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ÄKTAxpress User Manual - GE Healthcare Life Sciences

1. Prepare and Maintain Step Action 2 Change the required settings for the column displayed on the first page Advanced Setting Allimly Column Preparation Prevnwe Lee E pa Volume Remove Enano id fo 99099 Cv Pow Mate Metmove Ethanol 1 10 65 himin Voharne Water A5 5 p ana ev Fims Mote Water ASi 0 65 mimin Volume Siro Butter S4 al o 999899 cv Fion Rate Simo Butter 54 Sn Flow Rate Water ater Suip Y 0 65 mimin Vihane Water oer St 10 209389 cv Vome OP A7 5 Mm Piw Nate CIP 10 05 awmi Incubation Time stor CIP 0 9909 min Vome Water after CIP 445 id P o Cv Flow Rate Water ater CIP 45 l 0 65 mimin Volume Re Equabeation At gt 10 939999 CV Flow late Pe Equdteaton y 0 65 mimin The same type of settings is available for all columns For each procedure the flow rate and volume when performing the procedure can be changed For a list of possible procedures for the different columns see 4 3 2 System and column procedures in prepare and maintain methods on page 79 e Pressure Limit Upper pressure limit during column preparation see column instruction e Flow Rate Flow rate through the column during the procedure Examples Flow Rate Remove Ethanol Flow rate used when the ethanol is removed from the column Flow Rate Equilibration Flow rate used during the column equilibration e Volume Volume used to perform the procedure Examples Volume Remove Ethano
2. Note Do not put gel filtration columns in the right hand side column holder It will block the fraction collector and might get des troyed Location of liquid tubes and contain ers AKTAxpress overview 3 Column type Location Superloop A Superloop can be attached in the right hand column holder Note Do not put gel filtration columns in the column holder on the right hand side It will block the fraction collector and might get destroyed For detailed information on fittings and unions for connecting the columns see 6 4 9 Connecting columns and tubing on page 225 The system includes a tubing holder for organizing the tubing and a tube holder for tubes of two different sizes The tubes can be used for samples flow through fractionation and nonselected peaks if small volumes are expected A flask holder is available as an option It is mounted on the front of the system and used for example for samples bottles flowthrough fractionation bottles and bottles for nonselected peaks The flask holder can take a maximum weight of 3 5 kg The holders can be used independently of each other p35 3 AKTAxpress overview 3 2 Separation system overview 3 2 1 Location of the components j y Tubing holder apy Tube holder __ Flask holder Buffer and waste containers are usually large and may be stored directly on the floor or on suitable shelves
3. Static noise short and long term lt 40 AU Static drift 0 1 mAU h Hg lamp lifetime at 254 nm Lifetime in room temperature 7000 hours in cold room 2000 hours Hg lamp lifetime at 280 nm Lifetime in room temperature 3500 hours in cold room 2000 hours Conductivity monitor data Parameter Data Conductivity range 0 01 300 00 mS cm Accuracy Reproducibility short and long term Max 2 of full scale calibrated range or 0 1 mS cm whichever is greater Max 1 or 5 uS cm whichever is greater Noise Max 0 5 of full scale calibrated range typically 0 1 Response time Max 4 s 0 95 of step Internal volume 24 ul ep 421 10 Reference information 10 2 Technical specifications 10 2 2 Components specifications Pressure sensor Pressure sensor data data Parameter Data Pressure range 0 3 MPa Valve data Valve data Inlet valve Parameter Data Internal volume in out lt 33 ul Maintenance interval 10 000 positions 1 year Injection valve Parameter Data Internal volume in out Maintenance interval lt 17 ul 10 000 positions 1 year Loop valve and column valve Parameter Data Internal volume in out lt 16 ul Maintenance interval 10 000 positions 1 year Outlet valve Parameter Data Internal volume in out lt 15 ul Maintenance
4. ep 113 4 Methodology 4 7 Solution alternatives 4 7 2 Standard CIP Solution Suggestions Columns 4 7 2 Introduction Prerequisites Solutions for affin ity columns 28 4090 22 AB p114 Standard CIP Solution Suggestions Columns Standard CIP column procedures denoted CIP Columns contains two cleaning steps and can be run e included as a column post run procedure within a purification run or e within the Standard System and Column Procedures method plan in Prepare and Maintain The procedure can be combined with other procedures CIP column solution guidance is also provided in the instructions for each column and in the purification handbooks e The columns must be filled with water prior to CIP e Chelating columns must be stripped before CIP to avoid formation of metal ion salts that can damage the column The tables below list recommended solutions for the affinity columns supported by AKTAxpress Note If using chelating affinity columns the columns should be stripped before CIP to avoid formation of metal ion salts that can damage the column See 4 5 1 Descriptions of columns on page 95 for a list of columns supported by AKTAxpress Column Step Solution Chelating columns Step 1 1 M NaOH Step 2 Deionized water GSTrap columns Step 1 e 6 M GuaHCI or e 1 Triton X 100 Step 2 GST binding buffer 1 Recommended to pause 1 hour Solutions for
5. g Preparing theout Tubing from outlet valve ports F1 and F3 F11 let valve tubin PERS g Tubing from outlet valve ports F1 and F11 are used in the same way for all purification protocols Depending on the type of purification protocol to be run the outlet tubing F3 F10 are immersed into different flasks containers e Immerse the tubings from the outlet valve ports F1 and F3 F11 in appropriate flasks or containers as shown in the table below also shown on the Summary page in the Method Wizard in System Control Immerse outlet valve into the flask container for port tubing F1 WasteF1 Waste used for e g equilibration and loop wash F3 F10 See the Summary page F11 Metal ion waste 28 4090 22 AB p 210 Preparing the in jection valve waste tubing Operation 6 Tubing from outlet valve ports F2 and F12 Tubing from outlet valve ports F2 and F12 are used in the same way for all purification protocols These tubings are connected to the fraction collector and the injection valve according to the table below also shown on the Summary page in the Method Wizard in System Control Connect outlet valve port tubing to F2 FracCollF2 the fraction collector F12 LoopFracF12 injection valve port 7 to redirect intermediate peaks for storage in loops e Immerse the waste tubings from the injection valve in appropriate flasks or containers as
6. Indicators Status Standby The system is in standby All a RUN s active parts are turned off PAUSE The system can be turned on by press ing the On button Idle No activity The display shows the nae system unit number e g 12 28 4090 22 AB p48 AKTAxpress overview 3 Indicators Status Run A run is performed run PAUSE IL oun Pause The system is paused either i T omm manually or automatically by UNICORN ai Communication error There is no con Ta alain nection with the AKTAxpress PC driver Two segments flashing Error The system is paused and an er ror code is displayed For a description of the error codes refer to 9 6 Error code list on page 393 RUN Pause Display and PAUSE flashing The display alternates between the error code and the system unit number Lamp test All indicators are lit for 3 om seconds during the starting up se Pause EEE _ mA Program update A dialog is also shown 5 ipia on the UNICORN computer during the f e program update User control from The separation system is mainly controlled and monitored by UNICORN A the computer communication converter box is connected between the USB port in the computer and the UniNet port in the separation system as communication interface UNICORN can control up to twelve separation systems simultaneously More information about controlling the sepa
7. This section gives a general description of the Method Wizard in the Method Editor module and instructions for how to start the wizard The Method Wizard in Method Editor is a step by step guide for creating method plans It consists of a number of pages with questions and instructions on how to create the method plan The options on subsequent pages depends on the choices made on the initial main selection pages The Method Wizard in the Method Editor can be divided into two different parts e Inthe first part the main selections that define the method plan are made for example type of purification protocol and columns e Inthe second part called the Advanced Zone a number of parameter values in the method plan can be viewed and if preferred changed Note Avoid changing default parameter values in the advanced zone unless the result is clearly understood A Method plan is a set of method instructions created by the Method Wizard which define the frame of a run Additional information is though needed before running the method plan on a specific system see Using a method plan below The Method Wizard in the Method Editor can be used for creating two different types of method plans e Prepare and Maintain preparation and maintenance of columns and the system e Purify protein purification runs When creating a method plan the type of method plan to be created is first selected Different options are then presented de
8. 4 Possible undo actions when zooming e Undo zoom Right click in the window and select Undo zoom to undo the last zoom step e Reset zoom Right click in the window and select Reset zoom to reset all zoom steps at once Further instructions on how to change the viewing of the chromatogram can be found in How to view results in UNICORN AKTAxpress User Reference Manual Evaluation 7 7 4 Pooling fractions and adjusting the pools Introduction In the last purification step of each protocol fraction collection of peaks is performed The resulting fractions are shown in the chromatogram A theoretical pooling suggestion of the fractions can be provided in UNICORN If the extinction coefficient has been entered in the Method Wizard the protein concentration and amount will be calculated automatically when the chromatogram is opened This section describes the steps involved when creating the pooling suggestions and adjusting the pools Note When the fractionation of a peak is finished the subsequent fraction will appear to be very large in the chromatogram This is because the well position of the fraction collector is shown in the chromatogram If a new peak is detected only the delay volume will be collected in this well and the new peak will be collected in the next well Note The collection in the microplate is performed in a serpentine manner that is first in A1 A2 A11 A12 and then in B12 B11 B2
9. Eject Load fraction collector The separation systems are equipped with the following push buttons Button Function On Standby Reset In STANDBY mode Power on Reset of separation system incl pump synchronization In END mode Standby No power to active compon ents Pressing the button for 5 seconds in END RUN PAUSE mode Reset of separation system incl pump syn chronization Pause Continue In RUN mode Pause In PAUSE mode Continue Next breakpoint In RUN mode Go to next breakpoint in the running method Pressing the button in other modes will issue an error to UNICORN p47 3 AKTAxpress overview 3 2 Separation system overview 3 2 5 Indicators and controls Button Function Eject Load micro plate In END mode Eject or load microplate In PAUSE mode Eject or move the microplate to the position it had when the system was paused In RUN mode No function Indicators The separation systems are equipped with the following indicators Indicator Function Run O RUN Green indicator A run is active O PAUSE Pause Yellow indicator The system is in PAUSE mode OD D cD cD Status display Normal operation System unit number 1 12 for example 12 Error 3 digit error code for example E416 UV lamp broken Status indication The indicators can show the following status
10. Operation 6 Final setup and The final setup for both Prepare and Maintain and Purify method plans includes starting therun entering and viewing parameters on the Result Name page and the Summary page The table below describes how to make the final setup and how to start the run Step Action 1 If required edit the folder path and names of the result files Click Next By default the result files will be saved in Home folder Date System name 001 If there are several result files created on the same date the files will be named 001 002 and so on after the identical name If Main Folder is chosen on the Last Page in the Method Wizard the result file will be saved in Home folder Main folder Date System name 001 Fuki Name and Kusul Name Folder Path Home Fokder N aee Rosa File Name Syst tif une e p263 6 Operation 6 6 Starting a run 6 6 1 Starting a run using a method plan Step Action 2 The Summary page specifies the method requirements The tabs for each system specifies the required columns loops buffers solutions and so on and where they should be connected The total requirement of buffers and solutions is listed on the Summary tab We recommend printing the Summary list to save it Use the Summary list to make a final check of the system setup aL AEX Ship ot CIP ACAEX Fest Step AP AEX Wash Ruston baime Finan DS AGE Patter Water OSM Nah Rechoye A bihard ALA
11. Restrictor 0 2 MPa Eccentric Piston Timing belt C gt Inlet Pump Stepper heads motors Each piston is driven by a simple robust cam eccentric These cams are driven by stepper motors via timing belts The motor speed is varied to achieve linear movement which also produces the particular fluctuating motor sound This system guarantees e p403 10 Reference information 10 1 System description 10 1 1 Pump and related components 28 4090 22 AB p 404 an accurate low pulsation flow over the entire flow rate range independent of the back pressure When an increase in flow rate is programmed the motor speed accelerates gradually giving a soft start and building up speed to the flow rate required When a decrease in flow rate is programmed the motor speed reduces rapidly to the lower flow rate Pump heads The individual heads are identical but are actuated in opposite phase to each other by individual stepper motors controlled by a microprocessor Each outlet check valve houses a purge valve and a fingertight connector It is used for draining any unwanted liquid or to remove air from the system The purge valve is opened by turning it counterclockwise half a turn RT Purge valve j X Outlet check valve Rinse chamber outlet Inlet check valve The inlet to each pump head is fed from a common manifold Liquid is drawn up into the pump head through a non return check valve by the action of the piston
