Sartorius BioPAT Trace User manual

Operating Instructions
BioPAT®Trace | Multi Trace
2- | 2 × 4-Channel Online Analysis System
85032-541-53

BioPAT®Trace | Multi Trace Operating Instructions 3
Contents
Contents
1 About this Document ....................................................8
1.1 Introduction .........................................................8
1.2 Validity...............................................................8
1.3 Target Groups .......................................................9
1.4 Symbols Used.......................................................10
1.4.1 Warnings in Operation Descriptions ........................10
1.4.2 Other Symbols Used ........................................10
2 Safety Instructions .......................................................11
2.1 Intended Use ........................................................11
2.2 Personnel Qualification..............................................11
2.3 General Safety Instructions.........................................12
2.3.1 Electrical Power .............................................12
2.3.2 Energy Release..............................................12
2.3.3 Accessories, Consumables, and Spare Parts ................13
2.4 Conduct in an Emergency ..........................................13
3 BioPAT®Trace | Multi Trace Product Description........................14
3.1 The Basics of Electrochemical Measuring Technology .............15
3.2 Biosensor Principle .................................................16
3.3 Fluidic System and Sampling .......................................17
3.4 Data Transmission and Data Saving................................20
3.5 Equipment Supplied with the BioPAT®Trace | Multi Trace.........20
3.6 Consumables .......................................................21
3.6.1 BioPAT®Trace | BioPAT®Multi Trace Tubing Set.............21
3.6.2 Buffer Solution ..............................................21
3.6.3 Cleaning Solution ...........................................21
3.6.4 Calibration Standard Solutions (contents: 0.5 liters) ........22
3.6.5 Membranes .................................................23
3.7 Sampling Probes....................................................23
3.7.1 Dialysis Probe ...............................................23

4 BioPAT®Trace | Multi Trace Operating Instructions
Contents
3.8 Accessories for Sampling Probes ...................................24
3.8.1 Accessories for Dialysis Probe...............................24
3.8.2 Accessories for Dialysis Probe (1st generation,
no integrated Luer-Lock connection) ...................... 25
3.9 Accessories for the BioPAT®Trace | Multi Trace.................... 25
4 Design and Function ....................................................26
4.1 Setting Up the Device ............................................. 26
4.2 BioPAT
®Trace | Multi Trace Measurement Methods............... 29
4.2.1 Dialysis Measuring Method Using the Dialysis Probe ......30
4.3 Conductivity Cell Function .........................................33
4.4 Particle Filter Function............................................. 34
4.5 Diffusion Module Function ........................................ 35
4.6 Dialysis Probe Function............................................ 36
4.7 Enzyme Reactor Function ..........................................37
4.8 Temperature Correction............................................37
5 Transport................................................................38
6 Initial Installation........................................................39
6.1 Setup .............................................................. 39
6.1.1 Device Setup ............................................... 39
6.1.2 Tubing Set Connectors on the Device.......................41
6.1.3 Attaching the Tubing Set for the “Dialysis”
Operating Mode ...........................................46
6.1.4 Installing theTubing Set for the BioPAT®Multi Trace.......54
6.1.5 Connection and Removal of the Enzyme Reactor..........54
6.2 Connecting the Dialysis Probe.....................................56
6.2.1 Mounting the Membrane in the Dialysis Probe .............57
6.2.2 Mounting the Membrane for MeasuringMethanol and
Ethanol in the Dialysis Probe ...............................58
6.2.3 Installing the Dialysis Probe into theBioreactor............ 59
6.2.4 Sterilization of the Dialysis Probe Prior to Connection ..... 59
6.2.5 Connecting the Dialysis Probe to the Tubing Set............61
6.3 Cleaning the Dialysis Probe and Replacing theMembrane........ 62
6.4 Loading the Software and Network Connection....................63
6.4.1 Troubleshooting............................................66

