-jacketed versions. 4. Close the control box and use a screwdriver to lock the panel before continuing. Thermo Scientific Single-Use Bioreactor (S.U.B.) 23

24 Section 1.6 Initial Installation Preparation Temperature controller breaker Heater breakers (3) Pressure sensor display Load cell Motor controller switch (3-way) Figure 1.8 Control box for 1,000L resistive S.U.B. Temperature controller switch (2-way) Temperature controller breaker Heater breakers (3) (no heater, non-operational) Pressure sensor display Load cell Motor controller switch (3-way) Figure 1.9 Control box for 1,000L water-jacketed S.U.B. Temperature controller switch (2-way) Thermo Scientific Single-Use Bioreactor (S.U.B.) 24

25 Section 1.6 Initial Installation Preparation 2,000L Electrical Preparation 1. Using a flat head screwdriver, open the control box and locate the breakers for the pressure sensor, continuous power outlets non E-stoppable (2), continuous power outlets E-stoppable (2) (Figure 1.10). These breakers should be in the ON position during operation (blue indicator pressed in). For electrical schematics, please refer to the Equipment Turnover Packager (ETP) which is provided on a USB drive. Motor controller switch (3-way) Pressure Outlet 240 VAC continuous power non e-stoppable Outlet 240 VAC continuous power e- stoppable Figure 1.10 Control box for 2,000L jacketed S.U.B. 2. Verify that the three-way motor controller switch is in the middle position (for reference, the middle position is to enable the speed control keypad). 3. Close the control box and lock the panel using a screwdriver before continuing. Load Cell Preparation for 50L, 100L, 250L, 500L, and 1,000L S.U.B. Units Follow the steps below to prepare 50 to 1,000L S.U.B. units load cells for use. 1. For 50, 100, 250, 500 and 1,000L S.U.B. hardware units purchased with factory installed load cells, the load cells are shipped in the locked position for equipment protection (Figure 1.11). Thermo Scientific Single-Use Bioreactor (S.U.B.) 25

26 Section 1.6 Initial Installation Preparation Lockout nut 38.1mm (1 1/2 ) tri-clamp Lockout post A Delrin slip ring Figure 1.11 Load cells Drive shaft cap end Load cell lockout end Figure 1.12 Supplied wrench (SV50177D.05) 2. To unlock the load cells, remove and discard the delrin slip ring if it is present. Remove the tri-clamp. Loosen the Lockout Nut, using the small end of the supplied tool (Figure 1.12), until the nut is tight against the base or leg of the tank. Repeat this process for each load cell until all lockout nuts are disengaged from the Lockout Posts. Do not reinstall the tri-clamp. 3. At this point, the S.U.B. hardware is ready to be prepared for a cell culture run. CAUTION: Do not move unit, especially when filled, while load cells are unlocked as this can damage the load cells. 4. For sytems with load cell display screens, refer to Appendix C for information about how to calibrate the load cells. 5. To lock load cells that have been unlocked, hand-tighten the lockout nut onto the post. Use the supplied tool to turn the nut an extra 1/4 turn. WARNING: To avoid damaging the load cells, do not over-tighten the nut. Assemble a standard stainless 38.1 mm (1 ½ ) tri-clamp around the flanges. Complete this process for all load cells. Thermo Scientific Single-Use Bioreactor (S.U.B.) 26

27 Section 1.6 Initial Installation Preparation Load Cell Preparation for 2,000L S.U.B. Units 1. For 2,000L S.U.B. hardware units purchased with factory installed load cells, the load cells are shipped in the locked position (centering washer and nut are threaded up) for equipment protection (Figure 1.13 and 1.14). A 15/16 open-box wrench is recommended for unlocking the load cells. To unlock the load cells, loosen the centering washer and nut (5/8 nut) until the nut is threaded down on the hold-down bolt about half way. Figure 1.13 Unlocking load cells on a 2,000L S.U.B. Top of bolt (not a nut) Jam Nut Hold down bolts Figure 1.14 Load cells on a 2,000L S.U.B. 2. Next, loosen the jam nut on the base plate until the jam nut and centering washer/nut meet. Tighten both nuts together. Using an open-end wrench, loosen the hold down bolt (by moving it upward) to provide clearance below and above the cutout in the top plate (Figure 1.14 Section A-A). The clearance around (above and below) this nut should be from mm (1/8 ) to 4.76 mm (3/16 ). Once the proper clearance has been achieved, retighten the jam nut. Thermo Scientific Single-Use Bioreactor (S.U.B.) 27

28 Section 1.6 Initial Installation Preparation 3. At this point, the S.U.B. hardware is ready to be prepared for a cell culture run. 4. CAUTION: Do not move unit, especially when filled, while load cells are unlocked as this can damage the load cells. 5. For load cell calibration refer to Appendix C. Thermo Scientific Single-Use Bioreactor (S.U.B.) 28

29 Section 1.7 Installation and Setup 1.7 Installation and Setup All manual movements of mobile S.U.B. hardware (excluding the 2000L S.U.B.) should be over smooth surfaces with the S.U.B. empty and disconnected from all power and gas/feed sources. All load cells must be fully locked down in order to move a S.U.B. Refer to the previous subsection of this manual for illustrations. 1. Verify facility electrical supplies are sufficient to support the power requirements of the S.U.B. and ancillary components such as controllers or pumps. 2. Locate the outer support container in the area for the cell culture run. 3. If monitoring the volume, the unit may be located on a scale if load cells are not utilized. Other applications may measure all liquids going in and coming out. 4. Level the platform by disabling the swivel casters on the bottom of the outer support container. This is accomplished by threading the leveling feet (at the center of each caster) to the floor. 5. Verify the location of the ph/do controllers and assure cable/tubing lengths are satisfactory. WARNING: Electrical shock 6. Verify the main power is off and the emergency stop is pulled out. NOTE: THE EMERGENCY STOP DISCONNECTS ALL POWER TO THE SYSTEM. AN ALARM BUZZER WILL SOUND WHEN THE EMERGENCY STOP IS ACTIVATED. 7. Verify that the main motor power switch is in the off position. 8. Connect all electrical plugs to facility power. NOTE: 120 VAC - 250L S.U.B. to be connected to a dedicated 20A circuit. Refer to hardware/ electrical labels and schematics to ensure proper electrical voltage is connected to the S.U.B. The main power switch can now be turned on. 9. Resistive heating systems - Verify the temperature controller is off (the display should be flashing in the standby position). 10. For 1,000L water-jacketed units only, the water jacket ports are removed for shipping. Attach the ports to the tank using the tri-clamps provided (Figure 1.15). Thermo Scientific Single-Use Bioreactor (S.U.B.) 29

30 Section 1.7 Installation and Setup Figure 1.15 Attaching water jacket port using tri-clamp 11. For all water-jacketed systems, connect water inlet and outlet lines from the temperature control unit quick connects to the jacket (Figure 1.16). For 50 through 500 and 2,000L S.U.B. units, the inlet is typically on the left side when you are facing the connectors. For the 1,000L S.U.B. unit, the inlet is the lower connection, and the outlet is the upper. Inlet port Outlet port Figure 1.16 Water jacket ports for 50 to 500L units 12. Insert the sparge line support into the bottom of the S.U.B. unit (Figure 1.17). Figure 1.17 Sparge line support Thermo Scientific Single-Use Bioreactor (S.U.B.) 30

31 Section 1.7 Installation and Setup Drive Shaft 2,000L S.U.B. is supplied with a special drive shaft that differs in appearance and material when compared to the metallic shafts used in smaller S.U.B. sizes. The higher mechanical stress generated in the 2,000L S.U.B. requires a shaft made of carbon fiber composites (to reduce the weight of the long shaft) and special quick connect designs (to reduce joint fatigue in multiple-segment drive shafts). Before starting to load a drive shaft, verify that the drive shaft serial numbers match on all shaft segments (Figure 1.18). Do not interchange shaft segments. Always maintain a log history of the drive shaft and confirm that the it has sufficient life remaining. For warranty purposes, users must show documentation of proper drive shaft use. A sample log for documenting drive shaft use is provided in Appendix D of this publication. If the age or history of a drive shaft is questionable, it should be discarded. Multiple-segment drive shaft serial number with segment letter designations Air Line Preparation Porous Frit Sparger Figure piece drive shaft segment ends See tables 1.4 and 1.5, later in this chapter, for recommended air flow rates. The operating pressures at the level of the S.U.B. are of primary importance and these values must be adhered to. Please note flow rates in the graphics include both half and full volume applications. If you will be using a porous frit sparger in your S.U.B. BPC, whether alone or in a dual sparger setup, we recommend using the sparge line support (Figure 1.17) for all S.U.B. sizes. Simply attach this line support underneath the tank directly below where the porous frit sparger will be placed. This support piece holds the sparge line vertically so that the porous frit sparger itself is oriented in the vertical position for maximum effectiveness. The sparge line can be wound through the coil of the holder to keep the sparger oriented properly. WARNING: The S.U.B. BPC is not rated as a pressure vessel. The BPC should not be allowed to exceed the recommended pressure during inflation or operation. Gas pressure in the BPC should not exceed 0.5 psi (0.03 bar) at any time. Conditions of over pressure may result in BPC damage or personal injury. For reference the BPC will appear to be tight at 0.1 psi (0.007 bar). Thermo Scientific Single-Use Bioreactor (S.U.B.) 31

32 Section 1.7 Installation and Setup Cable Management System Setup If your system includes the optional cable management system, the short horizontal arm of the cable management system is removed for shipping and must be reattached, using a 7/16 wrench (Figure 1.19 through 1.21). Figure 1.19 Inserting cables into cable management system arm Figure 1.20 Seating the cable management system arm Figure 1.21 Attaching arm to cable management system vertical post Thermo Scientific Single-Use Bioreactor (S.U.B.) 32

33 1.8 General System Operation Information Section 1.8 System Operation Information BPC Preparation and Loading BPC Handling Instructions Each outer support container is designed for a specific S.U.B. BPC. Confirm the correct volume BPC is being used for the corresponding volume outer support container. Sections 2.2, 2.3 and 2.4 outline the installation and setup of the different volume S.U.B. BPCs please adhere to these instructions in the order in which they are presented. Take care if using a sharp object when opening outer polybags to avoid damaging the S.U.B. BPC. Do not drag containers over corners or sharp objects. Do not lift the container by the corners or top seams. Carefully coil tubing on top of the BPC to prevent puncturing the container with cable ties or clamps. Use cushioning between tubing and container in storage and transport. BPC Operating Information Aseptic Line Connection The most common recommended process for making connections to the tubing lines is with an aseptic tubing fuser. NOTE: Other connection options are available as a custom S.U.B. BPC assembly. Following the recommended tubing welder operating instructions, successful connections can be obtained for filling, supplementing, sampling, or dispensing from the S.U.B. BPC as needed. Working Volume Each S.U.B. is designed for a working volume range. The minimum working volume and the rated working volume are listed in the specifications tables (see Section 4). The total volume listed includes the headspace needed for proper aeration and gas management. Actual working volumes should not exceed the indicated rated working volumes by greater than 10%. In addition, working volumes less than 50% of the rated volume can result in damage to the S.U.B. BPC and hardware. Thermo Scientific Single-Use Bioreactor (S.U.B.) 33

34 Section 1.8 System Operation Information Aeration Exhaust vent filter The standard BPC is designed with special spargers that produce very efficient mass transfer of oxygen and typically will require much less gas inflow than conventional spargers. Gas inflow, in fact only need be limited to prevent foam generation and excessive pressure within the BPC. Gas flow rates supplied as overlay should also be reduced as much as possible or eliminated; this will minimize both liquid evaporation and demand on the exhaust filter. Ideally this will reduce the likelihood that gas inflows will exceed gas outflow of the system and reduce the occurrence of foam in the headspace that may plug the exhaust filter. For more information, refer to the Operating Pressure topic on the next page and section 1.10, Cell Culture Operating Instructions, of this manual. The exhaust vent filter used on the 50-1,000L S.U.B. is a Pall KA3 series filter utilizing hydrophobic PVDF membranes. To maintain a sterile connection, the standard BPC is supplied with the filter arrow pointing toward the BPC. This ensures that the filter vents are outside of the sterile connection. For users with more demanding applications, an optional vent filter heater can be used (see Section 5.3 for filter heater ordering information). The exhaust vent filters used on the 2,000L S.U.B. are Meissner UltraCap series filters utilizing hydrophobic PVDF membranes. These filters are provided in normal orientation with the flow arrow on the filter housing pointing away from the BPC. In the normal orientation, the filter has the maximum filter capacity. No side vents are provided; condensate must be managed by use of the condenser system or vent filter heater. Sampling The S.U.B. is equipped with a small volume sample port that is part of the S.U.B. BPC thermo-well. This small diameter (1.59 x 4.76mm [1/16 x 3/16 ]) silicone dip tube of 152.4mm (6 ) length allows low void volume samples to be taken for cell viability and density, as well as analyte analysis. This dip tube is supplied with a luer lock connector (SmartSite) that allows for direct sampling or attachment of various sampling manifolds by use of standard luer lock connection. Alternatively, manifolds can be welded onto the C-Flex sample line (3.18 x 6.35mm [1/8 x 1/4 ]) by tubing welder. For recommended systems for fluid transfer, see data sheet 016 (contact your sales representative for a copy). WARNING: Gas pressure within the S.U.B. BPC headspace should not exceed 0.5psi (0.03bar) at any time. Conditions of over pressure may result in BPC damage or personal injury. Thermo Scientific Single-Use Bioreactor (S.U.B.) 34

35 Section 1.8 System Operation Information Operating Pressure Agitation The S.U.B. BPC is not operating as a closed system, as it has both inlet and exhaust filters that are utilized to maintain a sterile environment for cells to grow without concern for contamination. However, conditions can be encountered when gas inlet flow rate may exceed exhaust flow rate. This may be encountered in the unlikely event of the failure of a pressure regulator on a gas feed, or when excessive foam within the bioreactor creates conditions of vent blockage. The S.U.B. BPC is not rated as a pressure vessel. All gas supplied to the bioreactor controller must be regulated to a pressure not to exceed manufacturer s recommendations (typically 10psi (0.69bar). Gas pressure in the S.U.B. BPC headspace should not exceed 0.5psi (0.03bar) at any time. More demanding applications may warrant an optional exhaust vent heater (See Section 4.3, Accessories and Options Specifications). If foaming is excessive in your cell culture process, it is best to reduce the operating volume of the process to 80% of maximum rated working volume of the S.U.B. system being used to provide greater head space volume. Single-use pressure transducers are available on custom S.U.B. configurations and are standard on the 1,000 and 2,000L systems. This technology combined with high-level control systems common with industrial applications can regulate gas pressure within the confines of the S.U.B. Extensive cell culture testing has not found an occurrence of overpressure sufficient to create a containment breach. Development testing of the BPC system has shown that in conditions of excessive pressure the polymer container will fail at the upper regions of the chamber where it is unsupported by the outer support container, minimizing the likelihood of the loss of bulk liquid. WARNING: The agitator should not be operated at volumes less than the stated minimum volume listed in the Specifications section of this guide. Agitation speed is adjusted by using the keypad interface on the electrical control panel, and must fall under the maximum mixing rates listed in the Specifications section. Agitation Control Interface for Units with A/C Motors and Electrical Box Enclosures The agitation control interface utilizes a red LED digital display to indicate stirring speed in units of revolution per minute (RPM). Power is supplied to the motor by a two position power switch that is illuminated in green when turned to the ON position (right position). The up and down arrows on the agitation keypad adjust the stirring speed. Due to the auto-restart capabilities of the S.U.B., the green start button on the keypad has been disabled; however the red stop button on the keypad is active. If the red stop button has been used to stop the motor, the controller can be reset and agitation restarted by using the main motor toggle switch on the left side of the control panel. For more information, see the illustrations in the control panel detail in section 1.9 of this manual. Thermo Scientific Single-Use Bioreactor (S.U.B.) 35

36 Section 1.8 System Operation Information Dispense and Harvest Hardware Operating Information Temperature (Units with A/C Motors, Electrical Box Enclosures, and Electric Resistive Heating Systems) The S.U.B. is equipped with a bottom drain line that allows for liquid harvest by means of peristaltic pump. Connection of the bottom drain line can be accomplished by use of a tubing welder or the connection of mm (1/4-3/8 ) quick connect that is provided. For S.U.B.s 50 to 1,000L, the bottom drain exits the S.U.B. BPC at the lowest vertical position on the side of the S.U.B. This allows for easy access for the user and minimizes the accumulation of cells in the area of the drain during the cell culture run. Manipulation of the S.U.B. BPC as the last few liters of media drain will minimize liquid hold-up within the S.U.B. The 2,000L S.U.B. is provided with a 25.4mm (1 ) style bottom drain near the center line of the tank bottom. Temperature setpoints can be adjusted via the temperature controller located on the front panel of the S.U.B. control box. This controller is pre-programmed to avoid overshoot during heat up and maintain a target temperature of +/- 0.5 C based on the set value (SV) display (green LED). (The set value of the PID temperature controller is factory preset at 2 C to prevent premature heat up). The process temperature is measured by means of a supplied RTD (pt-100) that is inserted into the thermo-well of the S.U.B. BPC. The temperature measured by the RTD is displayed with a red LED digital display as the present value (PV). For more detailed operating information on the controller, refer to the ETP. Enable/disable temperature controller by pushing left button. Adjust temperature set value by pushing up and down arrows located on the right. Process temperature value is illuminated in red. Flashing numeric display indicates the controller output is off (standby mode). NOTE: The heaters are not enabled unless the motor agitation is operational and should not be engaged until the BPC is filled to at least the minimum volume. Heating Performance Heating times for the Single-Use Bioreactor systems vary based upon liquid volume and temperature, ambient or heating liquid temperature, sparge rate and mixing rate. Users should adjust process liquid staging and seeding strategies to the unique aspects of the S.U.B. Process controllers and heaters are designed to provide optimum heat transfer and to minimize heat up times, while maintaining the material integrity of the polymer film construction of the S.U.B. BPC. (see Table 1.3) Thermo Scientific Single-Use Bioreactor (S.U.B.) 36

37 Section 1.8 System Operation Information Do not operate heater if the BPC is not at minimum liquid volume or greater. Care must be taken not to melt or damage the container or other components. Heaters should not be used to warm liquid above 40 C. In order to reduce the possibility of the tank becoming too hot, the heaters are supplied with a thermostat that will limit temperature to 65 +/- 5 C. The main power disconnect should be in the off position unless the S.U.B. hardware system is in active use. This will preserve the long term reliability of the heating system. NOTE: Users can reduce the heating time required to warm media by allowing media to warm to ambient temperatures before transferring into the S.U.B. BPC. WARNING: The heating element is provided with an over-temperature safety that will prevent direct damage of the heater element. The heater must not be operated when the outer support container is empty or when the S.U.B. is below minimum liquid operating volume. Serious harm to the operator or damage to the S.U.B. BPC will result. General temperature mapping has been performed for the S.U.B. systems by tracking thermal profiles within the liquid confines of the bioreactor. Testing conditions were analyzed using chilled media and low agitation rates. Gradients within the liquid did not exceed 0.5 C during heat up. Testing has also shown that temperature measurements in the S.U.B. when using the standard silicone thermo-well with 3.18mm (1/8 ) diameter temperature probes property represent bioreactor content temperatures. Users desiring exact temperature calibrations can order S.U.B. temperature/sample port. Using this port will allow users to simulate the temperatures seen by the RTD when used with the S.U.B. BPC. Thermo Scientific Single-Use Bioreactor (S.U.B.) 37

38 Section 1.8 System Operation Information Heating Times for S.U.B. Systems (ambient temperature of 25 C - values for jacketed systems assume a TCU heater size of 10W per batch liter) System Liquid Batch Volume (half/ full) Table 1.3 Approximate heating times for the S.U.B. Initial Liquid Liquid Target Time (half/full) Electric 50L 25L (minimum)/50l 5 C 37 C 5hr/7.5hr Jacketed 50L 25L (minimum)/50l 5 C 37 C 3hr/2.5hr Electric 100L 50L (minimum)/100l 5 C 37 C 5hr/7.5hr Jacketed 100L 50L (minimum)/100l 5 C 37 C 3.3hr/3hr Electric 250L 125L (minimum)/250l 5 C 37 C 6hr/8.5hr Jacketed 250L 125L (minimum)/250l 5 C 37 C 3.5hr/3hr Electric 500L 250L (minimum)/500l 5 C 37 C 8hr/11hr Jacketed 500L 250L (minimum)/500l 5 C 37 C 3.5hr/3hr Electric 1,000L 500L (minimum)/1,000l 5 C 37 C 9hr/12hr Jacketed 1,000L 500L (minimum)/1,000l 5 C 37 C 4hr/3.5hr Jacketed 2,000L 1,000L (minimum)/2,000l 5 C 37 C 4hr/3.5hr Protective Earthing (Units with A/C Motors) Replaceable Fuses (Units with A/C Motors) For units with A/C motors, protective earthing for the S.U.B. hardware system and the controller is provided through the ground terminal of the power plug. Source power to the controller must provide protective earthing to this terminal in order to minimize the hazard of a possible shock in the occurrence of a fault condition. Please refer to Appendix A for information about electrical receptacles. A ground wire is provided underneath the tank and must be tied to the controller before operation. Electrical components of the S.U.B. are equipped with circuit protection. The variable frequency drive used to power the mixer motor is protected by the use of midget quick blow fuses (Ferraz-Shuwmut, 10A for 50, 100 and 250L - #OTM10 and 15 A for 500L, 1,000 and 2,000L - #OTM15). Other components of the resistive heating system, such as the temperature controller and heating element, are protected with resetable breakers. In the case of an electrical fault condition, these safety devices are designed to protect the user from electrical shock and prevent electrical system components from being damaged. Fuses can be replaced and/or the breakers reset once the fault condition is resolved. NOTE: The normal on setting for these breakers is in the up position. A tripped breaker will be in the mid position. Off is in the fully down position. To reset a tripped breaker it must first be moved from the tripped or mid position to the off or fully down position before moving it the fully up or on position. Thermo Scientific Single-Use Bioreactor (S.U.B.) 38

39 Section 1.8 System Operation Information Scales and Weighing Systems Monitoring liquid volume within the S.U.B. during operation can be critical in cell culture applications that involve nutrient media feeds. This can also be a useful method for increasing the scalability of the S.U.B. by starting the process run at minimum operating volume. The ability to track operating volume by use of load cells or weigh scales allows the user the ability to control liquid volume and cell density as the bioreactor is increased to rated working volume during the process run. Load cells come standard on 1,000 and 2,000L S.U.B. units. For S.U.B. volumes of 50 to 500L, a load cell kit for weight/volume measurement is available as an option. Load cell kits can be installed at the factory or can be added later by a certified service technician. The load cell kit comes with three load cells, summing block, wiring and display with a choice of several interfaces. For more information, refer to the Specifications and Parts Information section of this manual. Refer to Section 1.6 for information about unlocking the load cells and Appendix C for load cell display calibration instructions. Please ensure that load cells are locked down for any movement of the S.U.B. unit. To lock the load cells before transporting a 50 to 1,000L S.U.B., follow the steps below and refer to Figure 1.11 and Hand-tighten the Load Cell Lockout Nut onto the Load Cell Lockout Post. You may need to use the small end of the supplied wrench to loosen the Load Cell Lockout Nut from the bottom of the base. 2. After nut is hand-tight against post, use the small end of the supplied wrench to turn it an extra ¼ turn. CAUTION: To avoid damaging the load cell, do not over tighten the nut. 3. Assemble a standard stainless 28.6mm (1 ½ ) tri-clamp around the flanges. 4. Repeat steps 1 through 3 for all load cells attached to the tank. To lock the load cells before transporting a 2,000L S.U.B., follow the steps below and refer to Figure 1.15 and Torque the both the bottom and top nut until they are locked together. 2. Since there is no access to the hold down bolt head, use the two locked nuts to turn the bolt until the bolt head is in contact with the top plate. CAUTION: To avoid damaging the load cell, do not over tighten the lockdown bolts. 3. Break the two nuts free, ensuring the bolt head is still in contact with the top plate. 4. Tighten the bottom nut to the base plate. 5. Tighten the upper nut to the bottom of the top plate. 6. Check to make sure bottom nut and bolt head engage the top plate. Thermo Scientific Single-Use Bioreactor (S.U.B.) 39

40 Section 1.9 Advanced Control Information 1.9 Advanced Control Information External Data Logging and Control Digital display weighing scales can be sourced from manufacturers such as Mettler-Toledo. Bench top scales are commonly used to measure the amount of bulk source media stored in a smaller volume BPC as it is transferred by peristaltic pump into the S.U.B. Floor scales can be used to measure the fluid content within the S.U.B. This is accomplished by rolling the S.U.B. onto the scale platform and leveling the S.U.B. skid once in position. The S.U.B. hardware systems are designed to allow advanced users to control all aspects of the operation of the bioreactor. Contact technical support for Thermo Scientific HyPerforma products general integration guidance. Thermo Scientific Single-Use Bioreactor (S.U.B.) 40

41 Section 1.10 Cell Culture Operating Instructions 1.10 Cell Culture Operating Instructions Checkpoints prior to Media Fill Operating Conditions for Cell Culture Applications Verify the following before proceeding to liquid fill: Load BPC into hardware following instructions provided in Section 2. All Kleenpak connector port heavy duty clamps (SV ) are closed and located as close as possible to the S.U.B. BPC. Exhaust filter is upright and secured using the holder. Clamp on drain tube is closed and located as close as possible to the S.U.B. BPC. Temperature RTD is completely seated in the thermo-well and secured. Air-filled BPC is properly oriented in the outer support container and BPC bottom tabs are secured. Optimal operating parameters for cell culture vary greatly between cell lines and media formulations. Tables 1.6 and 1.7 are provided as a reference for establishing safe upper operating control limits with the standard BPC design. Exceeding these operating limits may result in premature exhaust filter failure, excessive foaming, and excessive pressure build-up in the gas delivery line sets or the BPC. In many cell culture operations, the limits listed in tables 1.6 and 1.7 are excessive and should be further reduced when possible. When reducing gas flow rate limits, the following trade-offs should be expected: Reducing porous frit maximum operating limits will reduce system foaming but increase reliance on O 2 and/or the macro sparge (open pipe or drilled hole sparge) if reduced too far below system O 2 demand. Generally speaking, it is best to run the porous frit on air initially, and after the porous frit total flow rate limits are reached, substitute oxygen as shown in Graph 1.1. Reducing overlay maximum operating limits will reduce exhaust load (increase filter lifespan) but sacrifice sparger performance if reduced far enough to allow CO 2 buildup in the headspace. Reducing macro sparge maximum operating limits will reduce system foaming and exhaust load but also reduce system CO 2 stripping potential, leading to CO 2 build up in the culture solution which could result in large amounts of base addition to maintain the desired ph. Thermo Scientific Single-Use Bioreactor (S.U.B.) 41

42 Section 1.10 Cell Culture Operating Instructions If sparging seems to lose efficiency disproportionate to the cell culture density increase, increasing the headspace flow rate may help, if the efficiency loss is due to CO 2 buildup in the headspace. Gas Supply Setup for Dual Sparger Systems with Micro and Macro Delivery Systems The following graph depicts a dissolved oxygen (DO) management strategy. Refer to Tables 1.4 and 1.5 for gas flow rate recommended maximum values. In developing a gassing strategy for a S.U.B. using a dual sparger configuration, it is optional to have a cross over from N 2 to air when progressing from negative to positive DO control action (Graph 1.1 depicts a minor cross over). When progressing into higher positive DO control, it is optimal to scale the macro sparger (drilled hole or open pipe) air delivery so that sufficient CO 2 stripping is maintained as the micro (porous frit) sparger air flow rates increase. As a starting point, scale so that maximum macro sparger flow is achieved twice as early as the micro sparger, and then adjust for a given cell culture process. When micro sparger flow rate limits are reached, supplanting the micro sparger air flow with a steadily increasing ratio of O 2 allows for a higher degree of control while working within a total micro sparger gas flow rate limit. This approach will provide the best possible utilization of a dual sparger configuration. Graph 1.1 Dissolved oxygen control strategy Thermo Scientific Single-Use Bioreactor (S.U.B.) 42

