operator ˇs manual brx1000 (1000-liter working volume ... · systems and must be kept...

Operator’s Manual Dakota Systems Allen Bradley PLC Controls Package Dracut, MA BRX1000 (1000L Working Volume SIP/CIP Bioreactor)

Cytovance 1000L BR Operator's Manual_4Jun2010 page 1 - 91

1057 Broadway Rd Dracut, MA 01826 (978) 275-0600

www.dakotasystems.com

The contents of this document and all attachments are considered the property of Dakota Systems and must be kept confidential. Copying or distributing all or parts of this document to third parties without the written consent of Dakota Systems is strictly prohibited.

Operator’s Manual BRX1000 (1000-liter Working Volume Bioreactor)

_____________________________

Dakota Systems Project # 09-1793 June 4, 2010





Contents

Chapter Page CHAPTER 1. – System Description.............................................................................. 4

1.1. 1000-liter VW Base System Overview........................................................... 4 1.2. 1000-liter System Dimensions and Weights ................................................. 5 1.3. Dakota Modules from Sub-Suppliers ............................................................ 6

CHAPTER 2. – Vessel Modules ................................................................................... 7 2.1. Bioreactor Vessel ......................................................................................... 7

2.1.1. Design Pressures ...................................................................................... 8 2.1.2. Materials, Finishes, Design and Fabrication.............................................. 8 2.1.3. Vessel Top Head....................................................................................... 8 2.1.4. Penetrations and Ports.............................................................................. 8

2.2. Impeller Assembly (3-Bladed Pitched)........................................................ 10 2.3. Tank Baffles (Welded)................................................................................ 10 2.4. Gas Sparger Assembly (Sintered Frit) ........................................................ 10 2.5. Gas Overlay Assembly ............................................................................... 10 2.6. Contained Resterilizable Addition Valve Assembly .................................... 10 2.7. Illumination Assembly ................................................................................. 11 2.8. Contained Resterilizable Harvest / Drain Valve Assembly.......................... 11 2.9. Sample Valve Assembly ............................................................................. 12 2.10. Spray Ball Assemblies ................................................................................ 12 2.11. Sanitary Liquid Filled Pressure Gauge ....................................................... 12 2.12. Pressure Safety Devices ............................................................................ 12

CHAPTER 3. – Sensors & Probes.............................................................................. 13 3.1. pH probes (Gel Filled )................................................................................ 13 3.2. Dissolved Oxygen Probes (Polarographic) ................................................. 13 3.3. Vessel Temperature Sensor (RTD, Pt100) ................................................. 14 3.4. Sanitary Pressure Sensor........................................................................... 14 3.5. Gas Flow Controllers (TMFC) ..................................................................... 14 3.6. Weight Measurement Assemblies (Load Cells) .......................................... 15

CHAPTER 4. System Piping Modules ....................................................................... 16 4.1. Piping Frame and Vessel Support Skids .................................................... 16 4.2. Biowaste and Condensate Connections..................................................... 16 4.3. Piping Specifications .................................................................................. 16

4.3.1. Process Piping and Components ............................................................ 16 4.3.2. Clean Steam Piping and Components .................................................... 17 4.3.3. Non-Process Gas Piping and Components............................................. 17 4.3.4. Utility Piping and Components ................................................................ 18 4.3.5. System Utility Requirements ................................................................... 18

4.4. Pre-Filters, Regulators, & Isolation Valves ................................................. 19 4.4.1. Clean Steam ........................................................................................... 19 4.4.2. Process Air.............................................................................................. 19 4.4.3. Oxygen.................................................................................................... 19 4.4.4. Carbon Dioxide........................................................................................ 19 4.4.5. Nitrogen................................................................................................... 19 4.4.6. Chilled Water........................................................................................... 19 4.4.7. Chilled Water Return ............................................................................... 19 4.4.8. Exhaust ................................................................................................... 19





4.4.9. Drain (Clean and Biowaste)..................................................................... 20 4.4.10. Electrical .............................................................................................. 20

CHAPTER 5. – Control Modules................................................................................. 21 5.1. Vessel Pressure Control - Exhaust Gas Module........................................ 21 5.2. Aeration Control Module ............................................................................. 22

5.2.1. Gas Sparge and Overlay Systems (Independent)................................... 22 5.2.2. Gas Feed and Mixing .............................................................................. 23

5.3. Agitation Control Module ............................................................................ 30 5.4. Nutrient Feed Control Module..................................................................... 32 5.5. Dissolved Oxygen Control .......................................................................... 36 5.6. Control of the pH ........................................................................................ 39 5.7. Temperature Control Module ...................................................................... 43 5.8. Harvest Control........................................................................................... 50 5.9. Vessel Weight Control................................................................................ 51 5.10. Ad Hoc Profiling.......................................................................................... 51 5.11. CIP Piping Module ...................................................................................... 52 5.12. Pump Calibration and Totalization.............................................................. 53 5.13. Control of Discrete Devices During Process States.................................... 56 5.14. Control of Loops During Process States..................................................... 58 5.15. Alarms and Exception Handling.................................................................. 59

5.15.1. Generation of Alarms ........................................................................... 59 5.15.2. Alarm and Interlocks ............................................................................ 59 5.15.3. Enabling and Disabling of Alarm.......................................................... 61 5.15.4. Failure Handling ................................................................................... 65

5.16. Safety and Security .................................................................................... 65 5.16.1. Input Value Checking ........................................................................... 65

CHAPTER 6. – Start Up & Operation ......................................................................... 66 6.1. General Preparation ................................................................................... 66

6.1.1. Starting a batch ....................................................................................... 66 6.1.2. Operation and Preventative Maintenance of Instrumentation.................. 66

6.2. Start-up....................................................................................................... 67 6.3. Skid Operation Check................................................................................. 71 6.4. Pressure Test ............................................................................................. 72 6.5. Sterilization ................................................................................................. 73

6.5.1. Sterilization Procedure ............................................................................ 73 6.5.2. Sterilization Troubleshooting ................................................................... 76

6.6. Cultivation................................................................................................... 76 6.6.1. Preparing for Cultivation.......................................................................... 76

CHAPTER 7. – Troubleshooting................................................................................. 79 CHAPTER 8. – Cleaning & Maintenance.................................................................... 80

8.1. Cleaning ..................................................................................................... 80 8.1.1. pH and DO Probe Cleaning and Storage ................................................ 80 8.1.2. Cleaning the Bioreactor interior ............................................................... 80 8.1.3. Cleaning the Bioreactor Exterior.............................................................. 82

8.2. Maintenance ............................................................................................... 83 CHAPTER 9. –PLC Operation.................................................................................... 89

9.1. Operation.................................................................................................... 89 9.2. Hardware .................................................................................................... 89 9.3. Software ..................................................................................................... 90





CHAPTER 1. – SYSTEM DESCRIPTION This Operator’s Manual is for a 1000 Liter VW Bioreactor designed for the cultivation of mammalian cells as well as insect cells and other low shear organisms.

1.1. 1000-liter VW Base System Overview Dakota Systems has provided one (1) 1000-liter working volume Bioreactor for cultivation of mammalian and other low shear organisms. The 1350-liter bioreactor, designed for 1000-liter working capacity described in this document consists of the following modules: v Vessel Module with the following sub-assemblies: Ø Bottom Driven, Variable Speed

FlowServe Double Mechanical Seal Agitator Assembly;

Ø Two (2) Lightnin A-320 Impellers; 18 inch diameter,

Ø Sintered Stainless Steel Quick Change Type Gas Sparger Assembly;

Ø ASEPCO Contained Resterilizable Harvest / Drain Valve Assembly;

Ø ASEPCO Resterilizable Sampling Valve;

Ø Four (4) Resterilizable Addition Valve Assemblies;

Ø 4” Round View Port; Ø Sanitary Lamp w/ 100 W Bulb; Ø Sanitary Liquid Filled Pressure Gauge; Ø Vessel & Jacket Pressure Relief

Devices; Ø Two (2) Spray Ball Assemblies for CIP; Ø Port plugs and Sanitary End Caps for

spare vessel connections;

v Skid Piping Assembly with modules for: Ø Variable Speed Agitator Control Module; Ø Sparge Gas Inlet Piping (multi-ported ring sparger) with Sparge Gas (Air, CO2,,,N2 & O2)

Thermal Mass Flow Controllers, and 316L Stainless Steel Resterilizable Filter Housing with 0.2 Micron Absolute Filter;

Ø Overlay Gas Inlet Piping with Overlay Gas (Air) Thermal Mass Flow Controller, and 316L Stainless Steel Resterilizable Filter Housing with 0.2 Micron Absolute Filter;

Ø Gas Exhaust Piping with 0.2 Micron Steam Sterilizable Filter Assembly, Pressure Transducer and Automatic Back-Pressure Control Valve;

Ø Closed Loop Temperature Control Module with Jacket Circulation Piping, Utility Steam fed Heat Exchanger, and Thermostatic Steam Traps for Cell-Growth and Sterilization-In-Place;

Ø Two (2) Panel Mounted Watson Marlow pump modules for Base and Spare additions; Ø Connections for Two (2) Fixed Speed Peristaltic Pumps for Liquid Addition / Harvesting (one

assignable to the several loops and the other permanently powered); Ø Separate Condensate and Biowaste Drain Manifolds; Ø CIP Piping Module with Clean Steam / CIP Transfer Panel; Ø Load Cell Weight Measurement System;





v Process Mounted Sensors for: Ø Temperature (Vessel and Jacket); Ø pH (dual); Ø Dissolved Oxygen (dual); Ø Pressure; Ø Nitrogen (Sparge);

Ø Weight; Ø Air (Sparge & Overlay); Ø Oxygen (Sparge); Ø Carbon Dioxide (Sparge);

v Allen Bradley PLC System with WAGO I/O Modules for: Ø Temperature Measurement & Control; Ø pH Measurement & Control; Ø DO Measurement & Control; Ø Weight Measurement; Ø Agitation Measurement & Control; Ø Pressure Measurement & Control;

Ø Sparge Composition & Flow Control; Ø Overlay Gas Flow Measurement and

Control; Ø Pump Operation; Ø Sterilization-In-Place; Ø Cleaning-In-Place

v OIT with 10-inch Color Touch Screen;

1.2. 1000-liter System Dimensions and Weights

Dakota 1000-liter Bioreactor System

Length 120 inches

Depth 60 inches Height 143 inches Shipping Weight 3800 lbs Dry Weight 3200 lbs Operating Weight 5400 lbs





1.3. Dakota Modules from Sub-Suppliers The following list represents Dakota sub-suppliers for the major system components. Bioreactor Vessel Allegheny Bradford Corporation Harvest Valve ASEPCO Sampling Valve ASEPCO Diaphragm Valves Gemu (Process) Automated Valves Burkert (non-Process) Pressure Regulators Norgren or Tescom Plug Valves Swagelok Control Valves Badger pH & Dissolved Oxygen Probes Mettler-Toledo pH & Dissolved Oxygen Analyzers Mettler-Toledo Pressure Gauges (Sanitary) Anderson & Wika Pressure Transmitters ABB Pressure Switch Ashcroft Temperature Sensors (RTD) United Electric Mass Flow Controllers Brooks Instruments Weight Sensors / Terminals Vishay/BLH Agitator Motor / Gearbox SPX Corporation (Lightnin) Motor Controller Allen-Bradley Steam Traps Nicholson Lamp Assembly L.J. Star Stainless Steel Tubing BPE 2007 Spray Ball Sani-Matic Bioreactor Controller / OIT Allen Bradley SLC5 / PanelView Plus Mechanical Seals SPX Corporation (Lightnin) Impellors & Shaft SPX Corporation (Lightnin) Circulation Heat Exchanger Tranter Condensate Cooler Exergy High Foam Probe & Instrument Lumenite Control Circulation Pump Gould





CHAPTER 2.– VESSEL MODULES The Bioreactor and all of its attachments and internal components are specified and configured within the vessel module.

2.1. Bioreactor Vessel The Bioreactor is designed and fabricated to conform to the latest issue of Section VIII of the ASME Pressure Vessel Code. The vessel is marked with an ASME Code Stamp and supplied with copies of the Manufacturer’s Data Report, which is also registered with the National Board of Boiler and Pressure Vessel Inspectors. Vessel Design Specifications

VESSEL DATA 1000-liter BIOREACTOR

Total Capacity 1350.0 Liters

Maximum Working Capacity 1000.0 Liters Minimum Working Capacity 200.0 Liters

Diameter (ID) 36 inches Vessel Dimensions Height (internal) 84.6 inches Working Volume Vessel Height to

Diameter Ratio Total Volume

1.73 to 1

2.35 to 1 Jacket Type Dimpled Jacketed Bottom Head Yes Top Head ASME Flanged & Dished Bottom Head ASME Flanged & Dished Insulation 2” Inswool & SS Sheathing Vessel Design Pressure (MAWP) 45 psig & Full Vacuum @ -20 oF to 302 oF

Rupture Disk Rating (Max. Operating Temp & Press)

45 psig +/- 5% @ 121 oC

Jacket Design Pressure (MAWP) 125 psig & Full Vacuum @ -20 oF to 302 oF

Jacket Relief Valve Rating 125 psig

Interior

<20 µ-inch Ra (0.50 microns) followed by Electropolish

Vessel Surface Finish

Exterior 35 µ-inch Ra (0.89 microns)





2.1.1. Design Pressures The design pressure of the Bioreactor vessel is 45 psig and Full Vacuum. The jacket design pressures are 125 psig and Full Vacuum. The vessel and jacket are hydrostatically tested per ASME code requirements.

2.1.2. Materials, Finishes, Design and Fabrication All surfaces in contact with the culture liquid including the cover plate are fabricated of type 316L stainless steel. All internal and external vessel welds are ground smooth, and mechanically polished to match the surrounding finish. Welds are free of ripples, pits, undercutting and other surface defects. Surfaces in contact with product are free from pits, burrs, flash, folds, inclusions, crevices and nicks. All interior surfaces are mechanically polished to a finish of 20-microinch Ra (0.50 microns) or better followed by electropolish. The vessel exterior is mechanically polished to a uniform 35-microinch Ra (0.89 micron) or better finish on all visible surfaces.

2.1.3. Vessel Top Head The 1000-liter Bioreactor vessel has a lift off full opening ASME F&D top head for accessing the vessel’s interior. The cover is sealed to the vessel by swing bolts and a single Silicone O-ring. The vessel has a safety interlock to prevent operation of the agitator while the vessel manway or top head is open.

