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BioPAT® Trace

1. ssssssseeeeeee 65 Filtration probe with adapter RD 28 x s 2 eee eee 65 Placing the ferrule sabe beaded pt ke S RR bent s 66 Attaching the polypropylene membrane 0 006 66 Inserting the O rlfig s cca ent E eae ef E dea 67 Tightening thecap nut Joni eeeo e batir eredi es 67 Hydrophilizing set with pump cassette and tubing lines 70 Setting up and connecting the hydrophilization set 70 Filtration start window with operating panel for controlling the pump for hydrophilizing lesse 71 Filtration probe with tubing set 76 Connecting the tubing set 2 eee ee 76 Login for network settingS 0 ccc cece eee eee 78 Input field for network settings lessen 79 Start se ELITIOS maiii eai Santee Marites ue sedes Sedona Dads ce 81 Analog outputs aisi iiine ve ade indeed LEE xps 82 Start window indicating the selection and display ranges 85 AUtOStdTL uere reborn e Eds 86 Screen display in Standby mode 000000000ee 87 Display after reference measurement 000000 88 Figure 67 Figure 68 Figure 69 Figure 70 Figure 71 Figure 72 Figure 73 Figure 74 Figure 75 Figure 76 Figure 77 Figure 78 Figure 79 Figure 80 Figure 81 Figure 82 Figure 83 Figure 84 Figure 85 Figure 86 Figure 87 Figure 88 Figure 89 Figure 90 Figure 91 Prompt for sampling and connection to the three way stopcock
2. Standard levels glucose and lactate Here you indicate the concentrations of the calibration standard solutions that have been selected and hooked up for your application Measurements between calibrations Here you can enter how many measurements should be performed before an automatic calibration is run This feature only works however if the autocalibration function has been allowed Operation 103 104 Operation By pressing the Calibration button you can initiate a calibration at any time Any changes made to the automatic calibration data during running operations are not valid until the next calibration Measurements per standard This function is used to set the number of measurements per calibration standard One minute is required for each measurement of a calibration value That means that a normal calibration with 3 measurements per standard will take 6 minutes including rinsing times for changing the medium Output range glucose and lactate Here the output ranges of the analog outputs are set Proceed as follows The upper limit of the measuring range is always indicated The lower limit is preset to O For a required measurement range of e g 0 10 g L glucose glucose is set to 10 upper limit in the output range When selecting the voltage range of 0 10 V then 10 volts are equal to 10 g L glucose Autocalibration enabled Calibration delayed Measurements
3. 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 dialysable components develops in the fluid segment of the transport buffer Membrane Transport buffer 9 Step 1 Flushing Holding chamber Exit PrOD E p 0 f S ds or OF 9 0 o O o 0 j eee Step 3 Transporting the accumulated analyte to the measurement cell Figure 4 Principle of the flow diffusion technique BioPAT Trace Product Description When this segment is released by re triggering the valve the flow goes to the measurement 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 Bypass gt 1 ES Measurement Measurement Valve Holding cell Valve Holding cell chamber chamber Donor Donor Figure 5 Flow diagram to illustrate flow diffusion analysis Image on the left Valve control during the determination of the baseline and while loading Image on the right Valve control while unloading and during signal detection 2 4 Da
4. 0 cece eee eee eens 90 Connecting the external sample 0 c ee mannes 90 Correct position of the three way stopcock 91 LOG MENULOPTIONS aseo e or trece daly Ee Re ern udo Malta on 92 Device menu options sssesee Ie 93 Connecting the deviGe sous c vot re Red ep ec etd dx 94 Connection indICator ooo ceeded creer berries 94 Searching the netWork cssc ote ere REIS Ar ER E 95 Restart prompt window after Stop eee eee 96 Display menu options sss 97 Screen display of current calibration values 200 98 Screen display of status bar sarcsiaisetissrie tiraniei iai 98 Screen for adjusting the display of measured values 99 Display of the measurement curves 00 eee eee eee 100 Settings MENU OPUIONS uuo opa baw aiae eeu e 101 Entry screen for configuring the measurement parameters 102 Switching the analog output 0 eee eee 105 Entry screen for basic settings 0 00 cece eee eee 106 Selection window for language settings 00 107 Importing data from the ptd file into Excel Activate All Tiles iu i e tierno euge Ea da penati rece ey 112 Importing data from the ptd file into Excel Highlighting the ptd file iusso rc eem n 113 Importing data from the ptd file into Excel text conversion assistant step 1 00 cece eee ee 113 Importing data from the ptd file into Excel text c
5. Fields are aligned in columns with spaces between each field Start import at row 1 Io Fee gee CRESS Tw Preview of file C Weers Dan Kopec ARLI1004W7 Desttop Troce Runs12 06 9231 ptd 12 2012 231 02 16 028 74 000003 828 000001 663 6 12 2012 231 04 16 025 77 000003 817 000001 672 Figure 88 Importing data from the ptd file into Excel text conversion assistant step 1 Tips and Checklist 113 114 Tips and Checklist 6 Text conversion assistant step 2 In this step the Semicolon separator is selected circled in orange and confirmed by pressing the Next button circled in red enn Kom Tex guolfier Spoce l E oer Data preview Figure 89 Importing data from the ptd file into Excel text conversion assistant step 2 7 Text conversion assistant step 3 In this step the More option is selected circled in orange Temimponwasd step3ot3 TUO O O This screen lets you select each column and set the Data Format Column data format General Generel converts numeric values to numbers date values to dates and ali remaining Iot values to text Dee MOY T Do not pnport column skip m cmm sm Com Figure 90 Importing data from the ptd file into Excel text conversion assistant step 3 Here you can select More text import options A period is set as the decimal separator and a space as the separator for thousands circl
6. HE Sartorius biotech Operating Instructions BioPAT Trace 2 Channel Online Analysis System TA AE ucc Mu 2 Introduction Important Note The data and information contained in this manual were compiled with the greatest of care Notwithstanding the scrupulous attention paid to preparing this document no absolute guarantee of its accuracy can be given Please notify us if any information is missing that is important to you if you find any errors in the content or require more extensive information on specific components Reproduction translation and duplication in any form also in part require written permission from Sartorius Stedim Biotech GmbH Address of our Technical Documentation Department Sartorius Stedim Biotech GmbH Technical Editorial Department August Spindler Strasse 11 37079 Goettingen Germany e mail tech pubs sartorius stedim com Internet www sartorius stedim com Contents EG ue BUD I oriai PP 7 Listof ablesiniuscivvgecevsod eaghede apria kaata ai exert held DE dnd 10 Explanation Of Symbols x e sehe o teen o e XR Debo ER TR 10 1 Introduction coe sofia verear re mee mere ehe s 11 2 BioPAT Trace Product Description susuuseeeeeseesseeese 12 2 1 The Basics of Electrochemical Measuring Technology 13 2 2 Biosensory Principle once cadena canes SaL ANLA a a CERO KR RR 14 2 3 Fluidie System arid Sampling sse e b Re RE 15 2 4 Data Transmission a
7. After priming the device starts with the measure ments automatically if the autostart option was activated or switches to the Standby mode Getting Started 83 84 Getting Started If you are working with the dialysis method you must carry out a reference measurement at the right time in order to obtain conclusive results Proceed as described in Chapter 7 Operation on page 85 6 8 Decommissioning The device can be switched off at any time by pressing the power switch The calibration and reference values last stored remain in memory for the next start see Section P 7 1 2 1 Connect on page 93 If the measuring instrument is not operated for two days e g over the weekend the buffer filled system may remain standing In the event that the device will be out of use for longer periods of time the tubing set must be replaced before re starting operations For decommissioning in the methanol ethanol operating mode the enzyme reactor must be removed and stored at a cool temperature see Chapter 6 2 5 Connection and Removal of the Enzyme Reactor 7 Operation Switch on the BioPAT Trace at the power switch located on the back panel and run it on an external PC You will find the start icon on the PC s desktop n Double clicking it will start the application The user interface shown in Figure 63 appears Lg Figure 63 Start window indicating the selection and display ranges The device
8. a segment of the buffer solution is enriched with the analyte from the donor stream After time dependent enrichment in the diffusion module 6 the acceptor stream flows into the measurement cell 4 for assaying the analyte The measurement cell is fixated by a retaining plate which can be fastened or loosened with a knurled nut 5 Furthermore a rinsing line with return valve 8 is attached to the measurement cell Before the solutions are transported to waste they run through a drip count 7 26 Design and Function Figure 8 shows the rear view of the BioPAT Trace The device can be switched on and off by pressing the power switch 9 Using the calibration data the electrochemical signal of the biosensor is converted internally to a concentration value The measured value is output after the analysis is completed on a PC interfaced to the Ethernet connection 10 Optionally the signals can be output via the analog signal output 12 The following ranges are possible 0 10 V 0 20 mA 4 20 mA The USB interfaces 11 and the serial interface RS232 port 13 can also be used for data transfer currently not implemented 12a 12b 12c Figure 8 Rear view of the BioPAT Trace with the following elements Pos Function 9 Power switch 10 Ethernet connection 11 USB interface 12a b c Analog outputs 1 3 13 Serial interface Design and Function 27 28 The BioPAT Trace ca
9. 0 22 um Before the solution leaves the module a second membrane is used for degassing Thus the particle filter ensures that the acceptor stream in the BioPAT Trace stays free of particles and air bubbles To ensure proper operation of the measuring instrument the tubing set including the particle filter must be replaced regularly Design and Function 35 5 5 Diffusion Module Function The diffusion module is part of the tubing set The standard solutions or an external sample are pumped through the diffusion module to the analysis system A membrane enables diffusion of the substrate from the donor solutions both standard and sample into the buffer solution acceptor The different membrane materials enable dialysis cellulase stabile dialysis and gas diffusion for separating substrates Figure 13 shows a schematic diagram of this system Donor Figure 13 Schematic design and fixation of the diffusion module 36 The module is made of PMMA It has a diffusion area of 20 mm and ensures that a sufficient exchange of substances takes place between the donor and the acceptor stream The donor and acceptor streams flow in through the lower module inlet The drain is located at the top A In order to ensure reliable operation of the measuring instrument the diffusion module must be secured in the correct position on the retaining plate arrow pointing up Des
10. 5 and by tightening as far as possible Depending on the respective installation with the assembly kit s clamping device 7 the immersion depth of the filtration probe varies from 160 to 330 mm The dummy plugs 8 are used to seal the filtration probe during sterilization 6 2 7 2 Optional Accessories Two 19 12 mm reduction adapters M26 x1 with fine thread and RD 28 x s with coarse thread are available for the installation of the filtration probe into the 19 mm cover port amp E Figure 48 Filtration probe with Figure 49 Filtration probe with adapter M26 x1 adapter RD 28 xV s Getting Started 65 66 Getting Started 6 2 7 3 Assembling the Filtration Probe Push the Ferrule with the narrow opening first over the membrane support into the conical lower membrane retainer of the filtration unit Figure 50 The polypropylene membrane is removed from the packaging and carefully inserted until it stops into the membrane support in the already assembled PTFE Ferrule V c Figure 50 Placing the Ferrule Figure 51 Attaching the polypropylene membrane Pull the O ring over the outer diameter of the filtration unit Now the cap nut is carefully pulled over the inserted membrane and screwed into the filtration unit The screw connection is tightened using 2 open jawed wrenches until it reaches its mechanical stop position B Figure 52 Inserting the O ri
11. 5 5 5 6 5 7 5 8 5 9 Setting Up the DEVICE i5 deeem oo Rte tt dog eU Rie ea 25 BioPAT Trace Measurement Methods l liillleeeslsssesse 29 5 2 1 Dialysis Measuring Method Using the Dialysis Probe 31 5 2 2 Filtration Measuring Method Using the Filtration Probe 33 Measurement Cell Function 0 0000 cece eee eee 34 Particle Filter FUNCTION 20 porvi ceri eee aatem Rei 35 Diffusion Module Function 00 00 cece eee ees 36 Dialysis Probe FUNCUION 20 4 0 ca 4 cei ee Re krank eee es 37 Filtration Probe Function 0 00 cece eee eee ees 38 Enzyme Reactor FUNCTION w 2 50s40 0 elec ades verc ede ee eed 39 Temperature Correction usns semet eec x eR ideale oe dos E as 39 Contents 6 Getting Started iiie ia alate kieed as eee eden tees 6 1 Transporta sese ex beads ew naan Vasaew REOR RRRONE ERA A RA 6 2 Setup 6 3 6 4 6 5 6 6 6 7 6 8 6 2 1 6 2 2 6 2 3 6 2 4 6 2 5 6 2 6 6 2 7 Setting up the Equipment 0 ee eee eee Tubing on the Device 25e 254 cde dial POR cae eben Attaching the Filtration Tubing Set 0000 00 eee Attaching the Tubing Set for the Dialysis Operating Mode Connection and Removal of the Enzyme Reactor Connecting the Dialysis Probe 6 2 6 1 Mounting the Dialysis Membrane in the Dialysis Probe 6 2 6 2 Installing the Dialysis Probe into the Bioreactor 6 2 6 3 Sterilization of the Dialysi