43 Section 1.10 Cell Culture Operating Instructions Gas Flow Rates for BPCs with Open Pipe and Porous Frit Spargers Table 1.4, below, contains a listing of gas flow rates for all sizes of S.U.B.s with open pipe and porous frit spargers. This data should be used in specifying maximum volumes for mass flow controllers or rotameters. In optimal conditions (no condensation or fouling) the exhaust filters have a flow capacity of at least 20slpm at 0.1 psi. The total flow rate of gas into the system must be less than 20slpm per active exhaust filter. The values listed take into account the number of exhaust filters that are standard on each size of S.U.B. (1 6 filter for the 50, 100 and 250L and 2 6 filters for the 500 and 1,000L). The components of the 2,000L system are scaled up to allow flow ratings of 50slpm at 0.1psi. These values are a good rule of thumb, but are not absolute requirements. They are also not intended to be process gas flow settings. The process gas flow settings should be adjusted as discussed below with starting conditions not exceeding 50% of the listed maximum values in order to help avoid accidental foam-out of the S.U.B. exhaust filter. S.U.B. Range of Operating Parameters with Open Pipe and Porous Frit Spargers 50L 100L 250L 500L 1,000L 2,000L Temperature ( C) ± 0.1 Operating Volume (L) ,000 1,000-2,000 Agitation Rate (rpm) * Recommended Max. Gas Flow Rates Open Pipe Porous frit Overlay Open Pipe Porous frit Overlay Open Pipe Porous frit Overlay Open Pipe Porous frit Overlay Open Pipe Porous frit Overlay Open Pipe Porous frit (3) Overlay Air (slpm) O2 (slpm) CO2 (slpm) N2 (slpm) Total (slpm) Exhaust Load (slpm) Table 1.4 Operating conditions for cell culture applications (full and half volume) with open pipe and porous frit spargers *Note: 2,000L systems may run agitation up to 95rpm when operating at 90% or greater volume and using agitation rate governors, as listed in the specifications in Section 4 of this publication. Thermo Scientific Single-Use Bioreactor (S.U.B.) 43

44 Section 1.10 Cell Culture Operating Instructions Gas Flow Rates for BPCs with Drilled Hole and Porous Frit Spargers If foaming or exhaust filter load/lifespan is of primary concern, priority should be placed on tuning the system to operate primarily by adding O 2 through the porous frit. If CO 2 stripping is of primary concern, priority should be placed on running the macro sparge at flow rates sufficient to reduce or eliminate the need for base addition. Table 1.5, below, contains a listing of values for all sizes of S.U.B.s. This data may be used in specifying maximum gas flow rates for mass flow controllers or rotameters, when using drilled hole spargers (DHS) and porous frit spargers. In optimal conditions (no condensation or fouling) the exhaust filters have a flow capacity of at least 20 and 90slpm (standard liters per minute) at 0.1psig for the small and large standard equipped filter types, respectively. The total flow rate of gas into system must be less than the sum flow rate capacity of active exhaust filters.the values listed take into account the number and type of exhaust filters that are standard on each size of S.U.B. (one small filter installed in the 50 and 100L, one large filter installed in the 250 and 500L, two large filters installed in the 1,000L, and three large filters installed in the 2,000L vessels). These values are not absolute requirements. They are also not intended to be process gas flow settings. The process gas flow settings should be adjusted as discussed below with starting conditions not exceeding 25% of the listed maximum values to prevent unnecessary reduction of exhaust filter life span and foam generation. S.U.B. Range of Operating Parameters 50L 100L 250L 500L 1,000L 2,000L Temperature ( C) ± 0.1 Operating Volume (L) ,000 1,000-2,000 Agitation Rate (rpm) * Recommended Max. Gas Flow Rates DHS Porous frit Overlay DHS Porous frit Overlay DHS Porous frit Overlay DHS Porous frit Overlay DHS Porous frit Overlay DHS Porous frit Overlay Air (slpm) O2 (slpm) CO2 (slpm) N2 (slpm) Total (slpm) Exhaust Load (slpm) Table 1.5 Operating conditions for cell culture applications with drilled hole and porous frit spargers Note 1: See the integrator s reference guide and Appendix D for information about 2,000L agitation limits. Note 2: 2,000L S.U.B.s may require additional exhaust filters if running drilled hole and porous frit spargers at maximum capacity and overlay is run over 50slpm; the standard drilled hole plus porous frit dual sparge 2,000L B.P.C. is designed to safely handle an exhaust load of up to 270slpm. *Note: 2,000L systems may run agitation up to 95rpm when operating at 90% or greater volume and using agitation rate governors, as listed in the specifications in Section 4 of this publication. Thermo Scientific Single-Use Bioreactor (S.U.B.) 44

45 Section 1.10 Cell Culture Operating Instructions Manual Operation of Drilled Hole Spargers Table 1.6, below, provides guidelines for operating a BPC equipped with standard drilled hole and porous frit spargers when the user must manually set flow rates for the drilled hole sparger (DHS). Recommendations assume three manual flow rate adjustments of the drilled hole sparger during the course of a batch run, and are balanced with priority to maintain even to excessive CO 2 stripping throughout the culture operation. WARNING: Operating caution must be taken when manually setting drilled hole sparge gas flow rates if the drilled hole sparge is left inactive for any length of time. It is possible for liquid to flow between the drilled hole sparge and flow check valve if it is left idle. Never set an idle drilled hole sparge directly to values higher than 10% of their maximum recommended flow rate; pushing the water through the drilled hole sparge pores at higher flow rates may result in a damaging pressure spike. Once the drilled hole sparge has been purged it can safely be set to the target gas flow rate. Recommended Operation in Standard Dual Sparger BPC Configuration Using Manual Drilled Hole Sparger Operation (assumes 5 day log growth phase) 50L 100L 250L 500L 1,000L 2,000L Stage 1: Seed to 1/2 log growth (seed to day 2.5) Stage 2: 1/2 log growth to 3rd quarter log growth (day 2.5 to day 4) Stage 3: 3rd quarter log growth through stationary phase (day 4 and beyond) 0.015vvm vvm vvm vvm vvm vvm mid vvm mid vvm mid vvm mid vvm mid vvm mid vvm final vvm final vvm 0.05 final vvm final vvm final vvm final vvm Table 1.6 Manual Drilled Hole Sparge Flow Rate Operation Recommendation Note: Recommendation is based on a porous frit operated by a controller and CHO Gpex culture run using CDM4CHO with 6g/L Cell Boost 2 in a 250L S.U.B. at 250L liquid volume, 37 C, 50% DO, ph 7, and 124rpm agitation (approximately 19.7 watt/m3). Recommendation also assumes culture is capable of reaching near 10+E6 cells/ml density and has a log growth phase of approximately five days. Values for 100, 500, and 1,000L drilled hole sparger parts are estimates based on drilled hole sparger parameter scaling. End users should always verify performance in their specific operating environment. For more information on growth of specific cell lines in the S.U.B. system, please contact technical support. Thermo Scientific Single-Use Bioreactor (S.U.B.) 45

46 Section 1.10 Cell Culture Operating Instructions Media Fill 1. Select desired line set from the S.U.B. BPC for fluid introduction. 2. Make aseptic connection (tubing welder, quick connect or tri-clamp) and begin liquid fill. 3. Once approximately 20-30L of volume has been added, verify the position of the BPC in the outer support container, particularly the sparger and the drain line. Adjust positioning if necessary for proper fit. 4. It is recommended that the top corners of the BPC be pulled upward to reduce wrinkles during filling. NOTE: The BPC must be pulled at the top corners it will generally form wrinkles during liquid fill. If the film is not pulled from the top to remove wrinkles, film tensioning below the bearing port will result. 5. The stretch hooks can be removed from bottom corners of the S.U.B. BPC when fluid volume reaches 30% volume. This will eliminate any interference with the load cell readings. 6. Fill to desired liquid volume % of rated volume is recommended. 7. If employing a liquid batch-to-tank grounding cable with the stainless steel connector of the sample line, the sample line should be purged of air prior to probe calibration. Thermo Scientific Single-Use Bioreactor (S.U.B.) 46

47 Agitation (Units with Electical Control Boxes) 1. Once the media has reached half volume of the S.U.B., the agitation control can be turned on via the motor controller power switch, as shown in figure 1.21 Power reset switch Pressure display Motor speed control keypad Motor controller power switch Main switch Emergency stop button Figure 1.21 Front view of electrical control panel for S.U.B. units with water jackets (except for the 1,000L size) 2. Using the arrow keys on the motor speed control keypad, adjust the setpoint speed to the desired level (the red LED pressure display indicates the stirring speed in RPM). Adjust desired agitation rate within the recommended range per Table 1.4 or Table 1.5, depending on the sparge configuration. See figure for more information about various types of electrical control boxes. 3. Allow the speed to stabilize, then make fine adjustments if necessary. WARNING: Agitation must be stopped when volume falls below the minimum (1/2 of the rated volume), otherwise damage to the hardware or BPC may result. Only run agitation below 50% volume if you are using safety interlocks on the controller. See the Safety section in the front matter of this publication and Appendix D for more information about using safety interlocks and agitation speed governors on the controller. Thermo Scientific Single-Use Bioreactor (S.U.B.) 47

48 Section 1.10 Cell Culture Operating Instructions Temperature Control (Units with Electrical Control Boxes and Electric Resistive Heating Systems) 1. After the agitation has been set, turn on the temperature controller using the electric heater control keypad, using the left button (see figure 1.22 and 1.23). A flashing numeric display indicates the controller is off (standby mode). 2. Adjust temperature set value using the up and down arrows located to the right. 3. The process temperature value is shown in red. Power reset switch Display Motor speed control keypad Electric heater (PID) control Motor controller power switch External temperature control indicator Emergency stop button Main switch Figure 1.22 Front view of electrical control panel for S.U.B. units with electric resistive heaters (except for the 1,000L size) Thermo Scientific Single-Use Bioreactor (S.U.B.) 48

49 Section 1.10 Cell Culture Operating Instructions Power reset switch Motor speed control keypad Electric heater (PID) control for resistive heater units only Motor controller power switch External temperature control light Main power switch Emergency stop button Pressure display Load cell display Figure 1.23 Front view of electrical control panel layout for 1,000L S.U.B.s (all types) Buzzer Spare 1/4 liquid tight Continuous Power Use this outlet for devices that should not be turned off during and E-stop. Examples are recording devices, sensors, and controllers. The maximum for each receptacle is 1/2 amp. RTD input signal Emergency Shut-Off Power Use this outlet for devices that should be turned off when an operator activates an E-stop. Examples are pumps, motors, and any device that could harm an operator or ruin a batch during an E-stop. The maximum for each receptacle is 2 amps. Figure 1.24 Bottom view of electrical control panel (all sizes and types) Thermo Scientific Single-Use Bioreactor (S.U.B.) 49

50 Section 1.10 Cell Culture Operating Instructions Temperature Control (Water Jacketed Systems) Drive Shaft Rotation ph Probe Calibration DO Probe Calibration 1. Connection to an external temperature control unit (TCU) is made via the large quick connect couplings located on the vessel jacket. Ensure the inlet/outlet ports are connected properly; improper installation may result in poor heating/cooling performance. 2. Jacket should be purged of air any time the vessel jacket lines are reconnected. 3. Temperature set points are controlled by the TCU. Refer to the TCU manufacturer s guidelines for setup and operating instructions. Verify rotation of the drive shaft is operating in a counterclockwise motion when viewed from the top looking down. The S.U.B. is designed to mix in this direction only. In general, the ph probe calibration (post-autoclave) can be verified by pulling a sample and analyzing the ph on another calibrated ph meter. After polarizing for 6 to 8 hours, the DO probe can be calibrated in the S.U.B. using standard protocols. In general, turn on full air sparging (do not exceed 1lpm in 50L, 1lpm in 100L, 2lpm in 250L, < 4lpm in 500L, <6lpm in 1,000L, or <10lpm in the 2,000L) until the DO reading stabilizes; this is the 100% DO setpoint for the controller. A zero percent dissolved oxygen setpoint can be achieved by sparging with nitrogen or by unplugging the DO probe momentarily. Checkpoints Prior to Inoculation Verify the following before inoculation: Once S.U.B. is filled with media, disconnect BPC tabs from the S.U.B. hardware. Verify temperature and agitation are at the desired setpoints. ph probe is calibrated, autoclaved and connected via a Kleenpak Connector port. DO probe is autoclaved, connected via a Kleenpak Connector port, polarized (6 to 8 hours) and calibrated. Temperature RTD is completely seated in the thermo-well and secured. A method for making aseptic tubing connections should be available. Thermo Scientific Single-Use Bioreactor (S.U.B.) 50

51 Section 1.10 Cell Culture Operating Instructions Cell Inoculation Checkpoints In-Process Once the S.U.B. is operating at the targeted steady equilibrated state and has achieved temperature, the S.U.B. is ready for inoculation. Connect the inoculum addition line set to the seed culture vessel (equipped with the proper connectors/tubing) and transfer the inoculum into the S.U.B. Typically this is done with the tubing connection process (aseptic luer lock connection or tube welding) and peristaltic pump. Pump the desired volume of seed cells into the S.U.B. NOTE: For shear sensitive cultures, cells can be introduced by manipulating the addition port to direct the inoculum down the interior wall of the BPC and into the bulk fluid, reducing the shear on the cells. Custom line sets can be supplied with dip tubes which shorten the distance between the point of inoculum introduction and the bulk fluid level. Verify the following once or twice daily during the culture run: Visual verification of sparging (rising bubbles) through the sparger access window. Verification of process parameters such as temperature and agitation are at setpoint. BPC is not operating under pressure. Cap is tight on drive shaft. Temperature RTD is completely seated and secured. That no condensate accumulates in exhaust filter housing. If condensate has accumulated then this indicates that use of a filter heater or a Condenser System is required. NOTE: Noise may be emitted from the mixer assembly during operation. This noise may vary in intensity and frequency, but generally has no significant effect upon performance or overall durability of the S.U.B. BPC during the intended life of the product. Dispense and Harvest 1. Connect bottom drain tubing set to intended transfer line. 2. Open clamp positioned at the bottom drain port. 3. Begin drain by use of peristaltic pump. 4. When approximately 3-5 liters remain in the S.U.B. BPC, lift the S.U.B. BPC at the top hanging tabs located opposite of the bottom drain (this will pool media towards drain). 5. Hold bottom drain line near floor while lifting exhaust filter side of S.U.B. BPC to facilitate the drain of the final liter of harvest media. Thermo Scientific Single-Use Bioreactor (S.U.B.) 51

52 Section 1.10 Cell Culture Operating Instructions BPC Disposal S.U.B. Shutdown Preparation for Next Run Once the BPC is drained, the drive shaft can be removed and stored by reversing the steps used during assembly. The BPC can then be removed from the outer support container. Filters can be removed and integrity tested as needed according to the user s standard procedures. All product contact materials related to the S.U.B. can be disposed of in an appropriate waste container or incinerator. 1. Once run is complete, verify motor agitation is off and turn off the power to the outer support container by switching off the main power disconnect. 2. If the S.U.B. hardware has come in contact with caustic materials during the course of a run, rinse affected areas with a light water rinse, followed by normal routine cleaning (see Preparation for Next Run section). 3. Loose items such as the drive shaft, tools and RTD probes should be returned to their storage locations to prevent accidental damage. Between runs, the S.U.B. hardware (outer support container, probe shelf, drive shaft and mixer drive, etc.) can be wiped down with a sanitary wipe. The outer support container can also be cleaned with typical stainless steel cleaner. Store drive shaft in storage holder located near the handle of the outer support container. The S.U.B. hardware system can be cleaned to the extent of standard laboratory cleaning procedures. Care should be taken to ensure electrical connections have been disconnected and electrical enclosures are closed tightly. It is also recommended that excess water is not introduced under the heat shield or over the control panel. A wipe down with normal disinfectant solutions is sufficient. Avoid using excessive amounts of liquid. The unit must be allowed to fully dry prior to being brought back into operation. Thermo Scientific Single-Use Bioreactor (S.U.B.) 52

53 Section 1.11 Maintenance 1.11 Maintenance Routine Maintenance Environmental conditions, operating parameters and the ability of the user to adhere to the standard operating procedures described in this user s guide have significant impact upon the useful life of the S.U.B. hardware system. The following guidelines are based on standard operating conditions as outlined in the S.U.B. User s Guide. High wear items such as bearings, seals, O-rings, and sterilization valves common to conventional bioreactor systems have been purposefully considered in the design of the construction of the S.U.B. The S*.U.B. system is inherently robust and requires low levels of maintenance. Taking time between bioreactor runs to clean the exterior of the hardware will improve the appearance and overall longevity of the system. The drive motor is industrial grade induction motor with permanently sealed and lubricated gear box. The drive shaft is constructed to wear slightly with use and should be visually inspected after each run. The heating elements are designed to operate well within manufacturer s design limits and will provide consistent performance. Visual inspection of wear components and following the guidelines listed below will be sufficient to ensure dependable service. Replacement parts are available. Preventive Maintenance Schedule Replace the drive shaft after one year of service or refer to the following. Replace worn shaft head assembly when drive hex diameter at widest location measures equal to or less across the points (Figure 1.25). Refer to the Table 1.7 for new drive hex diameters and minimum drive shaft head hex diameters. Diameters are measured at widest location across the points. Refer to Table 1.8 and Appendix D for expected drive shaft longevity based on usage. If the drive cap becomes difficult to turn, lightly coat drive cap threads with food-grade anti-seize. Replacement of the drive motor is recommended every five years, or as needed. S.U.B. System New Hex Diameter Minimum Hex Diameter 50, 100 and 250L 14.9mm [0.587 ] +/ mm 14.4mm [0.566 ] 500 and 1,000L 21.3mm [0.839 ] +/ mm 20.8mm [0.820 ] 2,000L 21.3mm [0.839 ] +/ mm 20.8mm [0.820 ] Table 1.7 Shaft head dimensions Hex diameter measurement location Figure 1.25 Shaft head assembly Thermo Scientific Single-Use Bioreactor (S.U.B.) 53

54 Section 1.11 Maintenance Table 1.8 provides oeprating guidance and shaft replacement intervals when using the PIV-based agitation speed governor. If the 2,000L S.U.B. is operated without the protection of the prescribed PIV guidance, the maximum agitation speed should be limited to 75 rpm with a minimum mixing volume of 1,000L, and the drive shaft should be replaced every 3 months. See Appendix D of this publication for a log that may be used to track drive shaft usage. For warranty purposes, drive shaft usage must be documented. Usage-Based Drive Shaft Replacement Recomendations Replacement Interval 12 Months 6 Months S.U.B. Size 50L 100L 250L 500L 1,000L 2,000L* 100% 213 rpm 187 rpm 150 days 130 rpm 109 rpm 95 rpm 95% 210 rpm 184 rpm 148 rpm 128 rpm 107 rpm 93 rpm 90% 206 rpm 181 rpm 145 rpm 125 rpm 105 rpm 91 rpm 85% 202 rpm 177 rpm 142 rpm 123 rpm 103 rpm 90 rpm 80% 198 rpm 174 rpm 140 rpm 121 rpm 101 rpm 88 rpm 75% 194 rpm 170 rpm 137 rpm 118 rpm 99 rpm 86 rpm 70% 190 rpm 166 rpm 133 rpm 115 rpm 96 rpm 84 rpm 65% 185 rpm 162 rpm 130 rpm 113 rpm 94 rpm 82 rpm 60% 180 rpm 158 rpm 127 rpm 110 rpm 92 rpm 80 rpm 55% 175 rpm 153 rpm 123 rpm 106 rpm 89 rpm 77 rpm 50% 169 rpm 148 rpm 119 rpm 103 rpm 86 rpm 75 rpm 45% 164 rpm 143 rpm 115 rpm 100 rpm 83 rpm 72 rpm 40% 157 rpm 138 rpm 111 rpm 96 rpm 80 rpm 70 rpm 35% 150 rpm 132 rpm 106 rpm 92 rpm 77 rpm 67 rpm 30% 143 rpm 125 rpm 101 rpm 87 rpm 73 rpm 63 rpm 25% 134 rpm 118 rpm 95 rpm 82 rpm 68 rpm 60 rpm 20% 125 rpm 109 rpm 88 rpm 76 rpm 64 rpm 53 rpm * Assumes the use of agitation speed governors and safety interlocks on the controller Table 1.8 Drive shaft replacement schedule, based on a maximum recommended PIV of 40 Watts/ meter 3 Thermo Scientific Single-Use Bioreactor (S.U.B.) 54

55 Section 2 Operating Information This section covers the following topics: 2.1 Verification Procedures 2.2 BPC Loading Instructions (50, 100, 250L ) 2.3 BPC Loading Instructions (500, 1,000L) 2.4 BPC Loading Instructions (2,000L) 2.5 Probe Assembly 2.6 Sampling 2.7 Pall Kleenpak Connector Instructions Section 2 Calibration Procedures 2.1 Verification Procedures Mixer Speed Verification Temperature Controller Verification Pressure monitor Verification (when present) Load Cell Verification (when present) To verify the mixer speed, use a calibrated tachometer. Expect accuracy of +/- 1rpm. Speed scaling can be modified if the calibration needs to be adjusted. Please refer to Section 3.6 for details. To verify the temperature controller/rtd, use a S.U.B. silicone thermowell, the existing 3.175mm (1/8 ) OD RTD and a user supplied calibrated temperature bath. To verify the calibration of the pressure monitor, use a calibrated pressure standard. Pressures can be verified by clamping the BPC inlet line and supplying gas through the overlay gas filter. Expect accuracy of +/- 0.1psi. The monitor can be calibrated manually by referencing the monitor operator s manual supplied in the Equipment Turnover Package (ETP). It is recommended that the load cell manufacturer or a qualified technician verify the load cells on site. Refer to Appendix C for load cell calibration instructions. Expect an accuracy of +/- 0.5kg. Basic load cell default parameters are listed in the electrical schematic included with the Equipment Turnover Package (ETP). Thermo Scientific Single-Use Bioreactor (S.U.B.) 55

56 Section 2.2 BPC Loading L Volumes 2.2 BPC Loading for L Volumes Each outer support container is designed for a specific Single-Use Bioreactor BPC. Confirm the correct volume BPC is being used for the corresponding volume outer support container. The following section outlines the installation and setup of the S.U.B. BPC. 1. Remove irradiated bioreactor BPC from protective double polybags (Figure 2.1). 2. Load S.U.B. BPC from the top into the outer support container (Figure 2.2). 3. Orient S.U.B. BPC with bearing port up and toward motor drive with Kleenpak Connector probe ports facing bottom access cutout. Figure 2.1 BPC unpacking Figure 2.2 BPC loading Figure 2.3 Bearing port insertion 4. Place the bearing port into bearing port receiver, close the door and close the clamp (Figure 2.3). 5. Use the back access window to route the side and bottom ports through the opening in the outer support container (Figure 2.4). 6. Direct the sparger lines, bottom drain and sampling lines through the container access windows (Figure 2.5, Figure 2.6). 7. Pull the sparger lines through the bottom plate and loop them around the sparger line holder (Figure 2.5). 8. If a cable management system is available, attach the lines to the appropriate inlet ports (Figure 2.7, 2.8 and 2.9). 9. Connect incoming gas feed lines to both the overlay filter and the direct sparger filter (Figure 2.8, Figure 2.9). Thermo Scientific Single-Use Bioreactor (S.U.B.) 56

57 Section 2.2 BPC Loading L Volumes Figure 2.4 Bottom line access Figure 2.5 Sparge line setup Figure 2.6 Harvest line setup Figure 2.7 Line setup on cable management system Figure 2.8 Incoming line connection to overlay filter Figure 2.9 Incoming line connection to direct sparger filter Thermo Scientific Single-Use Bioreactor (S.U.B.) 57

58 Section 2.2 BPC Loading L Volumes 8. Inflate the S.U.B. BPC with air (inflation time approximately 20 min) through overlay filter, but do not exceed 25lpm or 0.5psi (0.34bar) internal BPC pressure. As BPC inflates, ensure it is properly oriented in the support container (port alignment, drain, sparge). WARNING: DO NOT EXCEED 0.5psi (0.034bar) within the BPC or the BPC could fail. For reference, the BPC will appear to be tight at 0.1psi (0.007bar). 9. As the container fills with air, manipulate the container to align the sparge lines in the sparge plate (Figure 2.10, Figure 2.11). NOTE: While a sparger line check valve is provided for each sparger line, it is not uncommon for some fluid to bypass check valves during typical use. Elevating the filter to ensure that it is not at the low point of the sparger line will reduce the chance that the filter is exposed to liquid. Figure 2.10 Sparger line positioning Figure 2.11 Sparger line alignment Thermo Scientific Single-Use Bioreactor (S.U.B.) 58

59 Section 2.2 BPC Loading L Volumes 10. Use the four bottom cut-outs located at the base of the support container as a reference to align hanging tabs on S.U.B. BPC (Figure 2.12). Figure 2.12 Hanging tab alignment 11. Attach the bottom corners (the number of hanging tabs varies from two to four, according to the vessel size) of the BPC s hanging tabs to the tab holders (pins) on the S.U.B. (Figure 2.13). Figure 2.13 Tab holder attachment 12. Position bottom-side drain, pulling out/downward to position port towards bottom edge of tank. 13. Align row of Kleenpak Connector probe ports within the access window (Figure 2.14). Figure 2.14 Kleenpak connector alignment IMPORTANT NOTE: Verify all port clamps are closed and located as CLOSE as possible to the body of the S.U.B. BPC. Thermo Scientific Single-Use Bioreactor (S.U.B.) 59

60 Section 2.2 BPC Loading L Volumes 14. Fill the S.U.B. BPC with air for approximately minutes to allow for drive shaft insertion (times will vary based on flow rate, inlet pressure and container volume). WARNING: The BPC must be at least partially inflated in order to insert drive shaft. 15. Connect the media ground clip to the stainless steel insert in the test line on the BPC. This step grounds the media inside the BPC and helps to eliminate electrostatic charge. (Figure 2.15) Ground clip connection Figure 2.15 Media ground clip connection Drive Shaft Insertion (50-250L Volumes) 16. Remove the latch pin from the safety cover over the mixing assembly and open the cover. Unscrew the threaded cap covering the hollow pass-through of the motor (Figure 2.16). Figure Motor assembly 17. Insert drive shaft through hollow pass-through of the motor assembly in the following manner (Figures ). Use two hands to load drive shaft through top of motor assembly; a slight back and forth twisting motion will aid in insertion and avoid stretching of the impeller tubing. When approximately mm (2-3 ) of shaft remains, twist back and forth slightly to engage impeller. Thermo Scientific Single-Use Bioreactor (S.U.B.) 60

61 Section 2.2 BPC Loading L Volumes When approximately mm (1-2 ) of shaft remains, twist back and forth slightly to engage bearing assembly. When approximately 6.4mm (0.25 ) of shaft remains, twist to align motor drive keyway with one of the four outer slots on the drive shaft head. Figure 2.17 Loading shaft Figure 2.18 Engaging impeller Figure 2.19 Bearing engaged Figure 2.20 Head aligned 19. Directly couple drive shaft to motor drive (Figures ). Place threaded cap on hollow pass-through and hand tighten clockwise. Tighten cap by placing spanner wrench on hollow passthrough and tighten cap using supplied torque wrench. NOTE: The torque wrench is a standard 3/8 square drive, and it is calibrated at the factory at 150in-lbs. Verify wrenches have been removed from system and placed in storage holders. Close safety access cover and insert latch pin. Thermo Scientific Single-Use Bioreactor (S.U.B.) 61

62 Section 2.2 BPC Loading L Volumes Figure 2.21 Replacing cap Figure 2.22 Tightening cap Figure 2.23 Replacing and latching cover Final Installation Steps 20. The air supply to the overlay can be turned off once the drive shaft has been inserted. 21. Optional: Wrap and secure vent filter heater on exhaust filter. Connect heater to controller and verify it is plugged into an appropriate 120 or 240 VAC outlet, then connect the power cord to the controller. NOTE: Controller is preset to 50 C. 22. Secure exhaust vent filter on its holder (Figure 2.24). NOTE: 500L BPCs and some custom containers are supplied with dual exhaust vents. Extended single and dual exhaust vent brackets are available (see section 4.3 for ordering information). Thermo Scientific Single-Use Bioreactor (S.U.B.) 62