12. The signal falls to a specified fraction of the most recent peak maximum minus the Delta_Peak value Stable_Baseline The signal is stable within the limits of the Delta_Base value for a specified time interval Stable_Plateau The signal is stable within the limits of the Delta_Plat value for a specified time interval Note If the flow rate is changed the shape of the peak will change as well Hence the slope values might need to be changed accordingly Example of using This example shows how peak collection is performed in AKTAxpress using watch watch combina conditions The following watch conditions are used tions 1 Greater_Than AND Slope_Greater_Than 2 Peak_Max with Factor 3 Less_Than_or_Valley OR Stable_Plateau The illustration below shows where the watch conditions are met mAU min 28 4090 22 AB p 66 Methodology 4 Example of using The Peak_Max factor is important for detecting a valley since Peak_End cannot be the Peak_Max detected unless Peak_Max has been detected factor The illustration below shows how two different Peak_Max factors affects the detection of a Valley and collection of double peaks Peak_Max factor 0 5 Peak_Max factor 0 8 Example of using The condition Stable_Plateau is met if the UV signal does not deviate by more than Stable_Plateau the Delta_Plateau value during the time interval specified for the watch The illustration b
13. Setting the con If the conductivity cell has been replaced a cell constant has to be set ductivity cell con stant The cell constant is shown on the packaging of the new cell Ifthe packaging is missing perform a normal calibration according to 8 5 2 Calibration of conductivity cell on page 331 To enter the cell constant of a new conductivity cell Step Action 1 Read the cell constant from the packaging of the new cell 2 In the UNICORN System Control module select the system with the new conductivity cell by clicking the system name In the menu select System Calibrate and choose Cond_Cell under Monitor ASI concert hen A a rem wk alk am He SEZI ra Famire t 1 Ramarao wet amwa renee ate ww he so n Enter the cell constant in the Reference value 1 field Click Read value 1 Read value 1 After a few seconds a new cell constant is calculated and saved in the system Click Close to finish the operation 28 4090 22 AB p334 Maintenance 8 8 6 Replacement procedures About this section This section describes replacement procedures of AKTAxpress WARNING T When using hazardous chemicals make sure thot the entire system has been flushed thoroughly with bacteriostatic solution for example NaOH and distilled water before service and maintenance Warning Waste disposal This symbol indicates that the waste o
14. The tubings can also be flushed using the following manual instructions not column or outlet tubings e PumpWash e SystemWash e LoopWash These instructions can be executed from the Pump Instructions dialog box by selecting Manual Pump in SystemControl The instructions are also included in the method plans where they are automatically executed Note The system is always in Pause mode during PumpWash Hence it is only possible to click END or CONTINUE during PumpWash For more information about the instructions click Help in the dialog box e p277 6 Operation 6 8 Procedures after a run 6 8 3 Standard cleaning of the system 6 8 3 Introduction Warnings and notes 28 4090 22 AB p278 Standard cleaning of the system Standard cleaning of the system includes cleaning of the system with three solutions and can be run e included as a post run procedure within a purification method denoted CIP System or e within the Standard System and Column Procedures prepare and maintain method plan denoted CIP System and Loops with NaOH Water Buffer The system will be automatically cleaned but the sample inlets and the outlets will not be cleaned If performing standard cleaning within a Purify method plan it is possible to include a procedure for cleaning the sample inlets between sample loading or after the run in the purification method see below If performing standard cleaning using the Standard Syst
15. e Select Gradient and Target 100 B and Mode A1 B1 e Click Execute to set the valve to position B1 Wait for the valve to turn a clicking sound before starting to draw liquid T Ado pape tthe a checked the pasamene tracks nll te p rod dang vahed aanl 4 Connect a male Luer syringe of at least 20 ml to a purge valve 5 Turn the purge valve counter clockwise half a turn to open it 6 Slowly draw buffer with the syringe When fluid starts to enter the syringe close the purge valve Check that there is no visible air left in the B1 or whichever chosen tubing T If required remove the syringe empty it and insert it in the purge valve again before continuing 8 To fill the B2 inlet tubing first switch the inlet valve e Select System Control Manual Pump e Select Gradient and Target 100 B and Mode A2 B2 e Click Execute to set the valve to position B2 Wait for the valve to turn a clicking sound before starting to draw Operation 6 Step Action 9 Repeat step 4 7 to fill inlet tubing B2 10 Stop the pump by clicking END in System Control p 221 6 Operation 6 4 Preparing the system for a run 6 4 7 Purging the pump and system 6 4 7 Introduction Purging the pump and system 28 4090 22 AB p222 Purging the pump and system Air remaining in the system can be removed by purging the pump and system by manually running Pump Wash or System Wash
16. p 391 9 Troubleshooting and corrective actions 9 5 Alarms and connection problems 28 4090 22 AB p392 Problem Solution UNICORN Connection status NO 1 No contact with the systems Check the cables see AKTAxpress Installation Guide Cables to the USB CAN converter UniNet cables between the systems and the USB CAN converter Check that the last system on the UniNet chain has the termination plug inserted see AKTAxpress Install ation Guide Remove and insert the USB cable to the USB CAN converter If problem persists restart the computer and reset the separation system UNICORN Connection status NO 2 No contact with the AKTAxpress driver Check the cables see AKTAxpress Installation Guide Cables to the USB CAN converter UniNet cables between the systems and the USB CAN converter Check that the last system on the UniNet chain has the termination plug inserted see AKTAxpress Install ation Guide Remove and insert the USB cable to the USB CAN converter Check the USB CAN driver installation see 9 7 Checking USB CAN driver on page 397 If problem persists restart the computer and reset the separation system UNICORN Connection status NO 3 Internal software error OCI Check the system installation by using the UNICORN CD supplied See Adding systems in UNICORN in AK TAxpress Installation Guide If trying to connect in view mode from a remote compute
17. 2006 42 EC Machinery Directive MD 2006 95 EC Low Voltage Directive LVD 2004 108 EC Electromagnetic Compatibility EMC Directive e p417 10 Reference information 10 2 Technical specifications 10 2 1 System specifications Parameter Data EMC This product meets the requirements of the EMC Directive 89 336 EEC through the following harmonized standards e EN 61326 emission and immunity e EN 55011 GR 1 Class A emission e This product complies with part 15 subpart B Class A of the FCC rules emission Operation is subject to the following two conditions This product may not cause harmful interference This product must accept any interference received including interference that might cause undesired operation Column holders Column holder data of AKTAxpress Parameter Data Column block capacity Max 5 small columns Column holders 2 left hand side and 2 right hand side designed for 30 mm columns Note The HiPrep 16 xx Column Wrap can be inserted in to the left hand side column holder when us ing one of the HiPrep 16 60 Sephacryl S 100 S 200 S 300 HR gel filtra tion columns 0 d lt 30 mm An optional column holder for 50 mm columns can be ordered separately 28 4090 22 AB p418 Microplate spe cifications Reference information 10 The 24 or 96 well microplates used in AKTAxpress must comply with the data in the tables b
18. B1 for 96 well microplates A1 A2 A5 A6 and then B6 B5 B2 B1 for 24 well microplates and so on To display all fractions used select Show all fractions in the Pooling Protocol dialog e p297 7 Evaluation 7 4 Pooling fractions and adjusting the pools Pooling fractions The table below describes how to view the suggested pool fractions and adjust the and adjusting the pools pools Step Action 1 If the results were opened via Recent Runs or Find tab in the Evaluation module by double clicking the chromatogram name e g Sample1 UNICORN will automatically display a suggested pooling of the fractions The pooled fractions are listed in a table below the chromatogram and the pooled peaks are numbered sequentially in the chromatogram If the results were opened in another way the pooling is not automatic ally performed Choose Operations Pool to pool the fractions before continuing below Note Only adjacent fractions will be pooled The fraction num bers for each pool are listed in the table as a range in re tention order for example A6 A7 and so on 28 4090 22 AB p298 If the extinction coefficient has been entered in the wizard in System Control concentration and amount are automatically calculated Otherwise enter the extinction coefficient manually by marking a pool and then typing the value in the extinction coefficient field The concen tration is calc
19. Internal leakage e Internal fault The item must be replaced contact a local GE Healthcare representative Troubleshooting and corrective actions 9 9 5 Alarms and connection problems Introduction This section specifies troubleshooting for when there are alarms and connection problems Alarms Find the solution for a specific problem in the table below Problem Solution UNICORN error See UNICORN help files and manuals Error code flashing on Check the meaning of the error code in 9 6 Error code the separation system list on page 393 Connection prob Find the solution for a specific problem below lems If the connection problem cannot be solved by the instructions below check the log file c unicorn bin p4can_drvx log where X is the system unit number Contact a local GE Healthcare representative and be prepared to send the log file Problem Solution No text on the sys e Check that the power cable is connected at the back tem s front display of the separation system e Check that the power socket has voltage present One segment on and e Ifthe AKTAxpress program update dialog does not system unit no or appear in the UNICORN computer the instrument 256 displayed update failed Contact Service Two segments flash e Check that the computer is turned on ing and system unit e Start UNICORN and check the connection status as no steady Bela
20. a conditioning procedure Select the positions occupied by the column type 1 5 f Select the positions occupied by the column type 1 5 Select 0 nm typ Select 0 Conditioning procedure Select the positions occupied by the column type 11 5 28 4090 22 AB p256 Operation 6 Conditioning the To condition the columns columns Step Action 1 Immerse the inlet tubing in flasks with buffer according to the check list on the Summary page 2 Run the method plan according to the standard procedure described in 6 6 1 Starting a run using a method plan on page 259 e p257 6 Operation 6 6 Starting a run 6 6 About this section In this section 28 4090 22 AB p258 Starting a run This section describes how to start a run using a method plan and how to run a system manually This section contains the sub sections below Sub section See Starting a run using a method plan 6 6 1 Starting a manual run 6 6 2 Operation 6 6 6 1 Starting a run using a method plan Introduction This section describes how to start a run using a saved method plan Selecting a meth Follow the steps below to select a method plan od plan Step Action 1 Click the System Control button located on the task bar Result The System Control module appears 2 Click the Instant Run button located on the tool bar Alternatively s
21. disconnect the two fingertight connectors of the ca pillary loop to be replaced 28 4090 22 AB p336 Maintenance 8 Step Action 4 Slide the loop out of the compartment 5 Insert the new loop and tighten the two connectors 6 Press the On button to reconnect the system to UNICORN 7 Set the injection valve to position Reinject and the loop valve to the po sition of the new capillary loop 8 Verify the function by flushing the replaced loop and check the connect ors for leakage e p337 8 Maintenance 8 6 Replacement procedures 8 6 2 Replacing tubing and connectors 8 6 2 Replacing tubing and connectors Maintenanceinter When required for example when a tubing has clogged or has been bent so that the val flow is stopped Reason for main Clogged tubing might contaminate or cause high back pressure and hence preventing tenance a successful purification run Required material Extra tubing is included in the accessory kit CAUTION Only spare parts supplied or approved by GE Healthcare may be used for maintaining and servicing the system Replacing tubing To replace the tubing Step Action 1 Set the system in Standby mode by pressing the On button This will disconnect the system from UNICORN RUN a 28 4090 22 AB p338 Maintenance 8 Step Action Loosen the connectors to remove the tubing The
22. used will also be cleaned CIP system then fill with buffer After the purification run the system is cleaned with 0 5 M NaOH followed by water and finally filled with affinity binding buffer The capillary loops used will also be cleaned p75 4 Methodology 4 3 System and Column Procedures 4 3 1 System and column procedures within a purify method Column prepara tions that can be included in a puri fication run 28 4090 22 AB p76 System procedure Description Clean sample inlets only available for proto cols starting with AC IEX After the purification run the sample inlets tubing are cleaned with the appropriate wash solution e g water or buffer from a separate bottle The system will pause and a message will appear on the screen Sample inlets should then carefully be immersed into the wash solu tion A number of column preparation procedures can be included in a purification run Column preparation Description procedure Equilibration Before sample loading the AC IEX columns are equilib rated with 5 CV affinity ion exchange binding buffer and the desalting gel filtration columns are equilibrated with 2 5 CV 2 CV desalting gel filtration buffer respectively Blank Run AC IEX columns Before the purification the affinity ion exchange are equilibrated with 5 CV affinity ion exchange binding buffer followed by 5 CV affinity ion exchange elution buff
23. your tag specifically binds to the IEX column 1 If an affinity tagged protease was used run an AC protocol and collect the flowthrough fraction which will contain the cleaved protein 28 4090 22 AB p60 Methodology 4 Consider when Questions to be considered before choosing a purification protocol choosing purifica tion protocol What purity is required The more purification steps that are performed the higher the purity of the target protein will be Usually an AC step is performed first to capture the protein and then an IEX step and or GF step can be included to refine purification further A DS step is usually included before the IEX step to exchange buffer before loading the sample onto the IEX column A DS step can also be included as the last step see below for information Protein loss increases with each successive purification step AKTAxpress is optimized to perform the supported protocols as efficiently as possible It is however up to the user to combine purification techniques columns and buffers to achieve the best purification for each specific target protein Should the protein be in any specific buffer to keep it stable and suitable for the next application If yes include a DS or GF step as the last step in the purification to exchange the buffer to the appropriate one What should the protein be used for after the purification Include the necessary purification steps that will