12. 6 2 4 Align and slightly open guide plate 4bff 19 Insert and pull down valve tubes into 46 20 22 valve slot Push guide plates in direction of valves 47 23 for protection Insert and engage pumps 47ff 24 26 Place measurement cell into recess and affix 48ff 28 29 with knurled nut Attach diffusion module 50 30 Insert drip counters 50 31 For dialysis only Connect three way stopcock 51 32 33 optional 116 Tips and Checklist Step Action Chapter Page Figure Checklist 4 Connect inlet and outlet lines 6 2 2 44 Connect tubing to transport buffer 44 Connect tubing to calibration solutions 44 Connect waste line to waste container 44 5 Switch on device 7 85 Switch on power switch on back of device 7 85 Wait for internal computer to boot 23 108 white LED turns red 6 Start PC software y 85ff 63 73 Double click Start icon on desktop 85 63 trace mon Wait for automatic connection setup or 7 and 85 72 connect device 7 1 2 1 93ff Start with selecting new tubing set 96 73 7 Set operating parameters 7 1 4 101ff 79 80 Settings menu Basic settings 7 1 4 2 105ff 82 Select measuring method 105ff 82 filtration dialysis Measurement parameters 7 1 4 1 102ff 80 81 Set measurement period 102 Enter calibration standard values 103 Create file for saving measured values 7 1 1 92 70 8 Connect sampling probe 6 2 60 and 60 76 X 44 45 6 2 7 9 57 58 Conn
13. D anon DECOMMISSIONING aen n RR HRERU S RR ERREUR CRURA Marana Contents 7 Operation 2 3 53 0 0 obs at one ehh Fe hd oe a SES de ditas Z3 Men Bar es Ex x eb x Eme cdd Bee maces MARE us 7 431 Menu ltem Log 2 08 esencia terre RRREX RUD XR PR GG 7 1 2 Menucltem Device isa ceu nde A rena 7 4 2 1 Conheet heb ani doe eS tn rese e ee tah e AR IS 7 1 2 2 Setting up Operation ee eee eee eee eee PAN2 3 StOP isc sc sect wo eie EIER REX ER PUO Ed basal an 7 43 M nu ltem Displays esae zon aun te eR Sex Ore RR P RE Ruds 7 1 3 1 Change View desde coe Se EX XR Eae E EA FAA Menu ltem Settihgs eriei cem met n eG reet RR TAAI Parameters ioc osecec keiner eb bre RE RR Td La 7 1 4 2 Basic Settings ocho eene cx aree er nn 7 1 4 3 Language esperto Rhe ERI Ex Eeseb NET x PAS 7 1 5 Menu Item Information 2 2 0 0 cece eee 7 2 LED Status Messages siehe IR ReIPERPTES EE RA 8 Tips and Checklist iore Etre bre RO Rye peer 8 1 Choosing Suitable Standard Solutions esses 8 2 Dialysis Mode Reference Measurements at the Start of Fermehtatlofi uenia tese tnde a etienne bog RU gn 8 3 Dialysis Mode Reference Measurements for Low Lactate Concentrations 0000 cece eens 8 4 Avoiding Contamination in the Tubing Set 8 5 Import Data to Excel 9 Troubleshooting 22er lur beca depre ta uA denar 10 Care and Maintenance 0 00000 c ccc RR RR 10 1 DEVICE 2 1obeere Ret eem e EO ae ppm bre doe dd e
14. clean and seated properly Remove the two Torx screws on the end of the dialysis probe using the Torx wrench supplied this allows the dialysis probe optic to be removed from the optic holder along with the dialysis membrane Afterwards clean the probe optic and the optic holder with a soft brush and water Getting Started 61 62 Getting Started 6 2 7 Connecting the Filtration Probe The filtration probe in line sampling probe is intended for harvesting microbe free filtrate from bioreactors and fermenters under sterile conditions A tubular microfiltration membrane made of polypropylene positioned inside the bioreactor using an in line probe serves as a sterile barrier The filtration probe is sterilized together with the bioreactor The sampling probe is installed in the reactor port according to METTLER TOLEDO INGOLD standard 12 and 19 mm cover port as well as 25 mm side port The microbe free filtrate is sampled continuously or discontinuously at the probe head that is accessible outside the bioreactor fermenter For pumping the filtrate the probe is connected to the filtration tubing set Figure 46 below shows the filtration probe for installation in the 12 mm cover port and with stopcock for installation in the 25 mm side port Figure 46 Filtration probes in 12 mm and 25 mm design When assembling and operating the filtration probe it is imperative that you follow the encl
15. draw an external sample and connect to three way stop cock Switch three way stop cock into right position Sample ready Figure 67 Prompt for sampling and connection to the three way stopcock Draw a cell free sample from the bioreactor at least 5 mL into a 5 mL syringe and connect it to the three way stopcock as shown in Figure 68 Check that the three way stopcock is in the correct position The arrows on the stopcock must point towards the green tube marking and the syringe 9 Figure 68 Connecting the external sample Only use the Sample ready function if you are sure that the filled syringe is connected and the three way stopcock is set to the proper position Otherwise erroneous measurements may result Figure 69 Correct position of the three way stopcock Sample ready is activated to measure the external sample Three measurements are taken and their average displayed as a result As described above you can then accept correct or discard measured values or you can perform another measurement on an external sample Before you continue working make sure that the three way valve was set back to the initial position see Figure 69 so that you can correctly measure Standard 1 during the next calibration A Make sure that the three way stopcock is set as displayed Otherwise all following calibrations will be erroneous and
16. for the BioPAT Trace as PDF file Then change the device s network address It can be found in the Network settings menu item EE sartorius st Start Download Network Logout Deutsch Figure 59 Login for network settings In order to do this you will need to log in with the password TuSh1Vf5Ja 78 Getting Started In most cases the factory set IP address will not be compatible with company networks Have a suitable IP address assigned by your network administrator otherwise the device may not be able to connect with the operating software Networking INTERFACE address 192 168 120 231 je netmask 255 255 255 0 e gateway 192 168 120 1 e RESOLV CONF search trace bs local e finmeenrvon 192 168 120 10 e send data Figure 60 Input field for network settings If you would like to work via a LAN or intranet that the factory set network address or the network name may not be compatible with your network If this is the case please contact your network administrator and change the BioPAT Trace s IP address in the form field After the network address has been changed the BioPAT Trace can only be accessed via this new IP address The last 3 characters of the IP address here 231 represent the number of the device that is displayed in the user interface Getting Started 79 80 Getting Started 6 3 1 Troubleshooting If you have entered dif
17. hydrostatic pressure at the dispensing location can improve the performance of the filtration unit The filtration probe must always be installed immediately before filling the bioreactor since drying out of the membrane results in a loss of hydrophilic properties 6 2 7 8 In line Sterilization of the Filtration Probe After installation the filtration probe is sealed with a dummy plug and sterilized together with the bioreactor During the entire process the probe must be completely covered with liquid Once sterilization is complete and the max operating temperature of the reactor of 40 C is reached the filtrate outlet must be opened and the filtration probe is connected to the BioPAT Trace via a tubing set for sampling Getting Started 75 76 Getting Started 6 2 7 9 Connecting the Filtration Probe to the Tubing Set The filtration probe is connected to the online analyzers through the filtration tubing set An adapter cable Luer UNF for connecting the filtration probe to the tubing set of the online analyzer BioPAT Trace is provided with the accessories As shown in the following Figures 57 and 58 it must be assembled between the probe UNF connection and the BioPAT Trace tubing set Luer connection Bo MN n e bi om uiid ni Figure 57 Filtration probe with tubing set Figure 58 Connecting the tubing set 6 3 Loading the Software and Network Connection The BioPA
18. 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 ona platinum electrode 2 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 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 BioPAT Trace Product Description 15 16 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
19. number appears in the header In this case it s 231 The asterisk indicates that a connection exists between the control PC and the BioPAT Trace If there is no connection to the measuring instrument it must be established see Section gt 6 3 1 Ethernet Connection on page 80 The following menu items can be selected from the task bar File Device View Settings and Information Operation 85 In the measuring window in the center of the screen the glucose and lactate measurements are plotted over time The glucose values are represented by blue dots and the lactate values by red dots The device status is displayed under the measurement Figure 64 Autostart window At the beginning the device is in default mode To the right of the measuring window there is an area for context based selection buttons Every time the device restarts completely fill the tubing set and run several preliminary measurements to render the sensor system stable before initiating the calibration routine This process is called priming alongside filling and preliminary measurements it also includes an initial calibration of the system After priming the device switches to Standby mode Depending on the measuring method selected the entire process takes between 40 and 60 minutes Another option is to configure the BioPAT Trace to start with the measurements immediately after the priming routine If a check mark is placed next to Sta
20. of the two selective principles makes the BioPAT Trace largely insensitive to matrix effects and extraneous materials CH OH H0O 0 2H 2e H O OH GOD OH H 310 7 H HOH CH 0H CH5O0H H o H O H OH B D Glucose Du y o gt K u H CooH OH H OH H OH D Gluconolactone D Gluconic Acid Figure 1 Enzymatic conversion of glucose with parallel anodic oxidation of HO HO OH HO wele ap LOD n ae or v t 02 4 c cH H50 H Oo HO Oo 2H 2e Figure 2 Enzymatic conversion of lactate with parallel anodic oxidation of H 0 14 BioPAT Trace Product Description The BioPAT Trace 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 H H AOD H C OH An F 05 o H203 R R R H Methanol R H Formaldehyde R CH Ethanol R CH Acetaldehyde H0 O 2H 2e Figure 3 Enzymatic conversion of methanol ethanol with parallel anodic oxidation of H 0 Figure 3 shows the enzymatic conversion of methanol or ethanol to formaldehyde or acetaldehyde The enzyme alcohol oxidase AOD which occurs in an enzyme reactor in immobilized form is used as catalyst The hydrogen peroxide H O formed
21. per group Standard 1 Glucose Standard 2 Glucose Measurements between calibrations Analog Out Glucose Measurements per Standard E Out 3 Mezawenents per extra seme a maximal count of measurements per standard Output range Glucose La Standard 1Lactate Standard 2 Lactate Analan NotI artate Lal e Figure 83 Switching the analog output Here you can switch the signals of the analog outputs between 0 20 mA and 4 20 mA or between 0 10 V and 2 10 V see Figure 83 Operation 105 7 1 4 2 Basic Settings In the Basic settings menu item you can enter data that is important for carrying out and documenting the measurements Figure 84 Before running the measurement you can enter the project name device location and user ID The most important item is the selection of the application used You can either select filtration or dialysis for the determination of glucose and lactate as well as methanol ethanol filtration and methanol ethanol dialysis Dialysis Filtration Methanol Ethanol Dialysis Methanol Ethanol Fri Feb 15 14 06 02 2013 E Adjust time I m oem Figure 84 Entry screen for basic settings 106 Operation Before working with a new tubing set always check to make sure that the right application has been selected Otherwise this can result in erroneous measurements or destruction of the tubing set Figure 85 Selection window f
22. start to gradually escape from the membrane Otherwise the membrane is hydrophilized insufficiently The escape of large air bubbles from the screw connections is an indication for damaged gaskets In order to maintain the hydrophilic properties of the membrane until its installation into the bioreactor it is recommended to store the membrane in a hydrophilization solution isopropanol 70 Immediately before installation into the bioreactor the isopropanol should be replaced with water in order to avoid evaporation and the loss of hydrophilization 6 2 7 6 Using the Hydrophilization Set The hydrophilization set is an auxiliary device which assists in setting up operation of the filtration probe It can be ordered optionally The polypropylene membrane used for the filtration probe is impermeable for aqueous media hydrophobic Therefore the membrane must be hydrophilized with 70 isopropanol solution prior to each use This hydrophilization can be conducted easily with the hydrophilizing set and the BioPAT Trace Getting Started 69 Figure 54 Hydrophilizing set with pump cassette and tubing lines Figure 55 Setup and connection of the hydrophilizing set 70 Getting Started 6 2 7 6 1 Description of Setup The hydrophilizing set consists of a pump cassette from which two PTFE tubes descend There is an UNF fitting with Ferrule for connection to the probe at one end of the tube The other e
23. with this new address automatically in the future proceed as described in Section P 6 5 Changing the Start Settings on page 81 Once you have entered the correct IP address and the device is acknowledged by the network it will connect to the control PC An asterisk appears in the view bar behind the name or number of the device Log Device Display Figure 73 Connection established 94 Operation IP Address or Name of Device 192 168 120 231 Figure 74 Searching the network Alternatively you can start a search for new devices on the network by clicking on the button Who is out there The results will be displayed after a little while You can highlight the desired device and select it with OK If no device can be found an error message appears Try to enter the IP address directly or contact your network administrator 7 1 2 2 Setting up operation If the tubing set has been replaced you must go to to the menu item Setting up operation Setting up operation deactivates all previous calibration and reference data and requires that the tubing system is re primed 7 1 2 3 Stop Stop is used for temporary decommissioning of the system e g when the monitored bioprocess is interrupted for short operating interruptions or when connecting enzyme reactors see Chapter 6 2 5 page 52 Select the menu item Stop to interrupt the measuring routine and ready status All pumps stop and th