63 Section 2.2 BPC Loading L Volumes Figure 2.24 Vent filter installation 22. Attach the overlay sparger line and any other lines to the cable management system, if available (Figure 2.25). Sparger lines routed to cable management system Figure 2.25 Optional cable management system on S.U.B. unit 23. Position and close a bar clamp (Part No. SV ) on the bottom drain line as close as possible to the BPC port (Figure 2.26). Figure 2.26 Bar clamp installation Thermo Scientific Single-Use Bioreactor (S.U.B.) 63

64 Section 2.2 BPC Loading L Volumes 24. Insert the RTD or selected temperature sensor into thermo-well (Figure 2.27, Figure 2.28). a. Place small amount of glycerol (0.5mL) in the well to aid in heat transfer. The glycerol will also act as a lubricant, which will help with probe insertion. b. Sensor should be inserted until base of probe meets the mouth of the thermo-well. Rotate the probe either clockwise or counter-clockwise to aid in insertion. c. Secure by twisting the luer lock collar if provided. The thermowell will stretch slightly when RTD is seated. Figure 2.27 Sensor insertion Figure 2.28 Sensor insertion 25. Optional: Connect pressure transducer to user-supplied monitoring device. 26. Continue to Section 2.5 for probe insertion instructions. 27. Close the bottom access hatch (500L only). The proper latch tension can be obtained by a combination of feel and visual inspection. When closing the latch the handle should begin to provide resistance to closing when the leading edge of the safety pin pass through of the latch handle aligns with the outside edge of the latch base (Figure 2.29). NOTE: When the latch is under-tensioned the safety pin pass through of the latch handle will be covered within the latch base and the handle will close very easily. If the latch is over-tensioned the handle will be excessively difficult to close. 28. The access doors must be closed and fully latched prior to filling the system with liquid. Safety pin pass through of latch set Latch retainer Figure 2.29 Latching the access door Thermo Scientific Single-Use Bioreactor (S.U.B.) 64

65 Section 2.3 BPC Loading 500-1,000L Electric Resistive Heater 2.3 BPC Loading for 500-1,000L Electric Resistive Heater Before beginning, verify that the outer support container is stationary with casters locked into place. BPC loading may require operators to step inside the bioreactor and the unit must be stationary for the safety of both operator and equipment. For ease in BPC loading, two operators may be required. For drive shaft insertion, a ladder or other means of elevation is required. 1. Open the door on the bioreactor support container and reach inside to open clamp on the bearing port receiver located below the motor (Figure 2.30, Figure 2.31). Figure 2.30 Opening the bioreactor door Figure 2.31 Close up of bearing receiver clamp Thermo Scientific Single-Use Bioreactor (S.U.B.) 65

66 Section 2.3 BPC Loading 500-1,000L Electric Resistive Heater 2. Remove irradiated BPC from protective double polybags (Figure 2.32). Do not remove the polybags from the line sets at this stage as the BPC may become difficult to manage. Do not allow the BPC or line sets to touch the floor. Figure 2.32 BPC in protective polybags 3. Reach into or step inside the outer support container with the front face (bearing port side) of the BPC oriented towards the motor (Figure 2.33). Figure 2.33 Bearing port orientation Thermo Scientific Single-Use Bioreactor (S.U.B.) 66

67 Section 2.3 BPC Loading 500-1,000L Electric Resistive Heater 4. Place the top line sets (still in polybags) over the top edge of the tank (Figure 2.34). This will keep the container from being restricted during the air inflation step. Figure 2.34 Line sets on edge of tank 5. Load the container bearing port into the receiver (Figure 2.35) and close the door and clamp (Figure 2.36). Figure 2.35 Bearing port in receiver Figure 2.36 Door clamped shut Thermo Scientific Single-Use Bioreactor (S.U.B.) 67

68 Section 2.3 BPC Loading L Electric Resistive Heater 6. Remove the bubble wrap from the sparge filters. Guide the sparge inlet lines and filters through the bottom cutouts in the outer support container (Figure 2.37). The operator can reach just below the tank to further extend the sparge lines from the cutouts (Figure 2.38). Figure 2.37 Sparger line insertion Figure 2.38 Sparger line extension 7. Pass the bagged drain line set and temperature/sampling port set through the large cut-out in the front of the outer support container (Figure 2.39). Extend the drain line set through the cut-out (Figure 2.40). Figure 2.39 Drain/sampling line set insertion Figure 2.40 Drain/sampling line extension Thermo Scientific Single-Use Bioreactor (S.U.B.) 68

69 Section 2.3 BPC Loading 500-1,000L Electric Resistive Heater Gas Supply Connections and Air Inflation The BPC should be partially inflated to allow for proper insertion of the drive shaft and to aid in proper alignment of the BPC in the outer support container. 8. Connect pressure transducer to monitor and once the display has stabilized, tare the monitor (the monitor should be allowed to warm up for 30 minutes before taring). Verify that the monitor reads zero. 9. Attach air supply to overlay gas inlet line. NOTE: Air pressure to the overlay gas line on the S.U.B. BPC should not exceed 3psi (less than 25lpm). WARNING: DO NOT EXCEED 0.5psi (0.34bar) within the BPC or the system could fail causing personal injury or damage to equipment. DO NOT leave the BPC unattended while inflating. 10. Begin air inflation through the overlay gas line. 11. While the BPC is inflating, attach the incoming gas supply to the sparger gas inlet line. NOTE: Air pressure to the sparger on the BPC should not exceed 8psi. Also, while a sparger line check valve is provided for each sparger line, some fluid may bypass check valves during typical use. Elevating the filter to ensure that it is not at the low point of the sparger line will reduce the chance that the filter is exposed to liquid. 12. Tare the load cell display before proceeding. 13. Attach the hanging tabs to the hooks on the bottom of the BPC (Figure 2.41, Figure 2.42) to the outer support container. Verify that the sparger filter and spargers remain in the correct position. It is recommended that users secure the hanging tabs on the front BPC panel first. This way the door will not be an obstruction when connecting the last set of stretch hooks. Figure 2.41 Hanging Tab and Hook Figure 2.42 Attaching Tab Thermo Scientific Single-Use Bioreactor (S.U.B.) 69

70 Section 2.3 BPC Loading 500-1,000L Electric Resistive Heater 14. While the BPC begins to fill with air, remove the protective packaging from the exhaust vent filters (Figure 2.43). Figure 2.43 Removal of protective packaging 15. Air filling of the container takes approximately minutes before drive shaft insertion can begin (times will vary based upon flow rate and inlet pressure). Thermo Scientific Single-Use Bioreactor (S.U.B.) 70

71 Section 2.3 BPC Loading 500-1,000L Electric Resistive Heater Drive Shaft Insertion for 1,000L Units The drive shaft is constructed in three or four pieces which must be assembled and inserted piecewise. Reminder: Operators should be elevated (i.e. ladder) to effectively assemble and insert the drive shaft. WARNING: Review ceiling height requirements in the Specifications section of this manual before trying to insert the drive shaft. 16. Prepare the hollow pass-through by first removing the latch pin on the safety cover (Figure 2.44), opening the safety cover (Figure 2.45) and removing the threaded cap (turn counterclockwise) of the mixing assembly (Figures 2.46 and 2.47). Figure 2.44 Latch pin removal Figure 2.45 Opening safety cover Cap Hollow passthrough Drive shaft head Motor drive keyway Latch pin Figure 2.46 Cap removal Safety cover Motor Drive shaft Figure 2.47 Motor and mixing assembly Thermo Scientific Single-Use Bioreactor (S.U.B.) 71

72 Section 2.3 BPC Loading 500-1,000L Electric Resistive Heater 17. Verify the three sections of the drive shaft are located in the drive shaft holders on the side of the outer support container. The sections will be referred to as the upper (section with the drive shaft head), middle (section with internal/external threads on each end) and lower (section with the square end). Lubricate the threaded ends with a light coat of food grade anti-seize with each use. 18. First insert the lower section through the hollow pass-through of the mixer drive (Figure 2.48). Slide latch pin from motor assembly into shaft to prevent it from falling into the tube (Figure 2.49). Assemble the middle and lower sections of the drive shaft by joining the segments with a twisting motion, fasten the two sections together (Figure 2.50). NOTE: Segmented shaft threads are left threaded (reverse threaded). Locate one wrench on the flat area in the middle drive shaft section, another wrench on the lower section and tighten the connection using a counterclockwise rotation (Figure 2.51, Figure 2.52). The shafts are reverse threaded to avoid loosening during operation. Once the sections are secure, return the wrenches to the tool holder. CAUTION: Do not over tighten; a snug fit is sufficient. Remove the latch pin. Figure 2.48 Inserting drive shaft Figure 2.49 Latch pin in shaft Figure 2.50 Section assembly Thermo Scientific Single-Use Bioreactor (S.U.B.) 72

73 Section 2.3 BPC Loading 500-1,000L Electric Resistive Heater Load the partially assembled drive shaft through the hollow pass-through and hold it in position with the latch pin. Obtain the upper section of the drive shaft and assemble it to the middle segment in the manner described previously. Figure 2.51 Section assembly Figure 2.52 Tightening of shaft connections 19. Using two hands, carefully guide the completed drive shaft into the BPC using a slight back and forth twisting motion (Figure 2.53). NOTE: It may be necessary for another operator to assist with drive shaft insertion. As one operator inserts the drive shaft, another operator should carefully manipulate the impeller when the end of the drive shaft begins to couple with the impeller. a. When mm (2-3 ) of shaft remains, twist slightly to engage the impeller. b. When mm (1-2 ) of shaft remains, twist slightly to engage the bearing assembly. c. When mm ( ) of shaft remains, twist to align motor drive keyway with one of the four outer slots on the drive shaft head. Figure 2.53 Drive shaft insertion Thermo Scientific Single-Use Bioreactor (S.U.B.) 73

74 Section 2.3 BPC Loading 500-1,000L Electric Resistive Heater 20. Directly couple the drive shaft to the motor by placing the threaded cap back on the hollow pass-through and tighten. 21. Tighten cap by placing spanner wrench counterclockwise on hollow pass-through and tighten using supplied torque wrench (Figure 2.54). NOTE: The torque wrench is a standard 3/8 square drive and it is calibrated at the factory at 150in-lbs. Figure 2.54 Tightening cap with wrench 22. Verify wrenches have been removed from the system and returned to the storage holders. 23. Close safety access cover and insert latch pin. Thermo Scientific Single-Use Bioreactor (S.U.B.) 74

75 Section 2.3 BPC Loading 500-1,000L Electric Resistive Heater Final Installation Steps Secure exhaust vent filters to top-mounted holders (Figure 2.55), or if using elevated dual exhaust filters, use the adapter piece and extended filter bracket (Figure 2.56). Figure 2.55 Vent filter Figure 2.56 Extended dual filter bracket 37. Fully extend the drain line set through the front cutout and attach the probe shelf. 38. Remove the polybag from the drain line set and position the line clamp as close as possible to the BPC port and close. Use a cable tie around the clamp to ensure the clamp cannot be accidentally opened. 39. Align the five Kleenpak ports through the front access window (Figure 2.57). Figure 2.57 Kleenpak port alignment Thermo Scientific Single-Use Bioreactor (S.U.B.) 75

76 Section 2.3 BPC Loading 500-1,000L Electric Resistive Heater 40. Insert RTD or selected temperature sensor into the thermowell (Figure 2.58). a. Place a small amount of glycerol (0.5 ml) in well to aid in heat transfer. b. Sensor should be inserted until base of RTD meets the mouth of the thermowell. c. Secure by twisting the luer lock collar if provided the thermowell will stretch slightly when RTD is seated (Figure 2.59). Figure 2.58 Sensor insertion Figure 2.59 Sensor insertion NOTE: Verify that all port clamps are closed and located as near as possible to the body of the S.U.B. BPC. Thermo Scientific Single-Use Bioreactor (S.U.B.) 76

77 Section 2.4 BPC Loading 2,000L Water Jacket 2.4 Loading 2000 L Water Jacket 1. Main power to the control panel should be switched on. Ensure that the drive motor is not running. Open both the front and rear doors on outer support container and use an elevated platform to open clamp on bearing port receiver located below the motor. Lower the hoist lifting frame to a position just above the top of the rear door by using the pneumatic control lever near the rear door. 2. Two operators should carefully remove irradiated BPC from protective double polybags (Figure 2.60). Do not remove the polybags from the line sets at this stage as the BPC may become difficult to manage. Do not allow the BPC or line sets to touch or drag on the floor. 3. Load the BPC through the rear access door, orienting the bottom of the container into the door first with the bearing port facing upward (Figure 2.61). Keep the container folded as supplied in the packaging, to allow the BPC to unfold naturally when lifted by the hoist. Figure 2.60 Removing the BPC Figure 2.61 Loading the BPC 4. Using the rear door for access, connect the retainer hooks of the hoist to the top of the BPC via the hanging tabs starting with the furthermost two tabs (Figure 2.62) and finishing with the closer two tabs (Figure 2.63). 5. Raise the BPC using the pneumatic lift, one operator observing from above with the second operator at ground level controlling the lifting valve (Figure 2.64). 6. Raise the hoist until the lift reaches full stroke. Once lifting stops the top level operator should hold the hoist frame and apply some minimal lifting force. This will assist the lifting device to pull in any Thermo Scientific Single-Use Bioreactor (S.U.B.) 77

78 Section 2.4 BPC Loading 2,000L Water Jacket Figure 2.62 Connecting furthermost retainer hooks Figure 2.63 Connecting closest retainer hooks Figure 2.64 Using the lift to raise the container remaining slack in the cable and ensure the lift device has been fully raised. Place the valve in the stop position. 7. Place the top line sets (in polybags) onto the lifting frame or over the side of the outer support container. This will help support the weight of the BPC and also keep the BPC from being restricted during the air inflation step. 8. Remove the black protective cap from the bearing port (Figure 2.65), load the BPC bearing port into the receiver (Figure 2.66) and close the bearing assembly door and latch it (Figure 2.67). Figure 2.65 Cap removal Figure 2.66 Bearing port loading Figure 2.67 Closing/latching door 9. Open the tubing set polybag and connect pressure transducer to monitor. Once the display has stabilized, tare the monitor (the monitor should be allowed to warm up for 30 minutes and the sensor connected for 10 minutes before taring). Verify that the monitor reads zero. Thermo Scientific Single-Use Bioreactor (S.U.B.) 78

79 Section 2.4 BPC Loading 2,000L Water Jacket Gas Supply Connections and Air Inflation The BPC must be partially inflated in order to aid in proper alignment of the BPC in the outer support container and is necessary to allow for proper insertion of the drive shaft. 10. Attach air supply to overlay gas inlet line at the top of the BPC. Operators should allow the container to fill to greater than half volume (typically > 20 minutes). NOTE: Air pressure to the overlay gas line on the S.U.B. BPC should not exceed 5psi (less than 100lpm). WARNING: DO NOT EXCEED 0.5psi (0.34bar) within the BPC or the system could fail, causing personal injury or damage to equipment. DO NOT leave the BPC unattended while inflating. 11. Feed the probe belt, sample line, and the subsurface addition lines through the front access door (Figure 2.68) 12. Remove sparge lines from the polybags and bubble wrap from sparge filters. Use the rear door to gain inside access to the floor of the hardware. Place a clamp on the bottom drain line at this time (Figure 2.69). Figure 2.68 Feeding lines through access door Figure 2.69 Clamping bottom drain line 13. Guide the sparger inlet line and filter through the bottom cutout in the outer support container. The plastic center tank insert of the floor provides the port locations for both the bottom drain (Figure 2.70) and the gas lines for the open pipe or drilled hole macro sparger (Figure 2.71). Opening the pivoting access cover in the bottom of the tank will allow access for loading the porous frit sparger gas line (Figure 2.72). Thermo Scientific Single-Use Bioreactor (S.U.B.) 79

80 Section 2.4 BPC Loading 2,000L Water Jacket Figure 2.70 Drain line support Figure 2.71 Macro sparger line support Figure 2.72 Porous frit sparger line loading Figure 2.73 Cutouts for porous frit spargers Three cut-out holes are provided in the tank for porous frit spargers. These holes are furthest from tank center line and align with the inside edge of this access cover door (Figure 2.73). NOTE: Air pressure to the sparger on the BPC should not exceed 8psi. Also, while a sparger line check valve is provided for each sparger line, it is not uncommon for some fluid to bypass check valves during typical use. We recommend elevating the sparger line filter as is feasible to help reduce this tendency. 14. Tare the load cell display before proceeding. 15. Using the positioning tab pins provided, secure the BPC by attaching the tabs on the bottom of the BPC onto the position tab pins (Figure 2.74, 2.75). Verify that the sparger filter and spargers remain in position while attaching the tabs. It is recommended that users secure the tabs on the front BPC panel first. This way the larger rear door will allow access when connecting the last set of tab pins. Thermo Scientific Single-Use Bioreactor (S.U.B.) 80

81 Section 2.4 BPC Loading 2,000L Water Jacket Figure 2.74 Container tab Figure 2.75 Securing the container tab to the pin 16. While the BPC begins to fill with air, remove the protective packaging from the exhaust (vent) filters. If the BPC is filled sufficiently with air the filters can be loaded into the holder. If using the exhaust condenser system, follow the setup instructions in the next few sections of this manual. If using elevated exhaust vent filters, use the corresponding extended dual vent filter bracket and filter heaters. Follow the setup instructions later in this chapter. Thermo Scientific Single-Use Bioreactor (S.U.B.) 81

82 Section 2.4 BPC Loading 2,000L Water Jacket Single-Use Exhaust Condenser Condenser System Functional Overview The condenser system is intended to be used as an accessory to the Single-Use Bioreactor (S.U.B.) as an alternative to vent filter heaters. Condenser systems are recommended for use with 2,000L S.U.B. tanks. The condenser s purpose is to prevent liquids and solids from condensing and collecting inside of the vent filters of the S.U.B. The condenser system cools the exhaust gasses leaving the S.U.B. chamber, condensing the moisture out of the saturated gasses coming from the S.U.B. The liquid condensate that is stripped from the exhaust gasses is then pumped back into the bioprocessing container chamber, creating a sterile loop and significantly reducing liquid loss due to evaporation. The condenser plate is chilled by a closed bath recirculating chiller which has sufficient capacity to cool two condenser plates simultaneously. Figure 2.76 shows a functional diagram of the condenser system. Exhaust line. from S.U.B. Filter straps Filter bracket assembly Exhaust vent filters Condenser bag gas outlet port Dual chambercondenser bag Condenser bag liquid drain ports Condenser return line back to S.U.B. Condenser bag gas inlet port Gripping tabs Alignment holes Dual headed peristaltic pump Closed Bath Recirculating Chiller Condenser plate assembly Condenser post assembly Post receivers Cart assembly Condenser Disposables Condenser Hardware Condenser System Figure Condenser System Overview Shown with 2,000L S.U.B. Thermo Scientific Single-Use Bioreactor (S.U.B.) 82

83 Section 2.4 BPC Loading 2000L Water Jacket When to Use the Condenser System 2,000L S.U.B. BPC with Single- Use Condenser System 2,000L S.U.B. BPC with Vent and Heaters Only Large 254mm (10 ) hydrophobic PVDF filters with a nominal 0.2 micron pore size were specified in order to increase the available surface area for off-gassing. In conjunction, the standard 2,000L S.U.B. is designed for use with a single use condenser system. This allows the S.U.B. to utilize a powerful phase-change type system which provides improved exhaust vent protection and reliability due to the ability to strip condensate and atomized materials that may be present from the off-gas stream of the S.U.B. This system has been shown to significantly reduce the fouling load on the vent filters that inherently increases operating back pressure as the cell culture run batch progresses. See the Single-Use Bioreactor Validation Guide for specific details. Some end-users may prefer to omit the Condenser System on the 2,000L S.U.B. under the expectation that this will allow for a more uniform installation (similar to smaller S.U.B. systems used in the upstream seed train), or will perhaps reduce system complexity and cost. The use of exhaust vent heaters and 254mm (10 ) filters will provide some impressive flow capacity over short periods (less than 5 days). However, the high sparge rates required during the scale-up of the S.U.B. to the 2,000L working volume may eventually create conditions of increased operating back pressure, usually due in part to blocking of the filter media. Depending upon the application, the user has the option of using both filters in parallel or initiating the run with a single filter, temporarily clamping off the line to the other filter (it being reserved as redundant back-up). NOTE: The single-use Condenser System has the potential to provide continuous and nearly indefinite protection against performance degradation of the exhaust vent filters (rated for continuous flow rates up to a total off-gassing rate of 50lpm and intermittent flows up to 100lpm). See the Single-Use Bioreactor System Validation Guide. Thermo Scientific Single-Use Bioreactor (S.U.B.) 83

84 Section 2.4 BPC Loading 2,000L Water Jacket Table 2.1 may help end-users specify the BPC configuration and operating parameter setup for custom 2,000L S.U.B. applications when not utilizing the exhaust condenser. Because the operating parameters of different cell cultures vary widely, a safety factor should be used to temper the data. As such, the data we used to generate a control base line are for reference only (filter fouling will vary and must be considered to ensure reliable performance). It is assumed no foam is present in the exhaust stream. S.U.B. System 2 each 254mm (10 ) Vents Run Duration Max. Combined Flow Rate Recommended Resulting Safety Factor 2,000L S.U.B. 7 days 40lpm 2x 2,000L S.U.B. 10 days 32lpm 2.5x 2,000L S.U.B. 14 days 27lpm 3x 2,000L S.U.B. 21 days Strongly Recommend: Single Use Condenser Table 2.1. Condenser system overview The above recommendations were generated using the test conditions shown in Graph 2.1. In this case a 2,000L S.U.B. was filled with 2,000L of DI water with a batch temperature of 40 C using a MKS vent filter heater at 60 C. Safety factor estimates are based on a maximum continuous internal S.U.B. BPC pressure not to exceed 0.1 psi, which corresponds to 40lpm with a single 254mm (10 ) vent ,000L S.U.B. BPC - Pressure Trending Direct Sparge Gas Loading, 2,000 L DI 40 C, Vent 60 C 0.20 Overlay Pressure (psi) Air Sparge (lpm) Single Meissner 10 Vent (0.2 micron PVDF) Graph 2.1 2,000L S.U.B. pressure trending NOTE: These results do not take into consideration a fouling safety factor. Thermo Scientific Single-Use Bioreactor (S.U.B.) 84

85 Section 2.4 BPC Loading 2,000L Water Jacket Also consider the size and type of tubing used to connect the exhaust vents to the S.U.B. BPC (when not using an exhaust condenser). Braid reinforced tubing provides the best protection against kinking or accidental pinching of the exhaust line. The 254mm (10 ) vents are supplied with 19.1mm (¾ ) hose barbs and this tubing diameter will allow condensate to return to the S.U.B. at total off-gas flow rates up to 30lpm (assuming the tubing is near a vertical orientation). Testing has shown that large diameter tubing will allow for lower exhaust gas velocities and if the vapor velocity is below 0.6m/s, gravity will allow the condensate formed in the tubing to return to the batch process (Graph 2.2). NOTE: Restrictive tubing connectors can create flow bottle necks and 12.7 mm (½ ) ID tubing is typically deemed too small for the 2,000L S.U.B. 2.5 Tubing I.D. Recommendation (inches) Flow Rate (lpm) Versus Vapor Exit Velocity (m/s) m/s Velocity Threshold Gas Vapor Velocity (m/s) Flow (lpm) Threshold Graph 2.2 Tubing diameter recommendation Thermo Scientific Single-Use Bioreactor (S.U.B.) 85

86 Section 2.4 BPC Loading 2,000L Water Jacket Various vent filter configurations are available on the S.U.B. depending upon the process scale and intended application. Graph 2.3 provides a reference for determining the relative capacity of different filters depending upon the amount of gas flow anticipated and the length of the run. In all cases, using a vent filter heater will reduce the chance of condensate blocking the filter but over time suspended solids carried in the exhaust stream will impede the flow of exhaust gas (resulting in increased back pressure). In addition, it is good practice to monitor the amount of foam present in the head space. In all cases, a vent filter heater have very little tolerance for handling the presence of foam in the exhaust stream. A small feed of antifoam (e.g., 10% simethicone, part number SH ) added directly to the liquid surface of the culture head space typically provides excellent foam control. Large systems (especially the 1,000 and 2,000L S.U.B.) can benefit from the use of a condenser system. It has been shown to increase system reliability at high flow rates (beyond 50lpm) and should warrant strong consideration when performing batch runs beyond 10 days. Results will vary; however, it is strongly recommended that end users select a vent filter configuration providing reserve capacity where possible. For example, dual vent configurations can be used independently with the second filter serving as a redundant backup (providing a quick reserve in case issues arise in process). Total Off-Gassing Load (LPM) Off-Gassing Management Guidelines for Single Use Bioreactor Condenser System vs. Exhaust Vent Heaters (Conditions: 37 C batch temperature, no foam in exhaust stream) Run Duration (days) Condenser System (2 X 10" vents) Vent Heater System (2 X 3" vents) Vent Heater System (2 X 10" vents) Vent Heater System (1 X 3" vents) Graph 2.3 Off-gassing management guidelines Thermo Scientific Single-Use Bioreactor (S.U.B.) 86

87 Section 2.4 BPC Loading 2,000L Water Jacket Condenser System Setup 1. Remove pre-assembled system from crate. 2. Position the system in a location adjacent to the S.U.B. that allows for connection to facility power. Lock the casters on the cart. 3. Remove reservoir cap of the chiller and add appropriate type and volume of fluid as per chiller user s guide. 4. Verify that the peristaltic pump and chiller power cords are connected to the power strip. 5. Plug in the system power strip. 6. Turn on power to the chiller. This will allow the chiller to prime. 7. Add fluid to the reservoir as needed to maintain minimum level. 8. Purge the chill plate by loosening the bleed plug on top of the plate. This is accessed using a hex wrench passing through the top tensioning plate of the chill plate assembly. Loosen plug only enough to allow trapped air to escape, then re-tighten. 9. Ensure that the reservoir is still at least at the minimum level. 10. The settings for the chiller and peristaltic pump are preset at the factory. These settings allow for the system to resume set point if the power is temporarily disrupted. Verify that the chiller and pump set points are at the recommended levels (5 C and 12 rpm). 11. Verify that the peristaltic pump is in place on the cart beneath the chill plate. Thermo Scientific Single-Use Bioreactor (S.U.B.) 87

88 Section 2.4 BPC Loading 2,000L Water Jacket Condenser System Loading Two operators are required to safely setup the exhaust system. Setup time is typically 2-3 min. 1. The first operator, located at the elevated position, should remove the condenser BPC carefully from the polybag packaging and then lower the assembly (directed in a vents first orientation) to the second operator located at ground level, standing to the rear but between the condenser cart and the S.U.B. (Figure 2.77). 2. The first operator at the upper position moves to ground level, opens both doors on the chiller plate, and loads the condenser BPC from the front, keeping the BPC in a saddle bag shape. The vents can hang freely (Figure 2.78). 3. The second operator routes the gas inlet lines around and behind the vent holders and inspects both lines to ensure they are connected to the S.U.B. and are not twisted or kinked, adjusting them as needed (Figure 2.79). Figure 2.77 Removing condenser Figure 2.78 Opening chiller plate Figure 2.79 Routing gas inlet lines 4. The second operator then holds the vent filters, places them into the vent filter holders above the chill plate (Figure 2.80). 5. The first operator then uses the Velcro straps to secure the filters in position (Figure 2.81). 6. The first operator then uses the grasping tabs to position the container using the two lower button pins on each side of the chiller plate (Figure 2.82). Thermo Scientific Single-Use Bioreactor (S.U.B.) 88

89 Section 2.4 BPC Loading 2,000L Water Jacket Figure 2.80 Placing vent filters Figure 2.81 Securing vent filters Figure 2.82 Positioning container 7. The clear side doors are then closed as the operator carefully manipulates first the gas inlet line and then the gas outlet line (Figure 2.83) to clear the doors as each is closed and latched (Figure 2.84). 8. The second operator then locates the gas inlet lines into the clips behind the vents (Figure 2.85). Figure 2.83 Moving gas lines Thermo Scientific Figure 2.84 Latching door Figure 2.85 Clipping gas lines in place Single-Use Bioreactor (S.U.B.) 89

