NEW BRUNSWICK SCIENTIFIC BioFlo 4500 User manual

BioFlo 4500
Benchtop
Fermentor/Bioreactor
MANUAL NO: M1254-0050
Revision H
September 27, 2002
NEW BRUNSWICK SCIENTIFIC CO., INC.
BOX 4005 •44 TALMADGE ROAD •EDISON, NJ 08818-4005
Telephone: [phone] •1-800-631-5417
Fax: [phone] •Telex: 4753012 NBSCO
Internet: http://www.nbsc.com •E-mail: [email protected]

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INTERNATIONAL OFFICES:
THE NETHERLANDS
New Brunswick Scientific BV
Kerkenbos 1101, 6546 BC Nijmegen
P.O Box 6826, 6503 GH Nijmegen
Holland
Tel: [phone]
Fax: [phone]
E-mail: [email protected]
BELGIUM
New Brunswick Scientific NV/SA
’t Veldeke 1
B-1970 Wezembeek-Oppem
België/Belgique
Tel: [phone]
Fax: [phone]
E-mail: [email protected]
FRANCE
New Brunswick Scientific SARL
3, rue des Deux-Boules
75001 Paris
France
Tel: [phone]
Fax: [phone]
E-mail: [email protected]
GERMANY
New Brunswick Scientific GmbH
In Der Au 14
D-72622 Nürtingen
Deutschland
Tel: [phone]
Fax: [phone]
E-mail: [email protected]
UNITED KINGDOM
New Brunswick Scientific (UK) Ltd.
Edison House, 163 Dixons Hill Road
North Mymms, Hatfield, Herts. AL9 7JE
United Kingdom
Tel: [phone]
Fax: [phone]
E-mail: [email protected]
CHINA
New Brunswick Scientific Co., Inc.
9A. Ai Bang Building
585 Ling Ling Road
Shanghai 200030, P.R. China
Tel: [phone]
Fax: [phone]
E-mail: [email protected]

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CAUTION!
This equipment must be operated as described in this
manual. If operational guidelines are not followed,
equipment damage and personal injury can occur.
Please read the entire User’s Guide before attempting to
use this unit.
Do not use this equipment in a hazardous atmosphere or
with hazardous materials for which the equipment was not
designed.
New Brunswick Scientific Co., Inc. (NBS) is not responsible
for any damage to this equipment that may result from the
use of an accessory not manufactured by NBS.

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Copyright Notice
New Brunswick Scientific Company, Inc.
Box 4005, 44 Talmadge Road
Edison, New Jersey 08818-4005
Copyright 2002 New Brunswick Scientific Co., Inc.
All Rights Reserved.
Reproduction, adaptation, or translation without prior written permission from
New Brunswick Scientific is prohibited.
Disclaimer Notice
New Brunswick Scientific Co., Inc. reserves the right to change
information in this document without notice. Updates to information in this
document reflect our commitment to continuing product development and
improvement.
Manual Conventions
NOTE:
CAUTION!
WARNING!
Bold Text in bold face type emphasizes key words or
phrases.
Caution messages appear before procedures
which, if caution is not observed, could result
in damage to the equipment.
Notes contain essential information that
deserves special attention.
Warning messages alert you to specific
procedures or practices which, if not followed
correctly, could result in serious personal
injury.

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Every Instrument manufactured by the New Brunswick Scientific
Co., Inc. is warranted to be free from defects in material and work-
manship. This apparatus with the exception of glassware, lamps and
electrodes (where supplied), is warranted for 1 year against faulty com-
ponents and assembly and our obligation under this warranty is limited
to repairing or replacing the instrument or part thereof, which shall
with-in 1 year after date of shipment, prove to be defective after our
examination. This warranty does not extend to any NBS products
which have been subjected to misuse, neglect, accident or improper
installation or application; nor shall it extend to products which
have been repaired or altered outside the NBS factory
without prior authorization from the
New Brunswick Scientific Co., Inc.
W A R R A N T Y

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FERMENTOR/BIOREACTOR
INFORMATION SHEET
On this page, record the information for your Fermentor/Bioreactor and retain this for future
reference.
Model Number:
Voltage:
Serial Number:
Controller Type:
The above information can be found on the electrical specification plate on the right side
panel of the console.
Purchased with the following installed options:

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CONTENTS
1OVERVIEW........................................................................................................................19
1.1 FEATURES ...................................................................................................................... 20
1.1.1 Vessels and Ports .................................................................................................. 20
1.1.2 Piping and Instrumentation .................................................................................. 20
1.1.3 Utilities.................................................................................................................. 20
1.1.4 Optional ML-6100 Controller .............................................................................. 21
1.1.5 Other Options........................................................................................................ 21
1.2 SPECIFICATIONS ............................................................................................................. 22
2BIOFLO 4500 FEATURES................................................................................................ 27
2.1 VESSEL .......................................................................................................................... 27
2.1.1 Viewing Window ................................................................................................... 28
2.1.2 Vessel Jacket ......................................................................................................... 28
2.2 VESSEL AND JACKET PENETRATIONS ............................................................................. 29
2.2.1 Standard Headplate .............................................................................................. 29
2.2.2 Optional Headplate Equipment ............................................................................ 30
2.2.3 Standard Vessel Upper Side Wall......................................................................... 30
2.2.4 Optional Vessel and Vessel Upper Side Wall Equipment..................................... 31
2.2.5 Standard Lower Side Wall .................................................................................... 31
2.2.6 Optional Lower Side Wall Equipment .................................................................. 31
2.2.7 Vessel Bottom: ...................................................................................................... 32
2.3 PROCESS PIPING & COMPONENTS .................................................................................. 32
3INSPECTION, VERIFICATION & UNPACKING OF EQUIPMENT ....................... 33
3.1 INSPECTION OF BOXES ................................................................................................... 33
3.2 PACKING LIST VERIFICATION ........................................................................................ 33
3.3 PHYSICAL LOCATION ..................................................................................................... 33
3.4 ENVIRONMENT............................................................................................................... 33
4GETTING STARTED ........................................................................................................ 35
4.1 POSITIONING THE FERMENTOR....................................................................................... 35
4.2 EXTERNAL COMPUTER SET-UP ...................................................................................... 36
4.3 COMPUTER CONNECTIONS ............................................................................................. 38
4.4 ALARMS......................................................................................................................... 38
4.5 BIOCOMMAND CONNECTIONS ....................................................................................... 39
4.6 ANALOG INPUTS ............................................................................................................ 40
4.7 EXTERNAL PUMP CONNECTIONS.................................................................................... 41
5ASSEMBLING THE FERMENTOR/BIOREACTOR ................................................... 43
5.1 BAFFLES ........................................................................................................................ 43
5.2 IMPELLERS ..................................................................................................................... 43
5.2.1 Rushton Impeller................................................................................................... 43
5.3 PLACING THE VESSEL ON THE CONSOLE ........................................................................ 44
5.4 HEADPLATE & MOTOR INSTALLATION .......................................................................... 45