2.1.4. Penetrations and Ports All process fittings are of sanitary design. Ports welded into the vessel cover and sidewall are internally beveled to achieve full drainability. Ports are provided with a stainless steel plug or cap, when not furnished with a valve or probe.





2.1.4.1. 1000-liter Bioreactor Ports and Connections

Qty Size Type Function Connection 1 1 ½“ Tri-Clamp Spare Capped 2 2” Tri-Clamp Cleaning Spray Ball 1 1 ½“ Tri-Clamp Light Sanitary Lamp 1 1 ½” Tri-Clamp Pressure Indication Sanitary Gauge 1 1 ½” Tri-Clamp Pressure Relief Rupture Disk 1 1 ½” Tri-Clamp Exhaust Exhaust Piping 1 1 ½” Tri-Clamp Spare Capped 1 1 ½” Tri-Clamp Sight Glass Sanitary Sight Glass 1 1 ½“ Tri-Clamp Spare Capped

Top

Hea

d

1 1 ½” Tri-Clamp Press. Transmitter Sanitary Press. Trans. 1 ½“ Tri-Clamp Gas Inlet Overlay Assembly 4 1” NA Fitting Liquid Addition &

Spare 4-Valve, Resterilizable, Addition Assembly with removable J-Tube plus 1 plugged

1 1 ½” NA Fitting Liquid Addition 4-Valve, Resterilizable, Addition Assembly with removable J-Tube Upp

er S

idew

all

1 4” Round Window

Tri-Clamp View Port Sanitary Sight Glass

1 1 ½” NA Fitting Temperature Sensor

Thermowell with RTD Probe

1 1 ½” NA Fitting Gas Inlet Replaceable SS Frit Sparger Assembly

2 1 ½” NA Fitting pH Sensor pH Probe and Housing 2 1 ½” NA Fitting DO Sensor DO Probe and Housing 1 ½” ASEPCO

Valve Weldment

Sample ASEPCO Sampling Valve

Low

er S

idew

all

1 2” NA Fitting Future Sample or Perfusion Return

Novaseptic Sampling System or Valve, Currently Capped

Jack

et 2 1 ½” Tri-Clamp Jacket Water Jacket Piping with

Relief Valve & Gauge

Bot

tom

1 2” ASEPCO Valve Weldment

Harvest / Drain ASEPCO Contained Resterilizable Harvest Valve Assembly





1 Bolted Flange

Agitator Double Sealed Agitator

2.2. Impeller Assembly (3-Bladed Pitched)

IMPELLER DATA 1000-liter BIOREACTOR

No. Impellers Two Impellor Type Lightnin A320 Impeller Diameter 18 inch Percent of Vessel ID 50 %

The impeller is constructed from 316L stainless steel and is polished to match the vessel interior. The impeller is welded to the shaft and can be removed and replaced as an assembly if a different type or size impellor is desired.

2.3. Tank Baffles (Welded) The Bioreactor has three (3) 316L stainless steel baffle plates attached to the inner straight wall of the vessel to provide additional turbulence / mixing for heat and oxygen transfer. These are welded to the vessel interior. Baffles are 6.3% of the vessel inner diameter, located approximately 0.02D off of the wall to allow for cleaning, and are spaced evenly at 120o

intervals.

2.4. Gas Sparger Assembly (Sintered Frit) The Bioreactor has a sintered stainless steel frit type sparger assembly to provide an even distribution of injected process gases into the culture media. The sparger consists of a tri-clamp gasket sealed, port adapter with a tri-clamp end connection. The sparge tube extends into the vessel through one of the NA style ports in the upper sidewall and ends in a tube with multiple holes that is positioned just below the impeller assembly. The sparger frit is a quick change item manufactured by Mott Corporation. The sparger frit has holes that average 0.2 micron pore size. The frit portion of the sparger is 1” diameter and is 4” long.

2.5. Gas Overlay Assembly The Bioreactor has an overlay assembly to permit the addition of process gases to the interior headspace above the Bioreactor media as an alternative to direct injection into the media. The overlay assembly consists of a tri-clamp ferrule welded into the vessel upper sidewall. The system is currently configured to supply only air to this port through its own Overlay Thermal Mass Flowcontroller.

2.6. Contained Resterilizable Addition Valve Assembly The contained addition valve assembly (better known as a steam-cross), is a common and widely acceptable method for making aseptic additions to a Bioreactor





The addition valve assembly consists of four (4) sanitary diaphragm valves: Addition valve, Blocking valve, Steam valve, and Drain valve. The Blocking / Drain valve array is connected to the Bioreactor’s upper sidewall through a 1-½’ NA style fitting using a welded tri-clamp sealed port adapter with integral J-tube. The J-tube directs the flow of added liquid to the vessel wall and minimizes splashing. Sterilization of the addition valve assembly takes place in three (3) stages: sterilization of the transfer line with the Addition valve, sterilization of the Blocking / Drain Valve array, and the sterilization of the connection between the Addition and Blocking valves. The second and third sterilization stages require approximately 20 minutes of steaming each. The addition valve assemblies are cleaned while cleaning the vessel using the blocking valve and the steam supply lines/valves. During the CIP phase, the Blocking valves are opened to clean the J-tube assembly.

1000-liter Bioreactor Addition Assembly

Bioreactor Addition Valve Assemblies

A B S D Media / Inoculation ½” ½” ½” ½” Base ½“ ½” ½” ½” Addition ½“ ½” ½” ½” Nutrient Addition ¾“ ¾“ ½” ½“

A = Addition Valve B = Blocking Valve S = Steam Valve D = Drain Valve

2.7. Illumination Assembly A non-invasive illumination assembly with 100-watt light bulb is installed onto one of the vessel’s 1 ½” tri-clover cover plate ports. The illumination assembly includes a cable that is wired to the main control and a timer is provided inside the control console to allow for the light to be automatically turned off after the timer times out.

2.8. Contained Resterilizable Harvest / Drain Valve Assembly An ASEPCO flush-sealing, radial diaphragm valve is welded to the bottom head of the vessel to provide complete drainability of the Bioreactor. The drain / harvest valve is supplied with a Class VI silicone diaphragm and a manual valve actuator.

One (1) additional hand operated diaphragm valve (HV-713) is provided to allow the entire assembly to be resterilized and cleaned in place. This valve is a zero static tee. The main through legs of the tee are 2” in size while the valve to the steam trap is ¾”. When opened, the drain valve allows steam to go to the thermostatic steam trap during the sterilization of the unit. There is also a bypass valve provided around the trap.

Bioreactor Harvest Valve Data

1000-liter Bioreactor

Harvest Assembly Location Bottom Center





Outlet Size 2” Tri-Clamp

Flow Coefficient (Cv) 72

For liquids Cv is equal to the flow (in GPM) that will produce a pressure drop of 1 psid.

2.9. Sample Valve Assembly An ASEPCO flush-sealing, radial diaphragm valve is welded to the lower sidewall of the vessel to provide uniform sample of the Bioreactor contents. The sample valve is supplied with a Class VI silicone diaphragm and a manual valve actuator. Three (3) additional hand operated diaphragm valves are provided to allow the entire assembly to be resterilized and cleaned in place: Sample Outlet (HV-722) valve, Steam valve (HV-724), and Condensate valve (HV-723).

2.10. Spray Ball Assemblies The Bioreactor is supplied with two (2) Spray Ball assemblies for cleaning of the vessel and its internal components. The selection and placement of the Spray Balls have been determined by Allegheny Bradford Corporation engineers to maximize the cleanability of the vessel. Spray balls may remain inside the vessel at all times (during growth) and are sterilized together with the Bioreactor although the spray balls may become clogged if there is a heavy foaming condition. CIP fluids exit the vessel through the Bioreactor’s drain valve assembly.

2.11. Sanitary Liquid Filled Pressure Gauge Local indication of the vessel pressure (and system pressure) is measured using a sanitary liquid filled pressure gauge. The pressure gauge is mounted onto a 1 ½” Tri-clamp port on the vessel cover plate. The pressure gauge has a range of -1.0 barg (-15 psig) to 4.0 barg (60 psig).

2.12. Pressure Safety Devices A 316L sanitary rupture disk, rated for 3.0 bar (45 psig), is mounted on the Bioreactor’s cover plate to vent the vessel in case of accidental over pressurization. A pressure relief valve, rated for 90 psig, is mounted on the jacket lines to vent the jacket in case of accidental over pressurization. The relief devices have permanently mounted tags, indicating manufacturers, model numbers, and serial numbers indicating conformance to test and to applicable ASME standards. Relief devices have been tested for proper operation, prior to installation (by the manufacturer). Vent and weep piping for both the vessel and jacket are directed to a safe location near the floor. Rupture Disk





CHAPTER 3. – SENSORS & PROBES The following process mounted, electronic sensors are provided with the Bioreactor system.

3.1. pH probes (Gel Filled ) Mettler-Toledo/Ingold pH sensors feature a double electrolyte reservoir design that prevents contamination of the sensor by broth diffusion into the reference junction. The pH sensors are designed to withstand repeated sterilization cycles without failure. Change of the zero point and slope of these electrodes is negligible after repeated sterilizations. The high isolation resistance in the head of the pH electrode is maintained after repeated sterilizations, even at temperatures as high as 135°C. This pH electrode is gel-filled and will never require replenishment of the internal electrolyte thus making the electrode essentially maintenance free. Features include: • sealed reference chambers • solid gel electrolyte • low maintenance • annular ceramic reference junction (reduces fouling) The gel-filled pH electrode is inserted into the Bioreactor media through a 25mm Ingold port in the lower sidewall of the vessel. A special adapter fitting is provided from Applikon to allow for the 13.5mm thread to fit into the 25mm fitting. The electrode is wired to a control console mounted pH Analyzer. Output from the analyzer is fed into the local control / OIT system providing a continuous pH measurement of the reactor’s contents. Upon detection of high or low pH, the system controller will carry out the customer’s pre-configured actions to return the system pH to the operator set point. The probe is designed to be sterilized with the Bioreactor. Calibration of the pH probe (two point calibration) is performed using the pH Transmitter/Analyzer. Current pH measurements are displayed on the system OIT as well as the local amplifier. There are two pH electrodes and amplifiers provided with the system. The operator may manually choose which probe is to be used for the control function.

3.2. Dissolved Oxygen Probes (Polarographic) Mettler-Toledo/Ingold DO sensors are designed to withstand repeated sterilizations without failure. The sensors are delivered factory tested and certified with the membrane module in place. The dissolved oxygen electrode is based on the principle of the Clark cell. It consists of a platinum electrode (cathode) and silver reference electrode (anode). A gas permeable silicon membrane separates the electrode from the medium. Oxygen molecules pass through the membrane and are reduced at the cathode while the anode is oxidized. Polarization creates a current that is proportional to the partial pressure of oxygen. The low permeability of the membrane used in this design enhances the lifetime of the electrode since oxidation of the





anode is minimized. The drift of the probe is negligible and response time is less than 30 seconds (98%). The slope of the DO sensor is temperature, pressure and media dependent so the probe must be calibrated under operating conditions. The body of the electrode is made of 316L stainless steel and is electropolished. The polarographic Dissolved Oxygen electrode is inserted into the bioreactor media through a 25mm Ingold port in the lower sidewall of the vessel. The electrode is wired to a control console mounted Dissolved Oxygen Analyzer. Output from the analyzer is fed to the local control / OIT system providing a continuous DO measurement of the reactor’s contents. Upon detection of high or low DO, the system controller will carry out the customer’s pre-configured actions to return the system DO to the operator set point. The probe is designed to be sterilized with the Bioreactor. Calibration of the DO probe (two point calibration) is performed using the DO Transmitter/Analyzer. Current dissolved oxygen readings are displayed on the system OIT as well as the local amplifier. There are two DO electrodes and amplifiers provided with the system. The operator may manually choose which probe is to be used for the control function.

3.3. Vessel Temperature Sensor (RTD, Pt100) The Pt100 temperature sensor is a platinum RTD probe. The resistance at 0°C is 100 Ω. The probe housing is made of a 316 stainless steel closed end tube, which immobilizes the RTD element and protects it against moisture. The housing also protects the fragile lead wires that are connected to the RTD element. The cable is a three-wire assembly to compensate for resistance of the cable and terminates inside a head mounted directly to the probe. The temperature transmitter that converts the resistance of the probe into 4-20 mA is located within the head of the probe assembly. The probe sheath has a diameter of ¼” and is designed to fit into a standard 25mm Ingold style thermowell. The 100-Ohm RTD / Thermowell assembly is inserted into the Bioreactor media through a 25mm Ingold port in the lower sidewall of the vessel. A 4-20 mA WAGO input card is provided in the control console to condition the probe’s signal thus providing a continuous measurement of the reactor’s temperature. The RTD / thermowell assembly is designed to be sterilized with the bioreactor. Current system temperature is displayed on the system OIT.

3.4. Sanitary Pressure Sensor Vessel pressure is continuously measured using sanitary pressure sensor / transmitter assembly. The pressure sensor is mounted onto a 1 ½” Tri-clamp fitting on the vessel cover plate. The measured pressure is fed into the PID Pressure Control loop and compared to the operator (or cascaded) set point. The pressure sensor / transmitters are factory calibrated and are ordered with certificates of compliance and material test reports.

3.5. Gas Flow Controllers (TMFC) The flow of gases (Air-Sparge & Overlay, Carbon Dioxide, Nitrogen and Oxygen) into the Bioreactor is measured by thermal mass flow controllers (TMFC). Flow measurements are transmitted to the Bioreactor Gas Flow Control Loops by a 4-20 mA analog signal where they are compared to





the operator (or cascaded) set point. TMFCs receive a 4-20 mA output signal from the Gas Flow Control Loop and will self adjust the flow through the flow tube sensor (50:1 turn down for all sparge gases and also 50:1 turn down for overlay air) with an internal fast cycling valve. The thermal mass flow meters and controllers are factory calibrated and ordered with certificates of compliance.

3.6. Weight Measurement Assemblies (Load Cells) The Bioreactor is supplied with a set of load cells to provide a continuous measurement of the vessel’s contents. The load cells are wired to a summing board mounted in the control console, and a display terminal that is mounted adjacent to the Bioreactor vessel. Output from the load cells is fed into the system weight control loop and is displayed on the OIT screen. Load cell assemblies have +/- 0.1% repeatability of the rated Full Scale capacity.





CHAPTER 4.SYSTEM PIPING MODULES

4.1. Piping Frame and Vessel Support Skids The Bioreactor has a 304 stainless steel frame designed to support the system piping modules, vessel, and electrical enclosures. The rugged frame construction is designed to withstand deformation due to rapid heating / cooling and possible damage during shipment or equipment installation.