90 Section 2.4 BPC Loading 2,000L Water Jacket 9. The first operator loads the peristaltic tubing into the pump, verifying there is sufficient slack at each end of the pump tubing, aligns tubing in the pump channel closes the pump ramp (Figure 2.86). 10. The pump is then started (pushing the red button, Figure 2.87). Operators verify that both the pump and chiller are enabled and running at the proper settings (we recommend that the pump setting be 12-30rpm and the chiller 5 C). Specified pumping system is qualified to run continuously (wet or dry) beyond 21 days. Figure 2.86 Loading pump tubing Figure 2.87 Starting the pump 11. After setup the operators should verify the following: The elbow fittings on the inlet and outlet of the condenser saddle bag are straight and level The gas inlet line and the condensate line are not twisted, pinched, or obstructed. Low spots in the gas inlet line should be avoided. Adjust the lines to avoid condensation pooling The pump union is loose on both ends of the pump and running smoothly in the peristaltic rollers. Specific performance questions can be answered by reviewing the Condenser portion of the S.U.B. Validation Guide or contacting technical support. Thermo Scientific Single-Use Bioreactor (S.U.B.) 90

91 Section 2.4 BPC Loading 2,000L Water Jacket Exhaust Vent Heater (optional) 1. A single operator can safely set up the exhaust vents and heaters in this configuration. 2. Clip each filter one at a time into the elevated vent filter holder system; carefully center the filter housing allowing the clip to secure it near the hose barb connections (Figure 2.88). 3. Verify the routing of the exhaust tubing, ensuring that it is not likely to cause kinks in the tubing. 4. Place the vent heaters around each filter ensuring the snap retainers are secured. Position the power leads to avoid interfering with the vent holder brackets (Figure 2.89). 5. Raise and rotate the vent holder bracket as needed. Make final inspection to ensure no kinks or low spots will occur in the tubing between the BPC and the filter (even if the BPC becomes pressurized) (Figure 2.90). 6. Connect the power to the vent heaters and verify operation of controllers. 7. Inspect the controllers set points (recommended 60 C). After 2-5 minutes of operation, verify the vent heaters are warm and near the desired temperature set points and that no alarm indicators are active. Figure 2.88 Clipping filter to holder Figure 2.89 Installing heaters Figure 2.90 Inspecting tubing Thermo Scientific Single-Use Bioreactor (S.U.B.) 91

92 Section 2.4 BPC Loading 2,000L Water Jacket Drive Shaft Insertion 1. The drive shaft is constructed by assembling either two or four pieces; these must be assembled and inserted in pieces in areas having low ceilings. For ceiling height recommendations for each type of drive shaft, see the Specifications in Section 4. Operators should be elevated (i.e. ladder) to assemble and insert the drive shaft. 2. The BPC must be filled with air to greater than approximately 50% volume to allow for unrestricted loading of the angled drive shaft. NOTE: After inflation, the impeller tubing should be hanging straight down inside the BPC, with the impeller near the bottom. 3. Verify the proper drive shaft sections and tools are available. 4. Prepare the hollow pass-through by first removing the latch pin on the safety cover (Figure 2.91), opening the safety cover, and removing the threaded cap (turning it counterclockwise) (Figure 2.92) of the motor and mixing assembly (Figures 2.93). Use the wrench and spanner provided, if required. Figure 2.91 Removing latch pin Figure 2.92 Opening safety cover Cap Hollow passthrough Drive shaft head Motor drive keyway Latch pin Safety cover Motor Drive shaft Figure 2.93 Motor and mixing assembly Thermo Scientific Single-Use Bioreactor (S.U.B.) 92

93 Section 2.4 BPC Loading 2,000L Water Jacket 5. Verify that the two or four sections of the drive shaft, all with matching serial numbers (Figure 2.94), are located in the drive shaft holders on the side of the outer support container. For the 4-piece drive shaft loading described here, the sections will be referred to as the upper (section with the drive shaft head), middle 2 each (section with internal/external quick connect on each end), and lower (section with the square end). IMPORTANT NOTES: Each time drive shafts are assembled and used in the S.U.B., operators must verify that sections have matching serial numbers. No lubrication is required with the quick connect assembly design. Multiple-section drive shaft serial number with section letter designations Figure 2.94 Close up view of the ends of 4-piece drive shaft sections 6. First insert the lower section through the hollow pass-through of the mixer drive (Figure 2.95). Once inserted, slide latch pin from mixing assembly into shaft to prevent it from falling into the impeller sleeve (tube), (Figure 2.96). Figure 2.95 Insertion of lower section of drive shaft Figure 2.96 Use of latch pin Thermo Scientific Single-Use Bioreactor (S.U.B.) 93

94 Section 2.4 BPC Loading 2,000L Water Jacket 7. To connect two shafts together, the button on the female side is depressed (Figure 2.97) and the sleeve is slid back, this will expose a red ring underneath the sleeve (Figure 2.98). This is a visual indicator that the sleeve is not in a locked position. Figure 2.97 Button used for connection Figure 2.98 Sliding sleeve exposes red not locked indicator 8. Place the female side of the quick connect over the male end (Figure 2.99). The connection is fully seated when the red of the male end is no longer exposed (Figure 2.100). 9. Slide the sleeve towards the connection, allowing the push button to lock into position, this will engage the locking mechanism and also cover the red indicator ring (Figure 2.101). NOTE: When fully connected, no red coloring should be visible. Figure 2.99 Quick connection Figure Red indicator ring Figure Sliding the sleeve into place Thermo Scientific Single-Use Bioreactor (S.U.B.) 94

95 Section 2.4 BPC Loading 2,000L Water Jacket 10. Remove the latch pin, load the partially assembled drive shaft through the hollow pass-through and hold it in position sufficient to allow the latch pin to be inserted in to the next set of holes. 11. Assemble the two middle sections of the drive shaft by joining the segment using the quick connects as performed in the previous steps. 12. Obtain the upper section of the drive shaft and assemble it to the other segment in the manner described previously. Once the sections are secure, remove the latch pin and return the wrenches to the tool holder. 13. Using two hands, carefully guide the completed drive shaft into the BPC using a slight back and forth twisting motion or a counterclockwise rotation (Figure 2.102). Do not push the drive shaft straight in. When mm (2-3 ) of shaft remains, twist slightly to engage the impeller. When mm (1-2 ) of shaft remains, twist slightly to engage the bearing assembly (Figure 2.103). When 6.4mm (0.25 ) of shaft remains, twist to align motor drive keyway with one of the four outer slots on the drive shaft head (Figure 2.104). Figure Insertion of drive shaft Figure Engaging bearing port Figure Drive shaft head alignment Thermo Scientific Single-Use Bioreactor (S.U.B.) 95

96 Section 2.4 BPC Loading 2,000L Water Jacket 14. Ensure the head is fully seated before directly coupling the drive shaft to the motor by placing the threaded cap back on the hollow passthrough and tightening it. Any spring-back indicates that the drive shaft is not properly seated in the impeller. Figure illustrates a drive shaft that is completely inserted into the impeller. NOTE: The cap should be easy to install when the drive shaft head is fully engaged in the hollow pass-through. Otherwise repeat steps 1 through 13 before installing the cap. Drive shaft end Figure Drive shaft fully inserted into impeller 15. Secure cap by placing spanner wrench on hollow pass-through and tightening using the supplied torque wrench (Figure 2.106). NOTE: The torque wrench is a standard 3/8 square drive and it is calibrated at the factory at 150in-lbs. Figure Tightening cap 16. Verify wrenches have been removed from the system and returned to the storage holders. 17. Close safety access cover and insert latch pin. Thermo Scientific Single-Use Bioreactor (S.U.B.) 96

97 Section 2.4 BPC Loading 2,000L Water Jacket Securing Access Doors 1. The access doors should be secured first with the latches. Proper tension is obtained by adjusting the threaded latch pin. Tension of the latch is adjusted by varying the position of the pin on the threaded shank. 2. The proper latch tension can be obtained by a combination of feel and visual inspection. When closing the latch the handle should begin to provide resistance to closing when the leading edge of the safety pin pass through of the latch handle aligns with the outside edge of the latch base. (Figure 2.107). NOTE: When the latch is under-tensioned the safety pin pass through of the latch handle will be covered within the latch base and the handle will close very easily. If the latch is over-tensioned the handle will be excessively difficult to close. 3. For maximum security insert pins (not included) into the respective latches. 4. Turn off the air supply to the overlay line. Insert drive shaft and close side access doors. Figure Latch access door Outside edge of latch base Safety pin pass through of latch set Latch retainer Safety pin pass through of latch-handle Thermo Scientific Single-Use Bioreactor (S.U.B.) 97

98 Section 2.4 BPC Loading 2,000L Water Jacket Final BPC Installation Steps 1. Verify the proper position of the exhaust filters and that the exhaust flow path is unobstructed. Connect the gas supply lines - verify the intended flow paths for overlay, porous frit spargers, and open pipe sparger. 2. Verify the overlay and direct sparger lines are well positioned and free of kinks or unintended clamps. Verify the rear access door is closed with proper latch tension. 3. Remove the polybag from the drain line set and verify redundant line clamps are in position. Use a cable tie around the clamp to ensure the clamp cannot be accidentally opened. 4. Ensure the front access door is latched and align the five Kleenpak ports through the front access window (Figure 2.108). This will be the lower cutout if hardware is supplied with two horizontal slots. Position clamps as close as possible to the BPC and close them on all tube ports (Figure 2.109). 5. Place clamps on subsurface lines as close to the port as possible, this will eliminate media from filling these lines prior to use (Figure 2.110). Figure Kleenpak port alignment Figure Clamp installation Figure Clamp positioning Thermo Scientific Single-Use Bioreactor (S.U.B.) 98

99 Section 2.4 BPC Loading 2,000L Water Jacket 6. Insert RTD or selected temperature sensor into the thermowell (Figure 2.111). Place a small amount of glycerol (0.5mL) in well to aid in heat transfer (Glycerol - Sigma G6279). Sensor should be inserted until base of RTD meets the mouth of the thermo-well. Secure by twisting the luer lock collar if provided. The thermowell will stretch slightly when RTD is seated. 7. Connect batch-to-tank grounding cable to the stainless steel connector of the sample line (Figure 2.112) NOTE: Verify all port clamps are closed and located as close as possible to the body of the S.U.B. BPC. 8. IMPORTANT: During media fill verify the position of all critical ports (drain, spargers, line sets, and probes) before more than 50L of liquid is filled into the container. This will still allow time for adjustments if required after the fill is initiated. 9. Typically two fill lines, 12.7mm (½ ) x 19.1mm (¾ ), and peristaltic pumps are recommended to fill the 2,000L S.U.B. in a timely manner. Figure Insertion of sensor into thermowell Figure Connection of grounding cable Thermo Scientific Single-Use Bioreactor (S.U.B.) 99

100 Section 2.5 Probe Assembly 2.5 Probe Assembly Preparation and Sterilization 1. Select the appropriate probe (see Section 1). Verify the presence of a Teflon support ring and O-ring on the probe and visually inspect them for damage. 2. Perform any required probe maintenance and calibrate the ph probe (see Probe Calibration within this section). 3. Insert the probe into the probe assembly through the threaded adaptor. 4. Verify the probe tip is not touching (more than 6.35mm [¼ ] gap) the membrane of the Kleenpak Connector before threading into probe adaptor. 5. Hand tighten the adaptor and verify the probe tip is not touching the membrane. 6. Place the probe assembly with probe into the autoclave tray for probe kits (Figure 2.113). 7. Autoclave the probe assembly using a validated sterilization cycle (approximately 30 minutes at 122 C). Thirty minute sterilization cycles are generally sufficient. Options of wet or dry cycle parameters can be used. Slow exhaust cycles are preferred as this minimizes stress on the probes during the temperature and pressure changes of autoclaving. 8. Allow sufficient time for probe assemblies to cool completely before seeking to connect to the S.U.B. BPC for probe insertion. 9. When stored properly, the autoclaved probe assemblies can be stored dry for short periods of time (< 24hr) without loss of sensor longevity, performance or sterility. NOTE: Before beginning probe insertion, please become familiar with the Kleenpak Connector procedure outlined in Section 2.7. Probe assembly Handle Autoclave tray for probe kits Figure Probe and autoclave tray Thermo Scientific Single-Use Bioreactor (S.U.B.) 100

101 Section 2.5 Probe Assembly Probe Insertion 1. Probe clips clip onto the outer support container and hang above the probe assembly (Figure 2.114). 2. The probe clip attaches to the tank and is retained using an adjustable ball detent (a spring-loaded screw) (Figure 2.114). 3. Install presterilized sensor and probe kit using aseptic connection methods as described in Section 2.7. The aseptic connection is completed prior to the bellows being collapsed (Figure 2.115). 4. Insert the probe by collapsing the bellows (Figure 2.116). Figure Probe clip Figure Asceptic connections NOTE: If the BPC is already filled with liquid, the best practice is to squeeze the bellows to expel air prior to collapsing it. Then insert the probe fully as described. 5. Position the probe clip in the desired horizontal location (Figure 2.117). The clip s adjustable ball detent (spring loaded screw) should be adjusted so the probe clip can be easily moved to the desired position while still securing the clip to the steel support piece. Figure Collapsed bellows Figure Positioning of probe clip Thermo Scientific Single-Use Bioreactor (S.U.B.) 101

102 Section 2.5 Probe Assembly 6. Swing the probe upward into the flexible spring. Release the probe assembly and verify the probe remains at the proper insertion depth and angle as the bellows expands to rest freely in the probe clip (Figure 2.118). Figure Connection of spring clip to probe NOTE: The fixed position of the probe clip and the flexible nature of the spring retainer allow for both proper angle (more than 10 degrees) and compression of the probe bellows. Probe Calibration Probe calibration is controller-specific; however, the following general rules apply: a. ph probes must be calibrated prior to steam sterilization and the calibration of the probe can be standardized by comparison of an off-line sample once the ph probe has been connected to the S.U.B. b. Dissolved oxygen probes are generally calibrated after steam sterilization. Once the probe is connected to the S.U.B. and is allowed sufficient time to polarize (6-8 hours of continuous connection to the power supply provided by a controller or polarization module), it can be calibrated. Thermo Scientific Single-Use Bioreactor (S.U.B.) 102

103 Section 2.6 Single-Use Bioreactor BPC Sampling 2.6 BPC Sampling Sampling Aseptic Sampling During operation of the S.U.B., samples may need to be taken for monitoring of various parameters established by the user. The following describes two techniques for sampling: aseptic sampling via gamma irradiated syringe and sampling utilizing a gamma irradiated manifold. Using standard luer lock on 60mL syringe or manifold: 1. Remove dust cover from SmartSite (Figure 2.119). 2. Clean SmartSite with sanitary wipe. 3. Connect sanitary luer lock type syringe (Figure 2.120). 4. Apply small amount of vacuum pressure by pulling out syringe plunger slightly. 5. Open pinch clamp and pull sample (approximately 30-60mL) using care not to allow any back flow. 6. Close pinch clamp and remove syringe (this will be a purge sample). 7. Clean SmartSite with sanitary wipe. 8. Connect sanitary luer lock type syringe. 9. Apply small amount of vacuum pressure by use of syringe. 10. Open pinch clamp and pull desired sample volume (approximately 10-20mL) using care not to allow any back flow. 11. Close pinch clamp and remove syringe (this will be a representative sample). 12. Clean SmartSite with sanitary wipe and replace dust cap. Figure Removal of dust cover from SmartSite Figure Connecting the syringe Thermo Scientific Single-Use Bioreactor (S.U.B.) 103

104 Section 2.6 Single-Use Bioreactor BPC Sampling Sampling with Sterile Manifold Use the steps below to attach a sample manifold, if purchased. 1. Remove manifold from protective polybag package. 2. Close all clamps on manifold lines. 3. Use sterile tubing welder to connect manifold to sample line (Figure 2.121). 4. Inspect welds and open flow path by pinching the welds. 5. Open two clamps at inlet and the clamp at purge container (100mL container). 6. Purge sample line by filling this container (recommended 30-60mL). 7. Close clamp nearest purge container. 8. Open clamp to sample container (50mL sample container) (Figure 2.122). 9. Allow container to fill with liquid by gravity (recommended 10-20mL). 10. Close clamps at sample manifold inlet. 11. Close clamp nearest the sample container. 12. Remove the filled manifold from S.U.B. by welding new manifold onto sample line (this will be used for taking the next sample). Figure Tubing welder Figure mL manifold Thermo Scientific Single-Use Bioreactor (S.U.B.) 104

105 Section 2.7 Pall Kleenpak Connector Instructions 2.7 Pall Kleenpak Connector Instructions Specifications These instructions should be read thoroughly as they contain valuable information gained by extensive experience. It is very important that all instructions are carefully followed and, where appropriate, should be incorporated into the user s standard operating procedures. If some of the procedures do not suit your needs, please consult Pall or your local distributor before finalizing your system. The Kleenpak connector has a maximum working pressure of 3bar (43.5psi) at 40 C in compatible fluids. WARNING: Operation outside the above specifications and/or with fluids incompatible with construction materials may cause personal injury and result in damage to the device. Receipt of Equipment Installation The male and female Kleenpak connectors are supplied in separate packages. There are several types of end fittings in order to match different tubing size requirements and to allow for different attachment possibilities to flexible tubing. To access full part number availability go to a. Store the male and female Kleenpak connectors in clean, dry conditions and wherever practical in the external packaging as delivered. b. DO NOT remove from the inner device bag packaging until just before installation. c. Male and female Kleenpak connectors are supplied protected by an inner and outer bag. Ensure that the packaging is undamaged. d. The assembly aid is provided non sterile and can be reused multiple times. It needs to be stored in clean and dry conditions between each use. The assembly aid is supplied separately and is available for purchase from your local Pall representative. Before installation, it is essential to verify that the Kleenpak connector is suitable for the liquid that it will be in contact with for the application and to follow the appropriate instructions listed below. a. Install the male and female Kleenpak connectors using compatible connections. Ensure that the tubing is attached firmly to the hose barb to prevent leakage during operation using cable ties or other methods. During tubing assembly, premature actuation of the male plunger is prevented by the anti-actuation ring. The antiactuation ring needs to remain in place until actual connection takes Thermo Scientific Single-Use Bioreactor (S.U.B.) 105

106 Section 2.7 Pall Kleenpak Connector Instructions place. The presence of valves on the tubing before the connector is recommended to prevent liquid contact with the Kleenpak connectors prior to use. b. If the connectors are to be autoclaved, orientate them with peel strips orientated upwards to prevent peel strip blockage by condensate. WARNING: The device must remain dry prior to connection of the male and female Kleenpak connectors. If there is fluid present in the line or around the devices, do not use. If the protective cap has been removed, do not use. Gamma Irradiation WARNING: These disposable Kleenpak connectors must not be in-line steam sterilized. Material design limitations will be exceeded when these devices are exposed to pressurized steam and they will be ruptured. a. Connect the male or female Kleenpak connector to the single use system. A valve or clamp needs to be installed close to the connector to prevent accidental wetting after the system is filled with liquid. b. Ensure that the protective cap is firmly in place. Autoclave paper or other radiation resistant material can be used to ensure that the cap does not become dislodged during handling. c. It is recommended that the entire assembly be placed in an inner and outer bag for protection prior to gamma irradiation. d. Treat with gamma radiation. The maximum allowable radiation dose is 50kGy (5Mrad). IMPORTANT: Pall recommends that the efficiency of the gamma irradiation cycle is validated using an appropriate method. e. These connectors have not been validated for repeated gamma irradiation exposure. Thermo Scientific Single-Use Bioreactor (S.U.B.) 106

107 Section 2.7 Pall Kleenpak Connector Instructions Autoclave Instructions a. Install the male or female Kleenpak connector to the equipment to be autoclaved. If the Kleenpzk connector is attached to a tank, the tank should be vented appropriately with a vent filter. b. Ensure that the protective cap of the Kleepak connector is firmly in place. Autoclave paper or other autoclavable and air/strem permeable material can be used to cover the cap loosely to ensure that the cap does not become dislodged during handling. c. The Kleenpak connectors shoud dbe allowed to vent during autoclaving. The venting strip should be oriented upwards to prevent blockage by condensates. WARNING: To avoid collection of condensate within the connectors, do not place the venting strip downwards during autoclaving. d. The connector should not be covered with heavy objects during the autoclave cycle. NOTE: The maximum temperature is 121 C for ACD part numbers and 130 C for KPCHT part numbers, The maximum exposure time is 75 mintues. Do not autoclave at a highter temperature or for a longer period of time. A slow exhaust cycle is recoommended. WARNING: To avoid collection of condensate within the connectors, do not place the venting strip downwards during autoclaving. IMPORTANT: Pall recommends that the efficiency of the autoclave cycle is validated using an appropriate method. Making the Connection WARNING: Do not use if fluid is in contact with the connector. Do not use if protective caps are loose or displaced. Thermo Scientific Single-Use Bioreactor (S.U.B.) 107

108 Section 2.7 Pall Kleenpak Connector Instructions FEMALE PART* Locking clips (on both sides) MALE PART* Protective caps Plunger Barrel O-ring Base Anti-actuation ring Thumb rest Figure Kleenpak connector schematic Making the Connection using the Kleenpak Connector Assembly Aid (a). Lift and pull tab off protective caps to remove caps from the Kleenpak connectors (Figure 2.124). Protective caps Figure Step (a) (b). Hold the barrel of the larger (male) Kleenpak connector above the base. Align the smaller (female) Kleenpak connector with the larger (male) Kleenpak connector Flat sides should be aligned Both peel away strips need to remain folded (Figure 2.125). Thermo Scientific Single-Use Bioreactor (S.U.B.) 108

109 Section 2.7 Pall Kleenpak Connector Instructions Female Connector Flat side where both folded over peel away strips exit Rounded side Barrel Anti-actuation ring Male Connector Figure Step (b) NOTE: If the Kleenpak connectors are not aligned properly, the connection cannot be made. (c). Once aligned correctly, press the two connectors together firmly until both locking clips snap together tightly (Figure 2.126). Locking clips (not visible) Locking clips Figure Step (c) (d). Support both the male and female Kleenpak connectors, and remove the anti-actuation ring from the male connector by pulling the tab towards the barbed end of the male Kleenpak connector (Figure 2.127). Figure Step (d) Remove the anti-actuation ring from the male connector (e). Support both the male and female Kleenpak connectors, and remove the anti-actuation ring from the male connector by pulling the tab towards the barbed end of the male Kleenpak connector (Figure 2.128). Thermo Scientific Single-Use Bioreactor (S.U.B.) 109

110 Section 2.7 Pall Kleenpak Connector Instructions Assembly aid (cross section view) Peel away strips Figure Step (e) WARNING: Kleenpak connector should stay securely in the assembly aid when properly installed. (f). Hold assembly aid in the palm of the hand with the Kleenpak connector facing outwards, and thumb supporting the Kleenpak connector in the assembly aid. Using the other hand, firmly grasp both peel away strips as close as possible to the body of the assembly aid to ensure a secure grip and pull both peel away strips simultaneously in a smooth continuous motion. Ensure that the Kleenpak connector is perpendicular to the peel away strips (Figures and 2.130). Figure Step (f) Figure Step (f) continued WARNING: Do not use if only one peel away strip is removed accidentally instead of two. Thermo Scientific Single-Use Bioreactor (S.U.B.) 110

111 Section 2.7 Pall Kleenpak Connector Instructions (g). With Kleenpak connector still secured in the assembly aid, push the thumb rest of the male Kleenpak connector down towards the base of the barrel (Figures and 2.132). Figure Step (g) Figure Step (g) continued Note that in order to establish a proper connection, the plunger inside the male Kleenpak connector must be fully inserted into the female Kleenpak connector. As a verification, repeat actuation until a hard stop is reached. If necessary, the Kleenpak connector may be removed from the assembly aid to complete the plunger movement. (h). Once the Kleenpak connector assembly is complete, the assembly aid may be removed. When assembly aid is removed, verify actuation until a hard stop is reached. (i). Start the fluid transfer (Figure 2.133). Figure Step (i) Thermo Scientific Single-Use Bioreactor (S.U.B.) 111

112 Section 2.7 Pall Kleenpak Connector Instructions Making the Connection without using the Kleenpak Connector Assembly Aid (a). Lift and pull tab off protective caps to remove caps from Kleenpak connectors (Figure 2.134). Protective caps Figure Step (a) (b). Hold the barrel of the larger (male) Kleenpak connector above the base. Align the smaller (female) Kleenpak connector with the larger (male) Kleenpak connector Flat sides should be aligned Both peel away strips need to remain folded (Figure 2.135). NOTE: If the Kleenpak connectors are not aligned properly, the connection cannot be made. Rounded side Female Connector Flat side where both foldedover peel awaystrips exit Barrel Anti-actuation ring Male Connector Figure Step (b) Thermo Scientific Single-Use Bioreactor (S.U.B.) 112

113 Section 2.7 Pall Kleenpak Connector Instructions (c). Once aligned correctly, press the two Kleenpak connectors together firmly until both locking clips snap together tightly (Figure 2.136). Locking clips (not visible) Locking clips Figure Step (c) (d). Support both the male and female Kleenpak connectors, and remove the anti-actuation ring from the male connector by pulling the tab towards the barbed end of the male Kleenpak connector (Figure 2.137). Remove the anti-actuation ring from the male connector Figure Step (d) (e). With one hand, support the male and female sides of the Kleenpak connector by wrapping fingers around both sides of the connector, next to the flange. Using the other hand, grasp both white peel away strips as close as possible to the flat side of connector to ensure a good grip and pull them out simultaneously in a smooth continuous motion. Ensure that the connector is perpendicular to the peel away strips shown in shown in illustration for Step (e). The perpendicular orientation must be maintained while the two strips are pulled simultaneously. Do not use if only one white peel away strip is removed accidentally instead of two (Figures and 2.139). NOTE: Do not impart perpendicular forces on the connector, as it can cause the connector to break. If a perpendicular force is present due to items attached to the Kleenpak connector, then the Kleenpak connector must be properly supported. Thermo Scientific Single-Use Bioreactor (S.U.B.) 113

114 Section 2.7 Pall Kleenpak Connector Instructions Figure Step (e) Figure Step (e) continued WARNING: Do not use if only one peel away strip is removed accidentally instead of two. (f). Push the thumb rest of the male Kleenpak connector down towards the base of the barrel until they meet (Figures and 2.141). Figure Step (f) Figure Step (f) continued Note that in order to establish a proper connection, the plunger inside the male Kleenpak connector must be fully inserted into the female Kleenpak connector. As a verification, repeat actuation until a hard stop is reached. (g). Start the fluid transfer (Figure 2.142). Figure Step (g) Thermo Scientific Single-Use Bioreactor (S.U.B.) 114

115 Section 3 Troubleshooting Section 3 Troubleshooting Symptom Remedy Symptom Remedy Single-Use Bioreactor will not operate. Check power supply. Verify main electrical plug connection at wall outlet, verify position of main power disconnect, and verify position of Emergency Stop switch. Verify condition of main electrical circuit breaker of facility. If protection breaker has been tripped, determine fault condition. The condition may exist where other electrical systems are requiring current loads beyond those allowed by the breaker. S.U.B. system should be placed on its own electrical circuit. Disconnect main power cord. Inspect electrical circuit breakers and fuses inside the electrical enclosure of the S.U.B. controller. Determine fault condition by visual inspection. If fault condition can not be determined by visual inspection, contact manufacturer. Single-Use Bioreactor temperature is below target or slow to respond. Check temperature controller and sensor. Verify the temperature probe (RTD) is not loose and has been fully inserted into the S.U.B. BPC thermo-well. Verify thermo-well has been filled with sufficient glycerol to aid in heat transfer. Verify that temperature controller is enabled. Indicator light (green LED labeled C1) will illuminate when temperature controller is cycling heater to maintain target temperature. Verify mixer is operating, safety interlock will not allow heaters to warm without the mixer in operation. Verify system is filled with sufficient volume of fluid. There must be enough volume of media (minimum volume) in the BPC to provide contact with the container. Add more media if the BPC is not touching heater area. Thermo Scientific Single-Use Bioreactor (S.U.B.) 115