24. 41 Figure 42 Figure 43 Figure 44 Figure 45 Figure 46 Figure 47 Figure 48 Figure 49 Figure 50 Figure 51 Figure 52 Figure 53 Figure 54 Figure 55 Figure 56 Figure 57 Figure 58 Figure 59 Figure 60 Figure 61 Figure 62 Figure 63 Figure 64 Figure 65 Figure 66 8 Contents Place of installation for three way stopcock 51 Three way stopcock and syringe mounted 0 51 Place of installation for the enzyme reactor 53 Securing the enzyme reactor anemia eb eek er e IA XAR dx 53 Fully installed enzyme reactor sso aerae cananan dinani 54 Replacing the membrane in the dialysis probe for the glucose lactate application nunnan rre n rere 55 Inserting the Membrane cess sioe e exta is 56 Installation of the probe optic 00 eee eee eee 56 Tightening the 2 SereWs sete tere SL Ra tede pee c 56 Inserting the friction washer and O ring 00005 58 Filling the probe with buffer solution 0 0005 58 Sealing the probe prior to sterilization 000 58 Connecting the dialysis probe to the tubing set 60 Connecting the dialysis probe to the tubing set 60 Filtration probe with two different membrane lengths 63 Components of the filtration probe for installation into the 12 mm cover port with PG 13 5 connection 64 Filtration probe with adapter M26x1
25. 7 1 3 1 Change View The presentation of the diagram can be modified in the menu item Change view Here you can configure dot sizes dot colors and the axis scales Figure 79 The default settings are the color blue for glucose and red for lactate A green dot color is preset for methanol or ethanol Bon Baur sea Veg Omie Dupin jeep interim 1 9371 1374 D e 1230 34 24b 1246 1240 1246 100 1305 1338 1315 1320 326 1330 133 0340 34 1250 1346 ime Standby Figure 79 Screen shot for adjusting the measured values display Operation 99 100 Operation The measurement display can be modified at any time during operation Press the right mouse button to display the input field for configuring the view Figure 80 The following additional options can be selected Moving the graph Hold down the right mouse button and move Zoom Pull the window from the upper right down to the lower left hold down left mouse button Return to Start screen Pull the window from the lower left up to the upper right hold down left mouse button D r 1230 1298 1248 1249 1298 1298 1300 1306 1310 131 1320 1925 1330 1335 349 1345 340 346 Tne Figure 80 Display of the measurement curves 7 1 4 Menu Item Settings D kd D 231 7 Dialysis Log Device Display Settings Information Parameters Basic Settings Language Figure 81 Set
26. Chapter 7 1 4 1 Parameters on page 102 The signal at the analog output is changed whenever there is a new measurement value and remains unchanged unless a there is a new measurement value b the device is rebooted c new tubing set is activated 6 7 Device Startup Before every startup check the BioPAT Trace to make sure that all modules installed on the device are functional The following conditions must be checked scrupulously on the device Are the two original pump cassettes installed properly s the tubing set connected completely and properly Is the enzyme reactor installed in the methanol ethanol operating mode s there a measurement cell in the holder Are the available volumes of transport buffer and the appropriate calibration solutions ready at hand If these checks are passed you can start the device Chapter 7 Operation on page 85 describes in detail the operating software functions necessary to start the device In principle the following procedure must be maintained 1 In the Basic settings menu select the measuring method Chapter 7 1 4 2 Basic Settings on page 106 2 In the Parameter menu configure the calibration standards and measuring and calibration frequency you want to use Chapter gt 7 1 4 1 Parameters on page 102 3 After you have connected all tubing and sampling lines start the filling routine Chapter gt 7 Operation on page 85 4
27. Error Error message Possible Causes Measured value varies greatly Air bubble inside the measuring chamber Measurement cell not properly inserted Tubing set out of date Filtration sampling probe sealed and or green valve of valve tube not properly inserted No connection to Trace_mon IP address set incorrectly PC IP address not automatically assigned Incorrect or defective power cord Device boots but program does not start Pump is not turning Remove pump cassette and clean motor shaft Pull tight pump tube No flow Transport buffer empty Calibration standard empty Filtration probe blocked Filter clogged Tubing set clogged Leaks in the tubing set Table 8 Problem description and troubleshooting on the BioPAT Trace Troubleshooting 121 Error message Calibration failed Possible Causes What should you do 1 The transport buffer is empty Is there still Connect new transport buffer enough transport buffer in the storage vessel and fill the tubing set Is the suction tubing still in the storage vessel Is buffer dripping into the drip counters 2 A calibration solution is empty Connect new calibration solution 3 Silicone tubes are not properly inserted into Check if the valve tubes are the valves secure and correct accordingly 4 There is an air bubble inside the measuring Remove the air bubble through chamber the rinsing line by using a syringe 5 Tubing set and ad
28. For further usage the enzyme reactor must be removed from the tubing set filled up with transport buffer and stored in the refrigerator at 4 8 C If air enters into the enzyme reactor there is the danger of a decrease in the capacity of the enzyme reactor resulting in lower sensitivity 54 Getting Started 6 2 6 Connecting the Dialysis Probe 6 2 6 1 Mounting the Dialysis Membrane in the Dialysis Probe Install a new dialysis membrane before every online measurement involving bioprocesses with sterile feeds as shown in the following Figure 37 If this rule is not observed a lack of sterility may jeopardize the entire cultivation process bottom section of the dialysis probe Membrane sandwich Lunette Screws M3 OP Silver side faces lunette Figure 37 Replacing the membrane in the dialysis probe for the glucose lactate application Getting Started 55 56 Getting Started The new dialysis membrane must be placed correctly and the probe lunette must be tightened firmly with the two screws see Figures 38 to 40 bs Figure 38 Inserting the Figure 39 Installation of the membrane probe lunette Figure 40 Tightening of 2 screws Pay particular attention to ensure that the membrane is seated correctly in the dialysis probe The white side of the membrane must be inserted facing the dialysis probe buffer touching The silver side
29. T Trace is run internally by a computer and can be operated as a full ranked member in intranets and LANs or can be directly interfaced with a PC In order to do this the operating software supplied must be installed on the designated PC The files needed for the installation of the operating software trace_mon are stored in the device s data memory and can be accessed via an internal device website To install the software proceed as follows Connect the BioPAT Trace to your network and navigate to the factory set IP address 192 168 120 231 in your web browser Here you have the option of downloading important files and the operating instructions onto your computer and of assigning the device an IP address appropriate for your own network If you do not want to make any changes if there is a direct connection with a PC via a cross over Ethernet cable for example then the device will work with the factory set IP address Make sure that your PC is not set to automatic address assignment when using a direct connection Getting Started 77 First download the other important files for the rest of the installation onto your computer by clicking the right mouse button They can be found in the Download menu item The following files can be found here setup_tracemon_x_x_x as setup file of the operating software _x_x_x version number ip set as the backup file of the boot file Operating instructions
30. agram of the online dialysis method For the dialysis method the internal diffusion module is used to calibrate the BioPAT Trace By contrast the dialysis probe in the bioreactor is always used for measuring the sample A reference factor between the membranes is then determined once Design and Function 31 32 Design and Function As a standard cannot be installed in the bioreactor the BioPAT Trace must be calibrated indirectly by way of the reference factor During this process the initial concentration in the bioreactor is used as the starting concentration for the dialysis probe Using the sample measurement values with an identical concentration the BioPAT Trace calculates the factor between the diffusion module membrane and the dialysis probe membrane Hence the calibration procedure for the dialysis method consists of three steps in total Step 1 Recording the calibration curves on the diffusion module with standards 1 and 2 Step 2 Measuring the external sample on the BioPAT Trace with the help of the calibration curves alternatively by directly entering the concentration values if known from the initial weight or by external analysis Step 3 Recording the calibration curves of the dialysis probe using the probe After measuring the standards for calculating the calibration curves the BioPAT Trace computes a factor from the gradients of each individual curve For all measurements carried ou
31. ampling Systems 4 4 1 Accessories for the Dialysis Probe Description Order No Installation kit Dialysis probe BPT0045 Adapter 25 12 mm for the installation of the 165 mm dialysis probe in 25 mm side port Ingold port 52 mm incl o rings 4 4 2 Accesories for the Filtration Probe BB 38242540 Description Order No Installation kit Filtration probe BPT0059 Tubeset for hydrophilization of filtration membranes BPT0042 Membrane protective cage for filtration probe with membrane BPT0038 length 130 mm for the installation in 25 mm side port insertion depth extended by ca 25 mm Membrane protective cage for filtration probe with membrane BPT0039 length 90 mm for the installation in 25 mm side port insertion depth extended by ca 25 mm Equipment Supplied Accessories and Consumables 23 4 4 3 Accessories for the Dialysis and Filtration Probe Description Order No Retaining nut PG13 5 with o ring and sliding ring BPT0061 Adapter 19 12 mm for the installation of the dialysis and filtration BB 8848630 probe in 12 mm cover port M26x1 incl o rings Adapter 19 12 mm for the installation of the dialysis and filtration probe in 12 mm cover port RD28x1 8 BB 34104704 O ring outside for adapter RD28x1 8 BB 39120830 O ring inside for adapter RD28x 1 8 Table 4 Accessories for sampling systems 4 5 Accessories for BioPAT Trace BB 39121054 Description Order No S
32. ate The measurement cell is shown in Figure 12 The fluid inlet flow runs perpendicular to the 2 channel sensor wall jet principle A viewing window allows the operator to monitor the air bubble free flow through the cell Any air bubbles can be removed as necessary by rinsing the measuring chamber For that purpose the fluid is injected through the rinsing line equipped with a return valve Contact springs intercept the electric signal measurable at the electrodes and transmit it to the BioPAT Trace The measurement cell is fastened to the device with a knurled nut A It must be ensured that no air bubbles are in the measuring chamber of the flow cell as these can influence the results After connecting the sampling probe and before starting each measurement series check the measurement cell for air bubbles through the viewing window Any existing air bubbles must be removed with the help of the rinsing line A Use only transport buffers or distilled water to rinse the measurement cell Other fluids can damage the sensor Use only fresh solutions and or a sterile filter attachment in order to prevent microbes from entering the tubing set Design and Function 5 4 Particle Filter Function The particle filter is part of the tubing set It fulfills two tasks in the BioPAT Trace First sterile filtration of the buffer solution is performed through the membrane with a pore size of
33. ate dean 102 TUDING Set a nein Sender Ryo paces Od Me meade ARA RE 11 Appendix oct eed a dede uode tart daga teda ae nee 11 1 Data Sheet 6 Contents List of Figures Figure 1 Figure 2 Figure 3 Figure Figure Figure Figure Figure Figure OS at BOT Figure 10 Figure 11 Figure 12 Figure 13 Figure 14 Figure 15 Figure 16 Figure 17 Figure 18 Figure 19 Figure 20 Figure 21 Figure 22 Figure 23 Figure 24 Figure 25 Figure 26 Figure 27 Figure 28 Figure 29 Figure 30 Figure 31 Enzymatic conversion of glucose with parallel anodic oxidation OTL Ol nd ees edi oit cas eben dde een es aces ad aches add Enzymatic conversion of lactate with parallel anodic oxidation OT ELO isst ist ide ROE redes CLER eee os s dtes Enzymatic conversion of methanol ethanol with parallel anodic oxidation of H O sssssssssssssesesees eese Principle of the flow diffusion technique 006 Flow diagram to illustrate flow diffusion analysis Front view of the BioPAT Trace with the following components Side view of the BioPAT Trace with the following components Rear view of the BioPAT Trace with the following elements Principle of sample infeed via the dialysis probe ON the BIOPAT TEaCe iusto egg ener RU Dae RR ER RIRT Flow diagram of the online dialysis method Flow diagram of the online filtration method Measurement ce