4.2. Biowaste and Condensate Connections Condensate from process or utility piping is removed from the system by one Biowaste header. Steam traps are brought to one (1) manifold mounted on the skid frame. Each condensate drain line has a 316L stainless steel thermostatic trap to insure that all lines receive the necessary flow of (clean or utility) steam. A separate valve is also used for draining the jacket, circulation pump and heat exchanger of water should service of these elements be necessary.

4.3. Piping Specifications System piping can be divided into the following categories:

• Process Piping • Clean Steam Piping • Non-Process Gas Piping • Utility Piping

The construction material of the in-line components varies with line service. Each of these sub-assemblies have their own characteristics and criteria that are described in the following sections. In General, transitions from larger pipe diameters to smaller diameters, eccentric reducer fittings are used with level side down on horizontal tubes. This design feature permits complete drainage of the tube on the upstream side of the reducer. In vertical tubes, reductions in diameter are achieved with concentric reducers.

4.3.1. Process Piping and Components Process Piping consists of all fittings that are in contact with the Bioreactor’s product. Piping which penetrates the vessel or piping on the sterile side of the gas inlet and exhaust filters is considered to be in product contact. 1. Process piping and fittings are constructed of 316L Stainless Steel with 20 µ-inch Ra

electropolished internal finish and 180 grit external finish. 2. Process piping is fully drainable. Horizontal pipe runs are sloped (typically 0.6o or larger) to

assist in drainage.





3. Process pipe fittings are either Tri-Clamp or Butt Welded. All welds within the process piping are orbital type and conform to the general standards set forth in BPE-2005.

4. Flex hoses, where used, have smooth, FDA approved Teflon liners with stainless steel

external braiding. 5. Gaskets and O-Rings are Silicone or EPDM. 6. Valve bodies are 316L forged stainless steel with either tube stub or Tri-Clamp end

connections. Manual and automatic valves are supplied with O-ring sealed bonnets. Removable diaphragm valves (i.e. addition valves) will have sanitary bonnet internals designed for autoclaving. Valve diaphragms are FDA approved EPDM. Valves are ported and oriented to ensure complete drainability that is essential for sterilization and cleanability. Ball valves and plug valves are excluded from product contact areas and are used only in utility and non-process gas piping.

4.3.2. Clean Steam Piping and Components Clean Steam Piping consists of all tubing that supplies (or removes) sterile steam (or condensate) to (or from) the Process Piping, and vessel valve assemblies. Clean Steam piping and fittings are constructed of 316L Stainless Steel with 25 µ-inch Ra internal finish and 180 grit external finish. Clean Steam pipe fittings are Tri-Clamp or Butt Welded. VCO fittings may be used for clean steam piping to and from the agitator seal housing if required. All welds within the Clean Steam piping are orbital type and conform to the general standards set forth in BPE-2007. Flex hoses, where used, have smooth, FDA-approved, Teflon liners with stainless steel external braiding. Gaskets and O-Rings are Silicone or EPDM. Valve bodies are 316L forged stainless steel with either tube stub or Tri-Clamp end connections. Manual and automatic valves are supplied with O-ring sealed bonnets. Valve diaphragms are FDA approved EPDM. Valves are ported and oriented to ensure complete drainability that is essential for sterilization and cleanability. Ball valves and plug valves are excluded from clean steam piping and are used only in utility and non-process gas piping and condensate drain lines. Nicholson stainless steel, thermostatic steam traps are used in clean steam condensate piping. The majority of these traps are Model DS-100, which have a 10-degree subcool point. The trap off of the vessel drain, the sample valve and the one off of the exhaust line are Model CDS-20, which have a special 3-degree subcool point. The traps will cycle more often thus not allowing as much condensate build up on these three lines.

4.3.3. Non-Process Gas Piping and Components Non-Process Gas piping consists of Air or Gas piping which is on the non-sterile side of the inlet and exhaust filter housings. 1. Non-Process Gas piping are 316 or 316L stainless steel.





2. Compression and threaded end fittings are permitted. 3. Ball Valves are 316L Stainless Steel and full bore.

4. 316 Stainless Steel Plug valves are utilized for lines smaller than ¾” OD.

4.3.4. Utility Piping and Components Utility piping carries plant steam, water (or other coolant), and instrument air to various portions of the Bioreactor system.

1. Instrument Air lines are 316 stainless steel, nylon, Teflon or polyethylene tubing with

compression or hose fittings.

2. Piping carrying plant steam, water, and non-sterile steam condensate is 316 stainless steel.

3. Nicholson stainless steel, thermostatic steam traps are used in utility steam condensate piping.

4.3.5. System Utility Requirements To ensure proper operation of the Bioreactors, the following utilities must be supplied to the system at the appropriate inlet pressures, flow rates, and voltages. Facility steam, water and air services should be pre-filtered before connection to the Bioreactor systems.

1000-liter Bioreactor Utility Requirements

SERVICE LINE FLOW RATE PRESSURE CONNECTION Clean Steam 170 lb/hr 35 psig (min) 1.5” Tri-Clamp Process Air 200 SLPM 80 psig ½”Compress. Oxygen 100 SLPM 30 psig ½”

Compression Carbon Dioxide 50 SLPM 30 psig ½”

Compression Nitrogen 100 SLPM 30 psig ½”

Compression Instrument Air 56 SLPM 80 psig ½”

Compression Water 15 GPM 50 psig 1.5” Tri-Clamp CIP Fluids 28 GPM 25 psig 1.5” Tri-Clamp Biowaste & Condensate Gravity Type-Open 1.5” Tri-Clamp Water Return 25 psig maximum 1.5” Tri-Clamp Exhaust Gas ½ psig maximum 1” Tri-Clamp Electrical 30 Amp (480

Volts) and 20 Amp (120 Volts)

480 Volts AC, 3 Phase, 60 Hz (non UPS) and 120 Volts AC, 1 Phase,

Conduit Conn.





60 Hz, UPS backed.

Remote Electrical for PLC Cabinet

15 Amp 120 Volts AC, 1 Phase, 60 Hz, UPS backed.

Conduit Conn.

4.4. Pre-Filters, Regulators, & Isolation Valves The following hardware should be installed by the customer external to the Bioreactor utility skid: • Clean Steam isolation valve. • Plant Steam isolation valve and steam trap. • Chilled Water supply and return isolation valves. • Process Gas (Air, Carbon Dioxide, Nitrogen and Oxygen) isolation valves, prefilters,

coalescing filter for air and two gauges for each service (upstream and downstream of the filters).

4.4.1. Clean Steam Clean steam is required for the sterilization of the Bioreactor’s interior and system components that are in direct contact with the process, such as the inlet filters, the exhaust filter, the sparger and overlay piping, and the re-sterilizable valve assemblies.

4.4.2. Process Air Air to the system should be oil-free and particulate-free at 85% relative humidity or less. We recommend that process air to the Bioreactor be of instrument grade or better.

4.4.3. Oxygen Oxygen supplied to the system should be particulate-free at 85% relative humidity or less. Facility feed piping and components should be cleaned for oxygen service prior to installation and connection to the Bioreactor.

4.4.4. Carbon Dioxide Carbon Dioxide supplied to the system should be particulate-free at 85% relative humidity or less.

4.4.5. Nitrogen Nitrogen supplied to the system should be particulate-free at 85% relative humidity or less.

4.4.6. Chilled Water Chilled Water is used for rapid cooling after sterilization and for cooling the vessel contents during growth. If the chilled water is heavily laden with particulates, a pre-filter should be installed.





4.4.7. Chilled Water Return The chilled water return allows the reuse of the water, via connection to a chiller. See table above for maximum return pressure. If this return pressure is exceeded then the jacket relief valve might release.

4.4.8. Exhaust The exhaust connection may be run to an open drain or to the outside environment if the gases exiting from the Bioreactor are not harmful and have no significant odor. Local regulations may require the exhaust to be run through a scrubber before exhausting to the atmosphere. Piping should be as short as possible and large in diameter to prevent excessive backpressure. See tables above for maximum backpressure.

4.4.9. Drain (Clean and Biowaste) The biowaste drain encompasses all waste streams, which are considered to have been in contact with the process and are therefore referred to as biowaste as well as other minor condensate returns from the jacket heat exchanger. Depending on the organism this may need to be sent to a kill system. The biowaste drain is also used for the disposal of the water in the jacket and piping to and from the jacket should it be necessary to drain the system for any repairs. This is necessary because the water return line is usually under pressure and will not allow the draining of the jacket.

4.4.10. Electrical The systems are wired in accordance with the National Electric Code for non-Hazardous and non-Corrosive areas. The main power source (which powers the agitator drive, circulation pump and a few other items) is 480 VAC, 3 Phase, 60 Hz. This power may be from an emergency generator system so that the system can be maintained in a running condition should there be a power outage. The system also suggests that the control circuitry is from an Uninterrupted Power Supply. This power is 120 VAC, 1 Phase, 60 Hz. There are two 120 VAC circuits required. The PLC requires its own supply and the control cabinet requires an additional 120 VAC supply. Both of these supplies can be (recommended) from a UPS.





CHAPTER 5. – CONTROL MODULES

This section explains the process control mechanisms of the Bioreactor systems.

5.1. Vessel Pressure Control - Exhaust Gas Module The exhaust gas assembly is designed to filter the gas leaving the bioreactor, minimize residual condensate from the exhaust gas stream, and to control the pressure within the vessel. The exhaust module consists of: • Sanitary Pressure Sensor mounted on the cover plate. • Steam-sterilizable, absolute filter, 0.2-micron pore size, with Tri-Clamp inlet, outlet and

drain connections to permit removal of the filter assembly during CIP. • Sanitary Tri-clamp cap to be installed in place of the filter during CIP. • Pressure regulating valve and current to pressure transducer.

The pressure of the vessel is measured by pressure transmitter PT-901 and controlled by manipulating pressure control valve PCV-911 (through I/P tag number PY-901), which controls the exhaust flow leaving the vessel.

Associated Control Loops

The vessel pressure control loop is designated PIC-901. The operator can select the following modes for this loop (see Figure 1 below):

• Automatic – PCV-911 (a fail closed valve) is throttled towards full closed (0 percent loop output) to increase the pressure measured at PT-901, and throttled towards full open (100 percent loop output) to decrease the pressure measured at PT-901.

• Manual—The loop output can be adjusted from 0 to 100 percent.

• Off—The loop output is fixed at 100 percent to fully open the exhaust valve.

• DO Cascade—This loop is a slave to the Dissolved Oxygen (DO) Loop. Therefore, its setpoint can be adjusted by the DO loop to increase or decrease the DO level via increasing or decreasing the vessel pressure.

Associated Discrete Devices

Valve TXV-902 is a fail closed valve and will be held open whenever the pressure control is active.

Transmitter Range

The transmitter range for the vessel pressure control subsystem is -15 to 60 PSIG for the 1000-L bioreactor.





Setpoint Range

The setpoint range for the vessel pressure control subsystem is 0 to 50 PSIG for the 1000-L bioreactor. However, because of the inlet gas pressures being limited to 30 PSIG and the vessel is ASME coded for only 45 PSIG it will not be possible to control the vessel pressure above approximately 25 PSIG.

Figure 1

5.2. Aeration Control Module

5.2.1. Gas Sparge and Overlay Systems (Independent) Gases are fed into the bioreactor to maintain an optimum environment for cell growth. The gas overlay and sparge assemblies permit the introduction of the process gases into the vessel headspace above the fluid level or directly into the culture media. Automated diaphragm valves are placed within each gas line to ensure sterilization and cleaning of the sparge and overlay assemblies.

Both gas inlet assemblies have the following components: • Automated Valves for injection of steam (and CIP fluid) into the sparge and overlay gas

piping.





• Manual Valves for controlling condensate drainage from the filters. • A manual valve for controlling the introduction of air/gas into the sparge and an automatic

valve for controlling the introduction of air/gas into the overlay gas piping. • Steam-sterilizable, absolute filter, 0.2-micron pore size, with Tri-Clamp inlet, outlet and

drain connections to permit removal of the filter assembly during CIP. • Sanitary Spool pieces to be inserted in place of the filter assemblies during CIP.

5.2.2. Gas Feed and Mixing The gas feed and mixing sub-systems are located on the non-sterile side of the bioreactor inlet filter housings. The addition of Air, Carbon Dioxide, Nitrogen and Oxygen to the bioreactor is automatically regulated in the following means:

• Thermal Mass Flow Controller. • Block Valve upstream of the Sparge filter(s).

When the system controller calls for the addition of a specific gas, the thermal mass flow controller is given a change in setpoint allowing gas to enter the flow measurement / control assembly. Following the flow measurement / control assembly, gases pass through a block valve before entering the sparge / overlay systems.

5.2.2.1. Sparge and Overlay Gas Flow Rates The following table identifies the operating range of the sparge and overlay flow controllers:

1000-liter Bioreactor Gas Flow Rates GAS SPARGE OVERLAY Air 0 / 4.0 – 200 SLPM * 0 / 4.0 – 200 SLPM * Oxygen 0 / 2.0 – 100 SLPM * -- Carbon Dioxide

0 / 1.0 – 50 SLPM * --

Nitrogen 0 / 2.0 – 100 SLPM * -- Flow Control Notes: * The Sparge and Overlay thermal mass flow controllers are capable of a 50:1 turndown. Attempting to set the TMFC below this minimum operating range could result in an inaccurate flow through the device.

5.2.2.2. Air Flow Control

Two of the mass flow loops control the mass flow rate of airflow into the bioreactor.


This air sparge control loop is designated FIC-121 (see Figure 2 below). The air overlay control is designated as FC-125 (see Figure 3 below). The sparge loop is a setpoint





controller for self-contained mass flow controller (MFC). An analog output from PLC / WAGO modules provides the setpoint to the MFC, which directly manipulates the mass flow. The air overlay is also a setpoint controller for self-contained mass flow controller (MFC). Again an analog output provides the setpoint to the MFC, which directly manipulates the mass flow.

The operator can select the following modes for the sparge loop:

• Auto—The MFC setpoint is adjusted from 0 to 200 slpm for the 1000-L bioreactor. The MFC will maintain the flowrate at the setpoint desired.

• Off—The loop output is fixed at 0 percent (or 0 slpm), fully shutting off the air sparge to the bioreactor.

• DO Cascade—This loop is a slave to the DO Loop. Therefore, its setpoint is adjusted by the DO loop to increase or decrease the DO level.