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5.5 SERVICE/UTILITY CONNECTIONS ................................................................................... 46
5.5.1 Steam Supplies ...................................................................................................... 50
5.5.2 Pure Steam............................................................................................................ 50
5.5.3 Utility Steam.......................................................................................................... 50
5.5.4 Exhaust.................................................................................................................. 50
5.5.5 Electrical............................................................................................................... 51
5.5.6 Drain..................................................................................................................... 51
5.5.7 Water & Water Return.......................................................................................... 52
5.6 PREFILTER & REGULATOR KITS (OPTIONAL)................................................................. 52
6PREPARATION ................................................................................................................. 53
6.1 INITIAL PREPARATION.................................................................................................... 53
6.2 WATER PRIMING ............................................................................................................ 54
6.3 CALIBRATING THE TOUCHSCREEN ................................................................................. 55
6.4 HARVEST/DRAIN VALVE ............................................................................................... 56
6.4.1 Asceptic Harvest/Drain Method A........................................................................ 57
6.4.2 Asceptic Harvest/Drain Method B........................................................................ 58
7STARTING THE SYSTEM............................................................................................... 61
7.1 START-UP...................................................................................................................... 61
7.2 DISPLAY GAUGE ............................................................................................................ 63
8ML-6100 CONTROLLER CONFIGURATION ............................................................. 65
8.1 CONFIGURING THE CONTROLLER .................................................................................. 65
8.2 SYSTEM INFORMATION .................................................................................................. 66
8.3 CONTROL MODE SELECTION AND NODE NUMBER ......................................................... 67
8.4 PASSWORDING AND LOCKING ........................................................................................ 69
8.5 SCREENSAVER OPTION................................................................................................... 72
9ML-6100 CONTROL LOOP OPERATIONS.................................................................. 73
9.1 ADDING A NEW LOOP TO THE DISPLAY ......................................................................... 73
9.2 CONFIGURING CONTROL LOOPS..................................................................................... 74
9.3 DELETING LOOPS ........................................................................................................... 81
10 ML-6100 TEMPERATURE CONTROL SYSTEM.................................................... 83
11 ML-6100 AGITATION CONTROL SYSTEM............................................................ 85
11.1 OPERATION .................................................................................................................... 86
11.2 DOUBLE MECHANICAL SEAL (OPTIONAL) ..................................................................... 87
12 ML-6100 AIR CONTROL SYSTEM............................................................................ 89
12.1 VESSEL PRESSURE ......................................................................................................... 90
12.2 EXHAUST GAS TREATMENT ........................................................................................... 91
12.2.1 Exhaust Air (Gas) Filters: .................................................................................... 91
12.3 OPERATION OF SPARGE SYSTEM .................................................................................... 92
13 ML-6100 TWO-, THREE- & FOUR-GAS CONTROL.............................................. 93
13.1 SETTING UP THE TWO-, THREE- OR FOUR-GAS MIXER OPTION .................................... 94

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13.2 OXYGEN ENRICHMENT .................................................................................................. 95
13.2.1 Set-Up Summary ................................................................................................... 95
13.2.2 Set-Up Procedure.................................................................................................. 95
13.3 TWO-GAS..................................................................................................................... 101
13.3.1 Control Action..................................................................................................... 101
13.3.2 Control Procedure .............................................................................................. 102
13.4 THREE-GAS.................................................................................................................. 103
13.4.1 Control Action..................................................................................................... 103
13.4.2 Control Procedure .............................................................................................. 103
13.5 FOUR-GAS ................................................................................................................... 104
13.5.1 Control Action..................................................................................................... 104
13.5.2 Control Procedure .............................................................................................. 104
13.6 VIEWING GAS MIX STATUS ......................................................................................... 105
14 ML-6100 PUMP CONTROL ....................................................................................... 107
14.1 PUMP CAPACITY .......................................................................................................... 108
14.2 TUBING SELECTION...................................................................................................... 108
14.3 LOADING TUBING INTO PUMP HEAD ............................................................................ 109
14.4 PUMP CALIBRATION..................................................................................................... 110
15 ML-6100 FOAM CONTROL ...................................................................................... 115
15.1 CHEMICAL FOAM CONTROL SYSTEM WITH PERISTALTIC PUMP ................................... 115
15.2 SETUP OF FOAM PROBE................................................................................................ 116
15.3 HIGH FOAM SAFETY SYSTEM....................................................................................... 117
15.4 PUMP 3 – ANTIFOAM SETUP ........................................................................................ 120
15.5 CONDUCTANCE PROBE CALIBRATION.......................................................................... 121
15.6 CONDUCTANCE PROBE GROUNDING ............................................................................ 122
16 ML-6100 PH CONTROL............................................................................................. 123
16.1 CALIBRATION OF PH PROBE......................................................................................... 124
16.1.1 Liquid-Filled Probes........................................................................................... 124
16.1.2 Gel-Filled Probes................................................................................................ 125
16.1.3 Calibration.......................................................................................................... 125
16.1.4 Recalibration After Sterilization......................................................................... 127
16.2 SETTING UP A CASCADE FROM PH TO PUMPS .............................................................. 128
17 ML-6100 DO CONTROL............................................................................................. 131
17.1 CALIBRATION OF DO PROBE ....................................................................................... 132
17.1.1 Electronic Method to Calibrate DO Zero Point ................................................. 133
17.1.2 Sparging with Nitrogen to Calibrate DO Zero Point ......................................... 134
18 ML-6100 LEVEL CONTROL ..................................................................................... 135
18.1 SENSOR ........................................................................................................................ 135
18.2 CONTROL ..................................................................................................................... 135
19 ML-6100 NUTRIENT CONTROL.................................................................................. 137
20 SAVING THE ML-6100 CONTROLLER CONFIGURATION ............................. 139