34. ation To do so replace the solution in the upright cylinder see Figure 55 with water and start the pump at the operating panel see Figure 56 by pressing the Pump on button Stop the process after approx 10 minutes by pressing the Pump off button Remove the hydrophilizing set from the filtration probe and install the probe into the bioreactor according to the operating instructions Make sure to observe the instructions regarding the filtration probe in the operating instructions Improper assembly pre treatment or cleaning of the membrane may cause measurement errors or contaminations of the bioreactor 6 2 7 6 4 Performing Filtration Probe Regeneration after Fermentation The filtration probe must be removed from the reactor port immediately after drainage and sterilization of the bioreactor We recommend replacing the membrane and PTFE Ferrule after each use In most cases however repeated use of the membrane 3 5 times is possible after previous cleaning In this case proceed as follows Remove all deposits attached to the filtration probe e g by using a soft brush under running water Next a weak base e g 0 5 N NaOH for removal of the protein contamination on the membrane is pumped through the filtration probe into the closed system in filtrate direction the cleaning cycle lasts approximately 4 hours To this end you can use the same equipment as for hydrophilization Getting Sta
35. can now be observed more easily In due course a standard reference measure ment overwriting both values can be performed 8 4 Avoiding Contamination in the Tubing Set During longer processes over a period of weeks biofilm can form inside the tubing set over time This can result in the lines clogging or in highly fluctuating measured values This applies in particular to storage tanks with ready for use diluted transport solution that are repeatedly refilled with fresh solution It is advantageous to completely replace the almost empty container with a new container with fresh solution to avoid a transfer of microbes The rinsing line of the measurement cell when removing air bubbles in the measuring chamber presents another opportunity for microbes to intrude into the tubing set Therefore it is imperative to use only fresh solutions and or a sterile filter attachment for this purpose In some long term cultivations it was observed that biofilm would form in the waste line and spread into the drip chamber In this case it is also recommendable to regularly replace the waste containers and to prevent the waste line from being immersed in the waste The waste containers can be further protected by adding disinfectant solution see Consumables Table 3 When using the filtration method the disinfectant solution can even be added to the transport buffer 0 5 v v directly This way the most effective pro tection again
36. cking OK Autocalibration enabled Select yes no If you allow autocalibration recalibration takes place automatically after every x number of measurements see measurements between calibrations Calibration delayed Normally a calibration is performed after a certain number of measurements Longer interruptions e g measurements every 6 hours for cell cultivations can lead to very long periods expiring between the most recent calibration and the next measurement 102 Operation Activating the delayed calibration function schedules the calibration to be performed shortly before the next measurement This way an up to date calibration is available Number of measured values in groups When using larger measuring intervals it can make sense to run several measurements with maximum frequency in quick sequence at a given point of time This way for example a group of 10 measurements may be carried out every 6 hours The number of measured values in the group can be set here No average is calculated Every measured value is displayed immediately after measurement If the time required for the performance of the group is longer than the measurement period then the next group starts with the new measuring interval For the determination of the number of measure ments between calibrations every measurement in a group is taken into account This must be given due consideration when selecting the calibration settings
37. ction 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 electro chemically active and thus many extraneous substances in a sample solution will not be detected Selectivity is dependent on the working 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 BioPAT Trace Product Description 13 2 2 Biosensory Principle The analytes glucose and lactate are detected by means of enzymatic reactions Figure 1 shows the enzymatic conversion of glucose to gluconolactone In the presence of water gluconolactone is immediately hydrolyzed to gluconic acid The hydrogen peroxide H O 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 HO is 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 system is a further selection The combination
38. e tubes on the device seated in their slots see Figure 21 22 Whilst doing so press alternately on the black buttons on the valve to ensure you also insert the rear hoses deep into the valve slot Then check that the tubes are fitted correctly in the valve in order to avoid measuring errors Figure 21 22 Tightening and inserting the pair of tubes 46 Getting Started Figure 23 Securing the valve tubes by pushing together the guide plates After you are done fitting all three valves push both guide plates towards the valve in order to secure the position of the tubes in the valve slot Figure 23 As the next step install the two pump cassettes on the motor shaft and press on the retaining plate firmly until you hear an audible click indicating that it has locked in Before installing the pump cassettes check if the motor shaft is clean if necessary wipe with a soft cloth Dirt particles or abrasion of previous applications can adversely affect or even completely prevent the proper functioning of the pump The lower pump P2 and the upper pump P1 are attached one after the other see Figures 24 25 Figure 24 25 Attaching lower pump cassette P2 and upper pump cassette P1 Getting Started 47 p Figure 27 Fully assembled Getting Started Figure 26 Proper placement of the pump cassette fastenin
39. e valves shut In this event the device will retain stored data on calibration and reference values Operation 95 96 Operation Click the Stop button and the BioPAT Trace switches to standby The program can now be closed The device can also be switched off at the power switch without losing any data When switching the device back on or re booting the control program the prompt screen shown in Figure 75 appears Continue measurement or start with new tubing set New tubing set Figure 75 Restart prompt window after Stop By clicking on the button Continue measurement the system switches directly to operational mode No rinsing cycles or calibrations will be carried out The former calibration values will be retained Do not activate Continue measurement unless you are certain that the same conditions prevail as before the pause e g in the event of brief interruptions Otherwise this may result in erroneous measurements In the case of longer interruptions e g overnight it is recommended to carry out a calibration before continuing with further measurements 7 1 3 Menu Item Display Settings Information Display Options Show Connection Print Diagram Calibration values Status SRR eee a DEAD ay ae yee Figure 76 Display menu options When the menu item Show connection is activated an asterisk indicates tha
40. ect filtration or dialysis probe to tubing 60 76 44 45 57 58 9 Starting the measurements 7 85ff 64 65 Confirm Fill button and wait for calibration 86ff 64 65 Alternatively Activate Start automatically 86ff 64 65 option device starts measurement automatically after filling and calibration Tips and Checklist 17 Step Action Chapter Page Figure Checklist 10 For methanol ethanol only 6 2 5 52ff 34 36 Connect enzyme reactor Install enzyme reactor into tubing set 52ff 34 36 11 Check measuring chamber 5 3 and 34 60 6 2 6 4 Check measuring chamber for absence 34 60 of air bubbles rinse if necessary 12 For dialysis only Perform reference 7 88ff 66 69 measurement Perform reference measurement with 88ff 66 69 external sample or known measurement value 13 Optional Connect analog output 6 6 82 62 to process control system Connect analog output 82 62 0 10 V 4 20 mA bzw 0 20 mA Table 7 Checklist for Setting up Operation 118 Tips and Checklist 9 Troubleshooting If the BioPAT Trace cannot be started up or malfunctions occur while running operations then it is generally best to call upon experienced users for maintenance and or the manufacturer s service technicians for repairs To avoid life threatening hazards from an electric shock work on the electrical equipment should only be carried out by qualified and authorized experts You should only try to re
41. ed in red Advanced Text Import Settings A EN X Settings used to recogniz numeric data Decimal separator Thousands separator I Note Numbers will be displayed using the numeric settings specified in the Regional Settings control panel Reset V Trailing minus for negative numbers e Cancel Figure 91 Import data of the ptd file into Excel More text import options After confirming this window by pressing OK confirm step 3 of the text conversion assistant by pressing Next The data from the ptd file are now available as an Excel spreadsheet Tips and Checklist 115 Checklist for Setting up Operation Step Action Chapter Page Figure Checklist 0 Install PC software 6 3 and 6 4 77ff 59 61 only on initial setup Access website of the device 77 Download installation program 77ff Start installation program 78ff Adjust IP address and subnet screen 79 59 61 1 Prepare probes 6 2 0and 55ff 37 58 6 2 7 Install membrane 55ff 37 40 64ff 50 53 For filtration only Hydrophilize membrane 69ff 54 56 with isopropanol Install probe in reactor 58ff 41 43 and sterilize if necessary 74ff 2 Prepare transport buffer and 622and 44 111 waste container 8 4 Dilute buffer concentrate and 6 2 2 42 prepare in bottles Prepare waste container with disinfecting 8 4 111 agent if necessary 3 Setup device tubing 6 2 2 to 42ff 17 33
42. er sterilization Re open the Luer connector after cooling e As before fill the probe with buffer solution by using the single use syringe and carefully check if it flows freely see Figure 42 Now the dialysis probe can be connected to the corresponding tubing set of the online analyzer f Getting Started 59 6 2 6 4 Connecting the Dialysis Probe to the Tubing Set The dialysis probe is connected to the online analyzers through the dialysis tubing set The inlets and drains on the probe are labeled with arrows on the side The labels on the tubing set face the same direction see the following Figures 44 and 45 a X N t Ne 4 Y es a NS A V s ui Pm r5 Vo o C uv 5 gt Figure 44 45 Connecting the dialysis probe to the tubing set remove any air bubbles from the measuring chamber at the online analyzer A When starting up the probe after sterilization 60 Getting Started 6 2 6 5 Cleaning the Dialysis Probe and Replacing the Membrane a b c e Before removing the dialysis probe from the bioreactor the tubing set is removed from the probe by removing the tube ends After harvesting the bioreactor or any dead autoclaving phase remove the dialysis probe from the reactor The subsequent cleaning of the probe and the adapter is carried out with a soft brush and water Check to make sure that the gaskets on the dialysis probe adapter are
43. f necessary reinstall the PC software trace mon from the website Table 9 Identifying and correcting calibration faults Troubleshooting 123 10 Care and Maintenance 10 1 Device If a measurement series is interrupted for a short time maximum of two days the system can continue to be operated without replacing the tubing set If the BioPAT Trace is to be decommissioned for longer periods of time longer than 7 days the tubing set should be replaced upon recommissioning The surface of the BioPAT Trace housing should only ever be cleaned with a wet cloth 10 2 Tubing Set Clogged or damaged tubing sets should be replaced with new ones Before every startup of the system check all connections made with Luer lock or UNF fittings for any leaks and tighten them as necessary 124 Care and Maintenance 11 Appendix 11 1 Data Sheet Measuring principle Enzymatic amperometric Measuring range glucose 0 10 40 g L Measuring range lactate 0 05 5 g L Measuring range methanol 0 5 20 g L Measuring range ethanol 1 40 g L Measurement frequency Up to 60 measurement values h Measurement deviation glucose lt 3 at 5 0 g L Measurement deviation lactate lt 3 at 2 5 g L pH range of the medium 4 8 9 2 Service life of the enzyme electrode 30 days or 5000 analyses Ambient temperature 15 35 C Ambient humidity 10 90 In
44. ferent or incorrect IP addresses on the device website and during the installation it will not be possible to establish a connection The following options are available First attempt to produce a connection to the internal device website via the standard IP address 192 168 120 231 using a direct PC connection and a cross over Ethernet cable If this is not possible a change to a known IP address must be forced using the boot file First switch the device off Then copy the boot file ip set onto a USB stick and connect it to the back of the device Then switch the device on Once the boot process is complete the device will now have the new IP address 192 168 120 232 Navigate to the new IP address 192 168 120 232 in your web browser After the device has been connected in this manner you can set the network address via your web browser as described above Terget type Application Target locator trace v 10 10 Toget VTRACE v roce mon exe TEER Sut CATRACE Sherteut key None Figure 61 Start settings 6 4 Installing the PC Software Run the file setup tracemon to install the operating software on your computer When doing so you will be asked to enter the new IP address In order to do this overwrite the suggested factory set IP address If you have not changed the IP address then use Next to confirm During the installation you will be asked to specify the storage locations for the program and the s