116 Section 3 Troubleshooting Symptom Remedy Symptom Remedy Symptom Remedy Mixer controller does not respond to user inputs. Allow speed to stabilize before using key-pad interface. Rapidly adjusting speed control in an excessive manner may require several seconds for speed stabilization. Wait ten seconds and then attempt to adjust speed at key-pad interface. Users should verify the position of the input select switch of the VFD. If the toggle switch is not in the middle position, the controller will not be able to receive control inputs from the control keypad on the front panel. Dissolved Oxygen (DO) readings are low or slow to respond. Check physical condition of the DO probe, calibration of the DO probe, flow rate of gas into S.U.B. DO probes require routine maintenance; replace damaged probe or membrane. Verify DO probe calibration relative to set-points of zero and span. Inspect line sets connected to direct spargers for restriction (closed tubing clamp, pinched line, low supply pressure). Direct membrane sparger does not seem to be working although gas is present. Allow sparger membrane to purge. If the S.U.B. is filled with liquid and allowed to sit idle for extended periods of time without gas being supplied to the sparger, liquid can accumulate between the membrane and check valve. Various media additives may restrict the membrane temporarily. Several minutes of gas pressure being supplied to the sparger should purge the membrane, allowing it to function properly. Certain operating conditions can create situations when the sparger membrane may become restricted due to insufficient line pressure from the bioreactor controller gas feed line. Increasing the flow rate to 1 liter per minute, or momentarily raising the pressure regulator outlet pressure to 5 psi (0.34 bar) may alleviate the problem. Alternatively, several seconds at this higher pressure will allow the membrane to purge pores that may be blocked due to the presence of accumulated liquid. Thermo Scientific Single-Use Bioreactor (S.U.B.) 116

117 Section 3 Troubleshooting Symptom Remedy Symptom Remedy Symptom Remedy DO readings are erratic or unstable. Adjust bioreactor controller to suite the volume of the S.U.B. system. Many different parameters can effect the ability of a bioreactor controller to effectively maintain a target setpoint during process control. Modern controllers utilize computer algorithms to adjust targeted parameters, the most common technique is that of tunable controller that uses variables of PID. Tuning these PID values to the specific characteristics of the system dynamics will in most cases stabilize process parameters to an acceptable level. It is recommended that S.U.B. users consult the user manual of the particular bioreactor controller being used. A grounding reference to the media can be created by using a grounding lead between the tank and the body of the stainless steel DO probe or to the stainless steel connector (if present) on the sample line of the S.U.B BPC. Most traditional systems utilize a condenser or exhaust vent heater to help manage condensate; is this necessary for the S.U.B.? Extensive testing has found that an exhaust heater or a condenser is not typically required under normal operating conditions for S.U.B.s 500L or smaller. However more demanding applications may warrant an exhaust vent heater which is available from your sales rep. I typically use level sensors to control volume and feed rate or supplement during a bioreactor run; how would I do this with the S.U.B.? Use load cells or a scale to control volumes based upon weight. The S.U.B. is not equipped with level sensors. However, the S.U.B. can be setup to allow supplement feeds and volumes to be managed by weight. Please refer to Section 4, Specifcations and Parts Information, for skid dimensions for sizing of scales. Thermo Scientific Single-Use Bioreactor (S.U.B.) 117

118 Section 3 Troubleshooting Symptom Remedy Symptom Remedy Symptom Remedy ph levels are questionable or out of range. Verify calibration of probe, utilize either media or gas buffers. ph levels can be managed in a similar manner to conventional bioreactors once calibration of the probe is verified by use of an off-line sample. Carbon dioxide gas sparged through the media or headspace, biocarbonate levels in the media, and the addition of liquid titrant solutions all serve to manage the ph balance of the bioreactor environment. See Section Probe Assembly instructions for more information on probe calibration. A grounding reference to the media can be created by using a grounding lead between the tank and the body of the stainless steel DO probe or to the stainless steel connector (if present) on the sample line of the S.U.B BPC. S.U.B. BPC seems overly tight or overpressure. Verify container is venting and inspect for cause of overpressure. Reduce inlet gas flow rate of overlay and direct sparge. Inspect exhaust filter for restriction or blockage. Excessive foaming should be avoided for several reasons. If foam levels are allowed to reach the exhaust filter, the filter will become restricted resulting in excessive internal pressure within the confines of the S.U.B. This may cause product failure and impending burst of the BPC. Noise emitted from the mixer assembly. No action required. The bearing port assembly supplied with the S.U.B. is an important component in maintaining a sterile environment during cell growth. The special seals used in the S.U.B. may generate some noise during operation, particularly after the first day of operation. This noise may vary in intensity and frequency but generally has no significant effect upon performance or overall durability of the BPC during the intended life of the product. Thermo Scientific Single-Use Bioreactor (S.U.B.) 118

119 Section 3 Troubleshooting Symptom Remedy We forgot to introduce the ph and DO probes prior to media fill; can we still make a sterile connection under these conditions? Yes, as long as the clamps were closed on the Kleenpak Connector probe ports before liquid fill. The Kleenpak connectors must be dry to make the connection of the probe assemblies. When media is already present in the S.U.B., follow the probe insertion procedures as outlined in Section 2.5: Probe Assembly. Some fluid may enter the bellows when the probe is inserted into a BPC already filled with media. Symptom Remedy I am not familiar with the use of the Pall Kleenpak Connectors and am concerned about making connections. What can I do to ensure a successful connection using this system? Familiarize yourself with the Pall Kleenpak Connector instructions found in Section 2.7 before beginning. When a connection is being made, visually evaluate the status of the four locking external clips and verify they are tightly secured (the snap should be audible for all four clips when pressing the connectors together). Always make sure the four locking clips are fully engaged for the male/female connection before removing the paper strips. A common cause for a leaky Kleenpak is an error in the final step of seating the tapered barrels of the male/female connector. There are a series of concentric rings inside the male connector (7.72mm [0.3 ] in front of the black O-ring) Visually verify that the four internal clips are on the last set of rings. Using both hands place connector flanges between index fingers and thumbs and squeeze until properly seated. Thermo Scientific Single-Use Bioreactor (S.U.B.) 119

120 Section 3 Troubleshooting Symptom Remedy Symptom Remedy We are not achieving the cell growth we expected in the S.U.B. while running under our normal bioreactor agitation and sparging rates. What should we do? Reduce agitation and sparging rates. Often low cell viability and cell growth can be attributed to excessive sparging or agitation. It is recommended that users reduce their sparge rate compared to what they might use in a conventional bioreactor. Gas flow rates supplied as overlay should also be reduced as much as possible. Too much gas creates excess foam and higher shear conditions. Provide only the level of agitation needed: low viability and lysed cells, reduce agitation speed cell aggregation and settling, increase agitation. Media formulation can also have a large affect on cell culture growth in the S.U.B. Surfactants such as Pluronic decrease shear and increase k L a, but at a cost of increased foaming. Thermo Scientific can offer custom media especially for the S.U.B. and customer s specific cell line(s). Excessive foam in the bioreactor headspace. Alter the liquid surface tension related to the culture media and/or sparge gas. A media supplement of antifoam can be used in the S.U.B. These serve to lower the surface tension of the media and will reduce the presence of foam. High sparge rates of air can result in the presence of excessive foam. Testing has shown that sparging with oxygen will typically result in dramatic reduction of foam in the headspace. Thermo Scientific Single-Use Bioreactor (S.U.B.) 120

121 Section 4 Specifications and Parts Information Section 4 Specifications and Parts Information This section covers the following information: 4.1 Hardware Features 4.2 BPC Specifications 4.3 Accessories and Options Specifications Thermo Scientific Single-Use Bioreactor (S.U.B.) 121

122 Section 4 Specifications and Parts Information 4.1 Hardware Features Design Features L S.U.B. with Water Jacket or Resistive Heater Side View of 250L Water Jacketed S.U.B. Front View of 250L Water Jacketed S.U.B. 1. Exhaust vent filter holder (optional) 2. Mixing assembly with shield 3. Bearing port receiver with clamp 4. Mixer motor 5. Stainless steel (304) outer support container 6. Electrical control panel (optional) 7. 3/8 Dimpled jacket or resistive blanket (side and bottom) 8. Leveling casters 9. Dimpled jacket bleed valve (water jacketed models only) 10. Quick connect water inlet/outlet ports (water jacketed models only) 11. Standard tool set: 3/8 x 150in-lb square torque wrench, load cell and motor cap lockout wrench 12. Drive shaft (stored) 13. Probe hanger bracket 14. Probe access windows 15. Cart assembly 16. Liquid sight windows 17. Bottom cut outs/pins for BPC attachment and alignment NOTE: Load cells are standard only on 1,000 and 2,000L S.U.B. units Thermo Scientific Single-Use Bioreactor (S.U.B.) 122

123 Section 4 Specifications and Parts Information Design Features 500-1,000L S.U.B. with Water Jacket or Resistive Heater Front/Top View Back View 1. Exhaust vent filter holder 2. Mixing assembly with shield 3. Bearing port receiver with clamp 4. Mixer motor 5. Stainless steel (304) outer support container 6. Electrical control panel (optional) 7. Water jacket (3/8 dimpled) or resistive blanket (side and bottom) 8. Probe-hanger bracket 9. Probe-access windows 10. S.U.B. BPC loading door and liquidsight windows 11. Bleed valve (water jacketed models only) 12. Cart assembly 13. Standard tool set: 3/8 x 150in-lb. square torque wrench, load cell and motor cap lockout wrench 14. Drive shaft (stored) 15. Quick connect water inlet/outlet ports (water jacketed models only) 16. Load cells (3) 17. Leveling casters 18. Bottom cut outs/pins for BPC attachment and alignment NOTE: Load cells are standard only on 1,000 and 2,000L S.U.B. hardware units. Thermo Scientific Single-Use Bioreactor (S.U.B.) 123

124 Section 4 Specifications and Parts Information Design Features 2,000L S.U.B. with Water Jacket Mixer motor 2. Mixing assembly with shield 3. Auxiliary E-Stop assembly 4. Stainless steel outer support container 5. Bag lift assembly 6. Standard tool set 7. Load cell display 8. Control panel 9. Load cell summing block 10. Load cell (3) 11. Probe access window 12. Probe clips 13. Water jacket 14. Bottom cut outs for BPC alignment 15. Sparge access plate 16. Jacket quick connect couplings 17. Pneumatic bag lift control Thermo Scientific Single-Use Bioreactor (S.U.B.) 124

125 Specifications for 50L S.U.B. Motor Agitation Impeller Reactor Geometry Rated Liquid Working Volume Minimum Liquid Working Volume 50L S.U.B. Specifications AC Motor DC Motor Resistive Jacketed Resistive Jacketed 50L 25L Total Reactor Volume (Liquid & Gas) 65.5L BPC Chamber Diameter BPC Chamber Shoulder Height Liquid rated working volume Fluid working volume (height/diameter) ratio Overall reactor geometry (height/diameter ratio) Tank baffles 13.75" (34.9cm) 31.5" (80cm) 20.5" (52.1cm) :1 Impeller (quantity X blade count) 1 x 3 Impeller scaling (impeller diameter/tank diameter) No 1/3 Impeller blade pitch (angle) 45 Impeller diameter 4.375" (11.11cm) Impeller - calculated power number (N) 2.1 Maximum mixing rate Nominal agitation rating - power/volume ratio Nominal agitation Nominal tip speed Counterclockwise mixing flow direction rpm 0.1hp/1,000 gal. (19.7W/1,000L) 160rpm ft. per min. (103.9cm/s) Down-Pumping Agitation shaft resolved angle 19.6 Agitation shaft centerline offset Overall drive shaft length Operational drive shaft length Drive shaft diameter Drive shaft poly-sheath outside diameter Impeller clearance from tank bottom Agitation motor drive (type, voltage, phase) AC motor only Agitation motor drive (type, voltage) DC motor only 0.75" (1.9cm) 30" (76.2cm) 23" (58.4cm) 0.5" (1.27cm) 1" (2.54cm) 4.63" (11.75cm) Induction, 208VAC, Brushless, 48VDC Motor power rating (AC Motor) 0.25hp (186.4kW) - Motor power rating (DC Motor) - 200W (0.268hp) Motor torque rating 82 in-lb. (9.5Nm) - Gear reduction 10:01 Programmable VFD, Remote Panel Interface, Power Fault Auto Restart Motor communication methods (for external controller) Standard Section 4 Specifications and Parts Information 0-10V, 4-20mA, ModBus - Thermo Scientific Single-Use Bioreactor (S.U.B.) 125

126 Section 4 Specifications and Parts Information Specifications for 50L S.U.B. 50L S.U.B. Specifications AC Motor DC Motor Resistive Jacketed Resistive Jacketed Programmable PID temperature controller Standard - Standard - Temperature Control Resistive Heater Fluid Jacket Solid state relay (discrete voltage signal) V AC/DC V AC/DC Heater power rating (total) 455.7W W - Heater power rating sides 360W - 360W - Heater power rating bottom 95.7W W - Approximate liquid heat-up time (5-37 C) 4.32hr hr - Jacket area: full/half volume - 4.4/2ft 2-4.4/2ft 2 Jacket Volume - 2.4L - 2.4L Jacket flow rate at 50psi - 136L per min L per min. Process connection - 1" Sanitary Tri-Clamp - - 1" Sanitary Tri-Clamp Nominal heating/cooling load (W) - 500W - 500W Approximate liquid heat-up time [5-37 C] - 1.7hr - 1.7hr Misc RTD or Thermocouple, 1/8 (3.18mm) OD RTD: Pt-100 (Standard) Support Container General Recommended Operating Parameters Overall Width 37.1" (94.2cm) with E-Box 22.25" (56.5cm) Overall Length 33.2" (84.3cm) with E-Box 30.3" (77cm) Overall Height Dry skid weight (mass) Wet skid weight rated working volume (mass) Ceiling height required for drive shaft loading Electrical power supply requirement (Voltage, Phase, Amp) 296 lb. (134.3kg) lb. (184.2kg) 255 lb. (115.7kg) lb. (165.7kg) 78.2" (198.6cm) 84 (213.4cm) 296 lb. (134.3kg) lb kg) 255 lb. (115.7kg) lb. (165.7kg) 120/240 VAC, Single, 20/10 A Dependent on Controller Tested system reliability (minimum) 0.9 at 90% ph & DO Probe Autoclavable type (Applisens, Broadley James, Metler Toledo) Operating temperature range Motor speed 12mm Diameter x mm Insertion Length x 13.5 PG (Pipe) Thread Ambient to 40 +/- 0.1 C (104+/-0.2 F) rpm Volume range 25-50L Maximum bag pressure Continuous operating time 0.5psi (0.03 bar) 21 Days Mixing Time at Nominal Volume Only Thermo Scientific Single-Use Bioreactor (S.U.B.) 126

127 Section 4 Specifications and Parts Information 50L S.U.B. Dimensions Ordering Information - 50L S.U.B. Size Description Part Number 50L Resistive, DC Motor, without Load Cell SUB L Jacketed, DC Motor, without Load Cell SUB L Resistive, 120VAC, AC Motor, without Load Cell SUB L Jacketed, 120VAC, AC Motor, without Load Cell SUB L Resistive, 240VAC, AC Motor, without Load Cell SUB L Jacketed, 240VAC, AC Motor, without Load Cell SUB Thermo Scientific Single-Use Bioreactor (S.U.B.) 127

128 Specifications for 100L S.U.B. 100 L S.U.B. SPECIFICATIONS AC Motor Section 4 Specifications and Parts Information DC Motor Resistive Jacketed Resistive Jacketed Motor Agitation Impeller Reactor Geometry Rated liquid working volume Minimum liquid working volume Total reactor volume (liquid & gas) BPC chamber diameter BPC chamber shoulder height Liquid rated working volume Fluid working volume (height/diameter) ratio 100L 50L 120L 17.25" (43.8cm) 37.5" (95.3cm) 26" (66cm) 1.5:1 Overall reactor geometry (height/diameter ratio) 1.9:1 Tank baffles Impeller (quantity X blade count) 1 x 3 Impeller scaling (impeller diameter/tank diameter) Impeller blade pitch (angle) 45 Impeller diameter No 1/3 5.75" (14.6cm) Impeller - calculated power number (N) 2.1 Maximum mixing rate Nominal agitation rating - power/volume ratio Nominal agitation Nominal tip speed Counterclockwise mixing flow direction 0.1 hp/1,000 gal. (19.7W/1,000L) 145.5rpm ft. per min. (110.7cm/s) Down-Pumping Agitation shaft resolved angle 19.6 Agitation shaft centerline offset Overall drive shaft length Operational drive shaft length Drive shaft diameter Drive shaft poly-sheath outside diameter Impeller clearance from tank bottom Agitation motor drive (type, voltage, phase) AC motor only Agitation motor drive (type, voltage) DC motor only 1.00" (2.54cm) 35" (88.9cm) 29" (73.6cm) 0.5" (1.27cm) 1" (2.54cm) 5.75" (14.6cm) Induction, 208VAC, Brushless, 48VDC Motor power rating (AC motor) 0.25hp (186.4kW) - Motor power rating (DC motor) - 200W (0.268hp) Motor torque rating 82 in-lb. (9.5Nm) - Gear reduction 10:01 Programmable VFD, remote panel interface, power fault auto restart Motor communication methods (for external controller) Standard V, 4-20mA, Modbus - Thermo Scientific Single-Use Bioreactor (S.U.B.) 128

129 Section 4 Specifications and Parts Information Specifications for 100L S.U.B. 100 L S.U.B. SPECIFICATIONS AC Motor DC Motor Temperature Control Resistive Heater Fluid Jacket Resistive Jacketed Resistive Jacketed Programmable PID temperature controller Standard - Standard - Solid state relay (discrete voltage signal) V AC/DC V AC/DC Heater power rating (total) 866W - 866W - Heater power rating sides 676W - 676W - Heater power rating bottom 190W - 190W - Approximate liquid heat-up time (5-37 C) 4.9hr - 4.9hr - Jacket area: full/half volume - 6.5/2.3ft 2-6.5/2.3ft 2 Jacket Volume - 4.5L - 4.5L Jacket flow rate at 50psi - 136L per min L per min. Process connection - 1" Sanitary Tri-Clamp - - 1" Sanitary Tri-Clamp Nominal heating/cooling load - 1,000W - 1,000W Approximate liquid heat -up time [5-37 C] (hr) hr hr Misc RTD or Thermocouple, 1/8 (3.18mm) OD RTD: Pt-100 (Standard) Support Container General Recommended Operating Parameters Overall Width 38.8" (98.5cm) with E-box 22.25" (56.5cm) Overall Length 35.9" (91.2cm) with E-box 33.8" (85.8cm) Overall Height Dry skid weight (mass) Wet skid weight rated working volume (mass) Ceiling height required for drive shaft loading Electrical power supply requirement (voltage, Phase, amp) 305 lb. (138.3kg) 525.5lb. (238.3kg) 354 lb. (160.6kg) 574.5lb. (260.6kg) 79.3" (201.5cm) 90 (228.6cm) 305 lb. (138.3kg) 525.5lb. (238.3kg) 354 lb. (160.6kg) 574.5lb. (260.6kg) 120/240 VAC, Single, 20/10A Dependent on Controller Validated system reliability (minimum) 0.9 at 90% ph & DO Probe Autocavable type (Applisens, Broadley James, Mettler Toledo) Operating temperature range Motor speed 12mm Diameter x mm Insertion Length x 13.5 PG (Pipe) Thread Ambient to 40 +/- 0.1 C (104 +/-0.2 F) rpm Volume range L Maximum bag pressure Continuous operating time 0.5psi (0.03bar) 21 Days Mixing Time at Nominal Volume Only Thermo Scientific Single-Use Bioreactor (S.U.B.) 129

130 Section 4 Specifications and Parts Information 100L S.U.B. Dimensions Ordering Information - 100L S.U.B. Size Description Part Number 100L Resistive, DC Motor, without Load Cell SUB L Jacketed, DC Motor, without Load Cell SUB L Resistive, 120VAC, AC Motor, without Load Cell SUB L Jacketed, 120VAC, AC Motor, without Load Cell SUB L Resistive, 240VAC, AC Motor, without Load Cell SUB L Jacketed, 240VAC, AC Motor, without Load Cell SUB Thermo Scientific Single-Use Bioreactor (S.U.B.) 130

131 Section 4 Specifications and Parts Information Specifications for 250L S.U.B. Reactor Geometry Impeller Agitation Motor Rated liquid working volume Minimum liquid working volume Total reactor volume (liquid & gas) BPC chamber diameter BPC chamber shoulder height Liquid rated working volume Fluid working volume (height/diameter) ratio Overall reactor geometry (height/diameter ratio) Tank baffles 250L S.U.B. Specifications AC Motor DC Motor Resistive Jacketed Resistive Jacketed 250L 125L 316L 23.5 (59.7cm) 45.5 (115.6cm) 36 (91.4cm) 1.5:1 1.9:1 Impeller (quantity X blade count) 1 X 3 Impeller scaling (impeller diameter/tank diameter) 1/3 Impeller blade pitch (angle) 45 Impeller diameter no (20cm) Impeller - calculated power number (N) 2.1 Maximum mixing rate Nominal agitation rating - power/volume ratio Nominal agitation Nominal tip speed Counterclockwise mixing flow direction rpm 0.1 hp/1000 gal (19.7 W/1000 liter) 118rpm ft/min (123.6 cm/s) down-pumping Agitation shaft resolved angle 19.6 Agitation shaft centerline offset Overall drive shaft length Operational drive shaft length Drive shaft diameter Drive shaft poly-sheath outside diameter Impeller clearance from tank bottom Agitation motor drive (type, voltage, phase) AC motor only Agitation motor drive (type, voltage) DC motor only 1.3 (3.3cm) 42 (106.7cm) 36 (91.4cm) 0.5 (1.27cm) 1 (2.54cm) (20cm) Induction, 208VAC, Brushless, 48VDC Motor power rating (AC motor) 0.25hp (186.4kW) - Motor power rating (DC motor) - 400W (0.536hp) Motor torque rating 102in-lbs (11.5Nm) Gear reduction 12.5:1 Programmable VFD, remote panel interface, power faults auto restart Motor communication methods (for external controller) Standard V, 4-20mA, ModBus - Thermo Scientific Single-Use Bioreactor (S.U.B.) 131

132 Section 4 Specifications and Parts Information Specifications for 250L S.U.B. Temperature Control Resistive Heater Fluid Jacket Misc 250L S.U.B. Specifications AC Motor DC Motor Resistive Jacketed Resistive Jacketed Programmable PID temperature controller Standard - Standard - Solid state relay (discrete voltage signal) V V - AC/DC AC/DC - Heater power rating (total) 1,566.8W 1,566.8W - Heater power rating sides 1,168.8W 1,168.8W - Heater power rating bottom 398W 398W - Approximate liquid heat-up time (5-37 C) 7.5hrs 7.5hrs Jacket area: full/half volume /5.8ft2-13.6/5.8ft2 Jacket volume - 8.6L - 8.6L Jacket flow rate at 50 psi - 136L/min - 136L/min Process connection - 1 sanitary 1 sanitary - tri-clamp tri-clamp Nominal heating/cooling load (W) 2,500 2,500 Approximate liquid heat-up time [5-37 C] 1.9hrs 1.9hrs RTD or Thermocouple, 1/8 (3.18 mm) OD RTD: Pt-100 (standard) Support Container General Recommended Operating Parameters Overall width 44.4 (112.8cm) (with e-box) 27 (68.5cm) Overall length (102.2cm) (with e-box) (96.9cm) Overall height Dry skid weight (mass) Wet skid weight rated working volume (mass) Ceiling height required for drive shaft loading Electrical power supply requirement (voltage, phase, amp) Tested system reliability (minimum) ph & DO probe autoclavable type (Applisens, Broadley James, Mettler Toledo) Operating temperature range Motor speed 425lbs (192.8kg) 976.2lbs (442.8kg) 493lbs (223.6kg) 1044lbs (473.6kg) 84.8 (215.5cm) 101 (256.5cm) 425lbs (192.8kg) 976.2lbs (442.8kg) 493lbs (223.6 kg) 1044lbs (473.6kg) 120/240 VAC, single, 20/10 A Dependent on Controller 0.9@90% 12mm diameter x mm insertion length x 13.5 PG thread Ambient to 40 +/- 0.1 C (104 +/- 0.2 F) rpm Volume range L Maximum bag pressure Continuous operating time 0.5psi (0.03bar) 21 days mixing time at nominal volume only Thermo Scientific Single-Use Bioreactor (S.U.B.) 132

133 Section 4 Specifications and Parts Information 250L S.U.B. Dimensions Ordering Information - 250L S.U.B.. Size Description Part Number 250L Resistive, DC Motor, without Load Cell SUB L Jacketed, DC Motor, without Load Cell SUB L Resistive, 120VAC, AC Motor, without Load Cell SUB L Jacketed, 120VAC, AC Motor, without Load Cell SUB L Resistive, 240VAC, AC Motor, without Load Cell SUB L Jacketed, 240VAC, AC Motor, without Load Cell SUB Thermo Scientific Single-Use Bioreactor (S.U.B.) 133

134 Specifications for 500L S.U.B. 500L S.U.B. Specifications AC Motor Section 4 Specifications and Parts Information DC Motor Resistive Jacketed Resistive Jacketed Motor Agitation Impeller Bioreactor Geometry Rated liquid working volume Minimum liquid working volume Total bioreactor volume (liquid & gas) 500L 250L 660L BPC chamber diameter 75.56cm (29.75") BPC chamber shoulder height 152.4cm (60.0") Liquid height at rated working volume cm (44.63") Fluid geometry at working volume (height/diameter) ratio Overall bioreactor geometry (height/diameter) ratio Tank Baffles 1.5:1 1.9:1 Impeller (quantity x blade count) 1 x 3 Impeller scaling (impeller diameter/tank diameter) No 1/3 Impeller blade pitch 45 Impeller diameter 25.1cm (9.875") Impeller - calculated power number (N) 2.1 Maximum mixing rate Nominal agitation rating (power/volume ratio) Nominal agitation Nominal tip speed Counterclockwise mixing flow direction rpm 19.7W/1,000L (0.1hp/1,000gal.) 100rpm 137.2cm/s (270fpm) Down-Pumping Agitation shaft resolved angle 19.6 Agitation shaft centerline offset 5.08cm (2.0") Overall drive shaft length 127.0cm (50.0") Operational drive shaft length cm (44.0") Drive shaft diameter 1.90cm (0.75") Drive shaft poly-sheath outside diameter 3.49cm (1.375") Impeller clearance from tank bottom 25.1cm (9.875") Agitation motor drive (type, voltage,phase) AC motor only Agitation motor drive (type, voltage) DC motor only Induction, 208VAC, Brushless, 48VDC Motor power rating (AC Motor) 372.8W (0.5hp) - Motor power rating (DC Motor) - 400W (0.536hp) Motor torque rating 9.5 Nm (82in.-lb.) - Gear reduction 10:1 Programmable VFD, remote panel interface, power fault auto restart Motor communication methods (for external controller) Standard V; 4-20mA; ModBus - Thermo Scientific Single-Use Bioreactor (S.U.B.) 134