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20.1 INTERNAL FLOPPY DRIVE ............................................................................................ 140
20.2 SAVING A CONFIGURATION.......................................................................................... 141
20.3 LOADING A CONTROLLER CONFIGURATION................................................................. 142
21 ML-6100 CASCADE CONTROL ............................................................................... 145
21.1 CREATING A CASCADE................................................................................................. 145
21.2 CONTROLLING DISSOLVED OXYGEN (DO) USING A CASCADE .................................... 147
22 ML-6100 TREND BUTTON........................................................................................ 149
22.1 CREATING A TREND GRAPH ......................................................................................... 149
23 ML-6100 GAUGE BUTTON ....................................................................................... 153
24 ML-6100 ALARMS BUTTON..................................................................................... 155
24.1 SETTING ALARMS ........................................................................................................ 155
24.1.1 Alarm Mode ........................................................................................................ 156
24.1.2 Visual & Audible Alarms .................................................................................... 157
24.2 ALARM SHUTDOWN ..................................................................................................... 157
24.3 RESPONDING TO ALARMS ............................................................................................ 158
24.3.1 Acknowledging the Alarm ................................................................................... 159
25 ML-6100 DEADBAND FEATURE ............................................................................. 161
26 ML-6100 USE WITH BIOCOMMAND ...................................................................... 163
27 PREPARING FOR ML-6100-CONTROLLED FERMENTATION....................... 165
27.1 SYSTEMS CHECK.......................................................................................................... 165
27.2 ADDING MEDIA TO THE VESSEL................................................................................... 166
27.3 STERILIZATION OF VESSEL USING HEAT-LABILE MEDIA............................................. 167
28 ML-6100-CONTROLLED AUTOMATIC STERILIZATION................................ 169
28.1 STERILIZATION (WITH LIQUID IN VESSEL) ................................................................... 169
28.1.1 Resetting Sterilization Parameters ..................................................................... 171
28.2 VALVE SEQUENCING.................................................................................................... 172
28.3 SHUTDOWN DURING STERILIZATION ........................................................................... 175
28.4 ENABLING STERILIZATION ........................................................................................... 176
28.5 ABORTING STERILIZATION........................................................................................... 176
28.6 STERILIZATION (VESSEL EMPTY)................................................................................. 178
29 INOCULATION & ADDITION VALVE (OPTIONAL).......................................... 179
29.1 STERILIZATION ............................................................................................................ 179
29.2 ADDITION & INOCULATION ......................................................................................... 180
29.2.1 Method A............................................................................................................. 180
29.2.2 Method B............................................................................................................. 181
30 SAMPLING VALVES (OPTIONAL)......................................................................... 183
30.1 STERILIZATION ............................................................................................................ 184
30.2 SAMPLING .................................................................................................................... 184

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30.2.1 Method A............................................................................................................. 184
30.2.2 Method B............................................................................................................. 184
31 SEPTUM PORTS (OPTIONAL) ................................................................................ 187
31.1 HEADPLATE SEPTUM PORT KIT ................................................................................... 187
31.2 SYRINGE PORT ASSEMBLY........................................................................................... 188
31.2.1 Luer Lok Syringe Adapter................................................................................... 189
31.3 OPTIONAL HOSE BARB PORTS ..................................................................................... 191
31.3.1 Hose Barb Addition Port Options....................................................................... 192
32 SHUTDOWN PROCEDURE ...................................................................................... 193
33 POST-RUN VESSEL CLEANING PROCEDURE................................................... 195
34 PREVENTIVE MAINTENANCE............................................................................... 197
34.1 CLEANING.................................................................................................................... 197
34.2 VESSEL REMOVAL ....................................................................................................... 197
34.3 THREE-MONTH INTERVALS ......................................................................................... 197
34.4 ONE-YEAR INTERVALS ................................................................................................ 198
34.5 AIR FILTER, INLET & EXHAUST ................................................................................... 199
34.6 BATTERY REPLACEMENT ............................................................................................. 200
34.7 MOTOR REMOVAL ....................................................................................................... 201
34.8 MOTOR INSTALLATION ................................................................................................ 202
35 TROUBLESHOOTING ............................................................................................... 203
35.1 TROUBLESHOOTING GUIDE .......................................................................................... 203
36 REPLACEMENT PARTS & ACCESSORY INFORMATION .............................. 205
36.1 DESCRIPTIONS & PART NUMBERS ............................................................................... 205
37 GLOSSARY................................................................................................................... 209
38 DRAWINGS .................................................................................................................. 211
38.1 CONTROL SCHEMATICS................................................................................................ 211
38.2 LIST OF DRAWINGS ...................................................................................................... 220
39 APPENDIX A: CORROSION RESISTANCE TABLES......................................... 223
40 APPENDIX B: GENERAL CHARACTERISTICS OF EPR.................................. 231
41 APPENDIX C: STANDARD PID VALUES ............................................................. 233
42 APPENDIX D: ADVANCED CONFIGURATION SETTINGS............................. 235
43 APPENDIX E: FOUR-GAS MIX ALGORITHM.................................................... 237
44 INDEX............................................................................................................................ 239