BioPAT®Trace | Multi Trace Operating Instructions 5
Contents
6.5 Installing the Software .............................................66
6.6 Changing the Start Settings ........................................67
6.7 Active Access Restriction | Log in..................................68
6.8 Assignment of Analog Outputs.................................... 69
6.9 Checklist for Starting-up Operation ............................... 70
7 Startup..................................................................73
7.1 Startup..............................................................73
7.2 Decommissioning...................................................74
8 Operation...............................................................75
8.1 Basic Commands ...................................................78
8.1.1 Start .........................................................78
8.1.2 Standby .....................................................78
8.1.3 Calibrate ....................................................78
8.1.4 Single Sample ...............................................79
8.2 Reference (for Dialysis only) ........................................79
8.2.1 Fast Reference (Only for Dialysis) .......................... 82
8.3 Menu Bar ...........................................................83
8.3.1 Menu Item “Device”........................................84
8.3.2 Menu Item “Display” ........................................87
8.3.3 Print Diagram ...............................................87
8.3.4 Calibration Values ..........................................88
8.3.5 Calibration History .........................................88
8.3.6 Status.......................................................88
8.3.7 Settings ....................................................89
8.3.8 Menu “Schedule”........................................... 96
8.3.9 Menu “Controller”.........................................100
8.3.10 Menu Item “Log in”........................................105
8.3.11 Menu Item „Information“..................................105
8.3.12 Data Transfer via TCP/IP...................................108
8.4 LED Status Messages.............................................109

6 BioPAT®Trace | Multi Trace Operating Instructions
Contents
8.5 Tips ................................................................110
8.5.1 Choosing Suitable Standard Solutions .....................110
8.5.2 Dialysis Mode: Referencing at the Beginning
of a Fermentation ...........................................111
8.5.3 Dialysis Mode: Referencing at Low Lactate
Concentrations..............................................111
8.5.4 Avoiding Contaminations in the Tubing Set ................112
8.5.5 Import Data to Excel .......................................113
8.5.6 Data Transfer to Excel Using an ExcelMacro...............115
9 Maintenance and Servicing ............................................116
9.1 Cleaning ...........................................................116
9.2 Decontaminating theTubing Set ..................................116
9.2.1 Contamination by Biofilm ..................................116
9.2.2 Causes of Contamination ..................................117
9.2.3 Decontaminating Connection Lines .......................117
9.2.4 Decontaminating the Transport Buffer .....................117
9.2.5 Decontaminating theTubing Set...........................117
9.2.6 Disposal Line | Disposal Tank ...............................118
9.2.7 Probe Sterilization..........................................118
9.3 Maintenance and Servicing Activities..............................118
9.3.1 Device......................................................118
9.3.2 Tubing Set..................................................118
10 Malfunctions...........................................................119
10.1 Error messages ....................................................119
10.1.1 Calibration Error........................................... 120
10.1.2 Calibration Implausible.................................... 120
10.1.3 Sensor Error................................................121
10.1.4 Error in Control ............................................121
10.2 Error and Possible Causes .........................................122
10.3 Error Message: “Calibration Failed”................................124
11 Transport...............................................................126

BioPAT®Trace | Multi Trace Operating Instructions 7
Contents
12 Storage and Shipping ..................................................127
12.1 Storage ............................................................127
12.2 Returning the Device and Parts....................................127
13 Disposal................................................................128
13.1 Information on Decontamination..................................128
13.2 Decommissioning the Device......................................128
13.3 Disposing of the Device and Parts .................................129
13.3.1 Information on Disposal ....................................129
13.3.2 Disposal ....................................................129
14 Technical Data .........................................................130
14.1 Measuring Data...................................................130
14.2 Dimensions and Weights .........................................130
14.3 Installation Conditions.............................................131
14.3.1 Ambient Conditions........................................131
14.3.2 Footprint ...................................................131
14.3.3 Storage Conditions.........................................131
14.4 Electrical Data .....................................................132
14.4.1 Power Supply...............................................132
14.4.2 Interfaces...................................................132
15 Conformity Documents ................................................133
16 Licensing or Trademark Information ...................................136