24. 150 ml of 100 degassed methanol e about 200 ml of water Standard System and prepare maintain the system and the selected columns Column Procedures Some of the procedures can also be included in a puri fication method See 4 3 2 System and column proced ures in prepare and maintain methods on page 79 for more information ep 141 5 Method Wizard 5 3 Using the Prepare and Maintain option 5 3 3 Selecting Standard System and Column Procedures options 5 3 3 Selecting Standard System and Column Procedures options Introduction Standard System and Column Procedures provides preparation and maintenance procedures for the system and the columns selected on the Prepare and Maintain page One page for choosing system procedures settings is always displayed One page per selected column type will be displayed Selecting System To select system procedures Procedures op 9 tong Step Action 1 Specify the Running Condition on the System Procedures page Note If selecting Cold Room the default flow rates are decreased to avoid high pressure due to the higher viscosity of buffers and solutions System Procedures Famnng lanen Room Temperance O Cod Room D Gunked Loading of Supmtoun Fal Sample Inlet Tubing Os Ose Oss Os Fa Rues irin Tubenge Oe Of Dhs Da Os Oas Ow Daw On Om Rarer Al Ninis insent a Mintoplate in the Fraction Collector O Remmuve Ethernet hum System and Love COOP Syamam
25. 20 ethanol To change the pump rinsing solution Step Action 1 Empty the liquid container and fill it with 20 ethanol Place the test tube for example in the right hand column holder 2 Repeat the procedure for all systems If required several systems can use the same liquid container Note Do not connect rinsing system tubing from several systems in serial The tubing from each system must be routed dir ectly to the liquid container Maintenance 8 8 3 Six monthly maintenance Aboutthis section This section describes the six monthly maintenance activities In this section This section contains the sub sections below Sub section See Checking UV lamp run time 8 3 1 Cleaning UV cell 8 3 2 Cleaning conductivity cell 8 3 3 ep311 8 Maintenance 8 3 Six monthly maintenance 8 3 1 Checking UV lamp run time 8 3 1 Maintenance inter val Reason for main tenance Checking UV lamp run time Every 6 months The UV lamp has a life time of typically Wavelength 254 nm Wavelength 280 nm In room temperature 7000 h 3500 h In cold room 2000 h 2000 h Checking UVlamp To check the UV lamp run time run time 28 4090 22 AB ep 312 Step Action 1 From UNICORN System Control select System Maintenance 2 In the Maintenance manager open AKTAxpress and Specific Result The UV lamp on time is shown in hours
26. 21 matofocusing 28 4090 22 AB e p 444 Index A Accessories 436 Adjust pools 299 Advanced zone changing parameters 173 changing parameters affinity ion exchange 174 changing parameters customized system procedures prepare and maintain 168 changing parameters desalting gel filtration 181 changing parameters fractionation purify 188 changing parameters included system procedures purify 185 changing parameters miscellaneous settings purify 190 changing parameters peak collection 186 changing parameters purge pump with methanol prepare and maintain 167 changing parameters standard system and column procedures prepare and maintain 168 introduction 165 172 peak collection 65 peak handling 64 Affinity tag description 85 GST tag 86 Histidine tag 86 removal processes 89 types 85 Affinity tag removal cleavage buffers 109 cleavage time 92 conditions 92 description of proteases 87 on column 238 protease calculation 239 Air bubbles 244 Air detection 247 Air sensor detection 247 sensitivity 247 Article numbers 436 B Background to AKTAxpress 20 Bar code reader 261 Basic operating principles 22 Blank run 76 82 254 Buffer alternatives 107 choosing 109 descriptions 107 for AC 107 110 for DS 107 111 Index for GF 108 111 for IEX 108 111 Cc Calibration conductivity cell 331 pressure 329 Capillary loops 45 filling 234 Cautio
27. 300 HR 320 ml HiPrep column prepacked with Sephacryl S 300 HR For high resolution polishing steps for proteins of sizes 10 1500 kDa and for sample volumes lt 13 ml Note Superdex columns give higher resolution than Sephacryl columns p99 4 Methodology 4 5 Column alternatives 4 5 1 Descriptions of columns Coupled columns The HiTrap Desalting 5 ml columns shall be used in pair coupled in series minimize the dead volume 2 x 5 ml HiTrap Desalting t gt P Further informa More information about the columns can be found in the Column list e g flow rates tion and pressure limits used Select Edit Column list in the Method Editor Click Advanced Parameters to view the AKTAxpress column parameters For further information see the instruction sheet for each column type or the handbooks in 4 8 Documents for further information on page 124 28 4090 22 AB p 100 4 5 2 Introduction About choosing columns Consider when choosing columns Choosing column for the AC step Methodology 4 Choosing columns This section describes how to choose columns The following information is included e considerations before choosing columns e guides for how to select columns for the different chromatography steps e anexample of how to select columns for the AC DS IEX GF protocol Choosing column for a chromatography step is normally independent of where in the protocol the step is included I
28. 4090 22 AB p342 8 Maintenance 8 6 Replacement procedures 8 6 2 Replacing tubing and connectors 28 4090 22 AB e p344 Label bold or designation A1 Description Buffer inlet switch valve 1 port NO Length mm 1500 Inner diameter mm 1 6 Material FEP B1 Buffer inlet switch valve 1 port NC 1500 1 6 FEP A2 Buffer inlet switch valve 2 port NO 1500 1 6 FEP B2 Buffer inlet switch valve 2 port NC 1500 1 6 FEP A3 Buffer inlet inlet valve port 3 1500 1 6 FEP A4 Buffer inlet inlet valve port 4 1500 1 6 FEP A5 Buffer inlet inlet valve port 5 1500 1 6 FEP A6 Buffer inlet inlet valve port 6 1500 1 6 FEP A7 Buffer inlet inlet valve port 7 1500 1 6 FEP A8 Buffer inlet inlet valve port 8 1500 1 6 FEP S1 red Sample inlet inlet valve port 12 500 1 6 FEP Maintenance 8 Label bold Description Length mm_ Inner Material or diameter designation mm S2 yellow Sample inlet 500 1 6 FEP inlet valve port 11 S3 blue Sample inlet 500 1 6 FEP inlet valve port 10 S4 green Sample inlet 500 1 6 FEP inlet valve port 9 SW1 Switch valve 350 1 6 FEP 1 port IN to in let valve port 1 SW2 Switch valve 350 1 6 FEP 2 port IN to in let valve port 2 AirS
29. 4090 22 AB p 40 Tubing Tubing designation Length Waste tubing W1 W3 1 5m Sample tubing 1 S4 0 5m Note The waste tubing and sample tubing S1 should never exceed these lengths Sample tubing S2 S4 may be longer if these inlets are used for buffer solution instead of sample Each sample has a dedicated sample tubing flowthrough outlet tubing and an outlet tubing for nonselected peaks except when running 2 or 3 step protocols starting with a DS step The tubings are marked with labels with a unique color as shown in the table Note See the Summary page for information on which flowthrough outlet tubing and outlet tubing for nonselected peaks is used for each sample Example The table below shows an example for a 2 step protocol Sample no Tubing designation Color 1 S1 F3 F7 Red 2 S2 F4 F8 Yellow 3 S3 F5 F9 Blue 4 S4 F6 F10 Green 3 2 4 Introduction Sample and buffer handling AKTAxpress overview 3 Functional description This section briefly describes the function of the components in the liquid flow path of AKTAxpress The functions of AKTAxpress can be divided into six categories e Sample and buffer handling e Pump and related items e Column handling e UVand conductivity detection e Intermediate fraction collection e Final fraction collection For a more detailed description of the components see 10 1 System description on page
30. 5 2 Calibration of conductivity cell on page 331 e Incorrect calibration solution Calibration solution 1 00 M NaCl not correctly pre pared Prepare a new calibration solution and recal ibrate the conductivity cell see 8 5 2 Calibration of conductivity cell on page 331 Faulty pressure curve Troubleshooting and corrective actions 9 Problem Possible cause and action Incorrect or unstable reading Cable not connected properly Check that the conductivity flow cell cable is connec ted properly to the rear of the system see the AKTAx press Installation Guide Air might be trapped in the pump Purge the pump according to the procedure in 6 4 7 Purging the pump and system on page 222 Find the possible cause and action for a specific pressure curve fault in the table below If the problem remains contact the local GE Healthcare representative Fault Possible cause and action Irregular flow Air bubbles passing through or trapped in pump Check that there is sufficient buffer in each liquid container Check all connections for leakage Use degassed solutions Purge the pump according to the procedure in 6 4 7 Purging the pump and system on page 222 Check valves not functioning There might be dirt in the check valves Clean the valves see 8 4 2 Cleaning check valves on page 320 Blockage or partial blockage of flow path Flush through to clear any blockage by runn
31. 5 Method Wizard 5 5 Advanced Zone 5 5 4 Advanced Zone for Purify Affinity lon Exchange AC IEX 2 Gener al parameters 28 4090 22 AB p 180 This page contains options and parameters related to flow rate volumes and pressure for the second AC IEX step as described below It is displayed when the AC IEX step is an intermediate last step of a protocol Flow Rate Flow rate during the affinity ion exchange step Pressure Limit Upper pressure limit during the affinity ion exchange step Equilibration between Samples e Volume Volume CV used to equilibrate the AC IEX column between samples e Flow rate Flow rate used during the equilibration step Peak Injection e Peak Injection Flush Volume The extra volume besides the peak volume used to flush the loops when loading the sample onto the affinity ion exchange column e Peak Injection Max Volume The maximum volume ml to be injected onto the IEX AC column The Peak Injection Max Volume is set to make sure that the sum of the peak volume and the Peak Injection Flush Volume is less than maximum sample loading of the column Wash out unbound sample The volume used to wash out unbound sample The Set Wash Concentration B can be selected to change the salt concentration B of elution buffer to be used in the wash step By raising the B concentration set to 0 by default loosely bound proteins will also be washed out Collect Flowthrough in To collect the flowthrough
32. 95 proteases 87 protocols 59 Description of the process steps 54 Documentation 24 Double loops changing peak collection parameters 68 connecting 231 definition 68 Drip plate emptying 274 329 E Eject 242 Equilibration 76 82 254 Error codes 393 Evaluation 293 Evaluation process normal 293 optional 293 Evaluation process overview 293 Exporting pooling protocol 301 Extinction coefficient 262 F Fault codes 393 Features 15 Filling Superloop 234 28 4090 22 AB p446 Filling the capillary loops 234 Flask holder set 35 Flow chart 53 Flow control 250 Flow path 38 Flow restrictor checking 308 351 Fraction collector description 414 loading a microplate 241 Fractions how to pool fractions 298 Functional description 41 G GST tag 86 H Handbooks 124 Help on line 30 Hints and directions for optimizing run parameters 192 Histidine tags 86 mport files 426 428 ndicators and controls 47 indicators 48 status indication 48 njection valve 408 nlet supply 207 nlet valve 407 nstallation requirements 19 nstallation test 327 soelectric point 262 L Labels 51 Leakage 243 389 Liquid flow path 38 Loop capillary 45 Loop valve 409 M ain folder 132 263 aintenance 304 CIP columns 284 288 CIP system 278 281 every 6 months 311 monthly 307 402 415 when required 318 Materials 424 Metal ion charge solution choosing 122 Meta
33. A O O ERORE 107 46 2 BUTTER suggestio S sansene o a a a aa 109 AT SOMMMIOMFAILELMOLIVES mans a e EAT EO A 112 4 7 1 Standard CIP Solution Suggestions SYSTOM ceeeecsscssssssssssssssssessssssssessssssssssusssssesecsssssssssunsuesseseeesse 113 4 7 2 Standard CIP Solution Suggestions COLUMNS eeeccccsscsssssssssssseesessessessssssssssesssssesecsssssssssusnsessesecesse 114 4 7 3 Customized CIP Solution Suggestions SYSTEM ccccccccsssssssssssseeesssssessssssssstusssssesscssssssssusssessssseesse 116 4 7 4 Customized CIP Solution Suggestions COLUMNS iceeccsccsssssssssessesssssscsssssssssusssessesssessssssssssssneseseeesees 117 4 7 5 Metal lon Charge Solution SUGGESTIONS cssssssseussssessccssssssssussueesssssessssssssssssussessessecssssssssisuseseeseeesss 122 A750 SUID SOUL OM SUGGESTIONS inneoin e aA ORANA 123 4 8 Documents for further INfOrMAtiON ssccssssssssssseessssessssssessssssesssssessnssesssesiinsiiasiiisinsiniantesnneneesee 124 5 Method Wizard eere erarnan ea aeaa aeea aaae aa aa aa oaao Eaa hand e a iaeaea ianei ahas 5 1 Method Wizard introduction 5 2 Working With method Plan Siarra aa aa i a 5 21 Creati NG GW method pliere e E O A A OE 130 552 2 SAVING AME MASEMOG Ho e A EAEE EETA E EEA EEEE IE E TA EES 132 5 2 3 Editing a method pO Rannan a aa i a a nn cesses 5 2 4 Deleting a method plan 5 3 Using the Prepare and Maintain Optiounen a iia 136 5 3 1 Selecting Prepare and Maintain OPtiON ee ecccccscccssssssssesess
34. AKTAxpress user documentation This section gives an overview of the user documentation for AKTAxpress The table below describes all user manuals included in AKTAxpress user documentation package User manual Content AKTAxpress User Manual How to use the system including con cepts methodology operation evalu ation troubleshooting and mainten ance AKTAxpress Cue Cards Short step by step instructions and tables for the daily work such as creat ing and running a method plan evaluat ing the results preparation and mainten ance of the system and columns AKTAxpress Installation Guide AKTAxpress Operating Instructions UNICORN AKTAxpress User Reference Manual UNICORN Administration and Technical Manual Information for the system administrat How to prepare for the initial installa tion to perform the installation to run the installation test and to expand an existing installation with additional sys tems Includes safety instructions that must be followed for the use of AKTAxpress Available in several languages e g English German Spanish French Italian and Swedish Detailed instructions on the use of UNICORN or about for example user administra tion network installation and troubleshooting Document struc ture User Manual Introduction 2 This user manual is divided into chapters Each chapter starts with a brief overview that presents the