45. g stud locked in place The proper placement of the pump cassettes should be verified Figure 26 After installing the pumps and valves on the front of the device attach the sensor drip chamber and diffusion module to the side panel Figure 27 shows a completely assembled side panel First insert the measurement cell into the measurement amplifier holder Figure 28 To this end unscrew the knurled nut on the upper retaining plate to allow it to be pushed upwards Next place the lower edge of the measurement cell in the recess in the lower border of the retaining plate Figure 28 Figure 28 Inserting the measurement cell into the device Leverage the measurement cell up onto the contact plug While doing so pull the upper retaining plate slightly upwards Press the measurement cell onto the holder and slide the retaining plate downwards Figure 29 The measurement cell is fastened to the device by tightening the knurled nut Figure 29 Fastening the measurement cell to the device Getting Started 49 Afterwards attach the rest of the components to the device Using the holding slits on the sides fasten the diffusion module to the mounting rail Figure 30 Make sure that the diffusion module is properly aligned with the arrow pointing upwards Figure 30 Fastening the diffusion module to the mounting rail Next insert the two drip counters into the holder designed for this p
46. hecklist 109 110 Tips and Checklist It is therefore recommended that you determine the reference value as soon as possible at the start of the cultivation and do so in a phase without fluctuations in the concentration of the medium For this purpose deactivate autocalibration and then set the measuring interval to 1 Afterwards track some measured values If they do not spread too widely and are as constant as possible you can trigger reference measurement Once the reference measurements are completed you should wait for some measurements and check if the recalculated values meet your expectations If necessary you can also repeat the reference measurement As soon as you are satisfied with the result you can readjust the measurement parameters and start with the cultivation 8 3 Dialysis Mode Reference Measurements for Low Lactate Concentrations In many cases there is a surplus in glucose and the medium is still lactate free at the start of a cultivation When performing reference measure ments the BioPAT Trace suggests a value once the reference measurement is completed This value is usually overwritten by entering the known value known due to the media composition or determined with an external analyzer If the available lactate concentration is still very low do not yet overwrite this value the reference factor for lactate is still 1 0 During the course of the cultivation the increase in lactate concentration
47. ign and Function loaded Buffer stream V a Analyte Figure 14 Functional principle of the dialysis probe 5 6 Dialysis Probe Function If a dialysis probe is used to feed the sample from the bioreactor into the BioPAT Trace then the analyte is transferred through a diffusion membrane into an internal pump driven buffer stream and transported to the measurement cell Figure 14 shows how the principal function of how the probe works The dialysis probe is suitable for all types of cultivation The use of an extremely robust diffusion membrane ensures a high level of sterility The different membrane materials enable dialysis cellulase stabile dialysis and gas diffusion for separating substrates The probe can be installed in the bioreactor as well as in the cover or in a side nozzle as well The application options for the dialysis probe depend on the reactor dimensions and the process conditions In general the dialysis probe is recommended for use on smaller bioreactors where any loss of volume during sampling is unacceptable The use of the dialysis probe as a sampling system ensures analysis at constant volumes Design and Function 37 5 7 Filtration Probe Function Figure 15 Filtration probe for sterile sampling from bioreactors Cell free substrate samples are removed from the bioreactor using the sampling probe on a filtration basis Figure 15 The hydrophilized probe i
48. is PC must also be connected to the local network or directly to the device using the cross over Ethernet cable supplied After connecting the device to the PC check to make sure that the system times of the two units are synchronized Proceed as described in Chapter p gt 7 1 4 2 Basic Settings on page 106 Getting Started 41 6 2 2 Tubing on the Device Depending on the type of operation intended the BioPAT Trace is equipped with corresponding tubing set fluidics For each of these types of operation you will find below the corresponding instructions for attaching the tubing set The corresponding tubing set consists of the components necessary for analysis Pump cassettes valve tubes measuring chamber diffusion module drip counters and connection lines for buffers standards waste as well as the sampling probe filtration dialysis The lines for buffers standards sampling probe and waste are premounted and packaged in foil bags It is recommended to only open these after the tubing set has been attached to the device in order to facilitate handling 42 Getting Started Figure 17 View of a tubing set for the Filtration operating mode In addition the dialysis tubing set is provided with a single use syringe with three way stopcock for the analysis of external samples Before you start attaching remove the two empty cassettes on the pump slots The empty cassettes can be reused a
49. iter Transport buffer concentrated 5x for microbial cultivations BPTOO60 Application Glucose Lactate 1 liter Transport buffer concentrated 5x Application Ethanol Methanol BPT0046 1 liter 20 Equipment Supplied Accessories and Consumables 4 2 3 Disinfectant Concentrate Description Order No Disinfectant concentrate 1 liter BPT0044 4 2 4 Calibration Standard content 0 5 liter Description Order No Calibration standard 0 1 g l Glucose 0 05 g l Lactate BPT0043 Calibration standard 0 5 g l Glucose 0 25 g l Lactate BPTOO11 Calibration standard 1 g l Glucose 0 5 g l Lactate BPTOO10 Calibration standard 2 g I Glucose 1 g l Lactate BPTOO09 Calibration standard 4 g I Glucose 2 g l Lactate BPTOO08 Calibration standard 10 g l Glucose 5 g l Lactate BPT0007 Calibration standard 20 g l Glucose 10 g l Lactate BPT0041 Calibration standard 0 2 g l Methanol BPT0051 Calibration standard 1 g l Methanol BPT0052 Calibration standard 5 g l Methanol BPT0053 Calibration standard 20 g l Methanol BPT0054 Calibration standard 0 5 g l Ethanol BPT0058 Calibration standard 2 g l Ethanol BPT0057 Calibration standard 4 g l Ethanol BPTOO56 Calibration standard 40 g l Ethanol BPT0055 Equipment Supplied Accessories and Consumables 21 4 2 5 Membranes Description Order No Membranes for Dialysis probe Glucose Lactate 5 pieces BPT0024 Membranes for Dial
50. justed application do not Adjust application properly match e g dialysis tubing set and filtration adjusted at the device 6 Atube is pinched e g when screwing the Remove pinch covers in the standard bottles 7 There is clogging in the tubing set When Remove clogging If this is not the buffer remains in the tubing set over possible use a new tubing set an extended period of time salts begin to crystallize out Crystallization usually occurs on the silicone tubes in the valves Clogging can also occur due to microbial growth 8 Leaks If the clog is not removed tubes may Correct leaks If this is not burst possible use a new tubing set 9 Sensor does not have proper contact When the Remove sensor and reinstall it sensor is not inserted properly the reverse side properly of the sensor does not have proper electrical contact with the sensor holder 122 Troubleshooting Possible Causes What should you do 10 The dialysis membrane was installed in Correctly install a new dialysis the probe in the incorrect position only for membrane into the probe DIALYSIS application 11 Three way stopcock was not properly adjusted Adjust the three way stopcock only for DIALYSIS application An external to the correct position sample was measured and the three way stopcock was not reset 12 The software versions of the BioPAT Trace and Use suitable software the PC are not compatible I
51. lified personnel only The symbols used in the operating instructions are specifically intended to draw attention to the safety precautions The symbol may not replace the text of the respective safety precaution Therefore the text must always be read completely Indicates useful information and user tips Warning of potential consequential damage Warning of dangerous electrical voltage gt eee Warning of materials that are health hazards or irritants General Safety Instructions 19 4 Equipment Supplied Accessories and Consumables 4 1 Equipment Supplied Description Order No Online Analytical System BioPAT Trace with power adapter BPT0001 power plugs Europe USA UK cross over ethernet cable operationg instructions and PC software trace_mon Table 1 Equipment supplied with BioPAT Trace 4 2 Consumables 4 2 1 Tubesets Description Order No Tubeset Dialysis Glucose Lactate BPTO003 Tubeset Filtration Glucose Lactate BPTOO04 Tubeset Filtration Glucose Lactate cellulase resistant BPTOO05 Tubeset Dialysis Ethanol Methanol BPTOO50 Tubeset Filtration Ethanol Methanol BPT0049 Enzyme reactor for Application Ethanol Methanol BPT0048 4 2 2 Transport Buffer Concentrate Description Order No Transport buffer concentrated 20x for cell cultivations BPTOO06 Application Glucose Lactate 0 5 l
52. ll with rinsing line 2 diedu Schematic design and fixation of the diffusion module Functional principle of the dialysis probe 0 Filtration probe for sterile sampling from bioreactors AOD enzyme redetar eno utes aw REC Lease eU doe 34 OA View of a tubing set for the Filtration operating mode View with fully installed tubing on the front pane for the Filtration operating mode eee eee eee Tightening and aligning of the pair of tubes in the guide plate Position of the valve tubes on the device 0006 Tightening and inserting the pair of tubes Tightening and inserting the pair of tubes Securing the valve tubes by pushing together the guide plates Attaching lower pump cassette P2 and upper pump cassette P1 Attaching lower pump cassette P2 and upper pump cassette P1 Proper placement of the pump cassette fastening stud locked in place nsscreenigpenrssiienrepeneias Fully assembled side panel 0 2 2 0 cece eee eee eee Inserting the measurement cell into the device Fastening the measurement cell to the device Fastening the diffusion module to the mounting rail Inserting the drip counters into the holder Contents Figure 32 Figure 33 Figure 34 Figure 35 Figure 36 Figure 37 Figure 38 Figure 39 Figure 40 Figure
53. ltration start window with operating panel to control the pump for hydrophilizing Getting Started 71 72 Getting Started 6 2 7 6 3 Performing the Leak Test Once hydrophilization has been completed a leak test must be performed via the user interface see Figure 56 To this end the Leak test control button is activated The hydrophilization setup depicted in Figure 55 is used for the leak test The free tube end of the hydrophilizing set must hang freely in the air above the liquid level in the upright cylinder see label in Figure 55 The test process takes 8 minutes Meanwhile air is pumped into the probe Afterwards the pump automatically draws the isopropanol back in through the membrane to ensure that the membrane does not become hydrophobic again During the test process the following should be observable After approx 6 minutes many small air bubbles should gradually start to escape from the membrane Otherwise the membrane is hydrophilized insufficiently f you observe large air bubbles escaping from the screw connections this indicates that the gaskets are damaged In order to maintain the hydrophilic properties of the membrane until its installation in the bioreactor it is recommended to store the membrane in 70 isopropanol solution Immediately before installation into the bioreactor the isopropanol must be replaced by water in order to avoid evaporation and the loss of hydrophiliz
54. measurement range of the BioPAT 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 60 analyses per hour maximum Due to its 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 can range between 15 and 35 C due to internal temperature correction The ambient humidity should not exceed 90 Analysis in the BioPAT 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 application serves as the biosensor BioPAT Trace Product Description Depending on the application the service life of the sensor system ensures 30 days or 5 000 measure ments The deviation from the average measurement is less than 3 for a measurement of 5 g L glucose and 2 5 g L lactate 2 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 measurement cell In the presence of an electrolyte solution the electrochemical conversion of the substance at the working electrode leads to the release or attra
55. medy malfunctions that are obviously attributable to operating or servicing errors yourself Under no circumstances should you try to troubleshoot any problems with electrical equipment If you have problems with BioPAT Trace start by consulting the table below It contains an overview of potential malfunctions Troubleshooting 119 Error Error message Possible Causes Calibration failed Calibration standard empty Transport buffer empty Air bubble inside the measuring chamber Incorrect tubing set used Measurement cell not properly inserted Wrong method selected Valve tubes inserted incorrectly or not inserted Dialysis only Three way stopcock incorrectly adjusted Tubing set out of date Leaks in the tubing set Calibration line blocked or tubing set clogged Software versions of device PC not compatible Valve yellow cannot be triggered Measured value too high or too low Calibration erroneous Standard parameter entry and actual standard do not match Air bubble inside the measuring chamber Transport buffer empty Leaks in the tubing set Dialysis only Reference measurement not performed Dialysis only Reference measurement not up to date No or negative measured value 120 Troubleshooting Measurement cell not properly inserted Transport buffer empty Tubing set clogged Leaks in the tubing set Tubing set out of date No sampling probe or wrong sampling probe connected