135 Specifications for 500L S.U.B. 500L S.U.B. Specifications AC Motor Section 4 Specifications and Parts Information DC Motor Resistive Jacketed Resistive Jacketed Temperature Control Resistive Heater Water Jacket Programmable PID temperature controller Standard - Standard - Solid state relay (discrete voltage signal) Heater power rating (total) Heater power rating sides Heater power rating Bottom V AC/DC W (4.42hp) W (3.51hp) 678W (0.909hp) V AC/DC W (4.42hp) W (3.51hp) 678W (0.909hp) Approximate liquid heat-up time (5-37 C) 10.5hr hr - Jacket area: full/half-volume (ft2) /0.780 (21.4/8.4) /0.780 (21.4/8.4) Jacket volume L L Jacket flow rate at 50psi (3.4 bar) - 136L/min L/min. Process connection - Nominal heating/cooling load (W) - 1" Sanitary Tri-Clamp 5,000W (6.705hp) - - 1" Sanitary Tri-Clamp 5,000W (6.705hp) Approximate liquid heat-up time [5-37 C] hr hr Support Container General Misc. Recommended Operating Parameters RTD or thermocouple, 1/8" (3.18mm) OD RTD: Pt-100 (Standard) Overall width 125.2cm (49.3") with E-Box 86.4cm (34") Overall length 124.4cm (47.8") with E-Box 45.7" (116cm) Overall height 251.1cm (98.9 ) Dry skid weight (mass) Wet skid weight rated working volume (mass) 304kg (670 lb.) 804kg (1,772.3 lb.) 354kg (780 lb.) 853.8kg (1,882.3 lb.) Ceiling height required for drive shaft loading 266.7cm (105 ) Electrical power supply requirement (voltage, phase, Amp) VAC, Single, 20A VAC, Single, 10A Tested System Reliability (Minimum) 0.9 at 90% ph & DO probe autoclavable type (Applisens, Broadley James, Mettler Toledo) Operating temperature range Motor speed 304kg (670 lb.) 804kg 1,772.3 lb.) 354kg (780 lb.) 853.8kg (1,882.3 lb.) Dependent on controller 12mm Diameter x mm Insertion Length x 13.5 PG (Pipe) Thread Ambient to 40 +/- 0.1 C (104 +/- 0.2 F) rpm Volume range L Maximum BPC pressure Continuous operating time 0.03bar (0.5psi) 21 days mixing time at nominal volume only Thermo Scientific Single-Use Bioreactor (S.U.B.) 135

136 Section 4 Specifications and Parts Information 500L S.U.B. Dimensions (inches [centimeters]) Top View Front View Ordering Information 500L S.U.B. Size Description Part Number 500L Resistive, DC Motor SUB L Water Jacketed, DC Motor SUB L Resistive, 240VAC, AC Motor SUB L Water Jacketed, 240VAC, AC Motor SUB Thermo Scientific Single-Use Bioreactor (S.U.B.) 136

137 1,000L S.U.B. Specifications 1,000L S.U.B. Specifications AC Motor Section 4 Specifications and Parts Information DC Motor Resistive Jacketed Resistive Jacketed Motor Agitation Impeller Bioreactor Geometry Rated liquid working volume 1,000L Minimum liquid working volume 500L Total bioreactor volume (liquid & gas) 1,320L BPC chamber diameter 95.9cm (37.75") BPC chamber shoulder height 200.7cm (79") Liquid height at rated working volume 142.2cm (56") Fluid geometry at working volume (height/diameter) ratio Overall bioreactor geometry (height/diameter) ratio Tank baffles 1.5:1 1.9:1 Impeller (quantity x blade count) 1 x 3 Impeller scaling (impeller diameter/tank diameter) Impeller blade pitch (angle) 45 Impeller diameter 32.1cm (12.625") Impeller - calculated power number (N) 2.1 Maximum mixing rate Nominal agitation rating (power/volume ratio) Nominal agitation No 1/ rpm 19.7W/1,000L (0.1hp/1,000gal.) 87rpm Nominal tip speed 146.1cm/s (287.6fpm ) Counterclockwise mixing flow direction Down-pumping Agitation shaft resolved angle 19.6 Agitation shaft centerline offset 5.08cm (2.0") Overall drive shaft length 167.6cm (66") Operational drive shaft length 152.4cm (60") Drive shaft diameter 1.90cm (0.75") Drive shaft poly-sheath outside diameter 3.49cm (1.375") Impeller clearance from tank bottom 32.1cm (12.625") Agitation motor drive (type, voltage, phase) AC motor only Agitation Motor Drive (Type, Voltage) DC motor only Induction, 208VAC, Brushless, 48VDC Motor power rating (AC motor) 372.8W (0.5hp) - Motor power rating (DC motor) - 400W (0.536hp) Motor torque rating 27.7 Nm (245 in.-lb.) - Gear reduction 15:1 Programmable VFD, remote panel interface, power fault auto restart Motor communication methods (for external controller) Standard V; 4-20mA; ModBus - Thermo Scientific Single-Use Bioreactor (S.U.B.) 137

138 1,000L S.U.B. Specifications 1,000L S.U.B. Specifications AC Motor Section 4 Specifications and Parts Information DC Motor Resistive Jacketed Resistive Jacketed Temperature Control Resistive Heater Water Jacket Programmable PID temperature controller Standard - Standard - Solid state relay (discrete voltage signal) Heater power rating (total) Heater power rating sides Heater power rating bottom V AC/DC 5,017.6W (6.729hp) 3,622.6W (4.858hp) 1,395W (1.871hp) V AC/DC 5,017.6W (6.729hp) 3,622.6W (4.858hp) 1,395W (1.871hp) Approximate liquid heat-up time (5-37 C) 12hr - 12hr - Jacket area: full/half-volume /14.9ft2-35.6/14.9ft2 Jacket volume L L Jacket flow rate at 50psi (3.4bar) - 136L/min L/min. Process connection - 1" Sanitary Tri-Clamp " Sanitary Tri-Clamp Nominal heating/cooling load - 10,000W - 10,000W Approximate liquid heat-up time [5-37 C] - 6.4hr - 6.4hr Misc. RTD or Thermocouple, 1/8" (3.18mm) OD RTD: Pt-100 (Standard) Support Container General Recommended Operating Parameters Overall width Overall length 143.8cm (56.6") with E-Box 139.2cm (54.8") with E-Box Overall height 284cm (111.8") Dry skid weight (mass) Wet skid weight rated working volume (mass) 467.2kg (1,030 lb.) 1,467.4kg (3,235 lb.) 539.8kg (1,190 lb.) 1,540kg (3,395 lb.) Ceiling height required for drive shaft loading 287cm (113 ) Electrical power supply requirement (voltage, phase, amp) VAC, Single, 30A Dependent on Controller Tested system reliability (minimum) 0.9 at 90% ph & DO probe autoclavable type (Applisens, Broadley James, Mettler Toledo) Operating temperature range Motor speed Volume range Maximum BPC pressure Continuous operating time 109.7cm (43.2") 133.9cm (52.7") 467.2kg (1,030 lb.) 1,467.4kg (3,235 lb.) 539.8kg (1,190 lb.) 1,540kg (3,395 lb.) Dependent on controller 12mm Diameter x mm Insertion Length x 13.5 PG thread Ambient to 40 +/- 0.1 C (104 +/- 0.2 F) rpm 500-1,000L 0.03bar (0.5psi) 21 days mixing time at nominal volume only Thermo Scientific Single-Use Bioreactor (S.U.B.) 138

139 Section 4 Specifications and Parts Information 1,000L S.U.B. Dimensions Top View Front View Ordering Information 1,000L S.U.B. Size Description Part Number 1,000L Resistive, DC Motor SUB ,000L Water Jacketed, DC Motor SUB ,000L Resistive, AC Motor SUB ,000L Water Jacketed, AC Motor SUB Thermo Scientific Single-Use Bioreactor (S.U.B.) 139

140 Section 4 Specifications and Parts Information 2,000L S.U.B. Specifications 2,000L S.U.B. Specifications AC Motor DC Motor Rated Liquid Working Volume 2,000L Motor Agitation Impeller Bioreactor Geometry Minimum Liquid Working Volume 1,000L Total Bioreactor Volume (Liquid & Gas) 2,575L BPC Chamber Diameter BPC Chamber Shoulder Height Liquid Height at Rated Working Volume 47" (119.4cm) 90.5" (229.9cm) 70.35" (178.7cm) Fluid Geometry at Working Volume (Height/Diameter) Ratio 1.5:1 Overall Bioreactor Geometry (Height/Diameter) Ratio 1.9:1 Tank Baffles Impeller (Quantity x Blade Count) 1 x 3 Impeller Scaling (Impeller Diameter/Tank Diameter) 1/3 Impeller Blade Pitch (Angle) 45 Impeller Diameter No 15.67" (39.8cm) Impeller - Calculated Power Number (N) 2.1 Maximum Mixing Rate (Revolutions per Minute) Nominal Agitation Rating (Power/Volume Ratio) Standard operation: 20-75rpm* 0.1hp/1,000 gal. (19.7W/1,000L) Nominal Agitation (Revolutions per Minute) 75 Nominal Tip Speed at 75 rpm Counterclockwise Mixing Flow Direction 305 ft./min. (154.9cm/s) Down-Pumping Agitation Shaft Resolved Angle 19.6 Agitation Shaft Centerline Offset Overall Drive Shaft Length (four-piece) Overall Drive Shaft Length (two-piece) Operational Drive Shaft Length Drive Shaft Diameter Drive Shaft Poly-Sheath Outside Diameter Impeller Clearance from Tank Bottom 2.63" (6.7cm) 82.9" (210.6cm) 82.9 (210.6cm) 72.5" (184.2cm) 0.75" (1.91cm) 1.38" (3.51cm) 15.67" (39.8cm) Agitation Motor Drive (Type, Voltage,Phase) AC Motor Only Induction, 208VAC, 3 - Agitation Motor Drive (Type, Voltage) DC Motor Only - Brushless, 48VDC Motor Power Rating (AC Motor) 0.5hp (372.8kW) - Motor Power Rating (DC Motor) - 400W (0.536hp) Motor Torque Rating 245 in.-lb. (27.7Nm) Gear Reduction 15:1 20:1 Programmable VFD, Remote Panel Interface, Power Fault Auto Restart Motor Communication Methods (For External Controller) Standard V, 4-20mA, ModBus - Thermo Scientific Single-Use Bioreactor (S.U.B.) 140

141 2,000L S.U.B. Specifications Temperature Control Support Container General Recommended Operating Parameters Water Jacket AC Motor Jacket Area: Full/Half-volume (ft2) 67.1/53.9 Jacket Volume (L) Jacket Flow Rate at 50psi (3.4 bar) Process Connection Nominal Heating/Cooling Load (W) Approximate Liquid Heat-Up Time [5-37 C] 44L 75L/min. DC Motor 1" Male NPT (National Pipe Thread) Nipple Provided with Hansen Quick Connect Check Valves 10W/L Misc. RTD or Thermocouple, 1/8" (3.18mm) OD RTD: Pt-100 (Standard) Overall Width Overall Length Overall Height Dry Skid Weight (Mass) Wet Skid Weight--Rated Working Volume (Mass) Ceiling Height Required for Drive Shaft Loading (four-piece drive shaft) Ceiling Height Required for Drive Shaft Loading (two-piece drive shaft) Electrical Power Supply Requirement (Voltage, Phase, Amp) 70.5" (179.1cm) with E-Box 6 hr 67.5" (171.4cm) 126.4" (321.1cm) 1,800 lb. (816kg) 6,200 lb. (2,812kg) 144 (365.8cm) 150 (381cm) 58.5" (148.5cm) VAC, Single, 10A - Tested System Reliability (Minimum) 0.9 at 90% ph & DO Probe--Autoclavable Type (Applisens, Broadley James, Mettler Toledo) Operating Temperature Range Mixing speed 12mm Diameter x mm Insertion Length x 13.5 PG thread Ambient to 40 +/- 0.1 C (104 +/-0.2 F) Standard operation: 20-75rpm, working volumes must stay above 50% during agitation* Volume range 1,000-2,000L Maximum BioProcess Container Pressure Continuous Operating Time 0.03 bar (0.5psi) 21 Days Mixing Time at Nominal Volume Only * WARNING: Mixing speeds must stay within the recommended operating parameters. Higher speed operation compromises system reliability and will void standard Thermo Fisher warranties. Under special circumstances, the 2000L S.U.B. may be run at speeds up to 95 rpm ONLY when the working volume is above 90%. When used at high speeds, your control strategy should include governors that regulate rpm based on liquid volume as well as safety interlocks that disable mixing when the liquid drops below the recommended volume. See Appendix D for more information about operating parameters, agitation speed governors and safety interlocks. Ordering Information 2,000L S.U.B. Size Description Part Number 2,000L Water Jacketed, DC Motor SUB ,000L Water Jacketed, 240 VAC, AC Motor SUB ,000L Mobile Stairs SV Thermo Scientific Single-Use Bioreactor (S.U.B.) 141

142 Section 4 Specifications and Parts Information 2,000L S.U.B. Dimensions O47.00 [119.38cm] TANK ID O50.48 [128.22cm] TANK OD [14.95cm] [161.80cm] AUXILIARY E-STOP 1/2 HP 15:1 RATIO GEAR MOTOR 135 [342.9cm] 11 3 MINIMUM CEILING HEIGHT [321.11cm BAG LIFT HEIGHT [260.15cm TANK HEIGHT 240VAC ETL/CE COMPLIANT [64.01cm] SMALL DOOR SENSOR PROBE MOUNT Thermo Scientific Single-Use Bioreactor (S.U.B.) 142

143 4.2 BPC Specifications Standard 50L-100L Dual Sparger BPC - Porous Frit and Open Pipe Back Face Front Face 1. Overlay Gas Sparger 9. Base Addition 2. Inoculum Addition Impeller 12. Exhaust Line 3-6. Probe Ports (4) Section 4 Specifications and Parts Information 8. Bottom Drain Harvest 7. Thermo-Well/Small Volume Sample 14. Porous Frit Micro Sparger 10. Feed Line 11. Feed Line 13. Open Pipe Macro Sparger Line Description Tubing Set (ID X OD X Length) End Treatment 1 Overlay Gas Sparger 1/4 (6.4 mm) ID X 7/16 (11.1 mm) OD C-Flex X 6 (15 cm) 0.2 micron hydrophobic vent filter with Emflon II 2 Inoculum Addition 1/4 (6.4 mm) ID X 7/16 (11.1 mm) OD C-Flex X 60 (152 cm) reduced to 1/8 (3.2 mm) ID X 1/4 (6.4 mm) OD C-Flex X 12 (30 cm) Plugged 3-6 Probe Ports (4) 1/2 (12.7 mm) Tube ports Pall Kleenpak connectors KPCHT Series (Female) 7 Thermo-Well/Small Volume Thermo-Well Adapter for 1/4 (6.4 mm) Diameter. 1/8 (3.2 mm) ID X 1/4 (6.4 mm) OD SterilEnz Pouch with injection site assembly Sample C-Flex X 18 (46 cm) 8 Bottom Drain Harvest 1/2 (12.7 mm) ID X 3/4 (19.1 mm) OD C-Flex X 60 (152 cm) reduced to 3/8 (9.5 mm) ID X 5/8 (15.9 mm) OD C-Flex X 12 (30 cm) splits to 1/4 (6.4 mm) ID X 7/16 (11.1 mm) OD C-Flex X cm) reduced to 1/8 (3.2 mm) ID X 1/4 (6.4 mm) OD C-Flex X 12 (30 cm) and 3/8 (9.5 mm) ID X 5/8 (15.9 mm) OD C-Flex X 12 (30 cm) Plugged 3/8 MPC Insert 9 Base Addition Feed Lines 12 Exhaust Line 13 Open Pipe Macro Sparger 14 Porous Frit Micro Sparger 12 mm dia (25 µm pores) Ordering Information for Standard 50L-100L Dual Sparger BPC - Porous Frit and Open Pipe 1/4 (6.4 mm) ID X 7/16 (11.1 mm) OD C-Flex X 6 (15 cm) reduced to 1/8 (3.2 mm) ID X 1/4 (6.4 mm) OD C-Flex X 60 (152 cm) 3/8 (9.5 mm) ID X 5/8 (15.9 mm) OD C-Flex X 60 (152 cm) splits to 1/4 (6.4 mm) ID X 7/16 (11.1 mm) OD C-Flex X 12 (30 cm) reduced to 1/8 (3.2 mm) ID X 1/4 (6.4 mm) OD C-Flex X 12 (30 cm) and 3/8 (9.5 mm) ID X 5/8 (15.9 mm) OD C-Flex X 12 (30 cm) 1/2 (12.7 mm) ID X 3/4 (19.1 mm) OD C-Flex X 6 (15 cm) splits to 1/2 (12.7 mm) ID X 3/4 (19.1 mm) OD C-Flex X 6 (15 cm) and 1 /2 (12.7 mm) ID X 3/4 (19.1 mm) OD C-Flex X 10 (25 cm) 1/4 (6.4 mm) ID X 7/16 (11.1 mm) OD C-Flex X 3 (8 cm) connected to Check Valve and 1/4 (6.4 mm) ID X 7/16 (11.1 mm) OD C-Flex X 48 (122 cm) 1/4 (6.4 mm) ID X 7/16 (11.1 mm) OD C-Flex X 6 (15 cm) connected to Check Valve and 1/4 (6.4 mm) ID X 7/16 (11.1 mm) OD C-Flex X 48 (122 cm ) for 50L size or 48 (122 cm) for 100L size Size Description Part Number 50L 50 L CX 5-14 Film BPC SH L 50L Aegis 5-14 Film BPC SH L 100L CX 5-14 Film BPC SH L 100L Aegis 5-14 Film BPC SH Thermo Scientific Single-Use Bioreactor (S.U.B.) 143 Plugged SteriEnz Pouch with injection site assembly 3/8 MPC Body Pall Kleenpak connector KPCHT series (female)pall Kleenpak 0.2 micron exhaust vent filter 0.2 micron hydrophobic vent filter with Emflon II Hydrophobic vent filter with Emflon II

144 Section 4 Specifications and Parts Information Standard 250L Dual Sparger BPC - Porous Frit and Open Pipe Impeller 1-4. Probe Ports (4) 6. Bottom Drain Harvest Front Face 7. Overlay Gas Sparger 8. Base Addition 12. Inoculum Addition 11. Exhaust Line 5. Thermo-Well/Small Volume Sample 14. Porous Frit Micro Sparger Back Face 9. Feed Line 10. Feed Line 13. Open Pipe Macro Sparger Line Description Tubing Set (ID X OD X Length) End Treatment 1-4 Probe Ports (4) 1/2 (12.7 mm) Tube ports Pall Kleenpak connectors KPCHT Series (Female) 5 Thermo-Well/Small Volume Sample 6 Bottom Drain Harvest Thermo-Well Adapter for 1/4 (6.4 mm) Diameter 1/8 (3.2 mm) ID X 1/4 (6.4 mm) OD C-Flex X 18 (46 cm) 1/2 (12.7 mm) ID X 3/4 (19.1 mm) OD C-Flex X 60 (152 cm) reduced to 3/8 (9.5 mm) ID X 5/8 (15.9 mm) OD C-Flex X 12 (30 cm) splits to 1/4 (6.4 mm) ID X 7/16 (11.1 mm) OD C-Flex X cm) reduced to 1/8 (3.2 mm) ID X 1/4 (6.4 mm) OD C-Flex X 12 (30 cm) and 3/8 (9.5 mm) ID X 5/8 (15.9 mm) OD C-Flex X 12 (30 cm) SterilEnz Pouch with injection site assembly Plugged 3/8 MPC Insert 7 Overlay Gas Sparger 1/4 (6.4 mm) ID X 7/16 (11.1 mm) OD C-Flex X 6 (15 cm) Hydrophobic vent filter with Emflon II 8 Base Addition 1/4 (6.4 mm) ID X 7/16 (11.1 mm) OD C-Flex X 6 (15 cm) reduced to 1/8 (3.2 mm) ID X 1/4 (6.4 mm) OD C-Flex X 60 (152 cm) Plugged 9-10 Feed Lines 11 Exhaust Line 12 Inoculum Addition 13 Open Pipe Macro Sparger 14 Porous Frit Micro Sparger 12 mm dia (25 µm pores) Ordering Information for Standard 250L Dual Sparger BPC - Porous Frit and Open Pipe 3/8 (9.5 mm) ID X 5/8 (15.9 mm) OD C-Flex X 60 (152 cm) splits to 1/4 (6.4 mm) ID X 7/16 (11.1 mm) OD C-Flex X 12 (30 cm) reduced to 1/8 (3.2 mm) ID X 1/4 (6.4 mm) OD C-Flex X 12 (30 cm) and 3/8 (9.5 mm) ID X 5/8 (15.9 mm) OD C-Flex X 12 (30 cm) 1/2 (12.7 mm) ID X 3/4 (19.1 mm) OD C-Flex X 6 (15 cm) splits to 1/2 (12.7 mm) ID X 3/4 (19.1 mm) OD C-Flex X 6 (15 cm) and 1/2 (12.7 mm) ID X 3/4 (19.1 mm) OD C-Flex X 10 (25 cm) 1/4 (6.4 mm) ID X 7/16 (11.1 mm) OD C-Flex X 60 (152 cm) reduced to 1/8 (3.2 mm) ID X 1/4 (6.4 mm) OD C-Flex X 12 (30 cm) 1/4 (6.4 mm) ID X 7/16 (11.1 mm) OD C-Flex X 3 (8 cm) reduced to Check Valve and 1/4 (6.4 mm) ID X 7/16 (11.1 mm) OD C-Flex X 48 (122 cm) 1/4 (6.4 mm) ID X 7/16 (11.1 mm) OD C-Flex X 6 (15 cm) reduced to Check Valve and 1/4 (6.4 mm) ID X 7/16 (11.1 mm) OD C-Flex X 48 (122 cm) Size Description Part Number 250L 250L CX 5-14 Film BPC SH L 250L Aegis 5-14 Film BPC SH SterilEnz Pouch with injection site assembly 3/8 MPC Body Pall Kleenpak connector - KPCHT Series (Female) Pall Kleenpak 0.2 Micron Exhaust Vent Filter Thermo Scientific Single-Use Bioreactor (S.U.B.) 144 Plugged Hydrophobic vent filter with Emflon II Hydrophobic vent filter with Emflon II

145 Standard 500L Dual Sparger BPC - Porous Frit and Open Pipe Impeller Section 4 Specifications and Parts Information 1-5. Probe Ports (5) Front Face 10. Inoculum Addition Feed Lines 6. Thermo-Well/Small Volume Sample 15. Porous Frit Micro Sparger Back Face 8. Exhaust Line 11. Base Addition 10. Inoculum Addition 14. Open Pipe Macro Sparger Line Description Tubing Set (ID X OD X Length) End Treatment 1-5 Probe Ports (5) 1/2 (12.7 mm) Tube ports Pall Kleenpak connectors KPCHT Series (Female) 6 Thermo-Well/Small Volume Thermo-Well Adapter for 1/4 (6.4 mm) Diameter. Sample 1/8 (3.2 mm) ID X 1/4 (6.4 mm) OD C-Flex X 24 (60 cm) SterilEnz Pouch with injection site assembly 1/2 (12.7 mm) ID X 3/4 (19.1 mm) OD C-Flex X 60 (152 cm) reduced to 3/8 (9.5 mm) ID X 5/8 (15.9 mm) OD C-Flex X 12 (30 cm) splits to Plugged 7 Bottom Drain Harvest 1/4 (6.4 mm) ID X 7/16 (11.1 mm) OD C-Flex X cm) reduced to 1/8 (3.2 mm) ID X 1/4 (6.4 mm) OD C-Flex X 12 (30 cm) and 3/8 MPC Insert 3/8 (9.5 mm) ID X 5/8 (15.9 mm) OD C-Flex X 12 (30 cm) 8 Exhaust Line 1/2 (12.7 mm) ID X 3/4 (19.1 mm) OD C-Flex X 4 (10 cm) splits to 1/2 (12.7 mm) ID X 3/4 (19.1 mm) OD C-Flex X 10 (25 cm) and (2) Pall Kleenpak 0.2 Micron Exhaust Vent Filter 1/2 (12.7 mm) ID X 3/4 (19.1 mm) OD C-Flex X 10 (25 cm) 9 Overlay Gas Sparger 1/4 (6.4 mm) ID X 7/16 (11.1 mm) OD C-Flex X 6 (15 cm) Hydrophobic vent filter with Emflon II 10 Inoculum Addition 1/4 (6.4 mm) ID X 7/16 (11.1 mm) OD C-Flex X 84 (213 cm) reduced to 1/8 (3.2 mm) ID X 1/4 (6.4 mm) OD C-Flex X 12 (30 cm) Plugged 11 Base Addition 1/4 (6.4 mm) ID X 7/16 (11.1 mm) OD C-Flex X 6 (15 cm) reduced to 1/8 (3.2 mm) ID X 1/4 (6.4 mm) OD C-Flex X 84 (213 cm) Plugged 3/8 (9.5 mm) ID X 5/8 (15.9 mm) OD C-Flex X 84 (213 cm) splits to Feed Lines 3/8 (9.5 mm) ID X 5/8 (15.9 mm) OD C-Flex X 12 (30 cm) 1/4 (6.4 mm) ID X 7/16 (11.1 mm) OD C-Flex X 12 (30 cm) reduced to SterilEnz Pouch with injection site assembly 3/8 1/8 (3.2 mm) ID X 1/4 (6.4 mm) OD C-Flex X 12 (30 cm) and MPC Body 14 Open Pipe Macro Sparger 1/4 (6.4 mm) ID X 7/16 (11.1 mm) OD C-Flex X 3 (8 cm) reduced to Check Valve and 1/4 (6.4 mm) ID X 7/16 (11.1 mm) OD C-Flex X 72 (183 cm) Hydrophobic vent filter with Emflon II 15 Porous Frit Micro Sparger 12 mm dia (25 µm pores) 1/4 (6.4 mm) ID X 7/16 (11.1 mm) OD C-Flex X 6 (15 cm) reduced to Check Valve and 1/4 (6.4 mm) ID X 7/16 (11.1 mm) OD C-Flex X 72 (183 cm) Hydrophobic vent filter with Emflon II Ordering Information for Standard 500L Dual Sparger BPC - Porous Frit and Open Pipe Size Description Part Number 500L 500L CX 5-14 Film BPC SH L 500L Aegis 5-14 Film BPC SH Thermo Scientific Single-Use Bioreactor (S.U.B.) 145

146 Standard 1,000L Dual Sparger BPC - Porous Frit and Open Pipe Impeller Section 4 Specifications and Parts Information 1-5. Probe Ports (5) Front Face 10. Inoculum Addition Feed Lines 6. Thermo-Well/Small Volume Sample 15. Porous Frit Micro Sparger Back Face 8. Exhaust Line 11. Base Addition 10. Inoculum Addition 14. Open Pipe Macro Sparger Line Description Tubing Set (ID X OD X Length) End Treatment 1-5 Probe Ports (5) 1/2 (12.7 mm) Tube ports 6 Thermo-Well/Small Volume Sample 7 Bottom Drain Harvest 8 Exhaust Line Thermo-Well Adapter for 1/4 (6.4 mm) Diameter. 1/8 (3.2 mm) ID X 1/4 (6.4 mm) OD C-Flex X 24 (60 cm) 1/2 (12.7 mm) ID X 3/4 (19.1 mm) OD C-Flex X 60 (152 cm) reduced to 3/8 (9.5 mm) ID X 5/8 (15.9 mm) OD C-Flex X 12 (30 cm) splits to 1/4 (6.4 mm) ID X 7/16 (11.1 mm) OD C-Flex X cm) reduced to 1/8 (3.2 mm) ID X 1/4 (6.4 mm) OD C-Flex X 12 (30 cm) and 3/8 (9.5 mm) ID X 5/8 (15.9 mm) OD C-Flex X 12 (30 cm) 1/2 (12.7 mm) ID X 3/4 (19.1 mm) OD C-Flex X 4 (10 cm) splits to 1/2 (12.7 mm) ID X 3/4 (19.1 mm) OD C-Flex X 10 (25 cm) and 1/2 (12.7 mm) ID X 3/4 (19.1 mm) OD C-Flex X 10 (25 cm) 9 Overlay Gas Sparger 1/4 (6.4 mm) ID X 7/16 (11.1 mm) OD C-Flex X 6 (15 cm) 10 Inoculum Addition 11 Base Addition Feed Lines Open Pipe Macro Sparger Porous Frit Micro Sparger 12 mm dia (25 µm pores) Ordering Information for Standad 1,000L Dual Sparger BPC - Porous Frit and Open Pipe 1/4 (6.4 mm) ID X 7/16 (11.1 mm) OD C-Flex X 84 (213 cm) reduced to 1/8 (3.2 mm) ID X 1/4 (6.4 mm) OD C-Flex X 12 (30 cm) 1/4 (6.4 mm) ID X 7/16 (11.1 mm) OD C-Flex X 6 (15 cm) reduced to 1/8 (3.2 mm) ID X 1/4 (6.4 mm) OD C-Flex X 84 (213 cm) 3/8 (9.5 mm) ID X 5/8 (15.9 mm) OD C-Flex X 84 (213 cm) splits to 1/4 (6.4 mm) ID X 7/16 (11.1 mm) OD C-Flex X 12 (30 cm) reduced to 1/8 (3.2 mm) ID X 1/4 (6.4 mm) OD C-Flex X 12 (30 cm) and 3/8 (9.5 mm) ID X 5/8 (15.9 mm) OD C-Flex X 12 (30 cm) 1/4 (6.4 mm) ID X 7/16 (11.1 mm) OD C-Flex X 3 (8 cm) reduced to Check Valve and 1/4 (6.4 mm) ID X 7/16 (11.1 mm) OD C-Flex X 72 (183 cm) 1/4 (6.4 mm) ID X 7/16 (11.1 mm) OD C-Flex X 6 (15 cm) reduced to Check Valve and 1/4 (6.4 mm) ID X 7/16 (11.1 mm) OD C-Flex X 72 (183 cm) Size Description Part Number 1000L 1000L CX 5-14 Film BPC SH L 1000L Aegis 5-14 Film BPC SH Pall Kleenpak connectors KPCHT Series (Female) SterilEnz Pouch with injection site assembly Thermo Scientific Single-Use Bioreactor (S.U.B.) 146 Plugged 3/8 MPC Insert (2) Pall Kleenpak 0.2 micron Exhaust Vent Filter Hydrophobic vent filter with Emflon II, Pressure Transducer Plugged Plugged Sterilenz Pouch with injection site assembly 3/8 MPC Body Hydrophobic vent filter with Emflon II Hydrophobic vent filter with Emflon II