8 BioPAT®Trace | Multi Trace Operating Instructions
About this Document
1 About this Document
1.1 Introduction
These instructions provide all the information necessary for operation of the
BioPAT®Trace | Multi Trace (hereinafter referred to as the device).
The instructions must be read, understood and used by all personnel using
the device.
t Before working with the device: Read the instructions carefully and
completely.
t Keep them in a safe and easily accessible place near the device’s site of
installation.These instructions are part of the device.
t If the instructions are lost: Request a replacement or download the latest
instructions from ourwebsite.
Masculine or feminine forms are used to facilitate legibility in these
instructions and always simultaneously denote the other gender as well.
1.2 Validity
These instructions apply to BioPAT®Trace | Multi Trace in the following
versions:
Device Type
BioPAT®Trace BPT0001
BioPAT®Multi Trace BPT00M
In general, these instructions apply to both devices (BioPAT®Trace and
BioPAT®Multi Trace).The procedural steps describe the standard tasks for
the BioPAT®Trace. Differences or deviating procedural steps for the BioPAT®
Multi Trace will be marked as such.

BioPAT®Trace | Multi Trace Operating Instructions 9
About this Document
1.3 Target Groups
These instructions are designed for the following target groups.The target
groups must possess the knowledge listed.
Target group Knowledge | responsibilities
User The user is familiar with the operation of the device and
the associated work processes. He knows the dangers
that can occurwhen working with the device and can
avoid these dangers.
The user has been trained in the operation of the
device.
The training takes place within the scope of startup and
is carried out by the operating engineer | laboratory
manager or the operator of the device.
Operator The operator of the device is responsible for compliance
with safety requirements and workplace safety
regulations.
The operator must ensure that all persons who work
with the device have access to the relevant information
and have been instructed in work with the device.

10 BioPAT®Trace | Multi Trace Operating Instructions
About this Document
1.4 Symbols Used
1.4.1 Warnings in Operation Descriptions
WARNING
Denotes a dangerwith risk that death or severe injury may result if it is
notavoided.
CAUTION
This symbol denotes a possible danger with risk that moderate or minor injury
may result if it is not avoided.
NOTICE
This indicates a hazard with a low risk which could result in property damage if
not avoided.
1.4.2 Other Symbols Used
tRequired action: Describes activities that must be carried out.
Theactions in the sequence must be carried out in succession.
yResult: Describes the result of the activities carried out.

BioPAT®Trace | Multi Trace Operating Instructions 11
Safety Instructions
2 Safety Instructions
2.1 Intended Use
The device is used for online monitoring of the analytes glucose, lactate,
methanol and ethanol in laboratory or industrial cultivations of micro-
organisms and cell lines.
The device may only be used indoors.
These instructions are part of the device. The device is intended exclusively
for use in accordance with these instructions.
Foreseeable Misuse
Any use beyond the intended use of the device can cause severe unfore-
seeable hazards and is the sole responsibility of the operator.
The following uses, for example, are deemed to be improper:
— Operation outside the permissible ambient conditions, e.g. extreme
temperatures, chemical vapors, moisture, shock, vibration or strong
electromagnetic fields
— Carrying out unauthorized modifications or other technical changes on
the device
— Connecting unsuitable devices
— Installation of unauthorized items on the device
2.2 Personnel Qualification
All persons working on the device must possess the necessary knowledge
(see Chapter “1.3 Target Groups”, page 9).
The activities described in these instructions are aimed at the target group of
operators. If individual actions must be carried out by other target groups, this
ill be indicated.