56. n cell that is only allowed contact with calibration solutions and in the dialysis probe that only has contact with the reactor contents The relationship of the two membranes to one another is expressed by a reference factor that is used for calculating the measured values You can conduct a reference measurement whenever you want in particular when changes occur in the process that may influence the diffusion speed e g as a result of temperature changes Reference measurements can also be carried out at regular intervals e g when samples are taken for external accompanying analysis The measured value is only corrected with a new reference factor when the new reference value was entered and confirmed by pressing Apply Up to then measured values that were determined in the meantime are always calculated and displayed with the current reference factor The device starts with a reference factor of 1 0 If the two membranes were permeable to the same degree then this value would be correct In practice however experience has shown that manufacturing related and ambient conditions affecting the probe can result in differences The comparison is activated as follows After clicking the Reference button the device determines the concentration in the reactor and suggests the corresponding values see Figure 66 Now using a reference sample you must check whether these values are correct and adjust them accordingl
57. n decrease of the reactor capacity and should therefore be absolutely avoided First start the filling process to fill the tubing set see Chapter 7 Figure 64 The progress of the filling in the display can be tracked by activating the status display see Chapter 7 1 3 The device must be stopped prior to or during the preliminary measurements to allow the reactor to be installed Stop the device with Stop see Chapter 7 1 2 3 If air enters into the enzyme reactor there is the danger of a decrease in the capacity of the enzyme reactor in association with a lower sensitivity In order to install the enzyme reactor remove it from the transport container and open the red cover caps Afterwards install it into the intended location in the tubing set see Figures 34 36 After installing the enzyme reactor restart the device with Setting up Operation see Chapter 7 1 2 2 Select New tubing set and start another filling process with the installed enzyme reactor pl h Figure 34 Place of installation Figure 35 Securing the enzyme for the enzyme reactor reactor Getting Started 53 Figure 36 Fully installed enzyme reactor The removal of the reactor is also carried out in stopped status Device Stop The reactor is closed with the red cover caps placed into the transport container and stored at a cool temperature between 4 8 C A
58. n optionally be attached to laboratory stands suspension bars or benchtop stands A corresponding stand clamp and a suitable benchtop stand are available as accessories Under no circumstances should a voltage source be connected to the analog data outputs at the back of the BioPAT Trace This also refers to a multimeter for resistance measurement If a multimeter is used to measure the voltage or current the contacts to the BioPAT Trace must be removed before changing the measurement range on the multimeter Otherwise there is the danger that the device will be damaged Design and Function 5 2 BioPAT Trace Measurement Methods Two online measurement methods are possible with the BioPAT Trace Filtration with a filtration probe Dialysis with a dialysis probe The simplest method is to directly measure a filtered sample of medium However because reactor medium is used the range of applications is limited to processes for which there s a sufficient reactor volume or which allow continuous feed Dialysis sampling is an option when processes are involved for which reactor volume does not allow enough sample material This method only removes low molecular substances from the reactor medium without reducing the volume of fluid For example Figure 9 shows a photographic representation of how the BioPAT Trace can be connected to a bioreactor by way of a dialysis probe A more detailed explanati
59. nd Data Saving sseeesleeseereeeee 17 3 General Safety Precautions 0 eee 18 4 Equipment Supplied Accessories and Consumables 20 41 Equipment Supplied sss 20 4 2 Consumables on eee a c E ahs ee eR aE NR T ER 20 4 2 1 Tubesetszi bi wei sr ro FEE ea xq eaa tma p dE HE 20 4 2 Transport Buffer Concentrate 2 0 ee eee eee eee 20 4 2 3 Disinfectant Concentrate 20000 0c cece cece 21 4 2 4 Calibration Standard content 0 5 liter 0 00 0 21 42 5 Membraries esce vaccum mage mh Reece nx Rd Habd e R 22 43 Sampling Systems i orencib reme ba Rx e Ee red ebbe p cep Ped d 22 4 3 1 Dialysis Probe delivery with installation kit 2 membranes and user MANU Al sarireki tov UrPIQSdm I UT ETE PUE 22 4 3 2 Single Use Dialysis Probe delivery with user manual 22 4 3 3 Filtration Probe delivery with installation kit 1 membrane and user manual 0c cece tEn EP eee eee eee eee RE 23 4 4 Accessories for Sampling Systems 0 c cece ee eee eee 23 4 4 1 Accessories for the Dialysis Probe 20000000 eee 23 4 4 2 Accesories for the Filtration Probe 000000005 23 4 4 3 Accessories for the Dialysis and Filtration Probe 24 4 5 Accessories for BioPAT Trace errre endt cece eee esses 24 Contents B 5 Design and Function 000 cece eee eee 25 4 5 1 5 2 5 3 5 4
60. nd of the tube has no fitting see Figure 54 6 2 7 6 2 Performing Hydrophilization After installing the filtration probe the polypropylene membrane must be hydrophilized with isopropanol 70 v v The probe is immersed into the 70 isopropanol solution inside an upright cylinder of sufficient length so that the membrane is entirely surrounded by liquid The tube end with the UNF fitting is connected to the filtration probe The free end of the tube is secured in the upright cylinder so that it hangs freely in the air above the liquid level see label in Figure 55 The pump cassette of the hydrophilizing set is attached to a motor shaft of the BioPAT Trace The second motor shaft remains unused As shown in Figure 56 the start window appears after starting the TraceMon program in the filtration mode The operating panel with control buttons for starting Pump on and stopping Pump off the pump as well as a control button for the leak test are on the right half of the user interface The hydrophilization process is started by pressing Pump on The isopropanol solution is sucked through the filtration probe and then pumped back into the upright cylinder The hydrophilizing period should last for at least 2 hours in order to achieve sufficiently high filtration during operation When the hydrophilization process is complete the pump is stopped by pressing the Pump off button fasc sate Figure 56 Fi
61. ng Figure 53 Tightening the cap nut Moisten the outer diameter of the filtration probe with demineralized water prior to assembly The screw connection of the filtration probe is tightened until it reaches its mechanical stop position Getting Started 67 68 Getting Started 6 2 7 4 Hydrophilizing the Polypropylene Membrane New polypropylene membranes are impermeable for aqueous media hydrophobic Thus the membrane must be hydrophilized after installation into the filtration probe Isopropanol 70 v v is used as hydrophilization solution The probe is immersed into the hydrophilization solution inside an upright cylinder of sufficient length that the membrane is entirely surrounded by liquid The alcohol is pumped through the probe into the closed system with a flow rate of 1 2 ml min by using a peristaltic pump connected to the sampling outlet of the probe The hydrophilizing period lasts at least 2 hours The probe can also be hydrophilized overnight without using the pump by storing it in 70 v v isopropanol 6 2 7 5 Leak Test Once hydrophilization has been completed a leak test is carried out In order to do this the hydrophilization setup described in point 6 2 7 6 2 is used Instead of drawing isopropanol through the membrane of the probe air is now pumped into the probe The flow rate should be equal to the flow rate of the previous hydrophilization Many small air bubbles should
62. nnection on red on Table 6 LED status messages 108 Operation 8 Tips and Checklist 8 1 Choosing Suitable Standard Solutions Various combinations of calibration standard solutions are available to optimally cover the expected concentration cycles We recommend connecting a higher concentrated standard than standard 1 Depending on the process conditions and the objective of the cultivation various concentration ranges have to be covered The concentration range should principally be between the concentrations of the standard solutions in order to ensure optimal measuring accuracy Furthermore we recommend always choosing a standard with very low concentration in order to properly determine measured values close to zero For regulation the concentrations of the calibration standards should not be too far from the regulation set point For regulation at 1g L for example it is more practical to use a combination of 0 5 g L and 4 g L than a combination of 1 g L and 10 g L for example 8 2 Dialysis Mode Reference Measurements at the Start of Fermentation It is crucial to perform reference measurements when using the dialysis method to compare the permeability of the membranes inside the reactor and outside the diffusion cell as well as to compensate for temperature differences between both membranes Thus reliable determination of the reference factor is very important for the quality of the measured values Tips and C
63. of the membrane faces the probe lunette media touching Incorrect insertion of the membrane may result in leaks in the dialysis probe head and jeopardize the sterility of the entire cultivation process Getting Started 57 58 Getting Started 6 2 6 2 Installing the Dialysis Probe into the Bioreactor Insert the friction washer and the O ring prior to installation in the fermenter Figure 41 5s Figure 41 Inserting the friction washer and O ring 6 2 6 3 Sterilization of the Dialysis Probe Prior to Connecting to the BioPAT Trace Before connecting the dialysis probe to the BioPAT Trace the probe must be sterilized along with the bioreactor Connect the dialysis probe to the tubing set as shown in Figure 42 The inlets and drains on the probe are labeled with arrows on the side The labels on the tubing set point in the same direction Figure 42 Filling the probe Figure 43 Sealing the probe with buffer solution prior to sterilization a Prior to sterilization fill the dialysis probe with buffer solution using the single use syringe and the Luer UNF adapter Figure 42 At the same time the probe can be checked for any leaks b For sterilization seal the probe by connecting the Luer connections Figure 43 This will prevent one sided overpressure from occurring at the membrane in the dialysis probe which could damage the membrane c Now sterilize the reactor d Aft
64. on of the sampling probes can be found in Chapters 5 6 Dialysis Probe Function on page 37 gt 5 7 Filtration Probe Function on page 38 Design and Function 29 Figure 9 Principle of sample infeed via the dialysis probe on BioPAT Trace 30 Design and Function 5 2 1 Dialysis Measuring Method Using the Dialysis Probe The dialysis measuring method using the dialysis probe involves the transfer of analytes through a dialysis membrane in the dialysis probe which separates the reactor contents medium from the acceptor stream At the same time the dialysis membrane functions as a sterile barrier Figure 10 shows a schematic of the flow system The calibration valve green and the three way stopcock can be used to choose between the standards and the manual probe The sample valve red and the bypass valve yellow can be used to select the dialysis probe and the diffusion module The upper part of the flow system represents the donor stream which is responsible for the infeed of the calibration solution into the diffusion module Depending on the task at hand the carrier stream accumulates the analyte in the diffusion module or in the dialysis probe External Sample Waste Standard 1 Standard 2 Measurement cell ou Diffusion module c Qv O vaez e Valve 3 e Check Valve rump e 3 way Stopcock Transport buffer Dialysis probe Figure 10 Flow di
65. onversion assistant step 2 0 0 e cece ee eee ee 114 Importing data from the ptd file into Excel text conversion assistant step 3 0 0 0 0 0 e cece eee eee 114 Importing data from the ptd file into Excel Further text import settings llle 115 Contents 9 List of Tables Table 1 Table Table Table Table Table Table Table Table 10 2 3 4 5 Table 6 7 8 9 0 Equipment Supplied with the BioPAT Trace Consumables for BioPAT Trace 2 200e cece eee eee ee Sampling Systems for BioPAT Trace 002e0e ee eee Accessories for sampling systeMS 0 0 0 eee eee Accessories for BioPAT Trace 0 2 00 e cece crete ees LED Status Messages io iso she cbr REOR nnii RETE R Ren Checklist for Setting up Operation s ciscsscirsiscisienrsiiras Problem Description and Troubleshooting on the BioPAT Trace Identifying and Correcting Calibration Faults Technical Data Sheet ror inet ae RR hme Explanation of Symbols Notes with useful information and user tips Warning of potential consequential damage Warning of dangerous electrical voltage A A A Warning of materials that are health hazards or irritants 10 Contents 1 Introduction BioPAT Trace is a 2 channel online analysis system used to determine glucose and L lactate Its applications cover both microbial fermenta
66. or language settings By placing a check mark next to Adjust time you can compare the system times on your PC and the BioPAT Trace The BioPAT Trace will then use the PC s system time 7 1 4 3 Language You can select your preferred language under the Language menu item Figure 85 7 1 5 Menu Item Information Select the menu item Information to display the current software versions of the BioPAT Trace and the control software Operation 107 7 2 LED Status Messages In order to be able to read the device status independently of the operating software on a connected PC directly on the BioPAT Trace the various operating statuses can be read using the LEDs on the front The following table 4 provides an overview of the LED signals Status Action LED1 green LED 2 color LED 2 status Setting up Operation and Decommissioning Boot process on white on Stop on cyan on Filling on cyan flashes Preliminary on green cyan flashes measurement Measurement Measurement loaded on green on Measurement unloaded on green flashes Breaks in Measuring Readiness readiness on yellow on Interval standstill on yellow flashes Rinsing Cycles Changing the medium on magenta on Rinsing probe on magenta flashes Calibrations Calibration loaded on blue on Calibration unloaded on blue flashes Reference loaded on blue on Reference unloaded on blue flashes Faults No network co