35. Example of location of different liquid containers T Buffers waste Samples fraction outlets WARNING Never place liquid containers on top of the separation sys tem If they become full and overflow liquid may enter the system causing a short circuit Control panel The separation system is mainly controlled and monitored via UNICORN The separation system is however also equipped with a control panel with controls and indicators 28 4090 22 AB p36 AKTAxpress overview 3 on the horizontal front bar The control panel enables limited control of the system as well as information feedback For more information about the indicators and controls see 3 2 5 Indicators and controls on page 47 p37 3 AKTAxpress overview 3 2 Separation system overview 3 2 2 Liquid flow path 3 2 2 Introduction The liquid flow path 28 4090 22 AB p 38 Liquid flow path This section contains an overview of the tubing and components configuration of the liquid flow path in AKTAxpress The illustration below shows the liquid flow path with the valves in the default positions 1 FrocCollf2 v jl a g Outiet Vole Qn Frocton Colector 3 2 3 Introduction Sample and buffer inlet tubing System and outlet tubing Tubing fittings Connecting the fittings Tubing lengths AKTAxpress overview 3 Tubing and connectors This section describes the tubing and co
36. HP HisTrap HP GSTrop 48 iml 5ml 5ml Use in DS step HiPrep 26 10 Desalting Which pi has the Which pl has the target protein target protein Use in IEX step RESOURCE QO RESOURCE S mi 6ml RESOURCE Q RESOURCE S imi iml Which M has the torget protein Which M hos the torget protein lt 70 kDa gt 70 kDa lt 70kDa gt 70 kDa HiLoad 16 600 H HiLoad 26 600 Superdex 200 pg Superdex 200 pg For comments on alternative column choices see the comments for each step described earlier Use in GF step HiLoad 16 600 HiLoad 26 600 Superdex 75 pg Superdex 75 pg ep105 4 Methodology 4 6 Buffer alternatives 4 6 About this section In this section 28 4090 22 AB p 106 Buffer alternatives This section contains general descriptions of different buffers and gives guidance on how to choose buffers for a protocol See also buffer suggestions in the column instructions This section contains the sub sections below Sub section See Description of buffers 4 6 1 Buffer suggestions 4 6 2 4 6 1 Introduction Affinity chromato graphy buffers Desalting chroma tography buffers Methodology 4 Description of buffers This section describes the different types of buffers needed and gives general guidance on how to choose buffers for different chromatography steps See also buffer suggestions in the column instructions Affinity chromato
37. Immerse the inlet tubing in flasks according to the check list on the Summary page Immerse outlet tubing F11 in a separate waste bottle Run the method plan according to the standard procedure described in 6 6 1 Starting a run using a method plan on page 259 p 291 7 Evaluation 7 Evaluation About this This chapter contains descriptions of how results from a run can be evaluated chapter In this chapter This chapter contains the sections below Section See Evaluation procedure overview 7 1 Finding and opening results 7 2 Viewing results 7 3 Pooling fractions and adjusting the pools 7 4 Creating pooling protocols 7 5 Printing report 7 6 28 4090 22 AB p292 Evaluation 7 7 1 Evaluation procedure overview Introduction This section is an overview of the normal procedure for evaluation of results after a run with AKTAxpress The normal evalu Normally 5 steps are performed after a run ation procedure Step Action 1 Find and open results see 7 2 Finding and opening results on page 294 2 View results and adjust the viewing see 7 3 Viewing results on page 296 3 Adjust pooling see 7 4 Pooling fractions and adjusting the pools on page 297 4 Create and print pooling protocol see 7 5 Creating pooling protocols on page 300 5 Print report see 7 6 Printing report on page 302 Optional evalu Optional actions can be performe
38. In the Method plan list in the Method Wizard select the method plan to be deleted Note If a folder is selected the folder and all method plans in the folder will be deleted Create of Change Method plan Note 2008 7 Attrity Got Fimen Hoel iap_ HP per N Hi mad TRAD Superis Lewen_ grade Alah Bane Tempetahier lose sanp Floan Howe Ie seiat pask Langast Froetionstinn weds Change pisie Promechars Gas foray orad Linan Piep htuanan nerpie Caan Prep 1a Siwo Fou nd Step faa Cobaan Post Pun tar Sitep Arapa Dod Step CP Renal Coar Click Delete Result A confirmation dialog appears Click Yes in the dialog Result The method plan is deleted and disappears from the Method plan list p 135 5 Method Wizard 5 3 Using the Prepare and Maintain option 5 3 About this section Overview 28 4090 22 AB p 136 Using the Prepare and Maintain option This section describes how to create a method plan for preparation and maintenance of the system and columns Depending on the prepare and maintain procedures to be performed different pages will appear in the Method Wizard The illustration below shows an overview of the possible workflows in the first part of the wizard For information about the Advanced zone see 5 5 2 Advanced Zone for Prepare and Maintain on page 167 When selecting The following pages are displayed Prepare an
39. OEE E eoe 267 6 8 Procedures aft r AT N kanndi AE E RAGA 273 6 8 Emptying dnp platessa a n e a e a 274 6 82 CIEAMING SVSUEMA QVE OW amni ee T A AERA RO 276 6 8 3 Standard cleaning of the Syste Naane E A E O EA 278 6 8 4 Customized cleaning Of the SYStOM ecccscssscsssssssseeessessecssssssssssmesssesesssssssssnssssseseecssssssssunssessesseesee 281 6 8 5 Cleaning Of columns OVErVICW scsssssssssesssscscssssssssssssesessssssssssssssssusessessecsssssssssussssssessesssssessssassseseeeees 283 6 8 6 St ndard cleaning OF COLUMNS ss ssvvvescexcosssassszsonveavsenvesvesusssssssesvvsnvenvvevssuyedtsesssossevasecasvesuasntdtelasaiscsccoeee 284 6 8 7 Customized cleaning OF COLUMNS sssssssessssssccssssssssssssessessccssssssssssssesssssesesssssssssssssssseseccesssssssuusecseseeesss 288 6 8 8 Metal ion stripping and recharging of affinity COLUMINS ccssecsscscccssssssssuseessessscssssssssssssessesssesee 290 PE VOUT OM e 2 AE E OE AE E EAE E EEE EEEE E 292 FL Eydluation Procedure Overview anran aE A REA AA A NEE 293 72 Finding andiopening res ltS miari i i a A E ATA 294 TeS VEWNG TESSU Siriara a EEA AONO EEA AOAR 296 7 4 Pooling fractions ANd adjusting the poolSissisnernniiepenennna e 297 7 5 Creating pooling protocols FiO PRIM GFE OM E ANER SAT EE AE E ET E OEA E bees Be Lelah nLo nK ol Aeae AEA E E EE E E E E E ase voeeesnsbeesosbnevetalyeteoedensae 8 1 Maintenance progra Mirasenunsne e E e EAE E Beiasaboavnnattedeasdaei
40. Slowly draw buffer A1 with the syringe When fluid starts entering the syringe continue to draw a few milliliters before closing the purge valve Check that there is no visible air left in the A1 tubing Repeat the steps 2 3 and 4 for the right pump head 28 4090 22 AB p368 Maintenance 8 Step Action 6 Check that the outlet tubing is not blocked 7 Connect a thin capillary or a column that will give sufficient back pressure see below 8 Reconnect the mains power cable to the system 9 Run at a flow rate of 1 ml min at a back pressure lt 0 5 MPa for 15 minutes 10 Run at 20 ml min at a back pressure of 2 3 MPa for 15 minutes 11 Finally perform system cleaning see 6 8 2 Cleaning system Overview on page 276 e p369 8 Maintenance 8 6 Replacement procedures 8 6 7 Replacing a damaged pump piston 8 6 7 Introduction Spare parts and tools required Replacing a piston 28 4090 22 AB p370 Replacing a damaged pump piston Typical symptoms of a damaged piston are observed as e excessive piston seal wear e unstable pressure e a reduction in the flow e noise as the piston moves The piston should be removed examined for damage or salt precipitation and then replaced with a new piston if necessary i If a damaged piston has been in operation the piston seal will be destroyed and should also be replaced The following spare parts and t
41. This section contains the sub sections below Sub section See System and column procedures within a purify method 4 3 1 System and column procedures in prepare and maintain methods 43 2 p73 4 Methodology 4 3 System and Column Procedures 4 3 1 System and column procedures within a purify method 4 3 1 Introduction When to run sys tem and column procedures within a purification run System proced ures that can be included in a puri fication run 28 4090 22 AB ep 74 System and column procedures within a purify method System and column procedures for preparing and maintaining the system and columns can be run within a purification method or separately in prepare and maintain methods see 4 3 2 System and column procedures in prepare and maintain methods on page 79 Including system and column preparation and or post run procedures within a purification run can be convenient if these procedures should be performed without any manual interference As a result the number of method plans to be run will decrease from three to one For example apart from performing purification it is possible to prepare the system and columns for the next run and or storage within one method plan Note In some cases however it is preferred to prepare the system and columns separately in a prepare and maintain method e g if charging new columns with metal ions outlets should be cleaned or many columns of the
42. a manual run at 10 ml min to fill the conductivity cell with the cal ibration solution Pump calibration solution through the cell until the conductivity signal is stable and then set the system to Pause e p331 8 Maintenance 8 5 Calibration procedures 8 5 2 Calibration of conductivity cell Step Action 4 In the menu select System Calibrate and choose Cond_Calib under Monitor 28 4090 22 AB p 332 Maintenance 8 Step Action Read the conductivity value displayed under Measured value and compare it with the theoretical value from the graph below mS cm HE EES ES SE cat a ee A HE EE HEH EET GRE EE HE EES ES WPS EGE REE BAG EE ER HEE EER En Gun PLES Pe GE E CE ES EB EERE EE E EE EE EE ERE ES eS EH EH HES TG E Ha A H EAH IEE me E ed ee ce eS eae ie ES Pa TE GEE EE GE HE GE i SE A SE ES SS EE ii EER EE EE EE TEES 20 25 30 C If the displayed value corresponds with the theoretical value No further action is required Proceed to step 8 If the displayed value differs from the theoretical value Proceed to step 6 Enter the theoretical conductivity value according to the graph in the field Reference value 1 Click Read value 1 Read value 1 The new cell constant is saved in the system Click Close to finish the operation e p333 8 Maintenance 8 5 Calibration procedures 8 5 2 Calibration of conductivity cell
43. and Column Procedures prepare and maintain method plan up to five columns of the same type can be cleaned simultaneously In this method standard system cleaning can also be included Note When cleaning chelating affinity columns the columns should be stripped before CIP to avoid formation of metal ion salts that can damage the column A Strip CIP procedure can only be performed in the Standard System and Column Procedures prepare and maintain method plan can not be included in a purification run WARNING When using hazardous chemicals take all suitable protective measures such as wearing protective glasses and gloves resistant to the chemicals used Follow local regulations and instructions for safe operation and maintenance of the system The following are required for cleaning the columns Cleaning solutions See the column instructions and or 4 7 2 Standard CIP Solution Suggestions Columns on page 114 A method plan for cleaning the columns Create a purify method plan in cluding CIP Columns Operation 6 The illustration below gives an overview of how to include CIP Columns in the purification method plan See 5 4 Using the Purify option on page 150 for detailed information Select the columns for which to include a OP Column procedure b the appropriate CIP Columns procedure for each of the selected columns 1p 285 6 Operation 6 8 Procedures after a run
44. and Longs vh NADH Wabe Rodier _ FE System Lone Desinta wih Fihanal O Sdect Al O Deselect At 28 4090 22 AB p 142 Method Wizard 5 Step Action Make sure that the Guided Loading of Superloop box is checked if the Superloop is going to be filled with protease solution or sample through the manual injection port in the injection valve during the run Note When starting the run help texts will appear to guide you through the manual operation Note If the Superloop has been loaded with protease solution or sample before starting the run do not check this box To Fill Sample Inlets with Buffer select the appropriate sample inlets to be filled by checking the 1 S4 boxes To Fill Buffer Inlets with Buffer select the appropriate buffer inlets to be filled by checking the A1 A8 and B1 and B2 boxes Select Rinse Outlets to rinse all outlets Result All outlet tubing F1 F11 including FracCollF2 will be washed with solution from inlet A1 Note A microplate must be inserted in the fraction collector to collect solution from FracCollF2 Select Remove Ethanol from System and Loops to remove ethanol from the system before starting the run Result The system flow path including the loops will be rinsed with water Select Wash System and Loops with NaOH Water Buffer to wash the systems and loops with solutions NaOH water and affinity ion exchange buffer Select Fill System
45. and current user name The log book provides a complete history of the run and is saved in the result file To customize the view panes right click in the respective view pane and select Properties For more information about customizing the view panes see the UNICORN user documentation By clicking Expand in the left hand upper corner more information on the current status of each system is displayed By clicking Collapse the information disappears e p267 6 Operation 6 7 During a run Manually select ing a peak for the next purification step 28 4090 22 AB p268 If Manual Selection was selected as procedure for how to select peak for the next purification step in the Method Wizard peak selection needs to be performed manually during the run Note The system is set to pause when the peaks from the current purification step has been collected in the capillary loops It is then possible to manually select a peak from one of the loops The table below describes how to select the peak to proceed with to the next purification step Operation 6 Step Action When the system has been set to pause display system message marks in the chromatogram if not already displayed e In System Control select View Properties in the menu bar Result The Properties dialog is displayed e Select the Curve Style and Color tab and click Filter Result The Filter Logbook dialog is displayed e Check the
46. and maintain methods 28 4090 22 AB p82 Column procedure Description Remove Ethanol Before the prepare and maintain procedures the columns are washed with water Fill Columns With Eth anol After the prepare and maintain procedures all used columns are washed with water and filled with 20 ethanol for storage This is performed to prevent bacteri al growth Equilibration The AC IEX columns are equilibrated with 5 CV affin ity ion exchange binding buffer and the desalting gel filtration columns are equilibrated with 2 5 CV 2 CV de salting gel filtration buffer respectively Re equilibrate The AC IEX columns are re equilibrated with 5 CV affin ity ion exchange binding buffer the desalting columns are re equilibrated with 5 CV desalting and the gel filtra tion columns are re equilibrated with 2 CV gel filtration buffer CIP Columns Cleans each column with the appropriate CIP solution followed by water See 4 7 Solution alternatives on page 112 for recommended solutions for different columns Blank Run AC IEX columns The affinity ion exchange are equilibrated with 5 CV af finity ion exchange binding buffer followed by 5 CV af finity ion exchange elution buffer and finally with 10 CV affinity ion exchange binding buffer General e A blank run should be performed before the first time the column is used and after long term storage e Ablank run is often performed to ma