147 Standard 2,000L Dual Sparger BPC - Porous Frit and Open Pipe Sparger 9. Media Fill 1-5. Probe Ports (5) 10. Inoculum Addition/Feed Line Impeller 9. Media Fill/Auxiliary Drain 8. Feed Line Section 4 Specifications and Parts Information 6. Thermo-Well 7. Bottom Drain Harvest Front Face 6. Small Volume Sampling Available with or without the condenser assembly Back Face 11. Base Addition 12. Overlay Gas Sparger 13. Exhaust Line 8. Feed Line 14. Condensate Return Line 9. Media Fill/Auxiliary Drain 15. Condenser Bag Assembly 16. Porous Frit Gas Sparger (3) Tri-Clamp Port 17. Open Pipe Gas Sparger Line Description Tubing Set (ID X OD X Length) End Treatment 1-5 Probe Ports (5) 1/2 (12.7mm) Tube ports Pall Kleenpak connectors (Female) 6 Thermowell/ Small Volume Sample 7 Bottom Drain Harvest 8 Feed Line 9 Media Fill 10 Inoculum Addition/Feed Line 11 Base Addition 12 Overlay Gas Sparger Thermowell Adapter for 1/4 (6.4mm) Diameter. 1/8 (3.2mm) ID X 1/4 (6.4mm) OD C-Flex X 24 (61cm) 3/4 (19.1mm) ID X 1 (25.4mm) OD C-Flex X 48 (122cm) splits to 1/2 (12.7mm) ID X 3/4 (19.1mm) OD C-Flex X 24 61cm) reduced to 1/4 (6.4mm) ID X 3/8 (9.5mm) OD C-Flex X 12 (30cm) and 1/2 (12.7mm) ID X 3/4 (19.1mm) OD C-Flex X 24 61cm) 1/2 (12.7mm) ID X 3/4 (19.1mm) OD C-Flex X 4 (10cm) splits to 1/2 (12.7mm) ID X 3/4 (19.1mm) OD C-Flex X 10 (25cm) and 1/2 (12.7mm) ID X 3/4 (19.1mm) OD C-Flex X 10 (25cm) 3/4 (19.1mm) ID X 1 (25.4mm) OD C-Flex X 84 (213cm) splits to 1/2 (12.7mm) ID X 3/4 (19.1mm) OD C-Flex X 24 (61cm) and 1/2 (12.7mm) ID X 3/4 (19.1mm) OD C-Flex X 24 (61cm) 1/2 (12.7mm) ID X 3/4 (19.1mm) OD C-Flex X 3 (8cm) reduced to 1/4 (6.4mm) ID X 7/16 (11.1mm) OD C-Flex X 6 (15cm) reduced to 1/8 (3.2mm) ID X 1/4 (6.4mm) OD C-Flex X 84 (213cm) 1/2 (12.7mm) ID X 3/4 (19.1mm) OD C-Flex X 3 (8cm) reduced to 1/4 (6.4mm) ID X 7/16 (11.1mm) OD C-Flex X 84 (213cm) reduced to 1/8 (3.2mm) ID X 1/4 (6.4mm) OD C-Flex X 12 (30cm) 1/2 (12.7mm) ID X 3/4 (19.1mm) OD C-Flex X 4 (10cm) reduced to 3/8 (9.5mm) ID X 5/8 (15.9mm) OD C-Flex X 84 (213cm) 13 Exhaust Line Condenser Bag Assembly 14 Condensate Return Line Condenser Bag Assembly 15 Condenser Bag Assembly Condenser Bag Assembly 16 Porous Frit Gas Sparger 12mm dia (25 µm pores) 17 Open Pipe Gas Sparger (3x) 12mm PDVF Porous Sparger Inserts connected to 1/4 (6.4mm) ID X 7/16 (11.1mm) OD C-Flex X 6 (15cm) converge to one 1/4 (6.4mm) ID X 7/16 (11.1mm) OD C-Flex X 72 (183cm) 1/4 (6.4mm) ID X 7/16 (11.1mm) OD C-Flex X 72 (183cm) reduced to Check Valve and 1/4 (6.4mm) ID X 7/16 (11.1mm) OD C-Flex X 72 (183cm) SterilEnz Pouch with injection site assembly 1/4 MPC Insert and Pall Kleenpak (Male) SterilEnz Pouch with injection site assembly and 3/8 MPC Body Pall Kleenpak connectors (Female) Thermo Scientific Single-Use Bioreactor (S.U.B.) 147 Plugged Plugged Kleenpak Emflon II Capsule and Pressure transducer Kleenpak Emflon II Capsule Kleenpak Emflon II Capsule 18 3 Tri-Clamp Port NA Gasket end cap, and clamp

148 Section 4 Specifications and Parts Information Ordering Information for Standard 2,000L Dual Sparger BPC - Porous Frit and Open Pipe Size Description Part Number 2000L 2000L CX 5-14 Film BPC without Condenser SH L 2000L CX 5-14 Film BPC with Condenser SH L 2000L Aegis 5-14 Film BPC without Condenser SH L 2000L Aegis 5-14 Film BPC with Condenser SH Standard BPC with Drilled Hole Sparger Drilled Hole Sparger Configuration Size Sparger Configuration Drilled Hole Sparger Configuration 50L 1 Porous Frit and 1 Drilled Hole Sparger 3.5 disk with 360 x 0.178mm holes 100L 1 Porous Frit and 1 Drilled Hole Sparger 3.5 disk with 570 x 0.178mm holes 250L 1 Porous Frit and 1 Drilled Hole Sparger 4.8 disk with 760 x 0.233mm holes 500L 1 Porous Frit and 1 Drilled Hole Sparger 6.75 disk with 980 x mm holes 1000L 1 Porous Frit and 1 Drilled Hole Sparger 6.75 disk with 1180 x mm holes 2000L 2 Porous Frit and 2 Drilled Hole Spargers 6.75 disk with 69- x mm holes (13,800 holes) Thermo Scientific Single-Use Bioreactor (S.U.B.) 148

149 Section 4 Specifications and Parts Information Standard 50L-100L Dual Sparger BPC - Porous Frit and Drilled Hole Sparger Front Face 2. Inoculum Addition 1. Overlay Gas Sparger Impeller 3-6. Probe Ports (4) 8. Bottom Drain Harvest 7. Thermo-Well/Small Volume Sample 9. Base Addition 12. Exhaust Line 14. Porous Frit Micro Sparger Back Face 10. Feed Line 11. Feed Line 13. Drilled Hole Macro Sparger Line Description Tubing Set (ID X OD X Length) End Treatment 1 Overlay Gas Sparger 1/4 (6.4 mm) ID X 7/16 (11.1 mm) OD C-Flex X 6 (15 cm) Hydrophobic vent filter with Emflon II, connected to 6 (15 cm) C-Flex 2 Inoculum Addition 1/4 (6.4 mm) ID X 7/16 (11.1 mm) OD C-Flex X 60 (152 cm) reduced to 1/8 (3.2 mm) ID X 1/4 (6.4 mm) OD C-Flex X 12 (30 cm) Plugged 3-6 Probe Ports (4) 1/2 (12.7 mm) Tube ports Pall Kleenpak connectors KPCHT Series (Female) 7 Thermo-Well/Small Volume Thermo-Well Adapter for 1/4 (6.4 mm) Diameter. Sample 1/8 (3.2 mm) ID X 1/4 (6.4 mm) OD C-Flex X 18 (46 cm) SterilEnz Pouch with injection site assembly 1/2 (12.7 mm) ID X 3/4 (19.1 mm) OD C-Flex X 60 (152 cm) reduced to Bottom Drain 3/8 (9.5 mm) ID X 5/8 (15.9 mm) OD C-Flex X 12 (30 cm) splits to 8 1/4 (6.4 mm) ID X 7/16 (11.1 mm) OD C-Flex X cm) reduced to Plugged 3/8 MPC Insert Harvest 1/8 (3.2 mm) ID X 1/4 (6.4 mm) OD C-Flex X 12 (30 cm) and 3/8 (9.5 mm) ID X 5/8 (15.9 mm) OD C-Flex X 12 (30 cm) 9 Base Addition 1/4 (6.4 mm) ID X 7/16 (11.1 mm) OD C-Flex X 6 (15 cm) reduced to 1/8 (3.2 mm) ID X 1/4 (6.4 mm) OD C-Flex X 60 (152 cm) Plugged 3/8 (9.5 mm) ID X 5/8 (15.9 mm) OD C-Flex X 60 (152 cm) splits to Feed Lines 3/8 (9.5 mm) ID X 5/8 (15.9 mm) OD C-Flex X 12 (30 cm) 1/4 (6.4 mm) ID X 7/16 (11.1 mm) OD C-Flex X 12 (30 cm) reduced to SteriEnz Pouch with injection site assembly 3/8 1/8 (3.2 mm) ID X 1/4 (6.4 mm) OD C-Flex X 12 (30 cm) and MPC Body 12 Exhaust Line Drilled Hole Macro Sparger 3.5 (8.9 cm) disk with 360 x mm (0.007 ) holes for 50L BPC or 570 holes for 100L BPC Porous Frit Micro Sparger 12 mm dia (25 µm pores) Ordering Information for Standard Dual Sparger BPC - Porous Frit and Drilled Hole Sparger 1/2 (12.7 mm) ID X 3/4 (19.1 mm) OD C-Flex X 8 (20 cm) connected to 1/2 (12.7 mm) ID X 3/4 (19.1 mm) OD C-Flex X 6 (15 cm) and 1/2 (12.7 mm) ID X 3/4 (19.1 mm) OD C-Flex X 10 (25 cm) 1/4 (6.4 mm) ID X 7/16 (11.1 mm) OD C-Flex X 3 (8 cm) connected to Check Valve and 1/4 (6.4 mm) ID X 7/16 (11.1 mm) OD C-Flex X 38 (97 cm) for 50L BPC or 45 (114 cm) for the 100L BPC 1/4 (6.4 mm) ID X 7/16 (11.1 mm) OD C-Flex X 6 (15 cm) connected to Check Valve and 1/4 (6.4 mm) ID X 7/16 (11.1 mm) OD C-Flex X 35 (89 cm) for 50L BPC or 42 (107 cm) for 100L BPC) Pall Kleenpak connector KPCHT Series (female) Pall Kleenpak 0.2 micron exhaust vent filter Meissner Steridyne 0.2 micron hydrophobic filter connected to 6 (15 cm) C-Flex Meissner Steridyne 0.2 micron hydrophobic filter connected to 6 (15 cm) C-Flex Size Description Part Number 50L 50L CX 5-14 Film BPC with Drilled Hole Sparger SH L 50L Aegis 5-14 Film BPC with Drilled Hole Sparger SH L 100L CX 5-14 Film BPC with Drilled Hole Sparger SH L 100L Aegis 5-14 Film BPC with Drilled Hole Sparger SH Thermo Scientific Single-Use Bioreactor (S.U.B.) 149

150 Standard 250L Dual Sparger BPC - Porous Frit and Drilled Hole Sparger Front Face Impeller Section 4 Specifications and Parts Information 1-4. Probe Ports (4) 6. Bottom Drain Harvest 12. Inoculum Addition 11. Exhaust Line 5. Thermo-Well/Small Volume Sample 14. Porous Frit Micro Sparger Back Face 7. Overlay Gas Sparger 8. Base Addition Feed Line 13. Drilled Hole Macro Sparger Line Description Tubing Set (ID X OD X Length) End Treatment 1-4 Probe Ports (4) 1/2 (12.7 mm) Tube ports 5 6 Thermo-Well/Small Volume Sample Bottom Drain Harvest Thermo-Well Adapter for 1/4 (6.4 mm) Diameter. 1/8 (3.2 mm) ID X 1/4 (6.4 mm) OD C-Flex X 18 (46 cm) 1/2 (12.7 mm) ID X 3/4 (19.1 mm) OD C-Flex X 60 (152 cm) reduced to 3/8 (9.5 mm) ID X 5/8 (15.9 mm) OD C-Flex X 12 (30 cm) splits to 1/4 (6.4 mm) ID X 7/16 (11.1 mm) OD C-Flex X cm) reduced to 1/8 (3.2 mm) ID X 1/4 (6.4 mm) OD C-Flex X 12 (30 cm) and 3/8 (9.5 mm) ID X 5/8 (15.9 mm) OD C-Flex X 12 (30 cm) 7 Overlay Gas Sparger 1/4 (6.4 mm) ID X 7/16 (11.1 mm) OD C-Flex X 6 (15 cm) 8 Base Addition 9-10 Feed Lines 11 Exhaust Line 12 Inoculum Addition Drilled Hole Macro Sparger 4.8 (12.2cm) disk with 760 x 0.233mm (0.009 ) holes Porous Frit Micro Sparger 12 mm dia (25 µm pores) Ordering Information for Standard 250L Dual Sparger BPC - Porous Frit and Drilled Hole Sparger 1/4 (6.4 mm) ID X 7/16 (11.1 mm) OD C-Flex X 6 (15 cm) reduced to 1/8 (3.2 mm) ID X 1/4 (6.4 mm) OD C-Flex X 60 (152 cm) 3/8 (9.5 mm) ID X 5/8 (15.9 mm) OD C-Flex X 60 (152 cm) splits to 1/4 (6.4 mm) ID X 7/16 (11.1 mm) OD C-Flex X 12 (30 cm) reduced to 1/8 (3.2 mm) ID X 1/4 (6.4 mm) OD C-Flex X 12 (30 cm) and 3/8 (9.5 mm) ID X 5/8 (15.9 mm) OD C-Flex X 12 (30 cm) 1/2 (12.7 mm) ID X 3/4 (19.1 mm) OD C-Flex X 12 (30 cm) splits to 1/2 (12.7 mm) ID X 3/4 (19.1 mm) OD C-Flex X 6 (15 cm) and 1/2 (12.7 mm) ID X 3/4 (19.1 mm) OD C-Flex X 6 (15 cm) 1/4 (6.4 mm) ID X 7/16 (11.1 mm) OD C-Flex X 60 (152 cm) reduced to 1/8 (3.2 mm) ID X 1/4 (6.4 mm) OD C-Flex X 12 (30 cm) 1/4 (6.4 mm) ID X 7/16 (11.1 mm) OD C-Flex X 3 (8 cm) connected to Check Valve and 1/4 (6.4 mm) ID X 7/16 (11.1 mm) OD C-Flex X 59 (150 cm) 1/4 (6.4 mm) ID X 7/16 (11.1 mm) OD C-Flex X 6 (15 cm) reduced to Check Valve and 1/4 (6.4 mm) ID X 7/16 (11.1 mm) OD C-Flex X 56 (142 cm) Pall Kleenpak connectors KPCHT series (female) SterilEnz Pouch with injection site assembly Size Description Part Number 250L 250L CX 5-14 Film BPC with Drilled Hole Sparger SH L 250L Aegis 5-14 Film BPC with Drilled Hole Sparger SH Thermo Scientific Single-Use Bioreactor (S.U.B.) 150 Plugged 3/8 MPC Insert Meissner Steridyne 0.2 micron hydrophobic filter connected to 6 (15 cm) C-Flex Plugged SterilEnz Pouch with injection site assembly 3/8 MPC Body Asceptiquick G Connector (genderless), (2) Meissner Ultracap 0.2 micron hydrophobic filters connected to 6 (15 cm) C-Flex Plugged Meissner Steridyne 0.2 micron hyrophobic filter connected to 6 (15 cm) C-Flex Meissner Steridyne 0.2 micron hyrophobic filter connected to 6 (15 cm) C-Flex

151 Standard 500L Dual Sparger BPC - Porous Frit and Drilled Hole Sparger Impeller 1-5. Probe Ports (5) Section 4 Specifications and Parts Information 7. Bottom Drain Harvest Front Face 9. Overlay Gas Sparger Feed Line 6. Thermowell/Small Volume Sample 14. Porous Frit Micro Sparger Back Face 8. Exhaust Line 10. Inoculum Addition 11. Base Addition 15. Drilled Hole Macro Sparger Line Description Tubing Set (Inner Diameter x Outer Diameter x Length) End Treatment 1-5 Probe Ports (5) 1/2 (12.7mm) Tube Ports Pall Kleenpak Connectors KPCHT Series (Female) 6 Thermowell/ Small Volume Sample 7 Bottom Drain Harvest 8 Exhaust Line Thermowell Adapter for 6.4mm (1/4 ) Diameter 3.2mm (1/8 ) (3.2 mm) x 6.4mm (1/4 ) C-Flex Tubing x 60cm (24 ) 12.7mm (1/2 ) x 19.1mm (3/4 ) C-Flex Tubing x 152cm (60 ) Reduced to 9.5mm (3/8 ) x 15.9mm (5/8 ) C-Flex Tubing x 30cm (12 ) Splits to 6.4mm (1/4 ) x 11.1mm (7/16 ) C-Flex Tubing x 30cm (12 ) Reduced to 3.2mm (1/8 ) x 6.4mm (1/4 ) C-Flex Tubing x 30cm (12 ) and9.5mm (3/8 ) x 15.9mm (5/8 ) C-Flex Tubing x 30cm (12 ) 12.7mm (1/2 ) x 19.1mm (3/4 ) C-Flex Tubing x 30cm (12 ) Splits to 12.7mm (1/2 ) x 19.1mm (3/4 ) C-Flex Tubing x 15cm (6 ) and12.7mm (1/2 ) x 19.1mm (3/4 ) C-Flex Tubing x 15cm (6 ) 9 Overlay Gas Sparger 6.4mm (1/4 ) x 11.1mm (7/16 ) C-Flex Tubing x 15cm (6 ) 10 Inoculum Addition 11 Base Addition Feed Lines Drilled Hole Macro Sparger 17.1cm (6.75 ) Disk with 1,180 Holes with a 0.445mm (0.018 ) Diameter Porous Frit Micro Sparger 12mm dia (25µm pores) 6.4mm (1/4 ) x 11.1mm (7/16 ) C-Flex Tubing x 213cm (84 ) Reduced to 3.2mm (1/8 ) x 6.4mm (1/4 ) C-Flex Tubing x 30cm (12 ) 6.4mm (1/4 ) x 11.1mm (7/16 ) C-Flex Tubing x 15cm (6 ) Reduced to 3.2mm (1/8 ) x 6.4mm (1/4 ) C-Flex Tubing x 213cm (84 ) 9.5mm (3/8 ) x 15.9mm (5/8 ) C-Flex Tubing x 213cm (84 ) Splits to 6.4mm (1/4 ) x 11.1mm (7/16 ) C-Flex Tubing x 30cm (12 ) Reduced to 3.2mm (1/8 ) x 6.4mm (1/4 ) C- Flex Tubing x 30cm (12 ) and 9.5mm (3/8 ) x 15.9mm (5/8 ) C-Flex Tubing x 30cm (12 ) 9.5mm (3/8 ) x 15.9mm (5/8 ) C-Flex Tubing x 8cm (3 ) Connected to Check Valve and 9.5mm (3/8 ) x 15.9mm (5/8 ) C-Flex Tubing x 190cm (75 ) 6.4mm (1/4 ) x 11.1mm (7/16 ) C-Flex Tubing x 15cm (6 ) Reduced to Check Valve and 6.4mm (1/4 ) x 11.1mm (7/16 ) C-Flex Tubing x 198cm (78 ) SterilEnz Pouch with Injection Site Assembly Plugged 3/8 MPC Insert (2x) Meissner Ultracap 0.2µm Hydrophobic Filters Connected to 15cm (6 ) C-Flex Tubing Meissner Steridyne 0.2µm Hydrophobic Filter Connected to 30cm (12 ) C-Flex Tubing Plugged Plugged SterilEnz Pouch with Injection Site Assembly 3/8 MPC Body Meissner Steridyne 0.2µm Hyrophobic Filter Connected to 15cm (6 ) C-Flex Tubing Meissner Steridyne 0.2 Micron Hyrophobic Filter Connected to 6 (15 cm) C-Flex Tubing Ordering Information for Standard 500L Dual Sparger BPC - Porous Frit and Drilled Hole Sparger Size Description Part Number 500L 500L CX 5-14 Film BPC with Drilled Hole Sparger SH L 500L Aegis 5-14 Film BPC with Drilled Hole Sparger SH Thermo Scientific Single-Use Bioreactor (S.U.B.) 151

152 Standard 1,000L Dual Sparger BPC - Porous Frit and Drilled Hole Sparger Impeller 1-5. Probe Ports (5) Section 4 Specifications and Parts Information 7. Bottom Drain Harvest Front Face 6. Thermowell/Small Volume Sample 9. Overlay Gas Sparger Feed Line 14. Porous Frit Micro Sparger Back Face 8. Exhaust Line 10. Inoculum Addition 11. Base Addition 15. Drilled Hole Macro Sparger Line Description Tubing Set (Inner Diameter x Outer Diameter x Length) End Treatment 1-5 Probe Ports (5) 1/2 (12.7mm) Tube Ports 6 Thermowell/ Small Volume Sample 7 Bottom Drain Harvest 8 Exhaust Line Thermowell Adapter for 6.4mm (1/4 ) Diameter3.2mm (1/8 ) (3.2 mm) x 6.4mm (1/4 ) C-Flex Tubing x 60cm (24 ) 12.7mm (1/2 ) x 19.1mm (3/4 ) C-Flex Tubing x 152cm (60 ) Reduced to 9.5mm (3/8 ) x 15.9mm (5/8 ) C-Flex Tubing x 30cm (12 ) Splits to 6.4mm (1/4 ) x 11.1mm (7/16 ) C-Flex Tubing x 30cm (12 ) Reduced to 3.2mm (1/8 ) x 6.4mm (1/4 ) C-Flex Tubing x 30cm (12 ) and 9.5mm (3/8 ) x 15.9mm (5/8 ) C-Flex Tubing x 30cm (12 ) 12.7mm (1/2 ) x 19.1mm (3/4 ) C-Flex Tubing x 30cm (12 ) Splits to 12.7mm (1/2 ) x 19.1mm (3/4 ) C-Flex Tubing x 15cm (6 ) and 12.7mm (1/2 ) x 19.1mm (3/4 ) C-Flex Tubing x 15cm (6 ) 9 Overlay Gas Sparger 6.4mm (1/4 ) x 11.1mm (7/16 ) C-Flex Tubing x 15cm (6 ) 10 Inoculum Addition 11 Base Addition Feed Lines Drilled Hole Macro Sparger 17.1cm (6.75 ) Disk with 1,180 Holes with a 0.445mm (0.018 ) Diameter Porous Frit Micro Sparger 12mm dia (25µm pores) Ordering Information for Standard 1,000L Dual Sparger BPC - Porous Frit and Drilled Hole Sparger 6.4mm (1/4 ) x 11.1mm (7/16 ) C-Flex Tubing x 213cm (84 ) Reduced to 3.2mm (1/8 ) x 6.4mm (1/4 ) C-Flex Tubing x 30cm (12 ) 6.4mm (1/4 ) x 11.1mm (7/16 ) C-Flex Tubing x 15cm (6 ) Reduced to 3.2mm (1/8 ) x 6.4mm (1/4 ) C-Flex Tubing x 213cm (84 ) 9.5mm (3/8 ) x 15.9mm (5/8 ) C-Flex Tubing x 213cm (84 ) Splits to 6.4mm (1/4 ) x 11.1mm (7/16 ) C-Flex Tubing x 30cm (12 ) Reduced to 3.2mm (1/8 ) x 6.4mm (1/4 ) C-Flex Tubing x 30cm (12 ) and 9.5mm (3/8 ) x 15.9mm (5/8 ) C-Flex Tubing x 30cm (12 ) 9.5mm (3/8 ) x 15.9mm (5/8 ) C-Flex Tubing x 8cm (3 ) (8 cm) Connected to Check Valve and 9.5mm (3/8 ) x 15.9mm (5/8 ) C-Flex Tubing x 246cm (97 ) 6.4mm (1/4 ) x 11.1mm (7/16 ) C-Flex Tubing x 15cm (6 ) Reduced to Check Valve and 6.4mm (1/4 ) x 11.1mm (7/16 ) C-Flex Tubing x 241cm (95 ) Pall Kleenpak Connectors KPCHT Series (Female) SterilEnz Pouch with Injection Site Assembly Plugged 3/8 MPC Insert (2x) Meissner Ultracap 0.2µm Hydrophobic Filters Connected to 15cm (6 ) C-Flex Tubing Meissner Steridyne 0.2µm Hydrophobic Filter Connected to 30cm (12 ) C-Flex Tubing Plugged Plugged SterilEnz Pouch with Injection Site Assembly 3/8 MPC Body Meissner Steridyne 0.2µm Hyrophobic Filter Connected to 15cm (6 ) C-Flex Tubing Meissner Steridyne 0.2µm Hydrophobic Filter Connected to 15cm (6 ) C-Flex Tubing Size Description Part Number 1,000L 1,000L CX 5-14 Film BPC with Drilled Hole Sparger SH ,000L 1,000L Aegis 5-14 Film BPC with Drilled Hole Sparger SH Thermo Scientific Single-Use Bioreactor (S.U.B.) 152

153 Standard 2,000L Dual Sparger BPC - Porous Frit and Drilled Hole Sparger Probe Ports (5) Section 4 Specifications and Parts Information 4. Media Fill/Auxiliary Drain 12. Overlay gas sparger 13. Feed Line 6. Inoculum Addition/Feed Line 5. Base Addition 15. Drilled Hole Sparger 1. Condenser Bag Assembly 4. Media Fill Impeller 14. Thermo Well/Small Volume Sampling 12. Overlay Gas Sparger 2. Exhaust Line 4. Media Fill/Auxiliary Drain 3. Condensate Return Line 13. Feed Line 16. Drilled Hole Sparger 17. Bottom Drain Harvest Tri-Clamp Port 16. Porous Frit Sparger Line Description Tubing Set (ID X OD X Length) End Treatment 1 Condenser Bag Assemblies (2) Condenser Bag Assembly (optional) 2 Exhaust Lines (2) Condenser Bag Assembly (optional) 3 Condensate Return Line Condenser Bag Assembly (optional) 4 Media Fill/ Auxiliary Drain Lines 5 Base Addition 6 Inoculum Addition/Feed Line 7 Overlay Gas Sparger (3X) 3/4 (19.1 mm) ID X 1 (25.4 mm) OD C-Flex X 84 (213 cm) splits to 1/2 (12.7 mm) ID X 3/4 (19.1 mm) OD C-Flex X 24 (61 cm) and 1/2 (12.7 mm) ID X 3/4 (19.1 mm) OD C-Flex X 24 (61 cm) 1/2 (12.7 mm) ID X 3/4 (19.1 mm) OD C-Flex X 3 (8 cm) reduced to 1/4 (6.4 mm) ID X 7/16 (11.1 mm) OD C-Flex X 84 (213 cm) reduced to 1/8 (3.2 mm) ID X 1/4 (6.4 mm) OD C-Flex X 12 (30 cm) 1/2 (12.7 mm) ID X 3/4 (19.1 mm) OD C-Flex X 3 (8 cm) reduced to 1/4 (6.4 mm) ID X 7/16 (11.1 mm) OD C-Flex X 6 (15 cm) reduced to 1/8 (3.2 mm) ID X 1/4 (6.4 mm) OD C-Flex X 84 (213 cm) 1/2 (12.7 mm) ID X 3/4 (19.1 mm) OD C-Flex X 4 (10 cm) reduced to 3/8 (9.5 mm) ID X 5/8 (15.9 mm) OD C-Flex X 84 (213 cm) 8-12 Probe Ports (5) 1/2 (12.7 mm) Tube ports 13 Feed Lines (2) (2X) 1/2 (12.7 mm) ID X 3/4 (19.1 mm) OD C-Flex X 4 (10 cm) splits to 1/2 (12.7 mm) ID X 3/4 (19.1 mm) OD C-Flex X 10 (25 cm) and 1/2 (12.7 mm) ID X 3/4 (19.1 mm) OD C-Flex X 10 (25 cm) Pall Kleenpak connectors (female) Plugged Plugged Kleenpak Emflon II capsuleand pressure transducer Pall Kleenpak connectors (female) SterilEnz Pouch with injection site assemblyand 3/8 MPC body Thermo Scientific Single-Use Bioreactor (S.U.B.) 153