67. osed instructions It is also extremely important that you follow the procedures described for hydrophilizing the polypropylene membrane for installation into the bioreactor leak testing in line sterilization and cleaning of the filtration probe Getting Started 63 6 2 7 1 Description of Setup The filtration probe for the installation into a 12 mm cover port consists of the filtration unit 1 with membrane support 2 cap nut 3 Ferrule 4 O ring 5 polypropylene membrane 6 assembly kit clamping device with friction washer and O ring 7 and dummy plugs 8 Figure 47 Components of the filtration probe for installation into the 12 mm cover port with PG 13 5 connection The polypropylene membrane 6 is fastened to the membrane support 2 a threaded pipe made of stainless steel The permeate created by the membrane is transported through a coil like channel to a total of 4 cross drilled holes They guide the filtrate along the shortest way possible to the collective drill hole of the filtration unit so that a remixing of the sample can be avoided The PTFE Ferrule 4 covers the end of the membrane and the disclosed part of the membrane support By tightening the cap nut 3 the Ferrule is deformed by pressure and provides a safe seal for the membrane end facing the membrane support 64 Getting Started The probe thread is protected against the intrusion of microorganisms by the O ring
68. rt automatically Figure 64 the device will automatically initiate the measurement after performing the initial calibration routine If the Fill button is pressed the device starts filling and the Fill button disappears 86 Operation Before you startup your system with autostart make sure that the tubing set is properly connected to the sampling outlet on the bioreactor When the system is ready to run the measurement the line on the lower right will appear Figure 65 In filtration mode two buttons become available after measurement ready mode has been achieved The one is the calibration button which can be pressed to run any new calibration measurement with the device and the other is the Start button that activates the measuring routine Leg Device Ouplay Settings Information Cucote Figure 65 Screen display in Standby mode In the dialysis mode a third button labeled Reference appears to the right of the measuring window If the Reference button is activated the device starts with a reference measurement to calibrate the attached dialysis probe Operation 87 Referen Glucose HESS Lactate 1 500 Apply Measurement Cancel Figure 66 Display after reference measurement 88 Operation Reference for dialysis only A reference measurement must be performed to compare the permeability of the two diffusion membranes in the diffusio
69. rted 73 74 Getting Started Once the cleaning process is completed fill the upright cylinder with demineralized water and carefully rinse the filtration probe with the connected hydrophilizing set so that the residues from the alkaline solution are removed In order to maintain hydrophilization of the membrane the probe must be stored in hydro philization solution 70 isopropanol until its next use If the filtrate flux of the filtration probe is insufficient despite intensive rinsing with alkaline solution a new membrane with the required PTFE Ferrule must be installed 6 2 7 7 Installation in the Bioreactor Prior to each installation of the filtration probe in the bioreactor s extraction ports all screw connections have to be checked and retightened if necessary The filtration probe O ring must be checked for damage and if necessary replaced as well Choose a port on the reactor that allows the probe to be positioned in an area with the greatest possible turbulence A high crossflow rate in the area of the filter surface leads to an extension of the filtration probe service life crossflow effect After installation make sure that the probe never comes in contact with moving parts e g stirrer stirrer shaft inside the bioreactor The polypropylene membrane always must be entirely immersed in the liquid during operation of the bioreactor Otherwise the filtrate may be drastically decreased High
70. s Probe Prior to Connecting to the BioPAT Trace 2 000005 6 2 6 4 Connecting the Dialysis Probe to the Tubing Set 6 2 6 5 Cleaning the Dialysis Probe and Replacing the Membrane 000000 eee ee Connecting the Filtration Probe 1 0 6 2 6 2 7 1 Description of Setup ise csse er temen khe wu 6 2 7 2 Optional Accessories 0 eee eee eee eee eee eee 6 2 7 3 Assembling the Filtration Probe 2000 6 2 7 4 Hydrophilizing the Polypropylene Membrane 6 2 725 Leak TESt sese De rear er RR RR RU CH ORA 6 2 7 6 Using the Hydrophilizing Set sossnrcsrerisisioriresevi 6 2 7 6 1 Description of Setup 0 0 0 eee eee 6 2 7 6 2 Performing Hydrophilization 6 2 7 6 3 Performing the Leak Test 0004 6 2 7 6 4 Performing Filtration Probe Regeneration after Fermentation 0c eee eee eee 6 2 7 7 Installation in the Bioreactor 00 c eee eee eee 6 2 7 8 In line Sterilization of the Filtration Probe 6 2 7 9 Connecting the Filtration Probe to the Tubing Set Loading the Software and Network Connection suus 6 3 1 TroubleshoOtifig ia idees EAR ORT RED NR ER ees Installing the PC SoftWare i iuueni rr mr ERR ORE UR TE EE Changing the Start Settlngs sisse rere Ra Rape ETRE REPRE EX Assignment of Analog Outputs sssssssseeseseeh Device StartUp suus exte eu t Ra e S EROR t Ree
71. s in the guide plate 6 2 3 Attaching the Filtration Tubing Set Before attaching the filtration tubing set the valve tubes must first be inserted into the pinch valves To keep them from getting mixed up each line is color coded The following rules apply The pair of tubes are color coded red yellow and green and are kept in place by two guide plates The top to bottom order follows the traffic light principle red at the top yellow in the middle green at the bottom The color coded line of the pair of tubes is always inserted into the front valve slot When inserting the tubing always guide the labeled side with your right hand After you have inserted the tubing set check that the result looks like in this picture As the first step take both guide plates in both hands and open them slightly in order to align the valve tubes in parallel see Figure 19 The red pair of tubes should be on top and the label on the right should be visible Getting Started 45 Now place the guide plates on the valves as such that the pair of tubes are positioned on their corresponding valve slots see Figure 20 When inserting the tubing make sure that the red yellow and green color coding is always located to the right of the valve and matches that valve s color coding During insertion hold both sides of the tubing tautly and press the pair down until both tubes are securely Figure 20 Position of the valv
72. s mounted inside the bioreactor and subsequently sterilized along with the medium The membrane module of the probe is positioned in the turbulent zone of the reactor near the agitator A tubular membrane made of polypropylene serves as a sterile barrier The filtrate flux does not exceed 2 mL min After sampling the cell free sample can be fed directly into the BioPAT Trace for analysis The filtration probe is used when a sufficient reactor volume is available 38 Design and Function Figure 16 AOD enzyme reactor 5 8 Enzyme Reactor Function The enzyme reactor is required for measuring methanol and ethanol Figure 16 Inside the reactor is the immobilized enzyme alcohol oxidase AOD There the corresponding alcohol is converted and hydrogen peroxide is formed The hydrogen peroxide is then detected in the measurement cell Due to the stability of the enzyme used A the ambient temperature may not exceed 25 C At higher temperatures it is recommended to use a climatic chamber 5 9 Temperature Correction The BioPAT Trace is equipped with a temperature sensor This is arranged in the proximity of the measurement cell All measured values are corrected by using a temperature dependent function Design and Function 39 40 Getting Started 6 Getting Started 6 1 Transport The BioPAT Trace including accessories is delivered in protective packaging Please save this packaging proper
73. s transport protection for the motor axle Only use tubing from the prefabricated original tubing set To ensure that all solutions flow freely never allow the tubing segments to be crushed or kinked Make sure that the valve tubes are firmly seated in their pinch valves in order to ensure that the tubing can be opened and closed without problems Getting Started 43 44 The inlet and outlet lines of the tubing set are packaged in foil bags and labeled The individual tubes are affixed with the following labels BUFFER Connect to the transport buffer WASTE Connect to waste Probe port 1 dialysis Probe port 2 dialysis Sample Probe port filtration Standard1 Cover label for standard calibration solution 1 Standard2 Cover label for standard calibration solution 2 Before filling tubing set make sure that tubing is connected to all solutions and that the vessels are sufficiently filled Tubing sets that are only partially filled or not filled at all can produce erroneous measurements Getting Started The transport solution is delivered as concentrate Observe the note for proper dilution on the packaging Incorrect or no dilution will lead to erroneous measuring values Figure 18 View of fully equipped front panel for the Filtration operating mode Figure 19 Tightening and aligning of the pair of tube
74. shipping is only possible in the original packaging Upon receipt check the delivery for completeness and any possible damage that may have occurred in transit Any transport damage should be reported within a week of delivery Complaints made after this date will not be accepted 6 2 Setup 6 2 1 Setting up the Equipment The BioPAT Trace must be setup near the bioreactor you wish to monitor at max distance of 2 m The location must be free of vibrations and protected against corrosive atmospheres and chemical contamination Additionally the BioPAT Trace should be protected against exposure to direct sunlight Follow the instructions and requirements listed below concerning the power connection Operating the equipment with the wrong supply voltage and or the wrong fuses can be life threatening The AC adapter supplied with the device is equipped with a power plug a mains adapter and a plug to the device Insert the device plug into the connection socket on the back panel of the BioPAT Trace and connect the other end to your mains outlet The device can be switched on and off by pressing the power switch When the device is switched on a green LED lights up on the front panel To connect the BioPAT Trace to the local network plug the Ethernet cable into the Ethernet connection on the back panel of the device The operating software supplied is used to control the device via a PC Likewise th
75. st contamination is achieved However this procedure cannot be used for the dialysis method since the transport solution penetrates the dialysis membrane and the disinfecting agent can then enter the reactor through the membrane directly Tips and Checklist 111 112 Tips and Checklist 8 5 Import Data to Excel The measured values of the BioPAT Trace are saved on the PC as a ptd file For further processing the data can be imported into Excel The data import of the ptd file into Excel is carried out in seven steps 1 Open the MS Excel program 2 Select File Load and select the directory in which the ptd file is saved 3 Activate AII files circled in orange w jP Figure 86 Importing data from the ptd file into Excel Activate All files 4 Highlight the ptd file circled in orange Confirm your selection by pressing the Load button circled in red Figure 87 Import data of the ptd file into Excel Highlighting the ptd file 5 Text conversion assistant step 1 The text conversion assistant will then start The file is read in three steps First select the field Separated circled in orange This step is completed by pressing the Next button circled in red The Text Wizard has determined that your data is Delimited Tf this i correct choose Next or choose the data type that best escribes your date such as commas or tabs separate each fled
76. t the control PC is communicating properly with the BioPAT Trace By selecting the menu item Print diagram the currently displayed diagram is printed out on an interfaced printer When the menu item Calibration values is selected a window opens that shows the current calibration values Figure 77 The axis intercepts a0 calibration gradient a1 and the reference factors Reference fr are displayed Operation 97 a0 0 001 al 10E10 Reference fr Figure 77 Screen shot of current calibration values 98 Operation Activating the option Reset reference resets the reference factors to the factor 1 0 only in dialysis mode When the menu item Status is selected an information bar appears at the bottom edge of the screen that displays further information on the current status in three fields see Figure 78 This function is of particular interest for long intervals or group measurements The number of pending group measurements or preliminary measurements is displayed in field 1 In field 2 a current time count counts down the time until the end of the current action in seconds The remaining time in the interval of the measurement period is displayed in field 3 in seconds 1 893 0 605 Mio MET Was UOS MM ORT CUN SEP MOS INZI 1622 MI IAU 12 ON GT HE Sup 10 Tmecute 27 Vr preperation for Mezeurament Figure 78 Screen display of status bar