47. at the rear of the unit and oosen the cable from the cable chute under the system UV monitor lamp 28 4090 22 AB p 354 Maintenance 8 Step Action Remove the UV monitor assembly from the system by moving it upwards until the unit is released from its holder Use a cross head screwdriver to detach the end plate by removing one and loosening the other of the two holding screws on the lamp housing to be removed Lamp housing end plate Slide the old lamp housing off the filter housing and discard it WARNING T The UV monitor has a mercury Hg lamp that contains small amounts of mercury The lamp must be handled with care and disposed of ac cording to national and local environmental reg ulations Hg On the new UV lamp housing detach the end plate e p355 8 Maintenance 8 6 Replacement procedures 8 6 4 Replacing UV lamp 28 4090 22 AB p 356 Step Action 7 Slide the new lamp housing onto the filter housing making sure that the cable is on the right side When sliding the lamp housing depress the two pressure pads on the filter housing in turn to facilitate the installation 8 Refit the lamp housing end plate 9 Slide the lamp housing firmly into place and check that the detector housing is in its right hand position Ot A v7 NJ i x gt mY Ow ras 4 i X y amp te at By a A amp Py R If this is not the c
48. columns Re equilibrate all column types Note If using chelating affinity columns the columns should be stripped before CIP to avoid formation of metal ion salts that can damage the column e Re equilibrate affinity and ion exchange columns only Strip columns Fill with Ethanol affinity columns only e Strip columns Recharge Blank Run affinity columns only e Strip columns Recharge Fill with Ethanol affinity columns only The table below describes each possible procedure separately These procedures are then combined to form the available column post run procedures above Column post run Description procedure Fill with Ethanol After the purification all used columns are washed with water and filled with 20 ethanol for storage This is performed to prevent bacterial growth p77 4 Methodology 4 3 System and Column Procedures 4 3 1 System and column procedures within a purify method 28 4090 22 AB p78 Column post run Description procedure CIP Columns Cleans each column with the appropriate CIP solution followed by water See 4 7 Solution alternatives on page 112 for recommended solutions for different columns Re equilibrate Equilibrates the affinity ion exchange column with affin ity ion exchange binding buffer If combined with other procedure equilibrates the affinity and ion exchange columns with 5 CV affinity ion ex change binding buffer the desalting column
49. columns are used to separate positively charged proteins The following cation exchange columns are supported in KTAxpress Supported cation IEX columns and volumes Characteristics HiTrap SP HP land 5ml RESOURCE S 1and 6ml Columns prepacked with SP Sepharose High Performance for high resolution purification Columns prepacked with SOURCE 15S Fast purification with high resolution Mono S 5 50 GL 1ml Column prepacked with MonoBeads for purification with the highest resolution Gel filtration GF columns separate proteins with differences in molecular size They are often used in a final polishing step Gel filtration separation results in high quality samples of homogenous size for example by separating protein monomers from protein dimers trimers and aggregates The following gel filtration columns are supported by AKTAxpress Supported GF columns and volumes Characteristics HiLoad 16 600 Superdex 75 prep grade 120 ml HiLoad column prepacked with Super dex 75 prep grade For high resolution polishing step for proteins of sizes 3 70 kDa and for sample volumes of lt 5 ml HiLoad 16 600 Superdex 200 prep grade 120 ml HiLoad column prepacked with Super dex 200 prep grade For high resolution polishing step for proteins of sizes 10 600 kDa and for sample volumes of lt 5 ml HiLoad 26 600 Superdex 75 prep grade 320 ml HiLoad column prep
50. gradient segments System Wash After Segment 1 3 Enable this option when performing step elution e g 40 100 B in 0 CV The system is filled with buffer with the set concentration System Wash before Clean after Elution Fills the system with 100 B buffer Tag Cleavage General paramet ers Method Wizard 5 Clean After Elution at 100 B Volume of elution buffer used to wash the column after the gradient segments Note This page replaces the AC IEX 1 General page when on column tag cleavage is selected in the first part of the Method Wizard This page contains options and parameters related to tag cleavage flow rate volumes and pressures as described below Note Several of the parameters are the same as for the AC IEX 1 General page but in some cases the parameters are used differently 0 T Load sane vuka of poese on af uuiunms 12 LEJ m 15 16 T tngble W aich Stable E aveire table Ime 1 D mo Deta Bare s W BUI mals Flow rate Sample Loading Sample loading flow rate e Wash Out Compl Sample loading Flow rate when washing out the unbound sample from the column s before the protease is injected onto the column s e Equilibration Cleavage Buffer Flow rate when loading the cleavage buffer onto the column before loading the protease solution e Protease Injection The flow rate used to inject the protease onto the column e Extra Wash Before Elution only appears if this was s
51. head Tighten the valves until fully finger tight and then use the wrench to tighten a further 1 3rd 110 of a turn CAUTION Do not over tighten the valves since damage to the internal components can occur 10 Refit the outlet tubing and the inlet manifold 11 Reconnect the mains power cable 12 Purge the pump carefully and check that the pumping action has been corrected see 6 4 7 Purging the pump and system on page 222 8 6 6 Introduction Spare part and tools required Maintenance 8 Replacing pump piston seal The pump piston seals should be replaced if There are signs of liquid leakage between a pump head and the panel The volume of the rinsing solution has increased or decreased CAUTION The pump head should only be disassembled by a trained person CAUTION Do not disassemble the pump head unless there is good reason to believe that there is an internal leakage Always make sure that sufficient spare components are available before attempting to replace a spare part Note It is not possible to reinstall a used piston seal after removal Note Always replace the piston seals on both pump heads at the same time Note It is recommended to use degassed liquids The following spare parts and tools are required Seal kit 18 1112 04 containing 2 piston seals and 2 rinse membranes 1 4 inch wrench 3 mm hex key Screwdriver 100 methanol for running in the new pisto
52. high or too low pressure A flow restrictor creating too low back pressure might cause air bubbles in the UV flow cell A flow restrictor creating too high back pressure will cause a high pressure alarm from the system In exceptional cases it might cause column rupture Flow restrictor FR 902 18 1121 35 CAUTION Only spare parts approved or supplied by GE Healthcare may be used for maintaining and servicing the system To replace the flow restrictor Step Action 1 Set the system in Standby mode by pressing the On button This will prevent runs from being started from UNICORN RUN eens ep 351 8 Maintenance 8 6 Replacement procedures 8 6 3 Replacing flow restrictor 28 4090 22 AB p352 Step Action 2 Remove the two capillary tubing from the old restrictor 3 Lift off the restrictor from the holder 4 Insert the new restrictor in the holder 5 Refit the capillary tubing to the new restrictor Make sure that the tubing from the conductivity cell is attached to the IN port on the flow restrictor 6 Press the On button to reconnect the system to UNICORN 7 Check the function see 8 2 1 Checking flow restrictor on page 308 Maintenance 8 8 6 4 Replacing UV lamp Maintenanceinter Replace the UV lamp when val e The UV signal trace has excessive noise e The runtime has exceeded the typical life time of the lamp The UV
53. in front of the fraction collector Step Action 1 Check that the system is in Idle mode with no active run Operation 6 Step Action 2 If the collector plate is outside the system press the Eject button to inject the collector plate makes it easier to access the drip plate Each time the button is pressed the sled switches between inside and outside the system 3 Carefully remove the drip plate by sliding it outwards gt 4 4 Empty any fluid into waste 5 Slide the drip plate back into position 6 Check the function by pressing the Eject button twice to unload and load the collector plate e p275 6 Operation 6 8 Procedures after a run 6 8 2 Cleaning system Overview 6 8 2 Introduction Warnings Cleaning sample inlets 28 4090 22 AB p276 Cleaning system Overview When running different types of samples or purification methods after each other the sample inlet tubings and the system flow path should be cleaned between the runs This will prevent buffer mixing sample contamination and protein precipitation When leaving the system for the weekend or for a longer time the cleaning procedure should be finished with 20 ethanol to prevent bacterial growth in the tubing Note Do not leave the system with salt buffer in the flow path It might damage the pump WARNING When using hazardous chemicals take all suitable protective m
54. longer Note Try to keep the glycerol concentration low in the protease solution since high glycerol concentration affects for example TEV activity negatively The table below provides guidance on appropriate conditions for on column cleavage using AKTAxpress If at then with units of protease per mg of protein using PreScission 4 C incubate 8 h 20 Protease using AcTEV room temperature incubate 8 h 200 1 Unit definitions e One unit PreScission will in batch cleave 2 90 of 100 ug of a test Glutathione S transferase fusion protein in cleavage buffer 50 mM Tris HCI 150 mM NaCl 1 mM EDTA 1 mM DTT pH 7 0 at 25 C at 5 C for 16 h e One unit AcTEV will in batch cleave 2 85 of 3 ug control substrate in 1 h at 30 E If AKTAxpress is used in a cold room it is possible to raise the temperature in the first step AC IEX column by using a column heater during the run The column heater is not controlled from UNICORN but can be placed on top of the system Extended tubing might be needed to attach the column to the column valve from the column heater Note Raise the temperature carefully if the protein is temperature sensitive See 4 6 2 Buffer suggestions on page 109 for guidelines on buffers for cleavage Methodology 4 Further informa See Instructions and Application notes from GE Healthcare 4 8 Documents for further tion information on page 124 and other
55. of how to create the Customized Column ized Column Pro Procedures method plan See 5 3 Using the Prepare and Maintain option on page cedures method 136 for detailed information plan Enter select parameters for whether to perform System Wash before the step flow rote inlet volume whether to pause before next step Cleaning steps 2 9 Cleaning the To run the Customized Column Procedures method plan columns Step Action 1 Immerse the tubing ends to be used in the correct containers see also the Summary page 2 Run the method plan as described in 6 6 1 Starting a run using a method plan on page 259 e p289 6 Operation 6 8 Procedures after a run 6 8 8 Metal ion stripping and recharging of affinity columns 6 8 8 Introduction Strip procedures Prerequisites Create a purify method plan in cluding Strip 28 4090 22 AB p290 Metal ion stripping and recharging of affinity columns Before recharging chelating affinity columns metal ions must be removed from the columns Stripping of affinity columns can be run e included as a post run procedure within a purification method only the affinity column in the first step of a protocol can be stripped or e within the Standard System and Column Procedures prepare and maintain method plan up to five affinity columns of the same type can be stripped simultaneously The following are required for stripping of the co
56. p228 Operation 6 Column valve Column block Column valve Bt Connection guide The connection guide on the next page describes how to use the unions supplied with the system to connect tubing with the columns Note HiPrep Desalting column require longer tubing Tubing specially made for connecting these columns is supplied with the system p 229 6 Operation 6 4 Preparing the system for a run 6 4 9 Connecting columns and tubing 1 16 Female a M6 Mole 1 16 Female i M6 Female 1 16 Female P M6 Mole i 350 mm tubing New models of HiTrap columns do not require the unions 1 The standard system tubing 190 mm should be replaced by the 350 mm capillary tubing available in the accessory kit when using a HiPrep 26 10 desalting column or gel filtration column in position 4 or 5 28 4090 22 AB p230 6 4 10 Introduction Required material for five double loops Where to place the extra capillary loops Operation 6 Preparing the system when using double loops This section describes how to connect an extra capillary loop to each of the five loops and where to mount the extra capillary loops on AKTAxpress Note It is a good idea to use double loops for all five loops because it is not possible to predict in which loops the peaks will be collected Note Parameters regarding peak collection must be changed in the Advanced Zone of the method plan when using double l
57. plate if necessary 2 Place the microplate on the sled and check that the labelling H and A for 96 well microplates or A for 24 well microplates match the labelling on the system 3 Check that the gap between the end of the tubing and the microplate is 1 2 mm 4 For better accessibility around the column block press the Eject button to load the collector plate 28 4090 22 AB ep 242 6 4 14 Introduction Checking the tubing Operation 6 Checking the tubing This section describes how to check the tubings before a run The tubings must be checked for e Air bubbles e Leakage If there are air bubbles or leakage the run might be adversely affected Especially after moving a system from room temperature to cold room it is important to tighten the connectors The material will shrink in cold room which might cause eakage To check the tubings follow the procedure below CAUTION To protect the piston seals in the pump the pump should not run with air in the inlet tubings Follow the instructions for purging the pump CAUTION Before the start of each run ensure that there is an adequate supply of eluent in the reservoirs Never allow the pump to run dry because this will affect the lifetime of the piston seals Step Action 1 Leakage inspection Start the flow manually from UNICORN Inspect the tubings for leakage Pay special attention to the tubing conne