The operator can select the following modes for the overlay loop:


• Off—The loop output is fixed at 0 percent (or 0 slpm), fully shutting off the air overlay to the bioreactor.





Figure 2

Figure 3 Associated Discrete Devices

In addition to the analog command to the mass flow controller, one discrete output opens valve FY-121 (air sparge) or opens valve FY-125 (for air overlay) downstream of the sparge and overlay mass flow controllers. This discrete output has two modes:

• Automatic (Unforced)—The output is energized while the system is in Active mode and the associated control loop is not in the Off mode. FY-121 and FY-125 operates with a 10 second timer to allow flow to exceed 5% of full range. If the PV drops below 5% of Air Sparge, this valve will close. The Air Overlay is set at 10% instead of 5%.

• Manual (Forced)—The output is forced manually from the OIT.

Transmitter Range

The transmitter range for the airflow control subsystem(s) is 0 to 200 slpm for the 1000-L bioreactor.





Setpoint Range

The setpoint range for the airflow control subsystem(s) is 0 to 200 slpm for the 1000-L bioreactor.

5.2.2.3. O2 Flow Control

The O2 mass flow loop controls the mass flow rate of oxygen into the bioreactor.


The O2 control loop is designated FIC-122 (see Figure 4 below). This loop is a setpoint controller for a self-contained mass flow controller (MFC). An analog output from the PLC / WAGO system provides the setpoint to the MFC, which directly manipulates the mass flow. The MFC reports the current mass flow to the system for display.

The operator can select the following modes for this loop:


• Off—The loop output is fixed at 0 percent (or 0 slpm), fully shutting off the oxygen sparge to the bioreactor.

• DO Cascade—This loop is a slave to the DO Loop. Therefore, its setpoint is adjusted by the DO loop to increase the DO level.


In addition to the analog command to the mass flow controller, one discrete output opens valve FY-122 downstream of the mass flow controller. This discrete output has two modes:

• Automatic (Unforced)—The output is energized while the system is in Active mode and the associated control loop is not in the Off mode. FY-122 operates with a 10 second timer to allow flow to exceed 5% of full range. If the PV drops below 5% of O2 Sparge, this valve will close.


Transmitter Range

The transmitter range for the O2 flow control subsystem is 0 to 100 slpm for the 1000-L bioreactor.

Setpoint Range

The setpoint range for the O2 flow control subsystem is 0 to 100 slpm for the 1000-L bioreactor.





Figure 4 5.2.2.4. CO2 Flow Control

The CO2 mass flow loop controls the mass flow rate of carbon dioxide into the bioreactor.


This CO2 control loop is designated FIC-123 (see Figure 5 below). This loop is a setpoint controller for a self-contained mass flow controller (MFC). An analog output from the PLC / WAGO system provides the setpoint to that MFC, which directly manipulates the mass flow. The MFC reports the current mass flow to the system for display.



• Off—The loop output is fixed at 0 percent (or 0 slpm), fully shutting off the carbon dioxide sparge to the bioreactor.

• pH Cascade—This loop is a slave to the pH Loop. Therefore, its setpoint is adjusted by the pH loop to increase or decrease the pH level.






In addition to the analog command to the mass flow controller, one discrete output opens valve FY-123 downstream of the mass flow controller. This discrete output has two modes:

• Automatic (Unforced)—The output is energized while the system is in Active mode and the associated control loop is not in the Off mode. FY-123 operates with a 10 second timer to allow flow to exceed 5% of full range. If the PV drops below 5% of CO2 Sparge, this valve will close.

• Manual (Forced)—The output is forced manually from the Force Digital Maintenance screen of the OIT.

Transmitter Range

The transmitter range for the CO2 flow control subsystem is 0 to 50 slpm for the 1000-L bioreactor.

Setpoint Range

The setpoint range for the CO2 flow control subsystem is 0 to 50 slpm for the 1000-L bioreactor.

Figure 5





5.2.2.5. N2 Flow Control

The N2 mass flow loop controls the mass flow rate of nitrogen into the bioreactor.


The N2 control loop is designated FIC-124 (see Figure 6 below). This loop is a setpoint controller for a self-contained mass flow controller (MFC). An analog output from the PLC / WAGO system provides the setpoint to the MFC, which directly manipulates the mass flow. The MFC reports the current mass flow to the system for display.



• Off—The loop output is fixed at 0 percent (or 0 slpm), fully shutting off the oxygen sparge to the bioreactor.


One discrete output opens valve FY-1.24 downstream of the mass flow controller. This discrete output has two modes:

• Automatic (Unforced)—The output is energized while the system is in Active mode and the associated control loop is not in the Off mode. FY-1.24 operates with a 10 second timer to allow flow to exceed 5% of full range. If the PV drops below 5% of N2 Sparge, this valve will close.


Transmitter Range

The transmitter range for the N2 flow control subsystem is 0 to 100 slpm for the 1000-L bioreactor.

Setpoint Range

The setpoint range for the N2 flow control subsystem is 0 to 100 slpm for the 1000-L bioreactor.





Figure 6

5.3. Agitation Control Module The Bioreactor has a bottom, center mounted, variable speed agitator assembly for mixing of the vessel’s contents. The agitator is designed for continuous duty and provides maximum containment of the Bioreactor’s product. The agitator assembly consists of the following components: agitator shaft, double mechanical seal assembly, gear reducer, motor, and variable speed AC drive. Dakota has provided an agitator system capable of achieving the following performance:

AGITATOR DATA 1000-liter BIOREACTOR

Operating Range 0 – 120 RPM *

Control Accuracy +/- 2 RPM (1.0% of Full Scale)

* Agitator VFD can provide a 20:1 turndown. The agitator is designed to rotate three Lightnin A310 impellors of a diameter of 18” at 120 RPM.





The agitator assembly is mounted onto the vessel with the use of a double mechanical seal housing. The agitator coupling will have FlowServe type-ST/QBM double mechanical seals that are pressurized using clean steam condensate. Seal pressure is maintained at 35 psig via a sanitary pressure regulator. A low-pressure switch is installed on the effluent line of the seal housing to ensure that a minimum seal pressure is maintained. The maintenance of the condensate fill is automatic, but the agitator should never be run without an adequate supply of Clean Steam to the unit. The bioreactor agitator has an inverter duty, high efficiency, TEFC, 1.0 SF, AC drive motors. Speed and torque characteristics conform to NEMA B requirements. The motor is rated for continuous duty and will not exceed the temperature rises for NEMA F type insulation according to NEMA Standard Publication MG1-1998.

MOTOR DATA 1000-liter BIOREACTOR

Power 3.0 HP Speed 1750 RPM Voltage 480 VAC, 3 phase, 60Hz

The agitator motor runs off of the main system power supply. No additional electrical utilities are required. Bioreactor agitation speed is controlled using a variable frequency drive mounted within the main control console. The VFD measures the output to the motor and provides a signal that is translated by the WAGO / PLC system into agitator shaft speed. This value is then input into the Agitator Control Module. The bioreactor VFD can provide a 20:1 effective turndown of the agitator speed. Agitation speed is displayed on the bioreactor’s OIT and when in local control, can be manually set by accessing the Agitator Loop Screen. The agitator control loop may also be configured to automatically adjust the stirrer speed in response to Dissolved O2.


The agitator speed control loop is designated SIC-601 (see Figure 7 below). The operator can select the following modes for this loop from the OIT:

• Auto— The loop setpoint is adjusted from 0 to 120 rpm for the 1000-L bioreactor.

• Off —The loop output is fixed at 0 percent to fully stop the agitator.

• DO Cascade—The loop is a slave to the DO Loop. Therefore, the DO loop will increase agitation speed to increase the DO level, and decrease agitation speed to decrease the DO level.


In addition to the analog speed command signal sent to the VFD, one discrete output enables the VFD. This discrete output has two modes:





• Automatic (Unforced)—The output is energized while the system is in Active mode and the associated control loop is not in the Off mode.


Transmitter Range

The transmitter range for the agitation control subsystem is 0 to 120 rpm for the 1000-L bioreactor.

Setpoint Range

The setpoint range for the agitation control subsystem is 0 to 120 rpm for the 1000-L bioreactor.

Figure 7

5.4. Nutrient Feed Control Module

The rate at which nutrient is fed to the Bioreactor is controlled by pulse width modulation (PWM) of the assignable pump. The pump plugs into a marine style outlet on the vessel side of the control console. Also provided on the same side of the control console is another receptacle that is powered with 115 VAC at all times the unit is turned on. This outlet may be used for various pieces of equipment but it is mainly recommended for use as a pump outlet





for the inoculation of the bioreactor. There are two separate Nutrient feeds, Nutrient 1 and Nutrient 2 (see Figure 8 and Figure 9 below).


The operator can select the following modes for the nutrient feed on an individual basis from the OIT:

• Manual—the PWM output is manually adjusted from 0 to 100 percent.

• Off —The PWM output is fixed at 0 percent to fully stop the pump. If no pump is assigned to the nutrient feed function, that loop is forced to this mode.

• Profile - The loop’s setpoint may be automatically adjusted based on ECT (elapsed culture time). When this is active the system asks the operator to put in a target setpoint, an ECT for the new setpoint to take effect, and if desired whether this new setpoint is to be reached in a ramp fashion or instantaneously. If instantaneous change is desired then 0 should be entered for the ramp time. If it is desired to reach the new setpoint over a set amount of time then that value should be entered for the ramp time (see Figure 10 below as an example of the Profile setup screen).


Nutrient Feed has an assignable pump (P-704). The associated discrete output has two modes:

• Automatic (Unforced)—The output is energized while the system is in Active mode and the associated control loop is not in the Off mode.


Control Range

The control range for the nutrient feed subsystem is 0 to 100% for the 1000-L Bioreactor.

Setpoint Range

The setpoint range for the nutrient feed subsystem is 0 to 100% for the 1000-L Bioreactor.





Figure 8





Figure 9





Figure 10

5.5. Dissolved Oxygen Control

The dissolved oxygen level (DO) of the vessel contents is measured by dual transmitters, AIT-301 and AIT-302. The operator selects the transmitter for control of the process from the Cascade Setup screen (see Figure 12 below). DO is controlled by manipulating various slave cascade loops (air sparge flow, oxygen sparge flow, vessel pressure and agitation). From the operator interface the operator can enable or disable cascade control for each slave loop.


The DO control loop is designated AIC-301 (see Figure 11 below). The operator can select the following modes for this loop:

• Off—The cascade slave loops’ setpoint are not adjusted.

• Automatic – To control the system’s measured DO level, the DO loop adjusts the cascade slave loops’ setpoint to achieve the operator-adjusted setpoint for DO.


No discrete devices are associated with the DO control mechanism.





Manipulation of Slave Loops

Adjustments are made for each slave loop as follows:

• The DO Loop output can vary from 0 to 100 percent.

• Each slave loop is assigned a Minimum DO Output % value and a corresponding Minimum Setpoint value. (Both values can be adjusted from the OIT.) If the DO loop output is less than or equal to the Minimum DO Output %, the slave loop setpoint is set to the Minimum Setpoint value.

• Each slave loop is assigned a Maximum DO Output % value and a corresponding Maximum Setpoint value. (Both values can be adjusted from the OIT.) If the DO loop output is greater than or equal to the Maximum DO Output %, the slave loop setpoint is set to the Maximum Setpoint value.

• If the DO loop output is greater than the Minimum DO Output % and less than the Maximum DO Output %, the slave loop setpoint is determined by linear interpolation.

Transmitter Range

The transmitter range for the DO subsystem is 0 to 100 % for the 1000-L Bioreactor.

Setpoint Range

The setpoint range for the DO subsystem is 0 to 100 % for the 1000-L Bioreactor.





Figure 11





Figure 12

5.6. Control of the pH

Dual transmitters AIT-401 and AIT-402 measure the pH of the vessel contents. The operator selects the transmitter for control of the process from the pH Cascade Setup screen (see Figure 14 below). pH is controlled by manipulating slave cascade loops, control of CO2 flow rate or injection of base. Through the OIT, the operator can enable or disable cascade control for each slave loop.


The pH control loop is designated AIC-4.01 (see Figure 13 below). The following modes can be selected for this loop from the OIT:

• Off—The cascade slave loops’ setpoints are not adjusted.

• Automatic—The pH Loop adjusts the cascade slave loops’ setpoints to maintain the pH measured by the system (1 to14.0 pH) equal to the setpoint.

• Profile - The loop’s setpoint may be automatically adjusted based on ECT (elapsed culture time). When this is active the system asks the operator to put in a target setpoint, an ECT for the new setpoint to take effect, and if desired





whether this new setpoint is to be reached in a ramp fashion or instantaneously. If instantaneous change is desired then 0 should be entered for the ramp time. If it is desired to reach the new setpoint over a set amount of time then that value should be entered for the ramp time. See Figure 15 below for the Profile setup.


The following discrete devices control the pH:

• Base Addition Pump —PWM discrete output that has the frequency/duration of its on-cycle modulated in an effort to raise the system pH.

• CO2 Flow —discrete output that turns carbon dioxide flow on and off and works in conjunction with the carbon dioxide Thermal Mass Flowcontroller.

Manipulation of Slave Loops

Each slave loop is adjusted as follows:

• The pH Loop output can vary from 0 to 100 percent.

• Each slave loop is assigned a Minimum pH Output % value and a corresponding Minimum Setpoint value. (The operator can adjust both values from the OIT.) If the pH Loop output is less than or equal to the Minimum pH Output %, the slave loop setpoint is set to the Minimum Setpoint value.

• Each slave loop is assigned a Maximum pH Output % value and a corresponding Maximum Setpoint value. (The operator can adjust both values from the OIT). If the pH Loop output is greater than or equal to the Maximum pH Output % value, the slave loop setpoint is set to the Maximum Setpoint value.

• If the pH loop output is greater than the Minimum pH Output % and less than the Maximum pH Output %, the slave loop setpoint is determined by linear interpolation.

Transmitter Range

The transmitter range for the pH control subsystem is 1 to14.0 pH for the 1000L Bioreactor.

Setpoint Range

The setpoint range for the pH control subsystem is 1 to14.0 pH for the 1000L Bioreactor.