77. t with the dialysis probe this factor is applied to calculate the true concentration 5 2 2 Filtration Measuring Method Using the Filtration Probe With the filtration measuring method the samples are drawn from the bioreactor through the filtration probe Here transferral of the sample into the buffer stream takes place by means of diffusion within the diffusion module The calibration valve yellow and the sample valve green are used to select between the calibration solutions and the filtration probe The bypass valve red is used to select the diffusion module and the bypass Figure 11 shows a schematic of the flow system Filtration probe Waste Standard 1 Standard 2 Diffusion module __ Measurement cell vv O valve 2 vv check vaive rump Transport buffer Pump 1 Figure 11 Flow diagram of the online filtration method With the online filtration method the diffusion module is always used for calibrating the BioPAT Trace and for measuring samples The BioPAT Trace calculates the regression curve including zero point and gradient from multiple measurements of calibration standards 1 and 2 In turn the sample concentration is calculated based on the calibration curves Design and Function 33 34 Figure 12 easurement cell with rinsing line 5 3 Measurement Cell Function The measurement cell contains biosensors for glucose and lact
78. ta Transmission and Data Saving The BioPAT Trace 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 20mA 0 to 10 V or 4 to 20mA the BioPAT 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 P 7 Operation on page 85 BioPAT Trace Product Description 17 18 General Safety Precautions 3 General Safety Precautions The BioPAT Trace was designed and built under consideration of hazard analyses additional technical specifications and according to a meticulous selection of harmonized standards to be complied with It is thus a state of the art device that ensures the highest degree of safety However this safety can only be achieved during practical operation when all the necessary measures are applied It is the operator s responsibility to plan these measures and monitor their execution In particular the operator must ensure that the BioPAT Trace is used for it s intended purpose only cf Chapter 2 BioPAT Trace Product Description on page 12 the BioPAT Trace is used only when functional and in proper working order the operating instructions are always kept legible and complete at the place of use the BioPAT Trace is serviced and repaired by adequately qua
79. tand BPT0012 Stand clamp BPT0013 Knurled screw for measurement cell replacement BPTO026 Power supply BPT0027 Power cable EU UK USA BPT0028 Table 5 Accessories for BioPAT Trace 24 Equipment Supplied Accessories and Consumables 5 Design and Function 5 1 Setting Up the Device The BioPAT Trace is an online analysis system in a compact metal housing Communication with the user is driven by an external computer As shown in Figure 6 and 7 the front and side panels are equipped with the elements of the pre assembled tubing set for analysis The actuator and sensor components are located on the outside of the housing A standard or sample solution is fed into the analysis system by a pump 1 b One of the pinch valves 3 allows a selection of the solutions to be pumped The buffer stream is driven by the upper pump 1 a A particle filter 2 removes smaller particles and air bubbles from the buffer stream Figure 6 Front view of the BioPAT Trace with the following components Pos Component 1a b Pump cassettes 2 units 2 Particle filter 3 Pinch valves 3 pcs Design and Function 25 Figure 7 Side view of the BioPAT Trace with the following components Pos Component 4 Measurement cell 5 Knurled nut for fastening the measurement cell 6 Diffusion module 7 Drip counters 8 Rinsing line with return valve In the diffusion module 6
80. terfaces RS232 Ethernet PC for control Operating system Windows XP Windows 7 Analog output 0 10 V 0 20 mA 4 20 mA Input voltage 100 120 220 240 V 50 60 Hz Dimensions in mm Wx H x D 120x 170 x 200 Weight Table 10 Technical Data Sheet 1 8 kg Appendix 125 Sartorius Stedim Biotech GmbH August Spindler Str 11 37079 Goettingen Germany Phone 49 551 308 0 Fax 49 551 308 3289 www sartorius stedim com Copyright by Sartorius Stedim Biotech GmbH Goettingen Germany All rights reserved No part of this publication may be reprinted or translated in any form or by any means without the prior written permission of Sartorius Stedim Biotech GmbH The status of the information specifications and illustrations in this manual is indicated by the date given below Sartorius Stedim Biotech GmbH reserves the right to make changes to the technology features specifications and design of the equipment without notice Status April 2013 Sartorius Stedim Biotech GmbH Goettingen Germany Printed in Germany on paper that has been bleached without any use of chlorine W Publication No SLL6006 e13042 Order No 85032 541 53 Ver 04 2013
81. the resulting measurements incorrect Operation 91 92 Operation 7 1 Menu Bar 7 1 1 Menu Item Log Figure 70 Log menu options The measured data are recorded as text in a ptd file and saved in the default subdirectory Data If you want to create a new file select the menu item New If you want to load and display measured data from an old file select the menu item Load The saved files can be easily processed e g in Excel For notes on importing as an Excel file see Chapter 8 5 7 1 2 Menu Item Device Display Settings Information Connect Setting up operation Stop id oe oe a Se oe ore ser oy Figure 71 Device menu options The user can choose from three sub items Connect Setting up operation and Stop see Figure 71 7 1 2 1 Connect By selecting the Connect menu item you have the option to connect a new BioPAT Trace Figure 72 To do so you must enter the IP address of the device If you have started the software by clicking on the start icon g on the desktop the standard IP address 192 168 120 231 will already be displayed Operation 93 Whoisthere Default IP Address or Name of Device 192 168 120 231 Figure 72 Connect device If your device has been assigned a different IP address e g by your network administrator then enter that address here If you would also like to be prompted
82. tings menu options In the Settings menu options you can configure the most important device settings for operation and communication Parameters allow the user specific configuration of measurement frequency calibration frequency concentration of the calibration standards etc Basic settings Here you can configure the device for the selected sampling method Likewise the user can enter information about the device and the project that later appear on the data output Language Here you can change the menu guidance language Operation 101 7 1 4 1 Parameters All relevant parameters for measuring operations can be configured in the Parameters sub item Figure 82 TE rati Calibration delayed Measurements per group Standard 1 Glucose Standard 2 Glucose Measurements between calibrations Measurements per Standard 100 Minutes Measurements per external Sample Analog Out Glucose maximal count of measurements per standard Measurement period m Output range Glucose Standard 1Lactate Standard 21 artate Figure 82 Entry screen for configuring the measurement parameters Measurement period Enter the desired measuring interval here When the value 1 is entered a measurement is performed every minute or at the maximum frequency If one analysis should be performed every hour then enter 60 The frequency can be changed at any time and becomes immediately effective by cli
83. tion processes as well as the cultivation of animal or human cell lines When analyzing substrates in cultivation media it is necessary for a sample to be removed from the bioreactor fermenter under sterile conditions The reproducibility and relevance of the sample taken must remain intact during this step The BioPAT Trace currently features the following sampling options Filtration probe and dialysis probe All systems enable sterile removal from the bioreactor The BioPAT Trace consists of a compact housing that is adapted to the application and ambient conditions All mechanical and fluid elements are arranged on the front and or on the side panels The electronics feature a modular design and are configured to communicate through several interfaces Analysis is carried out using a sensor unit consisting of an electrochemical biosensor System control and data analysis are run on an external computer The control software included is very easy to use Introduction 11 12 2 BioPAT Trace Product Description The BioPAT Trace 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 provides continual analysis independent of the type of cultivation batch fed batch continual cultivation The
84. ubsequent measurement data Next the software will start the device with the specified IP address and automatically connect to the BioPAT Trace 6 5 Changing the Start Settings If you change the IP address at a later point in time you should also update the start settings in the program link of the Start icon to make sure that the device still connects automatically To do this complete the following steps 1 Switch the screen view to your desktop 2 Click on the start icon with the right mouse button 3 Select the menu item Properties 4 Enter your new IP address at the place indicated in the adjacent figure 61 5 Confirm the change with Apply and then OK Getting Started 81 n Analog Figure 62 Analog outputs 82 Getting Started 6 6 Assignment of Analog Outputs The measured values remain available for further use as analog signals at the ports analog 1 Measurement channel 1 Glucose and analog 2 Measurement channel 2 Lactate When using the ethanol methanol method the signal is output on analog 1 The output analog 3 is not assigned 1 1 0 2 10V 2 1 0 2 10V 1 2 GND 2 2 GND 1 3 0 4 20 mA 2 3 0 4 20mA In the Parameters menu 7 1 4 1 the output format can be switched between 0 10 V and 2 10V or 0 20 mA and 4 20 mA The output ranges of the analog outputs are also configured here The output ranges of the analog outputs can be configured as described in
85. urpose Figure 31 Figure 31 Inserting the drip counters into the holder 50 Getting Started 6 2 4 Attaching the Tubing Set for the Dialysis Operating Mode To install the dialysis tubing set follow the same procedure as for the filtration tubing set Additional information In addition the dialysis tubing set is provided with a single use syringe with three way stopcock for the analysis of external samples If you want to use this option you have to place the three way stopcock between the connection with the green label and the line to calibration standard 1 see Figure 32 and 33 Figure 32 Place of installation Figure 33 Three way stopcock for three way stopcock and syringe mounted Getting Started 51 52 Getting Started 6 2 5 Connection and Removal of the Enzyme Reactor In the Methanol Ethanol operating mode an enzyme reactor with immobilized alcohol oxidase is required for the enzymatic conversion of analytes Since the enzyme is relatively heat sensitive and cannot be stored dry the reactor is delivered separately from the tubing set The enzyme reactor must be installed into the tubing set intended for this purpose Due to the storage conditions at 4 8 C this should only be carried out directly prior to initial operation Prior to installing the reactor the tubing set must be inserted into the BioPAT Trace and filled with buffer Dry setup may result i
86. y If you already know the concentration values e g from the initial weight you can enter these values directly Alternatively you can determine the concentrations in the reactor with an external sample using an off line analyzer Confirm the prompted measured values with Accept If you want to make changes enter the new values into the entry field with decimal point and confirm with Accept With the Discard function you can cancel the procedure and move back to the starting position As long as the input window is open and no new reference values were entered and confirmed the BioPAT Trace continues working with the current reference factor If determining a reference measurement using an external analyzer is going to take longer and the medium concentration is expected to change in the meantime you should wait for the reference measurement and only then take a sample for external analysis When the input window for the reference values appears the device has stored the currently measured concentration to compare it with subsequently entered values and determine the new reference factor If no off line analyzer is available for determining reference values you can also measure the sample on the BioPAT Trace To do so select the Measurement function and follow the further instructions prompted by the menu navigation see Figure 67 Operation 89 90 Operation Be prepared to type in values or
87. ysis probe Glucose Lactate cellulase resistant BPTO040 5 pieces Membranes for Dialysis probe Ethanol Methanol 5 pieces BPT0047 Membrane for Filtration probe length 130 mm BPT0025 Membrane for Filtration probe length 90 mm BPT0037 Table 2 Consumables for BioPAT Trace 4 3 Sampling Systems 4 3 1 Dialysis Probe delivery with installation kit 2 membranes and user manual Description Order No Dialysis probe length 132 mm BPTO014 Dialysis probe length 165 mm BPT0015 Dialysis probe length 212 mm BPTO016 Dialysis probe length 232 mm BPTO017 Dialysis probe length 332 mm BPT0018 Dialysis probe length 362 mm BPTO019 Dialysis probe length 432 mm BPT0020 4 3 2 Single Use Dialysis Probe delivery with user manual Description Order No Single Use Dialysis probe ContiTRACE BPTO002 22 Equipment Supplied Accessories and Consumables 4 3 3 Filtration Probe delivery with installation kit 1 membrane and user manual Description Order No Filtration probe for cover port 12 mm with retaining nut PG13 5 BPT0021 membrane length 130 mm Filtration probe for cover port 12 mm with retaining nut PG13 5 BPT0022 insertion depth extended by 100 mm membrane length 130 mm Filtration probe for side port 25 mm with shut off cock BPT0023 membrane length 130 mm Filtration probe for side port 25 mm with shut off cock BPT0036 membrane length 90 mm Table 3 Sampling systems for BioPAT Trace 4 4 Accessories for S

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