154 Section 4 Specifications and Parts Information Line Description Tubing Set (ID X OD X Length) End Treatment Thermo-Well/Small Volume Sample Drilled Hole Macro Sparger 6.75 (17.1 cm) disk with 690 x mm (0.023 ) holes (1380 holes) Porous Frit Micro Sparger 12 mm dia (25 µm pores) 17 Bottom Drain Harvest Thermo-Well Adapter for 1/4 (6.4 mm) Diameter. 1/8 (3.2 mm) ID X 1/4 (6.4 mm) OD C-Flex X 24 (61 cm) (2x) 3/8 (9.5 mm) ID X 5/8 (15.9 mm) OD C-Flex X 3 (8 cm) connected to Check Valve and 3/8 (9.5 mm) ID X 5/8 (15.9 mm) OD C-Flex X 72 (183 cm) 1/4 (6.4 mm) ID X 7/16 (11.1 mm) OD C-Flex X 6 (15 cm) reduced to Check Valve and 1/4 (6.4 mm) ID X 7/16 (11.1 mm) OD C-Flex X 72 (183 cm) 3/4 (19.1 mm) ID X 1 (25.4 mm) OD C-Flex X 48 (122 cm) splits to 1/2 (12.7 mm) ID X 3/4 (19.1 mm) OD C-Flex X cm) reduced to 1/4 (6.4 mm) ID X 3/8 (9.5 mm) OD C-Flex X 12 (30 cm) and 1/2 (12.7 mm) ID X 3/4 (19.1 mm) OD C-Flex X cm) SterilEn ztm Pouch with injection site assembly (2x) Meissner Steridyne 0.2 micron hyrophobic filter with 3/8 ID C-Flex tubing, (Y-connector and Quick Connects provided for joining lines) (2x) Meissner Steridyne 0.2 micron hydrophobic filter 1/4 MPC Insert and Pall Kleenpak (male) 18 3 Tri-Clamp Port NA Gasket end cap, and clamp Ordering Information for Standard 1,000L Dual Sparger BPC - Porous Frit and Drilled Hole Sparger Size Description Part Number 2,000L 2,000L CX 5-14 Film BPC with drilled hole sparger and condenser SH ,000L 2,000L CX 5-14 Film BPC with drilled hole sparger without condenser SH ,000L 2,000L Aegis Film BPC with drilled hole sparger and condenser SH ,000L 2,000L Aegis 5-14 Film BPC with drilled hole sparger without condenser SH Thermo Scientific Single-Use Bioreactor (S.U.B.) 154

155 Section 4 Specifications and Parts Information Custom BPC Options Category Options/Capability Notes Tubing Type C Flex, platinum cured silicone, PVC, Pharmed, PharmaPure Tubing Size Connectors Probe ports Ranges from 1/8 ID to 1 ID in various lengths Luers, CPC Quick Connects, SIP connectors, Triclamp, Kleenpak, Lynx, SmartSite, Clave, Lynx steam thru, CPC Steam Thru, Gore steam valve, Gore Mini TC, BioQuate (GE), PAW SterilEnz, end plug, etc. Additional ports: second row of four for 50 to 250L S.U.B.; second row of five for 500 and 1,000L S.U.B.s Pressure sensor: Pendotech and Finesse solutions (Pendotech comes standard on 500 and 1,000L). More information is available in the Tubing Selection Guide More information is available in the Tubing Selection Guide More information is available in the Connection System Selection Guide. Note: The only option for probe port connections is Kleenpak. The reusable probe port connection use a Kleenpak Connector. Disposable sensors DO and ph Finesse and PreSens Choice of qualified sensors available. Addition of ports/lines (other than 2nd row of probe ports) Port sizes Re-arrangement of lines on existing ports Sparger Dip tube lines Overlay and Sparge Line Filters Vent Filters Vent Filter Tubing Length Filters on Media and Supplement Inlets ph Mettler Toledo Limited engineer-to-order customization only. Limited engineer-to-order customization only. Limited customization possible, e.g., moving sample/thermowell port to a probe tube port, or swapping overlay inlet line with supplement line. Dual sparger (open pipe or drilled hole plus porous frit) standard. Limited customization possible. Filter options available from standard component library. Standard is Pall Kleenpak or Meissner Sterilenz 0.2 micron exhaust vent filter. Extended filter height above the S.U.B. bag is make-to-order. Limited engineer-to-order customization only. Choice of filters used to sterilize incoming media or supplements are available. Dependent on location in bag and compatibility with hardware Dependent on location in bag and fit with hardware ( e.g., 1 ID port on harvest line) Dependent on location in bag and fit with hardware Length cannot interfere with impeller and shaft. Note: Filters must be compatible with available vent filter heater configurations. Must be compatible with a vent filter bracket option Please Note: Not all options are available for all ports. No customization of port type and location, chamber dimensions or mixing assembly is possible. For additional information, please see the Selection Guides in the S.U.B. BPC Catalog. Thermo Scientific Single-Use Bioreactor (S.U.B.) 155

156 BPC Packaging for All Sizes (as Dry BPC Systems) Outer packaging Label Gamma Irradiation Shipping container Documentation Supplied flat-packed Two polyethylene outer layers Description Product code Lot number Expiry date on outer packaging and shipping container Irradiation (25 to 38 kgy) inside outer packaging Durable cardboard carton Section 4 Specifications and Parts Information Certificate of Analysis provided with each lot for each delivery 4.3 Accessories and Options Specifications This section includes information about the following accessories and options: AC and DC motors Load cells Drive shafts Cable management system Vent filter heaters Exhaust condenser system Miscellaneous items Thermo Scientific Single-Use Bioreactor (S.U.B.) 156

157 Section 4 Specifications and Parts Information AC and DC Motors AC and DC Motor options are available to help tailor the system to specific needs. The DC motor (see Figure 4.1) operates at lower voltage and, when integrated with a controller system that receives sensor feedback, provides more accurate speed control through a digital program transmitter. The DC motor comes with an encoder, but does not come with a motor control option from Thermo Scientific and must be specified by the end user. Figure 4.1 DC motor The AC motor (see Figure 4.2) may be used with the Thermo Scientific electric box, includes the variable frequency drive and is controlled using either the provided keypad or a controller specified by the end-user. Figure 4.2 DC motor DC Motor L S.U.B. DC Motor L S.U.B. AC Motor L S.U.B. AC Motor 250L S.U.B. AC Motor 500L S.U.B. General Brushless 48 VDC Induction 208 VAC, 3 Phase Power 200 W (0.268 hp) 400 W (0.536 hp) AC Motor 1,000-2,000L S.U.B kw (0.25 hp) kw (0.5 hp) Gear ratio 10:1 20:1 10:1 12.5:1 5:1 15:1 Thermo Scientific Single-Use Bioreactor (S.U.B.) 157

158 Section 4 Specifications and Parts Information Load Cells Load cells operate as a mechanical suspension and weighing system on the Thermo Scientific HyPerforma Single-Use Bioreactor (S.U.B.). FLEX- MOUNT Weigh Modules are used to enable weight measurements for 2,000L S.U.B. units (see figure 4.5). MTB load cells (see Figure 4.4) are used to enable weight measurements for all other S.U.B. sizes. Load cells may be purchased either at the time of equipment sale or as retro-fit kits for existing S.U.B. units. Units can be load cell equipped as an option for the L S.U.B. Load cells are standard on the 1,000L and 2,000L S.U.B hardware. Load Cell systems include three load cells, a summing block, a display, and wiring (see Figure 4.3). Figure 4.3 Load cell system overview Figure 4.4 Mettler Toledo MTB load cell on 50-1,000L S.U.B. units Figure 4.5 Load cell location on S.U.B. hardware Thermo Scientific Single-Use Bioreactor (S.U.B.) 158

159 Section 4 Specifications and Parts Information Load cells are typically radial mounted in sets of three. The mounting location varies slightly for each size in order to allow easy access to the bottom drain or sparging mechanisms and tubing. Figure 4.6 Mettler Toledo MTB load cell for 50L-1,000L SUBs Figure 4.7 FLEXMOUNT load cell for 2,000L S.U.B. Weigh Module Parameter S.U.B. Specification 50L & 100L 250L 500L 1,000L 2,000L Model number MTB-100 MTB-200 MTB-300 MTB FLEXMOUNT Rated capacity per load cell 100kg (220lb) 200kg (441lb) 300kg (661lb) 500kg (1102lb) 1100kg (2500lb) Load limit, safe See Specification 1700kg (3740lb) Maximum horizontal force Maximum top plate travel 0.98kN (220lb) 1.96kN (441lb) 4.1kN (920lb) 4.9kN (1101lb) 8.2kN (1840) 0.98kN (220lb) 1.96kN (441lb) 5.6kN (1250lb) 4.9kN (1101lb) Not applicable Not applicable 11.2kN (2500lb) ± 3.01mm (0.12in) ± 3.01mm (0.12in) Maximum uplift force Not applicable 22.2kN (5000lb) Safe dynamic load Weight (including load cell), nominal Material 70% of rated capacity 0.6kg (1.3ib) 304 stainless steel 7kg (15.5lb) Thermo Scientific Single-Use Bioreactor (S.U.B.) 159

160 Section 4 Specifications and Parts Information Ordering Information for Mettler Toledo Load Cell Kit Size Description Part Number 50L, 100L 3x load cell with summing box without display SV L 3x load cell with summing box without display SV L 3x load cell with summing box without display SV NOTE: 1,000L and 2,000L S.U.B. units include load cells as standard equipment. Load Cell Displays Harsh Mount Displays for L and 2000L S.U.B. Systems Figure 4.8 Mettler Toledo IND331 harsh mount load cell display for L and 2,000L S.U.B. systems Part Number SV SV SV SV SV Description Mettler-Toledo IND331 display, harsh mount style with analog interface (STD), 120VAC US line cord/plug Mettler-Toledo IND331 display, harsh mount style with Allen-Bradley RIO interface, 120VAC US line cord/plug Mettler-Toledo IND331 display, harsh mount style with Device Net interface, 120VAC US line cord/plug Mettler-Toledo IND331 display, harsh mount style with Ethernet/IP & Modbus TCP interface, 120VAC US line cord/ plug Mettler-Toledo IND331 display, harsh mount style with Profibus interface, 120VAC US line cord/plug Panel Mount Load Cell Displays (for 1,000L S.U.B. Units Only) Figure 4.9 Mettler Toledo IND331 panel mount load cell display for 1,000L S.U.B. systems only Part Number SV SV SV SV SV Description Mettler-Toledo IND331 display, panel mount style with analog interface (STD), 120VAC US line cord/plug Mettler-Toledo IND331 display, panel mount style with Allen-Bradley RIO Interface, 120VAC US line cord/plug Mettler-Toledo IND331 display, panel mount style with Device Net interface, 120VAC US line cord/plug Mettler-Toledo IND331 display, panel mount style with Ethernet/IP & Modbus TCP interface, 120VAC US line cord/plug Mettler-Toledo IND331 display, panel mount style with Profibus interface, 120VAC US line cord/plug Thermo Scientific Single-Use Bioreactor (S.U.B.) 160

161 Section 4 Specifications and Parts Information Drive Shafts The S.U.B. drive shaft is detachable and reusable. It is inserted into the BPC through the hollow pass-through of the motor assembly, into the bearing port, through the tubing sleeve inside the BPC, and into the polyethylene impeller. Drive shaft rods may be made of aluminum, stainless steel, or carbon fiber, depending on the size of the vessel and the strength requirements. Larger vessels include options for multiple-piece drive shafts to accommodate various ceiling heights. Ceiling height requirements are listed for each size vessel in the Specifications section of this publication. As a general rule, drive shafts should be replaced after 1 year of service, or as specified in the Maintenance section of this publication. You should always keep a log of actual drive shaft usage. Appendix D includes a form that can be used for this purpose. Description Part Number 50L 1-piece aluminum drive shaft SV L 1-piece aluminum drive shaft SV L 1-piece aluminum drive shaft SV L 1-piece stainless steel drive shaft SV L 2-piece stainless steel drive shaft SV L 2-piece stainless steel drive shaft SV L 3-piece stainless steel drive shaft SV ,000L 3-piece stainless steel drive shaft SV ,000L 4-piece stainless steel drive shaft SV ,000L 2-piece carbon fiber drive shaft SV ,000L 4-piece carbon fiber drive shaft SV Thermo Scientific Single-Use Bioreactor (S.U.B.) 161

162 Section 4 Specifications and Parts Information Cable Management System The Cable Management system includes the following components (see Figure 4.10): Internal channel for macro and micro sparge lines External channels for feed and base addition lines Harvest line hook Adjustable arm for external control power cable management (See Figure 4.11) Channels for Sparge Lines Channels for Feed and Base Addition Lines Adjustable Arm Harvest Line Hook Figure L S.U.B. with cable management system Figure 4.11 Close up of adjustable arm Thermo Scientific Single-Use Bioreactor (S.U.B.) 162

163 Section 4 Specifications and Parts Information Cable Management System Ordering Information Size Description Part Number 50L,100L Cable management system SV L Cable management system SV L Cable management system SV ,000L Cable management system SV Vent Filter Heaters The Vent Filter Heater system (see Figure 4.12) consists of the following components: Heater Controller Power Cord It includes a fully insulated resistive heating element with molded silicone foam. It is secured around the filter by use of snap retainers and fully encapsulates the exhaust filters for consistent temperature regulation. The temperature controller is preset for the HyPerforma Single-Use Bioreactor (S.U.B.) at 50 C and has an adjustable temperature range of C. Temperatures above 60 C are not recommended for the filters used on the S.U.B. hardware. The controller has programmable logic and is equipped with a low temperature alarm output. The vent filter heater system stands alone and does not integrate with the control system of the S.U.B. controller. Refer to the vent filter heater user s manual included in the S.U.B. ETP for more information. Contoured foam insulator covers the dome section to prevent heat loss Corrosion resistant, stainless steel snaps for easy installation or removal Thermocouple embedded in heater mat Molded ½ silicone foam conforms to housing Figure 4.12 Vent filter heater Thermo Scientific Single-Use Bioreactor (S.U.B.) 163

164 Section 4 Specifications and Parts Information Ordering Information - Filter Heater Kits for Use with Pall KA3 Venter Filters Sold as a kit which includes vent filter heater, controller with a water-tight closure, quick connects, and installation power cord. Description Additional Information Part Number NEMA rated Vent Heater with Programmable Controller ( VAC), power cord NEMA rated Vent Heater with Programmable Controller ( VAC), power cord Includes low temp alarm, preset temp 50 C, power cord with flying leads Includes low temp alarm, preset temp 50 C, power cord with flying leads SV SV Ordering Information - Filter Heater Kits for Use with Meissner Ultracap 10 Vent Filters Sold as a kit which includes vent filter heater, controller with a water-tight closure, quick connects, and installation power cord. Description Additional Information Part Number NEMA rated Vent Heater with Programmable Controller ( VAC) NEMA rated Vent Heater with Programmable Controller ( VAC) NEMA rated Vent Heater with Programmable Controller ( VAC) NEMA rated Vent Heater with Programmable Controller ( VAC) Includes low temp alarm, preset temp 50 C, 20 NEMA 5-15 power cord for U.S./Japan Includes low temp alarm, preset temp 50 C, 20 BS1363 power cord for United Kingdom Includes low temp alarm, preset temp 50 C, 20 CEE7/7 power cord for Europe Includes low temp alarm. Preset temp 50 C, 12 IEC320 power cord for 2,000 L SUB control box SV SV SV SV Bioreactor Condenser System The Thermo Scientific HyPerforma Single-Use Bioreactor (S.U.B.) Condenser System supports the effective use of the HyPerforma 2,000 L S.U.B. It is also available as an auxiliary product for all other S.U.B. systems. It efficiently condenses exhaust gases and transfers condensate back into the bioreactor, preventing potential vent filter blockage and reducing fluid loss due to evaporation. It is offered in both single and double chillplate formats. The Condenser System protects against filter blockage by condensing out moisture prior to exhaust gases reaching the vent filters. BPCs are not intended to operate under pressure, and fouled (blocked) exhaust filters lead to bag pressurization. While vent filter heaters may prevent condensate build-up in many instances, with larger bioreactors, such as the 2,000 L S.U.B., this becomes less effective, whereas condensing out the moisture first is a more reliable method for preventing liquid from reaching the filters. Thermo Scientific Single-Use Bioreactor (S.U.B.) 164

165 Section 4 Specifications and Parts Information The S.U.B. Condenser System (see Figure 4.13) consists of the following components: Cart and brackets convenient means of organizing and transporting key working elements of the Condenser System. Condenser (chill) plate disposable double chamber condenser bag is secured to the condenser plate to cool exhaust gases. Up to two plates can be used per system. Peristaltic pump to return condensate to bioreactor. Temperature control unit (TCU, also referred to as a chiller) circulates water to cool the condenser plate. Condenser disposables the BPC (double-chambered bag), tubing, and exhaust filters though which the exhaust gases flow and are chilled, and in which the condensate collects and is returned to the bioreactor. 9. Exhaust Vent- Filters 8. Condenser Bag- Gas Outlet Port 7. Dual Chamber Condenser Bag 6. Condenser Bag Liquid Drain Ports 1. Exhaust Line- From S.U.B. 3. Condenser Bag Gas Inlet Port 4. Gripping Tabs 5. Alignment Holes 10. Filter Straps 17. Condenser Plate Assembly 2. Condenser ReturnLine Back to S.U.B. 16. Dual Headed- Peristaltic Pump 15. Closed Bath Recirculating Chiller 11. Filter Bracket Assembly 12. Condenser Post Assembly 13. Post Receivers 14. Cart Assembly Condenser Disposables Condenser Hardware Condenser System Figure 4.13 Condenser disposables, hardware, and complete system Thermo Scientific Single-Use Bioreactor (S.U.B.) 165

166 Section 4 Specifications and Parts Information Item- Number Condenser Component Description 1 Exhaust line from S.U.B. vents S.U.B. BPC, maintaining a non-pressurized system 2 Condenser return line back to S.U.B. transfers condensate into bioreactor 3 Condenser bag gas inlet port receives S.U.B. exhaust for cooling 4 Gripping tabs enable operator to securely position condenser bag 5 Alignment holes match up with condenser plate posts to maintain bag position 6 Condenser bag liquid drain ports condensate flows through these ports to bioreactor 7 Dual chamber condenser bag gas circulates through integral channels for chilling 8 Condenser bag gas outlet port dry exhaust gases exit condenser bag toward exhaust filters 9 Exhaust vent filters vent exhaust gases to ambient atmosphere 10 Filter straps secures vent filters in position 11 Filter bracket assembly supports vent filters 12 Condenser post assembly supports condenser plate and filter brackets 13 Post receivers three alternatives for post position or additional posts 14 Cart assembly for organizing and transporting key working elements of the condenser system 15 Closed bath recirculating chiller (TCU) circulates chilled water for cooling condenser plate 16 Dual headed peristaltic pump returns condensate to bioreactor 17 Condenser plate assembly holds condenser bag for chilling exhaust gases Performance Guidelines and Comparison Choosing the best exhaust vent configuration for the S.U.B. is application specific. For most S.U.B. configurations it depends upon a combination of many variables, such as exhaust flow rate, process stability, reliability expectations, culture run duration or concern for evaporation loss. The accompanying graph (see Graph 4.1, below) provides a simple processbased reference for determining if a vent heater option will be sufficient or if a condenser system is preferred. Typically, any off-gassing flow rates approaching 50lpm or culture durations exceeding 10 days should consider utilizing the condenser system. Thermo Scientific Single-Use Bioreactor (S.U.B.) 166

167 Section 4 Specifications and Parts Information Total Off-Gassing Load (LPM) Off-Gassing Management Guidelines for Single Use Bioreactor Condenser System vs. Exhaust Vent Heaters (Conditions: 37 C batch temperature, no foam in exhaust stream) Condenser System (2 X 10" vents) Vent Heater System (2 X 3" vents) Run Duration (days) Vent Heater System (2 X 10" vents) Vent Heater System (1 X 3" vents) Graph 4.1 Condenser vs. exhaust filter heaters study [584.2mm] [1817.3mm] [1908.2mm] [1042.6mm] [1195.0mm] [1020.4mm] [720.3mm] [508.0mm] [819.2mm] [900.4mm] [962.0mm] [431.8mm] Figure 4.14 Condenser system dimensions Thermo Scientific Single-Use Bioreactor (S.U.B.) 167

168 Section 4 Specifications and Parts Information Condenser System Ordering Information Description Complete Condenser System (120 V) including cart, chill plate and mounting post with filter brackets, TCU, and pump Complete Condenser System (240 V) including cart, chill plate and mounting post with filter brackets, TCU, and pump Condenser assembly including chill plate and mounting post with filter brackets (Option: Allowing two chill plates per systems) Thermoflex 900 TCU (115 VAC/60 Hz) with necessary plumbing Thermoflex 900 TCU (240 VAC/50 or 60 Hz) with necessary plumbing Thermo Scientific Masterflex Pump (115 VAC/50 or 60 Hz or 230 VAC/50 or 60 Hz) Part Number SV SV SV SV SV SV Miscellaneous Items The miscellaneous items in this section are ancillary components that support the operation of the HyPerforma S.U.B. for cell culture production and enhance the overall performance of the complete system. Autoclave Tray and Probe Assembly The Autoclave Tray (see Figure 4.15) holds the electrochemical probes and bellows in place during the autoclave sterilization process. Design elements include the following: Fabricated from stainless steel Plastic handle provides for easy transport right out of the autoclave Positions probes on 15% incline for greater probe/membrane longevity Will restrain probe bellows from collapsing during sterilization Probe holder accommodates two probes Threaded Probe Attachment Cable Tie Probe Assembly Handle Molded Bellows Cover Figure 4.15 Autoclave tray and probe assembly Thermo Scientific Single-Use Bioreactor (S.U.B.) 168

169 Section 4 Specifications and Parts Information Porous Frit Sparge Line Support The porous frit sparge line support (see Figure 4.16) keeps the micro and macro sparge lines in a vertical position for optimal gas flow. This is placed in the bottom of the S.U.B. tank as described in section 1.7 of this user guide. NOTE: This is supplied as standard equipment on all S.U.B. tanks. Figure 4.16 Autoclave Tray and Probe Assembly Heavy-Duty Tubing Clamps Heavy-duty clamps are used for pinching off line sets that are not in use in order to prevent process fluids from escaping. Prior to sterile probe insertion, tubing clamps must be in place to close off probe ports. See Section 2 and 3 of this user guide for more information. Figure 4.17 Heavy-duty tubing clamps Miscellaneous Items Ordering Information Description Autoclave Tray Bioreactor Probe Assembly (non-sterile for use in autoclave) Porous Frit Sparge Line Support for L S.U.B. Tanks Porous Frit Sparge Line Support for L S.U.B. Tanks Heavy Duty Tubing Clamps (single) Heavy Duty Tubing Clamps (10 pack) Part Number SV SH SV50177B.19 SV50177B.14 SV SV Thermo Scientific Single-Use Bioreactor (S.U.B.) 169

170 Section 5 General Ordering Information Section 5 Ordering Information This section covers the following information: 5.1 Ordering Instructions 5.2 Ordering/Support Contact Information Thermo Scientific Single-Use Bioreactor (S.U.B.) 170

171 Section 5.1 Ordering Instructions 5.1 Ordering Instructions 5.2 Ordering/ Support Contact Information BPCs and hardware components for the Single-Use Bioreactor (S.U.B.) can be ordered directly from Thermo Fisher Scientific. These items include all components that have part numbers beginning with the following digits: SH SV SUB In the Americas and Asia 1726 Hyclone Drive Logan, Utah USA Tel: +1 (435) Toll-Free: +1 (800) In Europe Industriezone III, Industrielaan Erembodegem Belgium Tel: Fax: Technical Support Technical support for the Single-Use Bioreactor is available in a variety of formats. Some or all of the following may be appropriate depending on individual experience and circumstances. Technical Service Hotline Contact your Thermo Scientific sales representative for general product pricing, availability, delivery, order information and product complaints. Call Option 2 (US) or (EU) for direct and immediate response to overall product questions, and general product technical information (Technical Support). You can also contact Technical Support by at: techsupport.bioprocessing@thermofisher. com. Initial Setup and Operation Training Appropriate technical support is available to assist in the initial setup and operation of each Single-Use Bioreactor system. Inquire at the time of purchase. Training can be provided for startup and operation of the S.U.B. Contact your Thermo Scientific sales representative. Thermo Scientific Single-Use Bioreactor (S.U.B.) 171

172 Appendix A Installation of Female Electrical Receptacle for Units with A/C Motors and Electrical Boxes Appendix A Electrical Receptacle Installation Instructions 1. In order to complete the installation for units with A/C motors, the facility must be equipped with an electrical housing of sufficient size. Typically in the U.S. the plug will require a two gang box when using the adapter plate (supplied). For installations outside the U.S. (an adapter plate is not supplied) it is recommended that an electrical panel be modified to accommodate the cutout dimensions as shown in figure 1, below. 92mm 38mm 106mm 62mm 77mm 67mm 102.5mm 81mm 85mm Figure 1 Panel Cutout (4) 5.5mm DIA 2. Verify that electrical power has been disconnected and locked out for safety. 3. Verify the holes for mounting the receptacle housing are positioned properly. Center to center measurement of respective mounting holes is 85mm (3.35 ) tall and 77mm (3.0 ) wide. 4. Verify the condition of the three exposed wire leads, strip back to expose new wire if needed. 5. Connect the wire leads on the receptacle (shown in figure 2, below) using the screw terminals, paying strict attention to obtain the correct wiring position as it is labeled on the receptacle. Green (ground) White (common) Black in the US, Blue in the EU (hot) Thermo Scientific Single-Use Bioreactor (S.U.B.) 172

173 Appendix A Electrical Receptacle Installation Instructions Figure 2 Female Receptacle (blue for 240 VAC: yellow for 110 VAC) 6. If using the adapter mounting plate, secure it to the selected facility electrical housing as per drawing (Figure 3), otherwise proceed to step X #10X1 screws to mount plug to adapter plate. NOTE: The plug will be offset to the left or right if mounting to a 2-gang box with screws in opposite corners. If this is the case, then one of the holes in the plug will line up with the screw will need to be used in that hole. Use other 2X#10X1 screws to plug holes not used. 2X #8-32X 1 3/8 screws to mount to TYP 2-gang boxes. These boxes TYP have 2 screws in opposite corners. 4X #6-32X 1 1/4 screws to mount adapter plate to TYP 2-gang FS/FD box. NOTE: The plug will be offset to the left or right using 4X #10X1 screws. Use other 2X #10X1 screws to plug holes not used. Figure 3 Adapter Mounting Plate 7. Secure the electrical receptacle using four supplied screw fasteners. 8. Connect power back to the electrical circuit. 9. Test the circuit with multi-meter prior to making any connections to the electrical receptacle. Thermo Scientific Single-Use Bioreactor (S.U.B.) 173