58. pressure limit during the affinity ion exchange step Enable Watch Stable Baseline If Watch Stable Baseline is enabled the firstwash step will be completed when the baseline is stable or when the entire wash volume has been consumed The stability of the UV signal will be checked during the specified time interval The Stable Baseline condition is met when the signal fluctuates less than the set Delta Base value during the set Stable Time interval This page contains options and parameters related to gradient elution as described below It is displayed for protocols starting with AC IEX Note If tag cleavage was selected in the first step the Tag cleavage Elution parameters page will appear instead See Tag cleavage Elution parameters below for information on which parameters that can be changed ACAEX 1 Elution Graded Seyret 1 Taget Loncertiabon 10 m 10 xe bradet Lenan W 99999 EV v System Wash atter Sagmare 1 biraderi segment 2 Target Conoentiatien w poas Gentine erpa bd p saga Cy C System Wash aher Segmert 2 Guntient Segment 7 Target Concertation bead 100 28 Gradam Lang gt o 999933 CV T System wash ater Segment J Graders Segment 4 Taget Lorcerhahon w 1w 3B Iraderi Lergh W 933333 LY Clean after Eton al 100 XE L System Wash betore Clean ater Etion Denning Length p terey v Gradient Segment 1 4 Target Concentration and the Gradient Length are used to define two different
59. quently used that maintain run or be included as a preparation and or post run procedure in a canberuninboth purification run These are types of method plans Procedure For more information see Conditioning of columns i e procedures including equilibration or blank run as the last step 6 5 2 Conditioning of columns on page 254 Standard cleaning of the system 6 8 3 Standard cleaning of the system on page 278 Standard cleaning of the columns 6 8 6 Standard cleaning of columns on page 284 Stripping and recharging of columns 6 8 8 Metal ion stripping and rechar ging of affinity columns on page 290 28 4090 22 AB p 202 6 4 Preparing the system for a run Operation 6 About this section This section describes how to prepare the separation system for a run Warning In this section WARNING When using hazardous chemicals take all suitable protective measures such as wearing protective glasses and gloves resistant to the chemicals used Follow local regulations and instructions for safe operation and maintenance of the system A This section contains the sub sections below Sub section See Printing out a summary 6 4 1 Preparing buffers and solutions 6 4 2 Preparing outlet and waste tubing 6 4 3 Purging the pump and inlet tubing Overview 6 4 4 Filling inlets manually using a method
60. same type should be prepared Anumber of system procedures can be included in the purification run When choosing any of the procedures listed in the table below be aware of the additional time consumption for each protocol see 10 4 Typical run times on page 428 for information System procedure Description Preparation Remove Ethanol from In the beginning of the purification run the system is System washed with water The loops to be used during purific ation and the fraction collector tubing will also be washed Fill Sample Inlet In the beginning of the purification run all sample inlet Tubings with Buffer tubings to be used are filled with AC IEX binding buffer only available for proto from a separate bottle When the sample inlet tubings cols starting with have been filled the method will pause and a message AC IEX will appear on the screen Sample inlets should then carefully be immersed into the appropriate sample tubes flasks Methodology 4 System procedure Description Guided Loading of Su perloop only available for protocols with Su perloop sample loading or protocols where on column tag cleavage is included In the beginning of the purification run the superloop is filled with sample or protease solution When performing the filling the system will pause and messages appear to guide you through the operation Note This procedure should always be included unl
61. select options on the Column Post Run page Step Action 1 Select the requested columns on which to perform post run procedures by checking the boxes for the columns in the different steps First Chromatography Step Second Chromatography Step etc Colum Post Rue T hest Dacangtapaphy Step T secund Urumsopaptn Stev L Thra Channmagiaphy Step L Lans Cheomamograging Stap Noem Never CIP chetatng eaba urira the Anhi hva heen opad Note Ifyou do not wish to perform any column post run proced ures leave all boxes on this page unchecked Depending on the selected protocol the number of columns vary and different types of columns may occupy the different chromatography steps 28 4090 22 AB p 162 Method Wizard 5 Step Action 2 Select the appropriate post run procedure to be performed for each column See 4 3 1 System and column procedures within a purify method on page 74 for a description of the different column post run procedures 3 Click Next Result The Last Page is displayed 4 Continue with 5 2 2 Saving the method plan on page 132 e p163 5 Method Wizard 5 5 Advanced Zone 5 5 Advanced Zone About this section This section describes how to change parameter values in the Advanced Zone in the Method Wizard In this section This section contains the sub sections below Sub section See Advanc
62. selected options on the first page in Advanced Zone The parameters appearing on each page depend on the selections made in the first part of the Method Wizard The following sections describe options for the chromatography techniques system procedures peak collection fractionation and miscellaneous settings See the table below For parameters regarding See affinity ion exchange 5 5 4 Advanced Zone for Purify Affin ity lon Exchange on page 174 desalting gel filtration 5 5 5 Advanced Zone for Purify Desalt ing Gel Filtration on page 181 included system procedures 5 5 6 Advanced Zone for Purify In cluded system procedures on page 185 peak collection 5 5 7 Advanced Zone for Purify Peak Collection on page 186 fractionation miscellaneous settings 5 5 8 Advanced Zone for Purify Frac tionation on page 188 5 5 9 Advanced Zone for Purify Mis cellaneous settings on page 190 e p173 5 Method Wizard 5 5 Advanced Zone 5 5 4 Advanced Zone for Purify Affinity lon Exchange 5 5 4 Introduction Overview 28 4090 22 AB p174 Advanced Zone for Purify Affinity lon Exchange This section describes the parameters used in the affinity ion exchange step during a purification run Some of the default parameter values depend on the chosen column Note Make sure that the parameter values do not exceed the specification of the chosen column Tag cl
63. slope can be set The Stop Slope condition cannot be met unless the Peak_Max condition is met Stop Level can be met before Peak_Max The Peak_Max condition occurs when the UV signal has fallen to a fraction specified by Peak Max factor of the most recent peak maximum Note If the flow rate is changed the shape of the peak will change as well Hence the slope values might need to be changed accordingly Method Wizard 5 Minimum Peak Width sets the minimum fractionation time of a single peak The fractionation of the current peak continues at least until the Minimum Peak Width is reached even if the signal is fluctuating p 189 5 Method Wizard 5 5 Advanced Zone 5 5 9 Advanced Zone for Purify Miscellaneous settings 5 5 9 Introduction The Miscellaneous Settings page Loop parameters Air sensor con trolled sample loading paramet ers 28 4090 22 AB p190 Advanced Zone for Purify Miscellaneous settings This section describes the options and parameters available on the Miscellaneous Settings page in all protocols The content of the page differs depending on the protocol used The Miscellaneous Settings page contains a number of miscellaneous options Mescellaneuus Settings Fash Vos Empty Loops E W 100 ni Loop Wash Vokme ingly Loopt 2 Wot 1 99 ot Loop Wath Vohme 2 Wot 19 999 t lv Enantia as Sansor Conmatind Sampie Loading Mau S amgin ehane wa p sa m Maleza Weaght Line We Preton
64. system contains two types of tubing e 1 mm capillary tubing with fingertight connectors e 1 6 mm capillary tubing with M6 or UNF 5 16 air sensor pump inlet connectors including ferrules If the tubing has labels remove the labels to be used with the new tubing later Discard the tubing and connectors e p339 8 Maintenance 8 6 Replacement procedures 8 6 2 Replacing tubing and connectors 28 4090 22 AB p 340 Step Action 4 Cut the new tubing to the same length as the old tubing Use the cutting tool to get a straight angle cut Note When replacing system tubing use the original length to ensure that the correct delay volumes are maintained Inlet and outlet tubing may be shortened if required For a specification of the tubing see 8 6 2 Replacing tubing and con nectors on page 338 D a _ The cutting tool is included in the accessory kit Put the old labels on the new tubing Mount the connectors on the tubing For fingertight connectors 1 Slide the nipple onto the tubing For M6 and UNF 5 16 connectors 1 Slide the nipple onto the tubing 2 Slide the ferrule onto the tubing with the thick end towards the end of the tubing Maintenance 8 Step Action Attach the new tubing to the system e Insert the tubing with nipple into the port Make sure to insert the tubing all the way into the bottom of the port before tighten t
65. that should be puri fied using the affinity tag in the second or third step but first needs desalting and or ion exchange purific ation that start with a DS IEX step and in clude an AC step Group 3 protocols a protein where the tag has been re moved on or off column but further purification is necessary that start with a DS IEX GF step and do not include an AC step Group 4 protocols Note If an affinity tagged protease was used off column run an AC pro tocol instead and col lect the flowthrough fraction which will con tain the cleaved pro tein If further purifica tion is needed select a group 4 protocol 2 Choose protocol based on required sample characteristics Depending on the required sample characteristics to be achieved after purification and the considerations in the previous section include the appropriate chromatography steps that will fulfill your criteria The table below gives guidance on some sample characteristics achieved after a purification step and after the last purification step for a few example protocols Step Capture of Charge Size Buffer Effect on tagged homogeneity homogeneity exchange purity protein AC x 43 DS x N A IEX x Methodology 4 Step Capture of Charge Size Buffer Effect on tagged homogeneity homogeneity exchange purity protein GF x x Examples AC DS x x AC GF
66. together with other life time parameters M owed we mana x jriramainn Pump pistan strokes 1000 10 1 UV lamp on tme 2301 tire LIV inmp intansiy 12 irie vae total hane 13 Maintenance 8 Step Action 3 Read the run time Take suitable action considering the lamp life time e No action e Check that a new UV lamp is available or order replacement lamp e Replace the lamp see 8 6 4 Replacing UV lamp on page 353 e p313 8 Maintenance 8 3 Six monthly maintenance 8 3 2 Cleaning UV cell 8 3 2 Cleaning UV cell Maintenanceinter Every 6 months or when required val Reason for main A clean UV flow cell is essential for correct operation of the UV monitor tenance CAUTION Do not allow solutions containing dissolved salts proteins or other solid solutes to dry out in the UV cell Do not allow particles to enter the cell Damage to the UV cell might occur There are two procedures for cleaning the UV cell e Offline cleaning Use this procedure as preventive maintenance every 6 months e Inplace cleaning Use this procedure for general cleaning in case of problems see 9 3 Monitoring problems on page 382 Performing offline To perform offline cleaning of the UV flow cell cleaning WARNING T The UV monitor uses high intensity ultra violet light Do not remove the optical unit while the lamp is ON to prevent injury to eyes Required tool
67. using a double loop to collect large peak volumes select No at this option See 4 2 2 Peak collection parameters when using double loops on page 68 for more information on the parameters to change when using double loops Max Volume in Each Loop The maximum volume allowed in each loop Using volumes above 7 5 ml might cause sample loss due to band broadening Larger volumes can be collected using two loops connected in series See 4 2 2 Peak collection parameters when using double loops on page 68 for information on parameter values Max Number of Loops The maximum number of loops used to collect peaks in the affinity step Maximum two peaks will be collected in loops but more than two loops might be required to collect large peaks Method Wizard 5 Start Collection The peak collection starts when both the UV level exceeds the Watch Level Greater than value and the UV slope exceeds the Watch Slope Greater than value Note If the flow rate is changed the shape of the peak will change as well Hence the slope values might need to be changed accordingly Stop Collection The Peak_Max condition occurs when the UV signal has fallen to a fraction specified by Peak Max factor of the most recent peak maximum The Peak end is detected when the Peak_Max condition is met AND e the signal falls below the Watch Level Less than value OR e aValley is detected OR e aWatch StablePlateau condition is met The condition is met when the signal is st
68. will check e The liquid delivery by pumping liquid from buffer inlets to waste e The gradient formation by producing a linear gradient and a series of step gradients of acetone The principle flow path of the installation test Waste F1 Waste 1 Acetone Deionized water To run the installation test follow the instructions in AKTAxpress Installation Guide e p327 8 Maintenance 8 5 Calibration procedures 8 5 Calibration procedures Aboutthis section This section describes some calibration procedures of AKTAxpress In this section This section contains the sub sections below Sub section See Calibration of zero pressure 8 5 1 Calibration of conductivity cell 8 5 2 28 4090 22 AB p328 8 5 1 Introduction Maintenance inter val Calibrate zero pressure Maintenance 8 Calibration of zero pressure Problems in the pressure calibration can cause abnormal pressure readings in chromatograms For specification of the technical data and signal drift of the pressure sensor see 10 Reference information on page 401 The zero pressure reading of the pressure sensor can be calibrated The amplification is factory calibrated and cannot be changed When required and after replacing a pressure sensor To calibrate the zero pressure Step 1 Action In the UNICORN System Control module select the system to be calib rate
69. 05 HiTrap TALON crude 100 x 5 ml 28 9538 09 HisTrap excel 5 x 1 ml 17 3712 05 HisTrap excel 5 x 5 ml 17 3712 06 GSTrap HP 5x 1m 17 5281 01 GSTrap HP 100 x 1 ml 17 5281 05 GSTrap HP 5 x 5 ml 17 5282 02 GSTrap HP 100 x 5 ml 17 5282 05 GSTrap FF 5 x 1 ml 17 5130 01 e p441 10 Reference information 10 6 Ordering information Item Code no GSTrap FF 100 x 1 ml 17 5130 05 GSTrap FF 2x 1 ml 17 5130 02 GSTrap FF 1x 5 ml 17 5131 01 GSTrap FF 5 x 5 ml 17 5131 02 GSTrap FF 100 x 5 ml 17 5131 05 GSTrap 4B 5 x 1 ml 28 4017 45 GSTrap 4B 100 x 1 ml 28 4017 46 GSTrap 4B 5 x 5 ml 28 4017 48 GSTrap 4B 100 x 5 ml 28 4017 49 Desalting columns Item Code no HiPrep 26 10 Desalting 1 x 53 ml 17 5087 01 HiPrep 26 10 Desalting 4 x 53 ml 17 5087 02 HiTrap Desalting 5 x 5 ml 17 1408 01 HiTrap Desalting 100 x 5 ml 11 0003 29 Note All 100 packs are special packs delivered on customer order Includes connector package domed nuts and instructions lon exchange columns anion lEX Item Code no RESOURCE Q 1 x 1 ml 17 1177 01 RESOURCE Q 1 x 6 ml 17 1179 01 HiTrap QHP 5x 1 ml 17 1153 01 HiTrap QHP 5 x5 ml 17 1154 01 Mono Q 5 50GL 1x1ml 17 5166 01 28 4090 22 AB p 442 lon exchange columns cation lEX Reference information 10 Item Code n