12 BioPAT®Trace | Multi Trace Operating Instructions
Safety Instructions
2.3 General Safety Instructions
Failure to follow the safety instructions in this manual can have serious
consequences, e.g. exposure of individuals to electrical, mechanical or
chemical hazards.
t Before starting up the device: Carefully read the chapter entitled
“SafetyInstructions”.
t Only operate the device when it is safe and in perfect working order.
t Observe the maintenance intervals (Chapter “9.1 Cleaning”, page 116).
t Have any damage repaired immediately by the Sartorius Service.
2.3.1 Electrical Power
Electrical switching elements are installed in the device. Damage to the
insulation or individual components can be life-threatening. Contact with
parts under voltage represents a direct danger of death.
Work on and modifications to the electrical equipment of the device may only
be carried out by Sartorius Service personnel. The device may only be opened
by Sartorius Service personnel.
t Check the device regularly for defects, such as loose connections or
damage to the insulation.The device’s housing, power cord and all
connections must be undamaged.
t Note the device’s protection class (IP rating). The protection class
specifies the suitability of equipment for various ambient conditions, e.g.
moisture or foreign bodies (see Chapter “14.2 Dimensions and Weights”,
page 130).
t Make sure that the power connection is equipped with a ground lead.
t Do not use inadequately sized power cords.
t Keep live parts away from moisture. Moisture can cause short circuits.
t If the electrical equipment is defective, immediately turn off the power
supply and contact the Sartorius Service.
2.3.2 Energy Release
Energy supply lines may be incorrectly dimensioned and not protected
against impermissible fluctuations and faults.This can lead to energy being
released, e.g. electric surges or gases.

BioPAT®Trace | Multi Trace Operating Instructions 13
Safety Instructions
The power supplies used to operate the device must be designed in
accordance with the technical data (see Chapter 14.2, page 130). Energy
connections must be fitted with fully functional safety equipment:
— Earth leakage circuit breaker for mains connections (ground fault
circuitinterrupter)
— Fittings for shutting off water, compressed air or gas supplies
2.3.3 Accessories, Consumables, and Spare Parts
The use of unsuitable accessories, consumables and spare parts can
behazardous:
— Damage to the device
— Malfunction of the device
— Total failure of the device
t Only use accessories, consumables, and spare parts from Sartorius.
Sartorius can provide information on operational quality upon request.
t Only use accessories, consumables, and spare parts that are in technically
perfect condition.
The use of accessories, consumables, and spare parts not approved by
Sartorius is the sole responsibility of the operator.
2.4 Conduct in an Emergency
Malfunction may result in injury or property damage.
If there is immediate danger of personal injury or equipment damage, take
the device out of operation immediately:
t Unplug the power cord from the device(1).
t Have any malfunctions repaired immediately by the Sartorius Service.

14 BioPAT®Trace | Multi Trace Operating Instructions
BioPAT®Trace | Multi Trace Product Description
3 BioPAT®Trace | Multi Trace
Product Description
The BioPAT®Trace | MultiTrace analysis system is used for simultaneous
online monitoring of the analytes glucose and lactate in laboratory or
industrial cultivations of microorganisms and cell lines.
An additional application for the measurement of ethanol or methanol
isavailable.
The BioPAT®Trace | MultiTrace provides continual analysis independent of
the type of cultivation (batch, fed-batch, continuous cultivation).
The measurement range of the BioPAT®Trace | Multi Trace extends from 0.1
to 40 g/L for glucose, from 0.05 to 10 g/L for lactate, from 0.5 to 20 g/L for
methanol and from 1 to 40 g/L for ethanol.
Depending on the conditions, the measurement frequency is 30 analyses per
hour maximum. Due to the device’s structured design and easy-to-operate
concept, the user can perform routine analysis functions after just a brief
introduction.
The ambient temperature of the BioPAT®Trace | Multi Trace can lie between
15 and 35 °C due to internal temperature correction. The ambient humidity
should not exceed 90%.
Analysis in the BioPAT®Trace | Multi Trace is a combination of enzymatic
conversion and electrochemical detection. A 2-channel enzyme electrode
coated with oxidase or a combination of an enzyme reactor with a platinum
electrode (methanol | ethanol, (Glucose | lactate) application) serves as the
biosensor. Depending on the application, the service life of the sensor system
ensures 30 days or 5,000 measurements. The deviation from the average
measurement is less than 3% for a measurement of 5 g/L of glucose and
2.5 g/L of lactate. For more information please request the BioPAT®Trace:
Performance and Accuracy application note.