Figure 13





Figure 14





Figure 15

5.7. Temperature Control Module The system controller automatically regulates the Bioreactor growth and sterilization temperature. Heat is exchanged across the vessel shell by maintaining a temperature differential between the Bioreactor and jacket fluids (water to water) and by agitation within the vessel. An RTD sensor assembly inserted in the lower sidewall of the vessel continuously measures the Bioreactor’s temperature. Output from the sensor / transmitter is fed into the Bioreactor’s Temperature Control Loop TIC-201. The measured temperature is then compared to the loop set point that is either manually set by the operator or automatically set by the system controller. The controller will then send a calculated output (determined by a PID algorithm) to the Jacket Temperature Control Loop TIC-202. The Jacket Temperature Control Loop has its own RTD sensor mounted in the jacket outlet line as its input. The jacket loop controller can have its setpoint limited so that the jacket will never get too hot or too cold thus allowing the prevention of hot spots or cold spots along the jacket wall. This controller will then engage in either a heating or cooling mode of operation. Both modes operate by pulse width modulation (PWM) where heating or cooling is performed in short ON/OFF cycles. The greater the difference between the measured and set point temperatures, the greater the duration of heating or cooling.





The temperature control circuit is a closed circulation loop in which hot or cold water is pumped through the Bioreactor’s jacket. When heating is required, the circulation loop remains closed and the recirculating jacket water is heated with a steam fed heat exchanger. A fast acting valve is used to inject utility or plant steam into the heat exchanger. The frequency and duration to which the steam valve is cycled (pulsed Open/Closed) is determined by the Temperature Control Loop. When the vessel requires cooling, the temperature control output circuit is opened allowing fresh plant water to enter the jacket and the hot fluid exits the Bioreactor system via the water return piping. The frequency and duration of water injection is determined by the Temperature Control Loop. During cooling, the jacket circulation pump is constantly running and the heat exchanger’s steam injection valve is inactive. The temperature control system is capable of maintaining the Bioreactor temperature to within +/- 0.3 oC of the temperature set point over a range of 20 to 60 oC. Depending upon the temperature control loop configuration, the system is capable of adding or removing heat at the rate of 1 oC per minute (time averaged rate). Sterilization (Semi-Automatic) The Bioreactor is designed to be sterilized empty. Sterilization of the Bioreactor and process gas lines (inlet and exhaust) is automatically performed by the system controller with minimal system preparation. Sterilization of vessel valve assemblies (i.e. addition, sample, and harvest valves), are manually sterilized and can be re-sterilized at any time. The sterilization process is divided into five (5) periods of operation: Heat A, Heat B, Hold, Cool A and Cool B. The operation of each phase is factory configured, however the operator has the ability to access the controls to these phases via the OIT to modify the sterilization process. See Figure 18 below for SIP startup and initiation or aborting. From this screen you are able to toggle to a separate screen which shows the current temperature of all of the RTD’s within the system (see Figure 19 below). Heat A (Rapid Heating) – During this phase of sterilization clean steam is injected into the Bioreactor’s sprayball piping, entering the vessel through the top of the tank. Heated Air and vapor is ejected from the vessel through the exhaust system. The agitator is de-activated since there is no liquid in the vessel to agitate. The gas inlet line is closed allowing the vessel’s internal temperature to gradually increase. The jacket temperature control circuit is de-activated at this time. As the jacket water expands during this time it is allowed to go into the water return line through the backpressure control regulator near the outlet. Heat B (Sterilization) – This phase starts when the vessel temperature rises to 105 oC (or another operator determined temperature). Clean steam starts to be injected into the Bioreactor’s gas inlet piping, enters the vessel through the sparge, addition ports and overlay connections, and exits the Bioreactor though the exhaust piping and is directed to a thermostatic steam trap on the sterile and non-sterile sides of the exhaust filter. Steam will continue to be injected until the vessel reaches the sterilization temperature control point (typically 122 oC). In this reactor the temperature is maintained by controlling the incoming steam pressure. All of the valves will remain open and the steam will simply flow only as





required to keep the vessel temperature at the saturation temperature for the pressure set on the incoming pressure regulator. The vent valve on the exhaust filter should be opened at some time during the heat up in order to assure all of the air is ejected from the filter housing. Hold (Dwell) – The system will then begin the hold timer. In the event that the bioreactor temperature drops below by more than 1 degree C (default but may be changed on the SIP Settings screen) the SIP temperature before the cooling process starts, the SIP timer will be reset. If the system drops below this temperature more than three times then the operator will have to either restart the SIP or abort the process unless the problem is fixed and the cycle is able to complete without the temperature dropping below the setpoint. A “Continue” prompt will appear at the end of this phase in order for the operator to decide whether to continue and to set several manual valves in their proper positions. Cool A (Rapid Cooling) – At the end of the sterilization period, clean and plant steam injection are halted. Once the continue button is pushed the temperature control circuit is opened to allow plant water to flow through the bioreactor jacket and exit the system via the water return connection. The rate at which this cooling occurs is controlled by pulsing the water return valve (TXV-206) at a percentage of on-time that is pre-determined but adjustable by the operator. At the same time, air is injected into the Bioreactor overlay inlet (utilizing the valve PCV-922 and pressure regulator PCV-921) to prevent the formation of a vacuum within the vessel. The exhaust valve (PCV-902) is opened and the pressure control loop is activated in order to assure that some flow is passed through the exhaust filter in order to dry it for use after the system reaches the growth temperature. Cool B (Return to Growth Temperature) - When the vessel temperature drops to the Cool B Temperature set point, the sterilization will be considered over and the system is allowed to resume the standard temperature control protocol. . At this point the Start Inoculation button will appear so that once the media has been added to the empty vessel the button can be pushed and the Elapsed Culture Timer (ECT) will start.


The Bioreactor Temperature Control Loop is designated TIC-201 and the Jacket Temperature Control Loop is designated TIC-202 (see Figure 16 below). If the Bioreactor is in any mode other than SIP, the operator can select the following modes for the Bioreactor or the Jacket loop (from the OIT):

• Manual—The loop output is adjusted from 0 percent (full cooling) to 100 percent (full heating).

• Off—The loop output is fixed at 50 percent, fully closing the cooling valve and the heating valve.

• Automatic—The temperature loop adjusts its output from 0% (full cooling) to 100% (full heating) as a cascaded setpoint to the Jacket Temperature loop TIC-2.02 to maintain the temperature measured by the system at the operator-adjusted setpoint.





• Profile - The loop’s setpoint may be automatically adjusted based on ECT (elapsed culture time). When this is active the system asks the operator to put in a target setpoint, an ECT for the new setpoint to take effect, and if desired whether this new setpoint is to be reached in a ramp fashion or instantaneously. If instantaneous change is desired then 0 should be entered for the ramp time. If it is desired to reach the new setpoint over a set amount of time then that value should be entered for the ramp time (see Figure 17 below for Temperature Profile setup). Values must be entered for all four steps even though the last values may all be the same if there is only one or two steps required.

If the Bioreactor is in an SIP heating phase (Heat A, Heat B, or Hold- refer to description of sterilization phases above), the active phase will control the temperature by one of two methods (see Figure 18 below for SIP setup and initiation):

CIP Port Steam (Heat A Phase)—The temperature control of the jacket is turned off during SIP. All water circulation valves close, circulation pump P-01 stops. For vessel injection, CIP Supply Valve to Sprayballs FCV-551 opens fully.

Additional Steam (Heat B or Hold Phase) – Jacket circuit remains off. For vessel injection, Sparge Steam Supply Valve TXV-505, Port Steam Supply Valve TXV-766 and Overlay Steam Valve TXV-506 open and remain open until the Hold time is over.

The PLC system has full control of the temperature loop during SIP phases (even Cool A) -- the operator cannot control the automated temperature loop in any way during SIP other than changing the various setpoints and hold time.

Split-Range Control

During non-SIP Heating operation, the output of the PID calculation is used as an input to provide split-range control of the two pulse-width-modulated discrete outputs. This type of control uses the two outputs: Heating valve TXV-204 for heating, and Chilled Water Return Valve TXV-206 for cooling.

• TIC-202 output in range of 0% to -100.0%: Cooling valve ranges from 100% (full cooling) to 0% (closed), and heating valve closes.

• TIC-202 output = 0%: Cooling and heating valves close.

• TIC-202 output in range of 0% to +100.0%: Cooling valve closes, and heating valve ranges between 0% (disabled) to 100% (full heating).


When not forced, the following discrete devices are used in the control of the Bioreactor’s temperature.

Non-SIP-Heating

• The discrete output for the circulation pump P-01 is energized to enable the pump whenever the temperature loop is not Off and the system is in the Active mode.





SIP-Heating

• The discrete output for the circulation pump P-01 is de-energized to disable the pump.

Each of the above devices operates in two modes:

§ Automatic (Unforced) mode—The devices operate as described above. All operate in response to the mode of the temperature loop.

§ Manual (Forced) mode—The operator can manually force the device from the OIT. All forces are automatically removed when this screen is no longer displayed.

Transmitter Range

The transmitter range for the temperature control subsystem is 0 to 150.0 C for the 1000-L Bioreactor and for the Jacket.

Setpoint Range

The setpoint range for the temperature control subsystem is 0 to 90.0 C for the 1000-L Bioreactor.

Figure 16





Figure 17





Figure 18





Figure 19

5.8. Harvest Control

Any one of the three pumps can be assigned to be a Harvest Pump. Once this is done then the Harvest Control screen can be used to provide a setpoint to that pump. See Figure 20 below.


The Harvest control loop is not designated with a tag number. The following modes can be selected for this loop from the OIT:

• Off—The loops’ setpoint is not adjusted.

• Manual—The operator can provide a given setpoint (in percent of full flow) out to the assigned pump.





Figure 20

5.9. Vessel Weight Control

The weight transmitter WIT-802 measures vessel weight. The vessel weight is displayed on the Main Screen of the OIT. The vessel weight may be controlled by activating the Nutrient Pump P-704 as a function of the high alarm on the Weight System.


Pump output P-704 is a fail off device and its ON time will be adjusted by the Weight loop when control is active.

Transmitter Range

The transmitter range for the weight transmitter is 0 to 1375 kg for the 1000-L Bioreactor for a tare weight of 1000 kg.

5.10. Ad Hoc Profiling

The system is provided with a special button for causing a profile to be implemented at any time the operator desires. If this button is pushed the a screen appears in which the operator can choose between the Temperature Loop, the pH Loop, and Nutrient 1 or





Nutrient 2 loops to immediately start a profile of that loop’s setpoint. In this case there is no ECT time to enter only the Target Setpoint and the Ramp Time to get to the new setpoint. See Figure 21 below for setup of the Ad Hoc Profile screen.

Figure 21

5.11. CIP Piping Module The system is supplied with the necessary piping and hardware for automatic cleaning-in-place of the Bioreactor vessel, valve assemblies, and process piping. The operation of the Cytovance supplied CIP system that works in conjunction with this bioreactor will be described in a separate manual. The information provided here is simply the reactor portion of the system. Prior to cleaning, gas inlet and exhaust lines are isolated and their SIP filter housings are replaced with sanitary tube stubs in the case of the sparge and overlay filters and remove the exhaust filter from the vessel and replace it with a tri-clamp cap. Gas filter housings and DO/pH probes are removed from the system for cleaning-out-of-place. There are also spool pieces to connect the port steam lines to the outlet of the condensate return valve for each port in order to assure that the valves associated with all of the ports get cleaned properly. The cleaning fluid supply is connected to the Bioreactor’s CIP connection on the pipe-mounted transfer panel. The U-bend on the CIP / Clean Steam transfer panel is switched to the “Cleaning”





position thereby permitting the injection of CIP fluid into the Bioreactor’s clean steam lines without the possibility of intermixing or cross contamination with the facility clean steam supply. Before attempting to switch the U-bend tubing make sure the automated valve (TXV-767) just upstream of the transfer panel is closed. Position switches on the transfer panel are in place and provide the PLC system with an indication that the U-bend is in the correct position. The bioreactor’s harvest / drain valve is opened and connected to the CIP system’s return connection. Before activating the CIP system install all spool pieces in their correct location and the manual valves on the system should be set as required. When the preparations are complete and the system is ready for cleaning, the operator may activate the CIP mode. The CIP fluid’s normal flow pathway is to the bioreactor spray ball assemblies. However, at pre-determined intervals, the system’s controller will automatically open the clean steam valves in the sparge and overlay for cleaning of these process lines as well as the clean steam valve that feeds clean steam to the ports for cleaning of all of the addition ports and the sample port. Fluids injected into these lines flow back into the bioreactor vessel. The interval frequency and duration for cleaning of the gas inlet, ports and sprayballs may be modified by accessing the CIP controls on the OIT. Overlap times are provided between each flow path in order to never dead head the CIP pump. The overlap times are individually adjustable. See separate manual for adjustment of the CIP system in order to vary the cleaning fluids and rinse fluids and their times of contact with the vessel surfaces. The vessel and process piping are designed to be fully drainable so that at the end of the cleaning cycle, all fluids have exited the system. When cleaning is complete, the filter housings may be replaced and the CIP transfer panel is switched back to the “Cell Growth” position.

5.12. Pump Calibration and Totalization

On each pump screen there is a calibration constant to allow the user to enter the number of milliliters per minute that the pumps displace. This constant is used in the flow totalization for each pump.

Flow totals are calculated for each of the pumps. The totals are determined from the amount of time each pump is on and its calibrated flow constant. These totals are displayed on the Pump screen and may be reset at the start of each new batch or anytime desired by the operator.

To assign each pump to a particular type of pump or loop go to the Pump screen (see Figure 22 below) and assign pump by clicking on the assignment box at the top and using the scroll button on the lower right hand side of the Panel View Plus the system will scroll through the choices available. Once a pump has been assigned to a loop or function then the duty cycle for the PWM control should be assigned. This is usually in the range of 10 seconds but may be varied up and down from there. The longer this value is the longer the pump will last but it will also make the loop not able to control as well. For instance if 30 seconds is entered when the pump is being asked to supply a small amount of base for instance then the pump will run for only a small portion of the 30 seconds and then wait for the remaining time before adding again. Conversely if it is set too short then the pump will be required to turn on and off a large amount of times which will in turn make it fail faster.





Figure 22 To calibrate an individual pump simply select the pump to be calibrated from the pump calibration screen. Enter the seconds you wish the pump to run for in order to get a measurable amount of fluid. Then push the Start button and wait the number of seconds that was entered above. Once the pump has ran for this time then measure the amount pumped and enter it on the screen in terms of milliliters. See Figure 23 below for pump calibration.





Figure 23





5.13. Control of Discrete Devices During Process States The following table describes the control of discrete devices during the various process states.