70. 16 3 m 18 1142 38 FEP tubing i d 1 6 mm o d 1 8 3 m 18 1121 16 Desalt tubing ETFE tubing i d 1 6 mm 11 0004 04 o d 1 16 0 35 m Loop extension Kit The loop extension kit below is available Item Code no Columns Loop extension Kit including 5 capillary loops 5 1 16 female 1 16 female unions and double stick tape 28 9044 38 Column examples Affinity chromatography columns Item Code no HisTrap HP 5 x 1 ml 17 5247 01 HisTrap HP 100 x 1 ml 17 5247 05 HisTrap HP 5 x 5 ml 17 5248 02 28 4090 22 AB p440 Reference information 10 Item Code no HisTrap HP 100 x 5 ml 17 5248 05 HisTrap FF 5 x 1 ml 17 5319 01 HisTrap FF 100 x 1 ml 17 5319 02 HisTrap FF 5 x 5 ml 17 5255 01 HisTrap FF 100 x 5 ml 17 5255 02 HisTrap FF Crude 5 x 1 ml 11 0004 58 HisTrap FF Crude 100 x 1 ml 11 0004 59 HisTrap FF Crude 5 x 5 ml 17 5286 01 HisTrap FF Crude 100 x 5 ml 17 5286 02 HiTrap Chelating HP 5 x 1 ml 17 0408 01 HiTrap Chelating HP 1 x 5 ml 17 0409 01 HiTrap Chelating 5 x 5 ml 17 0409 03 HiTrap Chelating 100 x 5 ml 17 0409 05 HiTrap IMAC HP 5 x 1 ml 17 0920 03 HiTrap IMAC HP 5 x 5 ml 17 0920 05 HiTrap IMAC FF 5 x 1 ml 17 0921 02 HiTrap IMAC FF 5 x 5 ml 17 0921 04 HiTrap TALON crude 5x1ml 28 9537 66 HiTrap TALON crude 5 x 5 ml 28 9537 67 HiTrap TALON crude 100 x 1 ml 28 9538
71. 2 2 Editing a method plan 5 2 3 Deleting a method plan 5 2 4 p 129 5 Method Wizard 5 2 Working with method plans 5 2 1 Creating a new method plan 5 2 1 Creating a new method plan Introduction This section describes how to create a new method plan and the main selections available It is also possible to create a method plan by opening and editing an existing method plan and then saving it with a new name Creating a new Method plans are created in the Method Wizard that is opened from the Method Editor method plan in UNICORN See 5 1 Method Wizard introduction on page 126 To create a new method plan Step Action 1 In the Method plan list of the Method Wizard select New and click Next Result The Main Selection page appears Choose the requested type of method plan The Method Wizard provides two main selections The table below de scribes the method plan types that can be created from each selection 28 4090 22 AB p 130 Main selection Method Wizard 5 Step Action 3 On each new page select the appropriate parameters and click Next to continue 4 On the Last Page 132 or Note e Save the method plan See 5 2 2 Saving the method plan on page e Click Next to enter the Advanced zone to view or edit default values See 5 5 Advanced Zone on page 164 Do not change any values in the Advanced Zone unless the consequences are fully
72. 3 10 Reference information 10 5 Chemical resistance guide 28 4090 22 AB e p 434 Chemical Exposure lt 1 day Exposure up to 2 months Remarks Hydrochloric acid OK Avoid Silicone not resist gt 0 1M ant Titanium is af fected by long term use sopropanol OK OK Methanol OK OK itric acid diluted OK Avoid Silicone not resist ant itric acid 30 Avoid Avoid Elgiloy is affected by long term use Phosphoric acid OK Avoid Titanium alumini 10 um oxide and glass are affected by long term use Potassium carbon OK OK ate Potassium chlor OK OK ide Pyridine Avoid Avoid ETFE PP and PE not resistant Sodium acetate OK OK Sodium bicarbon OK OK ate Sodium bisulphate OK OK Sodium borate OK OK Sodium carbonate OK OK Sodium chloride OK OK Sodium hydroxide OK Avoid PVDF and borosilic 2M ate glass are af fected by long term use Reference information 10 Chemical Exposure lt 1 day Exposure up to 2 months Remarks Sodium sulphate OK OK Sulphuric acid di OK Avoid PEEK and titanium luted are affected by long term use Sulphuric acid Avoid Avoid medium concen tration Tetrachloroethyl Avoid Avoid Silicone PP and PE ene are not resistant Tetrahydrofuran Avoid Avoid ETFE CTFE PP and PE are not resist ant Toluene OK Avoid Pressure limit for PEEK decrease
73. 31 See 8 6 1 Replacing capillary loops on page 336 See 8 6 2 Replacing tubing and connectors on page 338 See 8 6 3 Replacing flow restrictor on page 351 See 8 6 4 Replacing UV lamp on page 353 See 8 6 5 Replacing check valves on page 358 See 8 6 6 Replacing pump piston seal on page 361 See 8 6 7 Replacing a damaged pump piston on page 370 See 6 4 4 Purging the pump and inlet tubing Over view on page 212 Maintenance 8 8 2 Monthly maintenance About this section This section describes the monthly maintenance In this section This section contains the sub sections below Sub section See Checking flow restrictor 8 2 1 Changing pump rinsing solution 8 2 2 e p307 8 Maintenance 8 2 Monthly maintenance 8 2 1 Checking flow restrictor 8 2 1 Checking flow restrictor Maintenanceinter Every month val Reason for main A flow restrictor creating too high back pressure will cause a high pressure alarm from tenance the system A flow restrictor creating at too low back pressure might cause air bubbles in the buffer solution in the flow path The position of the flow restrictor In addition a flow restrictor is integrated in the mixer Checking flow re To check the flow restrictor strictor Step Action 1 Immerse inlet tubing A1 into deionized water 2 Make sure that the pressure reading is
74. 4 6 Buffer alternatives 4 6 2 Buffer suggestions AC buffer sugges tions for GST tagged proteins 28 4090 22 AB p110 When performing suggested buffer step and gradient elu tion 20 mM sodium phosphate 0 5 M NaCl 500 mM im idazole pH 7 4 Note As a default gradient binding buffer is exchanged to 100 elution buffer during elution In the affinity tag removal protocols the affinity columns can be equilibrated with either AC binding buffer extra wash buffer or an alternative cleavage buffer before protease injection Suggested buffers for AC runs with GST tagged proteins when using the columns GSTrap HP FF 4B When performing suggested buffer binding PBS pH 7 4 140 mM NaCl 2 7 mM KCI 10 mM Na HPO 1 8 mM KH gt PO pH 7 4 Note 1 10 mM dithiothreitol DTT can be in cluded in the binding buffer to make sure that the column is in a reduced state en hancing GST binding cleavage using PreScis sion 50 mM Tris HCl 150 mM NaCl 1 mM EDTA 1 mM DTT pH 7 5 elution 50 mM Tris HCl 10 20 mM reduced glutathione pH 8 0 Note 1 10 mM DTT can be included in the elu tion buffer to make sure that the column is in a reduced state enhancing GST binding In the affinity tag removal protocols the affinity columns can be equilibrated with either AC binding buffer or an alternative cleavage buffer before protease injection DS bu
75. 402 The parts for handling samples and buffers are located at the lower part of the separation system They comprise two switch valves for buffer selection and gradient a general inlet valve for samples and buffers an injection valve and an air sensor Injection valve T asesan Switch valves Inlet valve Air sensor Buffers Buffers Samples Al A2 A3 A8 S1 to S4 B1 B2 Switch valves The switch valves are mainly used for gradient formation They have four buffer inlets two on each valve labelled A1 B1 A2 and B2 ep41 3 AKTAxpress overview 3 2 Separation system overview 3 2 4 Functional description Pump and related items 28 4090 22 AB p42 Inlet valve The flow from the switch valves continues to the inlet valve which provides six additional buffer inlets labelled A3 A8 The inlet valve also provides four sample inlet tubings labelled S1 S4 Injection valve A sample volume can also be injected manually into the flow path through the injection valve using a syringe The injection valve is located above the inlet valve Note Buffer containers sample tubes and other external hardware are not included at delivery Air sensor An air sensor is connected between the inlet valve and the pump It is used for e Detecting when the sample supply is empty during sample loading The inlet valve then switches to a buffer inlet e Detecting when the buffer supply is empty during a run The system i
76. 426 TOA TYPICAL TUN INES ea n a oSA AEE OAE ON R O i 428 10 5 CHEMICGTESISTAM CS guid eeraa n e RRO TAA 432 10 6 Ordenn gN OrmMmatiO Nimas aaan a AO E E OT 436 IGEN E EAE A A AEE ATASA 445 ep7 Preface 1 1 Preface About this This chapter contains regulatory contact and safety information chapter In this chapter This chapter contains the sections below Section See Important user information 1 1 Product information 1 2 Safety 1 3 p9 1 Preface 1 1 Important user information 1 1 Important user information Important userin All users must read this entire manual to fully understand the safe use of AKTAxpress formation Warning symbols The following warning symbol is used in the documentation WARNING T The Warning symbol highlights instructions that must be strictly followed in order to avoid personal injury Be sure not to proceed until the instructions are clearly understood and all stated conditions are met Caution notices The following caution notice is used in the documentation CAUTION The Caution sign highlights instructions or conditions that must be followed to avoid damage to the product or other equipment Be sure not to proceed until the instructions are clearly understood and all stated conditions are met Note signs The following note sign is used in the documentation Note The Note sign indicates information important for tr
77. 5 6 Operation 6 4 Preparing the system for a run 6 4 1 Printing out a summary Printing outthe The table below briefly describes how to create and print out the summary page Summary page Step Action 1 Select method plan e In UNICORN System Control click Instant Run Instant Run Result The Method Wizard for System Control is displayed e Select the required method plan from the list Click Next Method plan New id ACDS Result The Systems Samples page is displayed Select the System s on which to run the method plan and the Number of Samples for each system Go through the subsequent pages by clicking Next When the Summary page is reached click Print to print it and then click Cancel Note It is also possible to copy the information on the Summary page and paste it into Excel to save the information 28 4090 22 AB p206 6 4 2 Introduction Warning Prepare buffers and solutions Liquid quality re commendation when preparing buffers and solu tions Preparing the containers Vent opening on container caps Inlet supply for a purification run Operation 6 Preparing buffers and solutions This section describes in which buffer solution containers the inlet tubing from the system to the buffers and solutions should be immersed and where the containers should be placed WARNING A When using hazardous chemicals take all su
78. 6 8 6 Standard cleaning of columns CreateaStandard The illustration below gives an overview of how to include CIP in the Standard System System and and Column Procedures method plan See 5 3 3 Selecting Standard System and Column Proced Column Procedures options on page 142 for detailed information ures method plan including CIP the positions occupied by the column type 1 5 10800000 0 0 i 0 08 000000 i 0 Select the positions occupied by the column type 1 5 Select the positions occupied by the column type 1 5 the positions occupied by the column type 1 5 Cleaning the Run the purify method plan as described in 6 6 1 Starting a run using a method plan columns withina on page 259 Cleaning of the columns will be performed after purification purify method 28 4090 22 AB p286 Operation 6 Cleaning the To run the Standard System and Column Procedures method plan including cleaning columns using the of columns Standard System and Column Pro cedures method plan 1 IMPORTANT Connect the first column to port 1 in the column block the second column to port 2 and so on no matter the type of column used Step Action 2 Immerse the tubing ends to be used in the correct containers see also the Summary page 3 Run the method plan as described in 6 6 1 Starting a run using a method plan on page 259 e p287 6 Operation
79. 6 8 Procedures after a run 6 8 7 Customized cleaning of columns 6 8 7 Introduction Warnings and notes Prerequisites 28 4090 22 AB p 288 Customized cleaning of columns Customized cleaning of the columns includes cleaning of the system with up to nine solutions It is performed by running the Customized Column Procedures prepare and maintain method plan Up to five columns of the same type can be cleaned simultaneously The method is adapted to the columns used when setting up the method plan in the Method Wizard e g flow rate and pressure settings column volume etc Note When cleaning chelating affinity columns the columns should be stripped before CIP to avoid formation of metal ion salts that can damage the column WARNING When using hazardous chemicals take all suitable protective measures such as wearing protective glasses and gloves resistant to the chemicals used Follow local regulations and instructions for safe operation and maintenance of the system Note The columns must be filled with water prior to CIP It is possible to add an extra step with water in the beginning of the method The following are required for cleaning the columns e Cleaning solutions See the column instructions and or 4 7 4 Customized CIP Solution Suggestions Columns on page 117 e Amethod plan for cleaning the columns Operation 6 Create a Custom The illustration below gives an overview
80. 87 Protocols 59 descriptions 59 for KTAxpress 59 Pump description 403 synchronization 47 Pump piston replace 370 Pump piston seal replace 361 Pump pressure checking 324 Purging the pump with methanol 223 R Report printing 302 Reset the system 47 Results viewing 296 Run times 428 S Safety 13 Samples flow control 250 manual loading 246 preparing 245 tubing 245 Schematic drawing of the flow path 16 Software 397 Spare parts 436 Strip solution choosing 123 Superloop connecting 234 filling 234 pre filling with buffer 233 Switch valves 407 Syringe 215 218 368 System Control collapse 267 expand 267 e p447 Index T Tag cleavage see affinity tag removal 238 Technical specifications 416 Troubleshooting 372 Tubing checking 243 dimensions 39 fittings types 39 leakage 325 piston rinsing 349 replace 336 338 specification 342 Typographical conventions 26 U UNICORN accessing help functions 30 indicator colors 197 introduction 18 operation overview 29 28 4090 22 AB p 448 software modules 28 User documentation package 24 UV curve 382 UV cell cleaning 314 UV lamp checking run time 312 353 replacing 353 V Valves description 407 Viewing a run 267 View results 296 Ww Warnings 13 Waste disposal 335 Waste tubing 210 Watch conditions 65 Page intentionally left blank For local office contact information visit www ge