BioPAT®Trace | Multi Trace Operating Instructions 15
BioPAT®Trace | Multi Trace Product Description
3.1 The Basics of Electrochemical
Measuring Technology
An electrochemical detector measures the
current produced at a specific potential applied
to the electrodes by the analytes in a flow
conductivity cell. In the presence of an
electrolyte solution, the electrochemical
conversion of the substance at the working
electrode leads to the release or attraction of
electrons (oxidation or reduction). The size of the
measurable threshold current depends on the
parameters of Faraday’s law of electrolysis as well
as on Fick’s diffusion law and is proportional to
the concentration.
The selectivity of the sensitive detector is based
on the principle that not all substances are elec-
trochemically active and thus many extraneous
substances in a sample solution will not be
detected. Selectivity is dependent on the work-
ing electrode’s material and the applied
potential.
By combining this system with enzymes, the
selectivity of an electrochemical sensor can be
raised even higher – and the electrode is turned
into a biosensor.

16 BioPAT®Trace | Multi Trace Operating Instructions
BioPAT®Trace | Multi Trace Product Description
3.2 Biosensor Principle
The analytes glucose and lactate are detected by means of enzymatic reac-
tions. Figure 1 shows the enzymatic conversion of glucose to gluconolactone.
In the presence of water, gluconolactone is immediately hydrolyzed to glu-
conic acid. The hydrogen peroxide (H2O2) produced in the first step is
detected amperometrically through anodic oxidation that releases the two
electrons. Figure 2 shows the enzymatic conversion of lactate to pyruvate.
During this reaction as well, H2O2is formed and then detected
amperometrically.
Enzyme reactions take place according to the “key-lock principle”.They are
specific and thus highly selective.Therefore, the reaction at the enzyme sys-
tem is a further selection. The combination of the two selective principles
makes the BioPAT®Trace | Multi Trace largely insensitive to matrix effects and
extraneous materials.
β-D-Glucose
D-Glucono-δ-lacton D-Gluconic acid
Fig.1: Enzymatic conversion of glucosewith parallel anodic oxidation of H2O2
Lactate Pyruvate
Fig.2: Enzymatic conversion of lactate with parallel anodic oxidation of H2O2

BioPAT®Trace | Multi Trace Operating Instructions 17
BioPAT®Trace | Multi Trace Product Description
The BioPAT®Trace | MultiTrace biosensors consist of platinum thick-film
electrodes that are durably coated with the corresponding enzymes (glucose
oxidase (GOD) or lactate oxidase (LOD)).Then, enzyme and electrode are in
close proximity to each other, so that the reactions described above can be
recorded directly.
R=H Methanol
R=CH3Ethanol R=H Formaldehyd R=CH3Acetaldehyd
Fig.3: Enzymatic conversion of methanol | ethanol with parallel anodic oxidation of H2O2
Figure 3 shows the enzymatic conversion of methanol or ethanol to formal-
dehyde or acetaldehyde. The enzyme alcohol oxidase (AOD), which occurs in
an enzyme reactor in immobilized form, is used as catalyst. The hydrogen per-
oxide (H2O2) formed in the reaction is detected amperometrically. With this
method, the alcohol is converted in the enzyme reactor and the detection of
the formed hydrogen peroxide takes place on a platinum electrode.
3.3 Fluidic System and Sampling
To perform the respective analysis on a sample, representative material must
be transported to the biosensor. A fluidic system (tubing set) consisting of
tubing lines, pumps and valves is used for that purpose. Additionally, the
sample is also conditioned in the fluidic system, i.e. potentially interfering
components are removed, and diluted to the extent that it can be assayed
reliably by highly sensitive biosensors. This is done by separation through
adialysis membrane, which is integrated in every tubing set as a diffusion
module.