Device

Flo

w P

ath

1

Flo

w P

ath

2

Flo

w P

ath

3

Pre-

SIP

Hea

t A

Hea

t B

Hol

d

Coo

l A

Coo

l B

Pre-

Inoc

Gro

wth

Agitator (SIC-601) E E E 5 D D D D D 5 5 Water Return Valve (TXV-206) D D D 2 D D D 7 2 2 2

Clean Steam Supply Valve (TXV-767)

D D D E E E E E E E E

Heating Valve (TXV-204) D D D 2 D D D D 2 2 2 Water Feed Valve (TXV-205) D D D E D D D E E E E

Circulation Pump (P-01) D D D 1 D D D 1 1 1 1 Pumps P-001, P-403, P-704 D D D 3 D D D D D 3 3

Pump Outlet P-705 E E E E E E E E E E E Sparge Filter Steam

TXV-505 6 D D D D E E D D D D

Overlay Filter Steam TXV-506 6 D D D D E E D D D D

CIP to Spray Ball FCV-551 6 6 6 D E E E D D D D

Port Steam TXV-766 D 6 D D D E E E E E E

Air to Sparge FY-121 D D D 9 D D D D D 9 9

Oxygen to Sparge FY-122 D D D 9 D D D D D 9 9

Carbon Dioxide to Sparge FY-123

D D D 9 D D D D D 9 9

Nitrogen to Sparge FY-124

D D D 9 D D D D D 9 9

Overlay FY-125

D D D 9 D D D D D 9 9

Overlay Block TXV-509 (N.O.) E E E E E E E D D E E

Vessel Pressurize PCV-922 D D D D D D D E D D D

Exhaust Filter Condensate Drain TXV -901 D D D E E D D E E E E

Exhaust PCV-902 D D D D E E E D D D D





Discrete Output Table Legend: 1. On with temperature loop. 2. Pulse Width Modulated to control temperature when temperature loop is on. 3. Pulse Width Modulated to control pH, acid, base and nutrient addition. 4. All discrete outputs are disabled if the system is in the Inactive mode. 5. Discrete output for agitator is energized if the agitation loop is not OFF. 6. Alternated for CIP flow path control. Sprayball path always on. 7. Modulate via user set duty cycles. 8. Can be manually activated at this time. 9. Whenever respective gas loop is on. D = De-energized, E = Energized.





5.14. Control of Loops During Process States

The following table describes the control of the loops during the various process states.

Control Loop CIP

Pre-SIP/ Pre-Inoc G

row

th

Hea

t A

Hea

t B

Hol

d

Coo

l A

Coo

l B

Temperature TIC-201 OFF 1 1 OFF OFF OFF OFF ON Temperature TIC-202 OFF 1 1 OFF OFF OFF MAN ON

Pressure PIC-901 OFF 1 1 OFF OFF OFF ON ON pH Level AIC-401 OFF 1 1 OFF OFF OFF OFF OFF DO Level AIC-301 OFF 1 1 OFF OFF OFF OFF OFF

Agitation Speed SIC-601 3 1 1 OFF OFF OFF OFF OFF Air Sparge Flow

FIC-121 OFF 1 1 OFF OFF OFF OFF OFF

Oxygen Sparge Flow FIC-122

OFF 1 1 OFF OFF OFF OFF OFF

Carbon Dioxide Sparge Flow FIC-123 OFF 1 1 OFF OFF OFF OFF OFF

Nitrogen Sparge Flow FIC-124


Overlay Flow FIC-125


Nutrient Feed P-704 OFF 1 1 OFF OFF OFF OFF OFF Harvest OFF 1 1 OFF OFF OFF OFF OFF

Loop Table Notes

1. Operator configurable.

2. CIP to Sprayballs FY-551 is open at all times during Heat A in order to bring the vessel’s temperature up quickly.

3. Agitation Loop in Auto mode with output of 10 RPM.

4. Loops are all placed in the Off mode if the system mode is Inactive.





5.15. Alarms and Exception Handling

Alarms consist of information relayed to the user indicating failure or deviation of some portion of the system. These alarms are displayed and stored as described later.

Exception handling refers to those functions that deal with plant or process contingencies and other exception conditions. In the PLC system, exception handling detects deviation of the process from acceptable operating conditions by monitoring various process values, and detects the failure of control devices based on positive feedback or other predefined conditions. Exception handling includes safety interlocking and shutdown logic necessary to provide safe operation of process units, in other words, the safety of plant personnel, plant equipment, and the operating environment.

5.15.1. Generation of Alarms

When the PLC system detects a failure or abnormal event, alarms are generated and displayed, and where specified, the PLC system initiates interlocks or shutdown actions to protect personnel and equipment.

5.15.2. Alarm and Interlocks

Alarms and interlocks belong to one of four categories: setpoint deviation alarms, absolute value alarms, safety interlocks and status alarms.

Setpoint Deviation Alarms

Setpoint deviation alarms are determined by testing an actual process variable (an analog input or result of a calculation) against a predefined range calculated from the setpoint and an operator-adjusted deviation limit. If the process value is outside the acceptable range, an alarm is triggered.

Deviation alarms are activated 10 seconds after the process variable reaches its setpoint. This delay reduces false alarms on system startup.

The following loops are configured with deviation alarms: Temperature, Pressure, Air Overlay Flow, Air Sparge Flow, Carbon Dioxide Sparge Flow, Oxygen Sparge Flow, Nitrogen Sparge Flow, Agitation, pH, Weight and Dissolved Oxygen.

Absolute Value Alarms

Absolute value alarms are determined by comparing a process variable (an analog input or result of a calculation) against a pre-set limit. If the process value is greater than a high limit or less than a low limit for 10 seconds, the alarm is triggered.

The following loops are configured with absolute value alarms: Temperature, Pressure, Air Overlay, Air Sparge, Carbon Dioxide Sparge Flow, Oxygen Sparge Flow, Nitrogen Sparge Flow, Agitation, pH, Weight and Dissolved Oxygen.

Safety Interlock Alarms

The following safety interlock alarms are configured for the Bioreactor system:





• Temperature High-High—Heating valve TXV-204 closes, and Clean Steam valve TXV-767 closes.

• Pressure High-High—Exhaust valve PCV-911 and Exhaust Line Condensate valve TXV-901 goes to full open, air supply valve TY-121 closes, air overlay supply valve TY-125 closes, oxygen supply valve TY-122 closes, carbon dioxide supply valve TY-123 closes, and nitrogen supply valve TY-124 all close.

• Emergency Stop—De-energizes all discrete outputs (stopping the agitator, closing the steam and air inlet valves…etc.) and forces all loops to the Off mode. System state will resume after the emergency stop is reset, and attempt to control the process.

• Manway Opened—De-energizes the agitator (M-601) and Clean Steam valve TXV-767 closes.

Process Equipment Interlocks

Process equipment interlocks protect the product and quality of production by preventing the equipment from entering the Active mode phases out of sequence, as follows:

• The SIP state can run only before the Inoculation state or after completion of the Growth state.

• Inactive mode is not available during the SIP state The SIP must first be aborted prior to aborting the batch.





5.15.3. Enabling and Disabling of Alarm

The following table describes the when alarms are enabled and disabled during the various system states and the phases of the SIP state and operations.

Alarm Pre-SIP/ Pre-Inoc/ Inoc/ Growth/Harvest

Hea

t A

Hea

t B

Hol

d

Coo

l A

Coo

l B

Temperature Hi Hi Absolute E E E E E E Temperature Hi Deviation E D D D D D Temperature Lo Deviation E D D D D D Pressure Hi Hi Absolute E E E E E E Pressure Hi Deviation 2 D D D D D Pressure Lo Deviation 2 D D D D D

pH Hi Deviation 2 D D D D D pH Lo Deviation 2 D D D D D DO Hi Deviation 2 D D D D D DO Lo Deviation 2 D D D D D

Gas Flow Hi Deviation 2 D D D D D Gas Flow Lo Deviation 2 D D D D D

Agitation Speed Hi Deviation 2 D D D D D Agitation Speed Lo Deviation 2 D D D D D

Emergency Stop E E E E E E Agitator Fault E E E E E E

Manway Opened E E E E E E

Alarm Table Notes:

1. D = Disabled, E = Enabled

2. The alarm is enabled if the associated loop is in an automatic mode (Auto or Cascade) and the loop’s PV has reached SP after transition into automatic from a manual mode (Manual or Off). The alarm is disabled whenever the loop is changed from an automati c mode to a manual mode. Alarm will always be disabled if the system is Inactive since the associated loop will then be Off. Loop alarms are disabled for 5 minutes from the start of a phase.

There are three different screens associated with alarms. The Alarm Setpoint screen (see Figure 24 below) is where all of the loops have their appropriate alarm setpoints entered. The Alarm Summary screen shows all current alarms (see Figure 25 below) and shows whether they have been acknowledged or not. The Alarm History screen shows all of the alarms that have occurred (see Figure 26 below). From this screen these alarms can be sorted in various methods.





Figure 24





Figure 25





Figure 26





5.15.4. Failure Handling

This section explains the mechanisms for handling power and communications failures.

Power Failure

If wired in such a manor an Uninterruptible Power Supply (UPS) provides temporary power to the control system in case of a facility power failure.

Prolonged loss of building power shuts down all I/O modules in the system cabinet.

When power is restored, the following occur:

• The system comes up in the mode it was in before the power failure. For example, if the system was in Active mode when the power failed, it comes up in Active mode when power is restored.

• If the system comes up in Active mode, all loops and valves are in the same state (auto/manual, on/off, and so on) that they were in when the power failed.

• The PLC system returns to the mode and state it was in before the power loss. The PLC system also has an internal battery backup unit to prevent the loss of RAM (random access memory).

• All outputs are updated appropriately for the given process conditions.

• The OIT restarts automatically.

Communications Failures

If communication between the WAGO unit and the PLC system terminates for any reason—loss of power, cable failure, or communications hardware failure—an error message is displayed on the OIT. The PLC system logic continues to execute as long as no WAGO faults are detected.

5.16. Safety and Security

This section explains the safety and security mechanisms provided for the Bioreactor system.

5.16.1. Input Value Checking

Each analog input is limited within the range acceptable to the PID instruction, 0 to 16,383 counts. Operator data entry fields on the OIT screens is checked against allowable input ranges.





CHAPTER 6.– START UP & OPERATION

6.1. General Preparation

6.1.1. Starting a batch The normal operation of the equipment will involve entering setpoints, alarms, and control modes for all loops once a batch has been started on the 1000 L Bioreactor.

6.1.2. Operation and Preventative Maintenance of Instrumentation The instrumentation and sensors provided with the Bioreactor system require periodic calibration and/or preventative maintenance to ensure proper operation. For procedures regarding operation, installation and maintenance of the devices mentioned below, consult the OEM manuals provided in the Operator’s Manual Supplement Package. pH Electrode

• Calibrate the pH electrode, preferably with buffers pH = 4 and pH = 7. For information concerning the calibration routine (procedure) please refer to the pH transmitter manual provided.

• The gel-filled pH electrode is mounted in a holder that fits in a Tri-Clamp fitting on the lower sidewall of the vessel

• For this system there are two pH electrodes and two transmitters. It will be necessary to calibrate both systems and then chose which probe and transmitter is to be used for control initially. The controlling probe and transmitter can be changed at anytime during the process.

DO Electrode

• The DO electrode is calibrated after sterilization is completed, and the Bioreactor is cooled down to cultivation temperature and growth media has been added to the Bioreactor. For information concerning the calibration routine (procedure) please refer to the DO transmitter manual provided.

• If, during previous cultivation activities, any malfunction of the DO probe was observed, the membrane module should be replaced. Information about the replacement of this can be found in the information sheets provided.

• The DO electrode is mounted in a holder that fits in a Tri-Clamp fitting on the lower sidewall of the Bioreactor vessel.

• For this system there are two DO electrodes and two transmitters. It will be necessary to calibrate both systems and then chose which probe and transmitter is to be used for control initially. The controlling probe and transmitter can be changed at anytime during the process.





Temperature, Weight and Pressure Sensors

• Unlike the pH and DO probes, these sensors are more resistant to the effects of cell culture. They only require periodic calibration and maintenance. Consult the device manuals provided for recommended procedures and maintenance schedules.

6.2. Start-up The opening screen on the OIT allows the user to choose between the two Bioreactors currently configured to operate with the system (BRX1000). See Figure 27 below for appearance of this screen.

Figure 27

The Control Screen will allow you to traverse to all of the other screens including some additional screens not provided for at the bottom of the screen on most other screens. This screen is where a Batch is started, aborted or ended. It is also the screen where the Inoculation can be recorded so that the ECT (Elapsed Culture Time) can be calculated. It is also where you time stamp the Harvest of the batch so that the PLC





can record this time and date. See Figure 28 for this screen below. The screen capture below shows all of the buttons that can appear and their location. They are not always visible if not applicable at that time.

Figure 28

There are two primary screens from which the system operations can be observed. The Main Screen (see Figure 29 below) shows the system in a fashion similar to a P&ID and shows the values for the various parameters around that screen. The second type of view is called the Overview Screen (see Figure 30 below) and shows all of the variables in a table format along with the setpoints and current mode of operation of each loop. This screen also allows for the changing of the setpoints and setting up the profiles for either Nutrient 1 or Nutrient 2.





Figure 29





Figure 30

Check all Utility connections to be sure they are correctly connected and tight

1. Make sure the following connections are made and secure: a. Instrument Air b. Process gases c. Plant Steam supply d. Clean Steam supply e. Chilled Water supply f. Chilled Water return g. Biowaste Drain h. Condensate Return i. Exhaust Gas j. All four process gases (Air, Oxygen, Nitrogen and Carbon Dioxide) k. Electrical

2. Check main supply pressures. The skid will need the supply pressures listed

earlier in this manual (see section 4.3.5) to operate properly: a. Instrument Air (Minimum of 80 psig)





b. Process Gases (Air, Carbon Dioxide, Nitrogen & Oxygen) – All 30 psig c. Plant Steam Supply – 45 psig d. Clean Steam Supply – 20 psig e. Clean Steam Supply to Seals – 45 psig f. Chilled Water Supply – 45 to 50 psig g. Chilled Water Return – 25 psig maximum backpressure h. Condensate Return – Free Draining Preferred i. Biowaste Drain – Free Draining j. Electrical – specified on Name Plate on Control Console

3. Carefully turn on all utilities for skid and check for leaks a. Tri-Clamp Connections

i. If a leak is observed at a tri-clamp connection Snug clamp - DO NOT OVERTIGHTEN. (Note: Wing-nut tri-clamp connections are meant to be hand-tightened. Do not use tools to exert additional torque.)

ii. If the tri-clamp leak persists Shut down utility and replace gasket with like gasket

b. Pneumatic Tubing Connections i. If a leak is observed at a pneumatic connection

Make sure tubing is completely inserted and connection is secure ii. If pneumatic leak persists

Shut down supply Remove tubing and reface tube end by cutting square Re-insert tubing and turn on supply

4. Check all manual valves and place in safe position. Almost always this means that the valve is turned off.

5. Turn on skid power and PLC power 6. Perform skid operation check

6.3. Skid Operation Check

1. Verify that all probes, pH, DO and temperature probes, and/or port plugs are inserted and appropriately tightened. All ports on head plate and vessel walls must be appropriately closed by proper assemblies, sensors, or blind caps.