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1
1 O OV
VE ER RV VI IE EW W
The BioFlo 4500 fermentation system is an automatically sterilizable-in-place
fermentor/bioreactor. It consists of a console and a stainless steel fermentation
vessel. The console connects to all required services (air, gases, water, steam,
electricity, and drain). The vessel is ASME-Coded with ports and wells for the
insertion all the required sensors; to provide agitation and aeration; and to permit the
heating and cooling necessary for in-place sterilization of all process components
and to maintain growth conditions.
In a typical fermentation run, the fermentor/bioreactor is:
• filled through the filling port on the headplate
• raised to sterilization temperature while the contents of the vessel are being
agitated
• held at sterilization temperature for an operator-determined time period
• cooled to operating temperature
• maintained at fermentation temperature, pressure and sparge rate for an operator-
determined period
• harvested
• shut down.
Aseptic addition of culture medium and inoculum, as well as aseptic sampling, may
be accomplished by the use of the steam-sterilizable inoculation and sampling ports.
The BioFlo 4500 fermentation system is easily configured to meet the wide range
of culture requirements and operation modes for batch, fed batch, continuous
culture and perfusion processes. The BioFlo 4500 offers the versatility and
flexibility necessary to meet the complex demands of a wide variety of processes
for the culture of bacteria, fungi and yeast, as well as plant, insect, animal and
mammalian cells.
The versatility of the BioFlo 4500 is derived from its state of the art ML-6100
instrumentation. This instrumentation package may be configured as an
imbedded controller with touchscreen interface, a remote stand-alone system, or
can be controlled by third-party SCADA systems.
The ML-6100 allows a simple, intuitive user interface for operation and system
configuration. This allows the fermentor/bioreactor to be reconfigured in minutes
to meet virtually any control need. In addition, the incorporation of the ML-6100
in the BioFlo 4500 allows for real time interfacing with ancillary instrumentation.

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1.1 Features
1.1.1 Vessels and Ports
• Jacketed vessel with hemispherical bottom dish
• Four removable baffles
• Four built-in cartridge heaters in vessel jacket
• Rectangular viewing window
• Combined filling & light port on headplate
• Re-sterilizable poppet-type drain/harvest valve with quick-connect
fitting
• Four upper vessel wall 19mm Ingold-type ports
• Two 19 mm port in lower vessel wall, one with thermowell
• Three 25 mm ports in lower vessel wall for optional pH and DO
probes
1.1.2 Piping and Instrumentation
• Automatic sterilization in place (SIP) of vessel and all process-
related components
• Embedded NBS ML-6100 multi-loop process controller or third-
party SCADA system
• All related process components and piping are manufactured from
316L stainless steel
• Variable speed, top-entering agitation system
• SIP exhaust filter with upstream heat exchanger and inspection glass
• SIP inlet filter
• One switched auxiliary power outlet for remote fixed speed
peristaltic pump connection and control
• Thermal mass flow controller
• Three analog inputs for analyzers or other external devices
• Minimum 350 mMoles/L /hr oxygen transfer rate (OTR)
• Capable of minimum 1°C/minute temperature rise between 30°and
45°C
1.1.3 Utilities
• Connections for water, air, gases, steam and drain are provided on
either side of the base