18 BioPAT®Trace | Multi Trace Operating Instructions
BioPAT®Trace | Multi Trace Product Description
Conditioning of the sample is based on the method of flow diffusion analysis
(Figure 4). This method works with a time-dependent sample transfer. For
that purpose, the measurement line and the sample and |or the sample
infeed are separated from each other by a membrane.The measuring
distance is called the acceptor (receiving) and the sample side is called the
donor (giving).
During an analysis, the measurement line is flushed with a transport buffer,
which is an acceptor stream that can be stopped from behind the membrane
for a defined period of time.This procedure is carried out by triggering a valve,
which in turn opens up a bypass route.
Meanwhile, the analytes in the sample diffuse through the membrane and, at
the same time, are enriched in the holding chamber. In this way, a continually
increasing concentration of dialyzable components develops in the fluid
segment of the transport buffer.
Membrane
Transport buffer
Holding chamber
Outlet
Step 1: Rinsing
Step 2: Stop flow and analyte enrichment
Step 3: Transporting the accumulated material to the conductivity cell
Fig.4: Principle of the flow diffusion technique

BioPAT®Trace | Multi Trace Operating Instructions 19
BioPAT®Trace | Multi Trace Product Description
When this segment is released by re-switching the valve, the flow goes to the
conductivity cell, where it generates a signal. The intensity of the signal is a
measure of the concentration of the sample. One big advantage of this
enrichment method is the very broad measuring range that is achieved by
varying the diffusion time; it also protects the sensor against inferring
particles in the sample.
Bypass
Valve Holding
chamber
Measuring cell
Donor
Bypass
Holding
chamber
Measuring cell
Valve
Donor
Valve control during the determination
of the baseline and while loading. Valve control while unloading and during signal detection.
Fig.5: Valve control
PID Control
Feeding in a cultivation can be reliably controlled with the integrated PID
controller. The PID controller consists of three interacting single controller.
These are the proportional controller (P), the integral controller (I), and the
derivative controller (D). The control action of the controller is described by
adifferential equation:












The persistent control deviation of the proportional gain of the controller
canbe balanced with the integral gain. With the derivative gain of the
controller it is possible to react to fast changes of concentrations inside the
reactor. The PID controller is better suited for feeding processes than a simple
two-level controller.

20 BioPAT®Trace | Multi Trace Operating Instructions
BioPAT®Trace | Multi Trace Product Description
Adjusting the PID Controller
The principle of the PID controller is known since 1922 (Minorsky[1]), but
useful rules for adjustment were not available before 1942 (Ziegler and
Nicols[2]). Today most of the controllerwill be adjusted by empirical design.
This design can also be used in the field of biotechnology.
Literature
[1] Minorsky, Nicolas: Directional stability of automatically steered bodies;
J. Amer. Soc of Naval Engineers 34 (1922), pp. 280–309.
[2] Ziegler, J.G. and Nicols, N.B.: Optimum settings for automatic controllers;
Trans. ASME, 64 (1942), pp. 759-768
3.4 Data Transmission and Data Saving
The BioPAT®Trace | MultiTrace has a number of outputs, making integration
into data recording systems very flexible. Along with a standard analog output
for signal ranges from 0 to 20 mA, 0 to 10V or 4 to 20 mA, the BioPAT® Trace|
Multi Trace also features an Ethernet connection that connects the device to
a PC via a network. Using the software supplied, the measured results can be
visualized graphically and saved (see Chapter "8 Operation", page 75).
3.5 Equipment Supplied with the BioPAT®Trace | Multi Trace
Description Order no.
Device BioPAT®Trace with power adapter, power plugs
(Europe, USA, UK), cross-over Ethernet cable, operating
instructions and PC software (trace_mon)
BPT0001
Device BioPAT®Trace with power adapter, power plugs
(Europe, USA, UK), cross-over Ethernet cable, operating
instructions and PC software (trace_mon)
BPT00M