2. Verify that harvest and sample valves are closed 3. Add approximately 800 liters of high purity water to vessel. Vessel weight is displayed

as kilograms. 4. Verify that filter housings contain properly sized filter elements 5. Close the filter housing drain and vent valves if not already closed 6. Start a batch and make sure transfer panel is set to the clean steam (CS) position. 7. Turn on power to motor drive and control agitation at 50 RPM 8. Turn on automatic temperature control with a setpoint of 30°C:

Control output should go to 100% (assuming that water is colder than 30°C) 9. Open HV-510 – Air Sparge Inlet block valve near sparge filter. 10. Turn on Air Sparge supply at 50 liters per minute:





Air flow should stabilize within two minutes at 50 slpm ± .3 slpm 11. Turn on automatic backpressure control with a setpoint of 5 psig:

Pressure should stabilize within five minutes at 5 psig ± 0.5 psig 12. Check system for leaks 13. Turn off all control loops

The reactor is now prepared for the pressure test.

6.4. Pressure Test The pressure test is performed to verify that the Bioreactor system has no leaks. Make sure that all of the preparation steps described in the previous sections are carried out. NOTE: Since the air that is forced into the Bioreactor is normally at room temperature, even a small increase in pressure can be expected after closing the air inlet valve (air will continue to expand after heating up).

1. Fill the vessel to its maximum working volume 2. Start the temperature control loop, put in a setpoint of 28°C and wait until the

temperature in the Bioreactor reaches setpoint. 3. Set the motor speed at 50 RPM 4. Set a vessel pressure on the Pressure loop to 15 psig 5. Verify that the exhaust line drain, inlet filter drain(s), and addition supply (ies) are

off/closed 6. Open air sparge inlet block valve (HV-510) if not already on. 7. Turn on airflow control and enter a setpoint of 100 SLPM. 8. Open air sparge inlet block valve (HV-510) if not already on. 9. Monitor the pressure in the Bioreactor until it reaches 15 psig; now close the air block

valve (HV-510). 10. Monitor the pressure over a time interval of approximately one hour (temperature

control must be on and stable). Control valves are not necessarily tight shut off devices so the exhaust line may be broken downstream of the control valve and a cap placed over the exhaust line outlet if necessary.

11. The test is finished successfully when, during the elapsed time, the pressure does not drop more than 0.5 psig

12. If the test fails check all the connections in order to determine the location of the leak. Repeat the pressure test again until it is completed successfully

13. Open-air supply block valve (HV-510) and remove cap from exhaust line if installed. 14. The pressure setpoint can be stepped down until it is safe to turn the pressure control

off which will open the back pressure control valve and, in turn, will allow the remaining pressure to escape





6.5. Sterilization THE VESSEL IS DESIGNED TO BE STERILIZED EMPTY. MEDIA WILL BE FILTER STERILIZED INTO THE VESSEL UTILIZING ONE OF THE FOUR-VALVE ADDITION PORT ASSEMBLIES NOTE: A pressure test is recommended before each sterilization process and should be completed before the sterilization continues. The pressure hold might also be run before CIP in order to assure that the vessel spool pieces have been hooked up properly.

6.5.1. Sterilization Procedure

1. General preparation steps:

a. Turn on all utility supplies and regulate to appropriate pressures b. Enter the SIP Settings screen on the OIT and check or change values according

to pre-determined (validated) process parameters. c. The contained addition assemblies contain four manually operated diaphragm

valves and are connected to the drain through a steam trap. During sterilization, the addition valve may be replaced with a spool piece. The addition valve, together with tubing and liquid addition bottle, is sterilized separately in an autoclave. (See connections below for this)

2. Pressure Hold – See above instructions. It is recommended to do a pressure hold test before each SIP.





3. Heat A (Rapid Heating) – Before starting the sterilization process the following manual valves should be placed in the appropriate position:

HV-207 Jacket Drain Valve Closed HV-915 Exhaust Filter Vent Valve Closed HV-711 Vessel Drain Valve (Asepco) Open HV-721 Vessel Sample Valve(Pharmenta) Open HV-716 Clean Steam Low Point Drain Open HV-510 Sparge Block Valve Closed HV-601 Seal Steam Condensate Drain Open HV-715 Vessel Drain Port Sterilize Closed HV-713 Vessel Drain Port Cond. Drain Open HV-717 Vessel Drain Port Trap Bypass Closed HV-504 Overlay Filter Condensate Drain Open HV-512 Sparge Filter Condensate Drain Open

HV-731 Media/Inoculation Addition Valve Closed (it is expected that this valve will be at the autoclave at this time with the reagent tubing and bottle. In this case flex hose should be attached directly to the Media/Inoculation Block Valve – HV-733. As an alternative a triclamp cap could be placed on the inlet toe valve HV-733 and the steam valve would be left closed until ready to attach the addition valve with reagent bottle and tubing.) (See diagram above for valve positions)

HV-733 Media/Inoculation Block Valve Open HV-732 Media/Inoculation Steam Valve Open or Closed (If

sterilizing the port as proposed above then the steam valve will remain closed until the addition valve with reagent bottle and tubing are ready to attach)

HV-734 Media/Inoculation Cond. Valve Open HV-741 Base Addition Valve Closed (See

comments above for HV-731) HV-743 Base Addition Block Valve Open HV-742 Base Addition Steam Valve Open or Closed

(See comments above for HV-732) HV-744 Base Addition Cond. Valve Open HV-751 Addition Valve #1 Closed (See

comments above for HV-731) HV-753 Addition #1 Block Valve Open HV-752 Addition #1 Steam Valve Open or Closed

(See comments above for HV-732) HV-754 Addition #1 Cond. Valve Open HV-761 Addition Valve #2 Closed (See

comments above for HV-731) HV-763 Addition #2 Block Valve Open HV-762 Addition #2 Steam Valve Open or Closed

(See comments above for HV-732) HV-764 Addition #2 Cond. Valve Open

HV-724 Vessel Sample Steam Valve Open HV-723 Vessel Sample Cond. Valve Open

HV-722 Vessel Sample Valve Closed





Assure that the transfer panel is in the clean steam position or else the sterilization will not start.

Start sterilization on the OIT by calling up the SIP Screen and pushing the

start button 4. Heat B (Sterilization) – This phase starts when the vessel temperature rises to

105 oC (or another operator determined temperature). The unit will move into Heat B automatically once the Heat B temperature is reached.

At some point after Heat B has started the exhaust filter vent valve below should

be opened for a short time:

HV-915 Exhaust Filter Vent Valve Open for short time 5. Hold (Dwell) –The controller will continue to hold the steam injection valves

open to maintain the sterilization temperature for the sterilization period (the inlet pressure clean steam regulator should have been adjusted to the correct pressure in order to hold the vessel and all of the lines attached to the correct hold temperature. The timer will begin counting down once the hold temperature has been reached and the “start cool down” button has been pushed. Should the temperature drop below the setpoint by more than a user defined difference then timer will reset for two times. After that an alarm will happen the system will abort sterilization. The condensate lines temperatures will all be logged for inspection should this occur so that it can be analyzed as to what might be causing the malfunction.

A “Start Cool down” prompt will appear at the end of this phase in order for the operator to decide whether to continue and to set the following manual valves in their proper positions:

HV-711 Vessel Drain Valve (Asepco) Close HV-715 Vessel Drain Port Steam Valve Open HV-733 Media/Inoculation Block Valve Close

HV-732 Media/Inoculation Steam Valve Open (if in place at this time)

HV-743 Base Addition Block Valve Close HV-742 Base Addition Steam Valve Open (if in place at this

time) HV-753 Addition #1 Block Valve Close

HV-752 Addition #1 Steam Valve Open (if in place at this time)

HV-763 Addition #2 Block Valve Close HV-762 Addition #2 Steam Valve Open (if in place at this

time) HV-601 Seal Steam Outlet Block Valve Close HV-721 Sample Block Valve Close

HV-724 Sample Steam Valve Open HV-504 Overlay Filter Condensate Drain Close HV-512 Sparge Filter Condensate Drain Close





6. Cool A (Rapid Cooling) –Once the “start cool down” button is pushed the

temperature control circuit is opened to allow chilled water to flow through the bioreactor jacket and exit the system via the water return connection. The rate at which this cooling occurs is controlled by pulsing the water return valve (TXV-206) at a percentage of on-time that is pre-determined but adjustable by the operator. Set the Pressure Loop to Auto and enter a setpoint of 15 to 20 psig. At the same time, air is injected into the Bioreactor overlay inlet to prevent the formation of a vacuum within the vessel. Once the vessel has cooled sufficiently the following valves should be moved to position shown:

HV-510 Sparge Block Valve Open if Sparge flow is

desired.

7. Cool B (Return to Growth Temperature) - When the vessel temperature drops to the Cool B Temperature set point (47 °C default), the sterilization temperature control circuit will close and the system is allowed to resume the standard temperature control protocol.

NOTE: Should it be necessary to stop the sterilization process at any time this may simply be accomplished by pushing the “Abort” button on the sterilization screen. The unit may also be placed in a Hold state from the SIP screen, wait until the unit cools sufficiently and the vessel has no pressure before trying to correct the problem or fixing the defective part.

6.5.2. Sterilization Troubleshooting NOTE: A “Tempil Stick™” that melts at 115°C or 121°C is a useful tool in the troubleshooting process for the sterilization cycle.

1. Make sure the clean steam supply pressure is at least 20 psig. 2. Make sure steam supply valve(s) are functioning properly 3. Make sure drain valve is open 4. Make sure all steam traps are functioning properly

6.6. Cultivation After completion of the sterilization process, the Bioreactor can be prepared for cultivation (growth). Since the vessel was sterilized empty the media must be introduced to the vessel in a sterile manner. Since the media for the Bioreactor is more than likely heat labile it must be filter sterilized instead of heat sterilization. All actions related to the process must be performed in an aseptic manner. It will now be necessary to add the media utilizing a sterile technique through one of the addition valve arrays. This media may be either filter sterilized into the bioreactor or transferred from a media vessel.

6.6.1. Preparing for Cultivation The following preparation steps should be followed:





1. Addition of sterile media from either a sterile filter or sterile media vessel. 2. A sample may be taken and the pH checked with a separate bench pH meter. If the

sample disagrees with current reading on the system pH probe then the zero may be adjusted to move the value to the correct reading.

3. Calibration of the DO electrode a. When the vessel contents (media) have reached process temperature the DO

electrode may be calibrated b. Aerate the Bioreactor for such a period that the signal of the DO electrode is

stable c. Carry out a one-point calibration (e.g. at 100% DO). For more information

concerning this calibration procedure please refer to the DO transmitter operations manual included in your system documentation package

d. NOTE: One can check the response of the electrode and gather slope information by performing a two-point calibration using Nitrogen as your zero reference gas. Also in most cases since the probe is an amperometric device the zero can be checked by simply unplugging the probe from the cable and checking the reading for a zero value.

4. Inoculate the vessel using one of the contained addition connections.

a. This procedure assumes that a peristaltic pump is to be used to make the transfer. It is also possible to simply use a pressure transfer from one container or vessel to the bioreactor. Bring the inoculum along with presterilized tubing and the addition block valve, HV-7X1 to the bioreactor. Attach the valve to the addition valve on the vessel, HV-7X3. Attach the flex hose from valve HV-7X2 to the port on the addition block valve, HV-7X1. (See diagram below)

b. Open steam valve (HV-7X2) and drain valve (HV-7X4) for at least 15 minutes

allowing steam to sterilize the transfer connection and then close these valves. c. Place appropriate peristaltic tubing into chosen inoculation pump. d. Open the addition valve, HV-7X3.





e. Open the blocking valve, HV-7X1 slowly. Make sure the drain and steam supply valves are fully closed!

f. Run pump until all of the inoculum is inside the vessel. g. When inoculation is complete, shut the pump off. Close blocking valve and

addition valve h. Open steam valve and drain valve for at least 15 minutes allowing steam to

sterilize the transfer connection i. Remove addition valve and re-sterilize if required for another addition.

5. Connect the addition bottles to the other addition ports a. Sterilize connection as in 4 above. See diagram above. b. Prime addition lines by placing the pump in “Manual”. Pump fluid until it reaches

the vessel port. 6. pH control using the contained addition assembly

a. If desired to have a totalized addition of acid and base it is necessary to calibrate the pump before proceeding.

b. Make sure the pH control is OFF c. Attach the autoclaved base bottle using the attached addition valve and steam

through the connection as described above in Item 5. d. Prime the pump by opening the addition valve and the drain valve and switching

the pump to manual mode. Stop when the liquid reaches the drain valve. Close the drain valve.

e. Slowly open the block valve

7. Sampling a. Sampling is accomplished by using the Pharmenta sampling valve system. The

system utilizes a four-valve assembly very similar to the four-valve assembly used above for inoculation.

b. Open the steam supply valve (HV-724) and the condensate drain valve (HV-723). Make sure that the ASEPCO valve and the sample outlet valve are completely closed before opening the steam supply valve.

c. Close the steam valve and the drain valve after a sufficient time for sterilization of the port.

d. There are several non-aerosol sampling devices on the market which could be attached to the sample outlet valve (HV-722) before this sterilization takes place. Some of these allow steaming through the device before taking the sample. In any case once the connection is sterile then the sample may be taken into the container by closing the steam and drain valves and opening the Pharmenta valve (HV-721) and the Sample Outlet valve (HV-722).





CHAPTER 7.– TROUBLESHOOTING The intent of this Chapter is to give guidance in the process of isolating problems. It is not intended to provide complete, detailed instructions on electrical or mechanical maintenance or repair. CAUTION: The interiors of controls enclosures may contain lethal voltages! Ensure equipment lockout/tag out procedures are instituted and followed prior to opening any enclosure! Only qualified personnel should perform maintenance on “live” circuits! Electrical Ensure all connectors are dry and tightly connected Ensure supply voltage is available at the facility source. Visually check the interior of the control panel for any illuminated blown fuse indicators Ensure the main circuit breaker is in the “ON” position Attempt to isolate the problem by system or loop Investigate any common components or subsystems that may be shared by the affected equipment. Typically, following this process will isolate the problem. If additional assistance is required please contact Dakota Systems for further support. If necessary for any reason to return to the Inactive mode without completing a CIP process (although this is not encouraged) it may be possible to do so by opening up the Bioreactor Control cabinet for the unit and pushing the momentary button that is labelled Reset near the bottom of the box.





CHAPTER 8. – CLEANING & MAINTENANCE

8.1. Cleaning

8.1.1. pH and DO Probe Cleaning and Storage Proper cleaning is essential for the working integrity of the system and is vital to avoid problems during process runs and cultivation. After ending the cultivation (the reactor content may be inactivated) the reactor is drained, remove the pH and DO electrodes and clean them thoroughly according to the manufacturer’s guidelines. Be sure to remove all remaining cell culture broth from the electrode surface. Store the pH electrode in pH 4 buffer or KCl solution. The DO electrode should be stored wet in DI or WFI water in order to avoid membrane solution evaporation. For further instruction regarding the handling of pH and DO electrodes please refer to the equipment manuals in your document package Blind the electrode ports with appropriately sized Tri-Clamp caps Normal vessel cleaning procedures are defined in Standard Operating procedures. The following is an example: § DO NOT USE BLEACH ON STAINLESS STEEL

8.1.2. Cleaning the Bioreactor interior

a. Prepare CIP skid per separate operation instructions. b. Close Clean Steam valve just upstream of the Transfer Panel and then switch position

of transfer panel spool piece. c. Place the following spool pieces into their appropriate places:

SP-01 In place of Overlay Filter – F-01 SP-02 In place of Sparge Filter – F-02 SP-07 Connect Tee on Sample Steam line (DD) to Sample Valve (HV-722). SP-XX In place of drain lines from HV-734, HV-744, HV-754 and HV-764.

There should be four of these spool pieces and all should be placed in order with the shortest one going closest to the vessel window and the others proceeding around the vessel in order of length.

d. Remove Exhaust Filter – F-03 and place triclamp cap on vessel port. e. Clean filters separately. f. Go to CIP Control button on OIT and set Path A, Path B, Path C and overlap Times.

See Figure 1 below. g. Place manual valves in proper position per the following list:

HV-731 Media/Inoculation Addition Valve Closed (if present) HV-733 Media/Inoculation Block Valve Open HV-732 Media/Inoculation Steam Valve Open





HV-734 Media/Inoculation Cond. Valve Open HV-741 Base Addition Valve Closed (if present) HV-743 Base Addition Block Valve Open HV-742 Base Addition Steam Valve Open HV-744 Base Addition Cond. Valve Open HV-751 Addition 1 Addition Valve Closed (if present) HV-753 Addition 1 Block Valve Open HV-752 Addition 1 Steam Valve Open HV-754 Addition 1 Cond. Valve Open HV-761 Addition 2 Addition Valve Closed (if present) HV-763 Addition 2 Block Valve Open HV-762 Addition 2 Steam Valve Open HV-764 Addition 2 Cond. Valve Open HV-711 Vessel Drain Valve (Asepco) Open HV-713 Vessel Drain Port Cond. Valve Closed HV-716 Clean Steam Low Point Drain Closed HV-715 Vessel Drain Port Steam Valve Opened for short time at end HV-724 Vessel Sample Steam Valve Open HV-721 Vessel Sample Valve(Pharmenta) Open HV-723 Vessel Sample Cond. Valve Closed HV-722 Vessel Sample Block Valve Open HV-510 Sparge Block Valve Closed

h. Set up various CIP screens per CIP System manual and start CIP process. i. Run system for validated amount of time to effectively clean the vessel or time required

by internal CIP cleaning protocols. j. These steps should be repeated for each of the CIP fluids that cleaning process

requires (i.e. Acid, Detergent, and rinse steps) k. Rinse vessel at least three (3) times with process water (RO, DI or WFI) with the





agitator running. l. Rinse cycle is complete when rinse water effluent has reached a pre-determined

evaluation parameter (e.g. Conductivity) m. After vessel and contents have completely drained the system may be closed off and

clean steam line returned to normal position on the transfer panel as well as removal of all spool pieces and replacement with cleaned filters.

Figure 32

8.1.3. Cleaning the Bioreactor Exterior The exterior surfaces of your Bioreactor do not require the same level of regulated cleaning as do the interior surfaces. Given that many Bioreactors are located in controlled environments, they do not accumulate dust and dirt common to other pieces of equipment. The following items are intended as guidelines for the exterior cleaning of the equipment

1. Mop up all spills of any kind on or near the equipment immediately! 2. Do NOT use any product containing bleach on stainless steel 3. Completely dry exposed surfaces with a clean, soft, dry, lint-free cloth 4. Should “dusting” of the equipment appear necessary a clean, soft, lint-free cloth may

be moistened with process water to remove particulates 5. Water spots on equipment may be removed with a clean, soft, lint-free cloth moistened





with process water

8.2. Maintenance Maintenance instructions for individual installed components can be found in their respective maintenance manuals and/or instruction sheet in the documentation package prepared for this equipment by Dakota Systems. Trained, qualified individuals familiar with the care, handling and maintenance procedures for bioprocess equipment including Bioreactors should perform general equipment maintenance. A Maintenance screen is provided on the OIT (can be reached from the Control Screen – see Figure 33 below). This screen will provide for various maintenance functions of the Panel View Plus including setup of the touch screen to allow for various height operators. If there is a significant variation in operators heights then a compromise will have to be arrived at. This screen will also give the maintenance person the status of various communications within the PLC.

Figure 33

Depending on the intended use of the equipment, facility guidelines, in-house maintenance capabilities and single or multiple product use status, maintenance requirements will vary from one piece of equipment to another. The following is a general outline of the issues requiring consideration for a regular maintenance regime:





§ Calibration - Individual component calibration should be completed on an annual basis for normal use. Equipment that is used on two shifts should consider semi-annual calibration. Equipment used on three shifts should consider quarterly calibration. The need for calibration during product changeover procedures should be considered. Items requiring calibration include, but are not limited to: Mass f low controllers, pressure gauges, pressure transmitters, temperature probes, load cell system.

§ Elastomer replacement – Bioprocess equipment systems typically have “wear components” that need replacement on a regular basis. It is recommended that al l elastomers be replaced with new, identical equipment at least once per year. Systems experiencing heavier than normal use (2 or 3 shifts daily) should consider increasing that frequency to semi-annual or quarterly replacement. The elastomers that should be considered for replacement include, but are not limited to diaphragms for diaphragm valves, o-rings, and vessel gaskets. Different elastomers may have different schedules for replacement. It is recommended that all elastomers undergo an inspection process during maintenance procedures even if they are not scheduled to be replaced.

§ Steam traps – thermostatic steam traps are used on various lines on your process equipment. Depending on the particular model number and the manufacturer these units will either be serviced or discarded and replaced. Disposable steam traps are sealed units that cannot be serviced and must be replaced. The determination to replace one of these units is made based on process experience or an operational field test. Units that are not sealed should be opened and inspected during your regularly scheduled maintenance. It is recommended this procedure be done on at least an annual basis. Units that are found to be corroded should be evaluated for replacement. Some units can be cleaned allowing correct functioning to be restored. Units that cannot be made to function correctly after cleaning should be replaced or the bellows assembly and gasket should at least be replaced on serviceable traps.

§ Filter elements/housings – there are several filters on your bioprocess skid. All filters should be inspected and evaluated for replacement during you maintenance protocol. Filter elements should be scheduled for regular replacement based on the type and location. Consideration should be given to the replacement of all disposable filter elements for each new “run” of an identical product in the equipment. It is essential that steri le filter elements be replaced between product runs for different products. All filter housings should be examined for corrosion, pitting, and the “fit” of the associated gasket or o-ring. Defective or damaged seals should be replaced with new equipment. Corroded housings should be cleaned or replaced. Pitted or otherwise defective housings should be evaluated for process impact and considered for replacement.

§ Operational inspection/verification – Several items on your Bioreactor should be checked for correct operation during a maintenance phase. These include, but are not l imited to:

Circulation Pump P-01

Agitator Seals

Pressure Regulators PCV-931, PCV-601, PCV-921

Current to Pressure Transducer PY-901

§ Review operational history with equipment operator

§ Check on and around equipment tak ing notice of active leaks or signs of leakage.

§ Check for blown or corroded fuses

§ Check for burned out l ight bulbs

§ Inspect and listen to all pneumatic tube connections to insure that there is no leakage of air. The use of bubble solution may be appropriate to determine if air is leaking. Bubble solution or any other liquid should NOT be used inside the control panels





§ Check all cables for signs of wear at “strain” points. Examine all strain-relief apparatus to insure it is performing as expected

§ Check shaft for “run out” to determine if the gearbox needs to be serviced

§ Listen to motor and gearbox for normal operation sounds

§ Check gearbox to be sure it is full of oil and contamination free. Water or other contaminants in the gearbox will damage the gearbox and may signal problems with the gearbox or mechanical seals

§ Operational checkout – the final step in any good maintenance program is the operational checkout. The Digital Forces Screen may be used to assure that each digital device turns on and off properly. When you leave this screen all devices will be returned to their automatic position (which may be OFF if not running a process). See Figure 34 for the bioreactor and Figure 35 for the CIP skid for the appearance of this screen). This step insures that the equipment is operational when maintenance is complete and insures that the maintenance process itself has not caused equipment failure in some way. While the items included in this step are set by the owner, the following are typical:

§ Run the steri lization cycle. Be sure the cycle completes without incident, variation or failure.

§ Use the control system to set various process parameters; evaluate system response § Physically check system for air or liquid leaks during test operation. § Listen to motor and gearbox for correct operational sounds





Figure 34





Figure 35 Tuning of various loops of the Bioreactor and the CIP skid

There are two screens provided for entering the P, I and D values for the various loops within the system. These screens also allow for the setting of any deadbands that are desired for any loop and for the PWM period for the temperature loop. Suggested values will be provided for these in a separate table. Figure 36 shows this screen for the bioreactor and Figure 37 shows it for the CIP skid.





Figure 36

Double Mechanical Seal Assembly and Maintenance

1. If the seal assembly is already installed it can be extracted via opening the vessel and removing the upper part of the shaft (it is a left hand thread so it will unscrew in a clockwise direction). A bar maybe placed through the hole at the top of the shaft in order to provide some leverage to break the two parts of the shaft apart. Remove the upper shaft and impellor and then at the bottom of the vessel the 4” triclamp fitting may be loosened in order to lower the assembly from the vessel. It will be good if the shaft seal and gearbox assembly is supported in some fashion in order to prevent it from falling to the floor when the triclamp is loosened. The disassembling process is the reverse of the following steps which were written as if the assembly is already disassembled.

2. Place the gearbox on blocks high enough to be able to get a hand under the hollow bore end. Make sure blocks are stable so that when assembly is completed it will not fall over. Refer to drawing 14BD5300.





3. Place the snap ring (item 7) onto the lower portion of the lower shaft (item 4). Then place the lower shaft and snap ring through the gearbox and at the same time installing the key (item 9).

4. Thread in the 3/8" bolt (item 10) with the washer (part of gearbox) into the bottom of the shaft and tighten until snap ring is tight against top of gear box bearing. This should lock the bottom shaft into place within the gear box.

5. Place the seal housing assembly (item 2) over the shaft and line up the holes with the gearbox holes. Install the four ¼” cap screws (item 11) and lock washers (item 8) that hold the seal assembly and the gearbox together.

6. Install and tighten the six (6) set screws that lock the seal assembly onto the shaft.

7. Move this assembly under the vessel drive opening if the assembly is ready to be installed at this time. If not then proceed to step 15 in order to assure that the upper shaft fits properly.

8. Place the o-ring (item 6) on the upper part of the shaft (item 3) above where it will actually reside in its final resting place. The o-ring will be stretched more than normal even when it is in its final place just to assure it is not pushed out of the groove that will exist between the shafts when they are screwed together.

9. Place the upper shaft into the vessel and slide it through the agitator port if the vessel is available and it is ready to be assembled into the vessel.

10. Screw the two shafts together until they stop.

11. Unscrew the shafts a small amount and slide the o-ring (item 6) down into the groove that exists between the shafts. Then retighten the shafts using a bar through the hole in the upper part of the shaft (this step can be done after mounting the assembly into the vessel if it is easier to reach the hole with the bar, but it would probably be good to see what happens to the o-ring when the shafts are screwed together to assure it does not come out of the groove and it seals well). The action of the agitator once it is rotating will tend to tighten the left hand thread.

12. Mount the whole assembly upward into the vessel port if the vessel is available using the 4” sanitary clamp such that the outlet steam connections are properly located. The assembly and the port should be match marked (with punch pin) so that it will be possible to always locate it properly.

19. Attach steam condensate inlet and outlet lines and tighten their clamps.

20. For disassembling from the vessel proceed with above instructions in reverse order.





CHAPTER 9.–PLC OPERATION

9.1. Operation The PLC system is supplied with its own operation manual and should be considered an integral part of this manual. Become familiar with all sections of the PLC manual in order to adequately operate the bioreactor.

9.2. Hardware The PLC hardware consists of the following items: Description Item Number Configuration Location 405TX N-Tron 5-port Ethernet

Switch PLC Cabinet

NTPS-24-1.3 N-Tron Ethernet Power Supply

PLC Cabinet

1747-L552 Allen Bradley SLC 5/05 PLC Processor, 32 K Memory, Ethernet Communications

PLC Cabinet

1746-P1 Allen Bradley Power Supply, SLC 500, 120/240 VAC, 2 Amps

PLC Cabinet

SST-PFB-SLC Daniel Woodhead Profibus Module for SLC 500

PLC Cabinet

1746-A4 Allen Bradley SLC 500 – 4 Slot Chassis

PLC Cabinet

750-333 WAGO, Profibus Fieldbus Coupler

Each Bioreactor Console and CIP Skid Console





9.3. Software The development of the PLC system and Data Logging application used the following software (software not necessarily supplied with system): Description Emerson Item Number Configuration Software Description RSLogix 500 Version 6.00 Rockwell

Programming Software

For the programming and documentation of the custom PLC real-time control logic

RSView Studio Version 3.20 Rockwell Programming Software

For programming and developing screens for the PanelView HMI’s.

Wonderware Active Factory

Version 9.1 Invensys Data Collection Software

For collecting data from the PLC and

operator ˇs manual brx1000 (1000-liter working volume ... · systems and must be kept...

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