81. AKTAxpress User Manual Page intentionally left blank Table Of Contents Table Of Contents Dy PRETACES cie3 ohdss cos AAE T A a cadancestiaastss same cetesticasesese decals ase acede deus inane E AATE 9 1 1 Important user information 1 2 PrOGUCEINTOFMOGLION wased hea a a e e A aan nea ac EI SIEV raaa e E T E AO EREN OAO 13 PARo lo Uos ao p PIES EEE E E E AE EAEE 14 2 1 ntrod cing AK TAX KOSS 5 ii menean aai ire a T ee iaer E E A EE EES 15 2 2 Purification of recombinant proteins 20 2 3 Basic operating PFINCIDIESs iinne REE EROE OE 22 2 4 KTAxpress user COCUMENTATION eessssscssssssssssessssssssessssssssssssssssssssssssesnssssssseeesnssssasesestnsssssessensssssesseeeeensen 24 3 AKTAXPreSS OVE A a a N RN k 27 S UNICORN OVE A a N E 28 5 2 SCPALALIOM SV E OVELVI QW a E A A A 31 3 2 1 LOCATON OF THE Components irsneiii tiiir AON vets 32 32 2 Maid NON pateren iaa REA O TOA UEA 38 3 2 3 TUDING ANd CONNECTOTS pne E A N OA NOONE a 39 32A Functional descriptio N secin onnen ena a O E A ERA 41 3 2 9 INGICATOLS GNA CONTOlS ariana e A TE A A N 47 32 oI No ere 51 3 3 PUPIFICALION PLOCESS OVER VIO Wi aieia E REA REO ERA 52 3 3 1 PROCESS TOW CROT aoire aK E AEEA EEEE E EEEE A EA 53 3 3 2 DESCHIPLION of THE PLOCESS Stops iian en eE A UGEN UER e 54 4 Methodology scsiscicscsccccdasssccscscssnetecsetecseusseascstcastsvasdives seteescsivtsdscsoudacddten rer EEEE desdsesdstbdetstvesdsdedscsibssteentes 58 41 PFOTOCOL
82. Column type and the Column Positions for the attached columns Cuttomized Column Procedures Cohen AESOURLE Q of Global Nemnbes of Steps w C wash Coins vaih Wates befara CIP Bniet ASI 2 Select the Number of Steps 3 Optional To perform a column wash before the cleaning step s select Wash Columns with Water before CIP Enter the Flow Rate and Volume for the wash See 4 7 4 Customized CIP Solution Suggestions Columns on page 117 for information 28 4090 22 AB p 148 Method Wizard 5 Step Action 4 For each step make the following selections if there is more than one cleaning step additional pages appear by clicking Next e To perform a system wash before the cleaning step select System Wash e Enter an appropriate Flow Rate according to the instructions supplied with the column e Select the buffer Inlet to be used e Enter an appropriate Volume in column volumes according to the instructions supplied with the column See also 4 7 4 Customized CIP Solution Suggestions Columns on page 117 for information on solutions flow rates and volumes 3 Enter a pause time in Pause before Next Step if incubation of the column in the chosen solution is required 4 Proceed to 5 2 2 Saving the method plan on page 132 p149 5 Method Wizard 5 4 Using the Purify option 5 4 Overview 28 4090 22 AB ep 150 Using the Purify option When using the Pu
83. Columns Fill with Ethanol 25 CIP Columns Re equilibrate 20 Re equilibrate 5 Reference information 10 Column procedure Typical run time RT CR min Strip Columns Fill with Ethanol 10 Strip Columns Recharge Blank Run 40 Strip Columns Recharge Fill with 20 Ethanol DS Fill with Ethanol 25 CIP Columns Fill with Ethanol 50 CIP Columns Re equilibrate 25 IEX Fill with Ethanol 5 CIP Columns Fill with Ethanol 30 CIP Columns Re equilibrate 20 Re equilibrate 5 GF Fill with Ethanol 485 CIP Columns Fill with Ethanol 1130 CIP Columns Re equilibrate 650 1 Run times are approximate and with default settings Sample loading time is not included p 431 10 Reference information 10 5 Chemical resistance guide 10 5 Chemical resistance guide Introduction commonly used chemicals in liquid chromatography Note This section specifies the chemical resistance of AKTAxpress to some of the most AKTAxpress is intended to be used with water based solutions only Organic solvents are not recommended due to the mixer design Assumptions made Note The ratings are based on the following assumptions The synergy effects of chemical mixtures have not been taken into account Room temperature and limited overpressure is assumed stated all concentrations are 100 Chemical influences are time and pressure dep
84. E Ehun Budhe UP DS UF Secun Step FA Clean Sange Irist F Clean Samgie Iniets Al a 4a At a5 AB A7 As a B a 32 3 f HieTrop HP 1 Hitap HP_t_o Hilosd_16 00_Sup Make a final check of the system setup using the list from the Summary page Click Run to start the run on the selected systems 28 4090 22 AB p264 6 6 2 Introduction Manual control System settings Operation 6 Starting a manual run This section describes how to run a system manually For more information see the UNICORN user documentation Besides running method plans on the systems it is also possible to control the system manually for example starting and stopping the pump switching valve positions and so on Follow the steps below to control the system manually Step Action 1 Select Manual Pump in System Control A dialog box containing manual instructions appear om a tor r _ m a oJ hm oe ee ee atant e 2 e Select an option in the Instructions field and an instruction e Select parameter or set a suitable value in the Parameters field and click Execute to start the instruction 3 For information about the instructions in a dialog box click Help Each system has a set of default system settings which can be changed e Tochangeasystem setting select System Settings in System Control The system must be in End mode Make the appropriate changes o
85. E R OE 353 le TeS la RA e e NE ETE S AE EEEE EETA 358 8 6 6 REPIGCING Hump pIStOn Sedlari AE E A EE 361 8 6 7 Replacing a damaged PUMP PISTON eecssccscsssssssssssseessssesssssssssssssessesseseessssssssssusssssesecsssssssssusseesesseees 370 9 Troubleshooting and Corrective actionS e essesseesessesseesessesseessessesresseeseosesseesnssrsneesseosessesseeseeses 371 9 1 Introduction to TFOUDIESHOOTING cecessssssssssssssssssssssssssccsssssssssssssssssecsssssssssssussssssesscsssssnssssussseeseesssstsssssusecseseees 372 9 2 Problems duning OTU ieii NA A ETNE AON aA 375 9 3 Monitoring Pro hEm n re Geirio EAA E EOE TE as 382 o Az LEAKAGE KOl IERES IE IEE E TEE EE E EEEE AES uct 389 9 5 Alarms and connection Problem Sirrini annii i a a i 391 JO ETON COE liS teniri ea EEE EEE EE A OA ual des 393 9 7 CHECKINGAUSB GAN GRIV ELS s sdcccsseecsticstivesescecodsecssccovssctube eto scccorssssotsosean Meee OTETO ERO 397 10 Reference information 10 1 System description 10 11 Pump and r lated components enni atau AA A A N E 403 AKOPA INSIREN TATE E ASEEN E EEAO EES AS 10 13 MONTO Sa arerioaren o neies EEEa POE ea EEEa ata PEIES ATEEN EANA REE TETEH 10 1 4 Fraction collector 10 2 Technical Specifications reiron aN TO RNA 415 1052 1 System SpECINCAtON Srnie iii E O AE ERRE 416 10 22 Components SpECITICOtONS sinisinta aa ai AE a KAA 420 10 2 3 Wetted Material ne r 20 8 E E E O A a ae as 424 10 3 Import MEMO ea a E N E a
86. GESCHIPLIOMS and Choit ssmiiseniiiiiesisiiiiiiiiti a A NEE 59 716 2 Pak RandiNg nanata E E EEE A EL E A A 64 4 21 PEAK collection Paramete Sivens a EE AREN 65 4 2 2 Peak collection parameters when using double lOOPS a cesssssssssessesssssccsssssssssunssesseseesssssssssunsessees 68 4 2 3 Peak fracti nation DATEAIMELETS tes gissscascccsinseissevsessTebbeslecssss saves cossnantaticbusnssshesosbovonensuvesavonssssstoveoonenvasuavbons 71 AS SVSLEMPANA COMUMM PROCEM ULES ssciccscvesscssssssccessssssesusrssleauaseesccnvsssdaseqesssbsosoovasotauscssnsbStavbae anne aasdbovnas Bs 73 4 3 1 System and column procedures Within a purify MEthOd ccessssessessccscccssssssseeesssescessssssssssnseesees 74 4 3 2 System and column procedures in prepare and maintain MEthOdS cessssessesccccccssssessseeses 79 FA APTI LUGE EIN OWG ss ra TE E E TEOR E OA REO 84 4 4 1 Description OF affinity tags isssemiieerann niii O AARON 85 442 DESCrIDLION Of ProtledSE Sinnen a a E O R 4 4 3 Description of the affinity tag removal processes ep3 Table Of Contents 4 4 4 Conditions for affinity tag CleAVAGEC ssssssssssssesssssscssssssssssssesssssessssssssusssussssessessssssssssesessesseesssssssssunsessess 92 GS Columa GIS RMA Ve Sysas at A AETA AE 94 45 Us Descriptions Of COUMANS a a OE E ale A 95 4 5 2 Choosing ColUMN Sasiia i a E AE O E E A 101 46 BUTTER alternative Snes a a a a O CE EA 106 ASG L DESCHIPLIOMN OF DUT OLS nae AAN N
87. HP are used These can be changed in the Advanced Zone Note Any HiTrap means that any HiTrap affinity column can be used The same default values as for HisTrap HP are used These can be changed in the Advanced Zone Method Wizard 5 Step Action 5 Select the type of column procedure to be performed by choosing the appropriate radio button See 4 3 2 System and column procedures in prepare and maintain methods on page 79 for a description of the possible column procedures for each column 6 Select the Column Positions for the column 7 Select Fill Columns with Ethanol to fill the column s with ethanol after the run if the column s is not going to be used for a few days 8 When options for one column type have been selected click Next to display the settings for the next column type Repeat steps 2 7 to select settings When all the requested options have been made the Last Page is dis played Proceed to 5 2 2 Saving the method plan on page 132 ep 145 5 Method Wizard 5 3 Using the Prepare and Maintain option 5 3 4 Selecting Customized System Procedures options 5 3 4 Customized Sys tem Procedures options 28 4090 22 AB p 146 Selecting Customized System Procedures options To select Customized System Procedures options Step Action 1 On the Customized System Procedures page select the Sample Inlets S1 S4 and the Buffer Inlets A2 A8 B1 and B2 to be c
88. High Performance to be charged with the metal ion of choice for high resolution purification of histidine tagged pro teins HiTrap IMAC FF 1and5 ml HiTrap column prepacked with IMAC Sepharose 6 Fast Flow to be charged with the metal ion of choice For purification of histidine tagged proteins at high flow rates GSTrap HP 1and5 ml GSTrap FF 1and5 ml GSTrap 4B 1and5 ml HiTrap column prepacked with Glutathione Sepharose High Performance For high resolution purification of GST tagged proteins HiTrap column prepacked with Glutathione Sepharose Fast Flow Often gives high binding capacity for GST tagged proteins HiTrap column prepacked with Glutathione Sepharose 4B A softer medium that needs to be run at a lower flow rate but often gives high binding of GST tagged proteins Desalting DS columns separate molecules with large size differences Desalting is a quick method for size separation and is often used to remove salts from proteins or to change buffers The following desalting columns are supported by KTAxpress lon exchange columns Methodology 4 Supported DS columns and volumes Characteristics HiPrep 26 10 Desalting 53 ml HiPrep column prepacked with Sepha dex G 25 Fine For fast buffer ex change and desalting of samples with a volume of lt 13 mlt 2 x HiTrap Desalting 5 ml 10 ml see also 4 5 1 Descriptions of columns on page 95 HiTrap colum
89. IEX Make sure to use an IEX column with sufficient capacity for the protein amount Choosing column Recommended column choice for the GF step for the GF step Which sample volume Which My has the torget protein HiLoad 16 600 HiLoad 16 600 Superdex 75 pg Superdex 200 pg Comments on alternative column choices Which M has the torget protein lt 70kDo gt 70 kDa HiLoad 26 600 HiLoad 26 600 Superdex 75 pg Superdex 200 pg Use in GF step e p103 4 Methodology 4 5 Column alternatives 4 5 2 Choosing columns 28 4090 22 AB p 104 Step Comments on alternative column choices GF To get sufficient separation it is important to choose a gel filtration column with appropriate selectivity GF Superdex columns give higher resolution than Sephacryl columns Preferably use columns with higher resolution in late polishing purific ation steps GF If the protein forms for example a dimer the true M will be twice as large as the theoretical My received from the sequence information Example of choos ing column com binations for a four step protocol Methodology 4 Example of recommended column combinations when running an AC DS IEX GF protocol Amount of target protein Which target Which torget protein tag protein tag Histidine cst GSTrap 4B i 1ml f Use in AC step HisTrop
90. IN of the pump head Tighten the valves until fully finger tight and then use the wrench to tighten a further 1 3rd 110 of a turn Note Do not overtighten the valves as damage to the internal components can occur 11 Refit the outlet tubing and the inlet manifold 12 Purge the pump carefully and check that the pumping action has been corrected see 6 4 7 Purging the pump and system on page 222 e p323 8 Maintenance 8 4 Maintenance when required 8 4 3 Checking pump pressure 8 4 3 Checking pump pressure Introduction Problems in the pump can cause abnormal pressure readings Examples of pump problems Air trapped in the pump heads Leaking connections Leaking pump piston seal Check valve malfunctioning Piston damage Checking the To check the pump function pump function Step Action 1 Disconnect the Waste tubing from the injection valve port 1 and connect a capillary tubing giving a counter pressure of about 1 MPa at 1 ml min to port 1 2 Set the injection valve to position Waste 3 Start a flow of 1 ml min or less and observe the pressure curve over several minutes 4 If the pressure pulsation seems abnormal e Purge the pump heads see 6 4 7 Purging the pump and system on page 222 e Check the tubing connectors for leakage e Check the piston seals check valves and pistons e Check the number of piston strokes by selecting System Mainten ance in System Contro
91. Inlet valve 190 16 FEP port IN to air sensor Pump Air sensor to 230 1 6 FEP pump mani fold Mix1 Pump head 2 120 1 ETFE o mixer Mix2 Pump head 1 120 1 ETFE o mixer Press Mixer to pres 160 ETFE sure sensor Mixinj Pressure 260 ETFE sensor to in jection valve port 5 e p345 8 Maintenance 8 6 Replacement procedures 8 6 2 Replacing tubing and connectors 28 4090 22 AB p346 Label bold Description Length mm_ Inner Material or diameter designation mm ColV Injection 350 1 ETFE valve port 6 to column valve IN Clin Column valve 350 T ETFE port 6 to column 1 Clout Column block 190 1 ETFE port 1 to column valve port 12 C2in Column valve 350 1 ETFE port 5 to column 2 C2out Column block 190 1 ETFE port 2 to column valve port 11 C3in Column valve 350 1 ETFE port 4 to column 3 C3out Column block 190 1 ETFE port 3 to column valve port 10 C4in Column valve 350 1 ETFE port 3 to column 4 C4out Column block 190 350ifus 1 ETFE port 4 to ing a HiPrep column valve 26 10 desalt port 9 ing or GF column in this position Maintenance 8 Label bold Description Length mm_ Inner Material or diameter designation mm CSin Column valve 350 1 ETFE port 2 to column 5 C5out Column block 190 350ifus 1 ETFE port 5 to ing a HiPrep column valve 26 10 desalt port 8 ing or GF column in this position B
92. Loops Outlets with Ethanol to fill the system in cluding the loops and the fraction collection tubing with ethanol after the run if the system will not be used for a few days Click Next Ifno columns were selected on the Prepare and Maintain page the Last Page will be displayed Proceed to 5 2 2 Saving the method plan on page 132 e If columns were selected on the Prepare and Maintain page one page selected column type will be displayed if several columns should be prepared maintained additional pages one column ap pear by clicking Next Proceed with Selecting Column Procedures options below e p143 5 Method Wizard 5 3 Using the Prepare and Maintain option 5 3 3 Selecting Standard System and Column Procedures options Selecting column procedures op tions 28 4090 22 AB p 144 To select column procedures follow the instructions below Step Action 1 For each column type a separate page with available options will be displayed Select options according to the steps below Cohen Chelate Oest Helia HP 1 atikbal o Eades Sup gt Blow Pim 2 If the column is filled with ethanol select Remove Ethanol to fill the column with water 3 Select the type of Column to be used Chelating GST or IEX 4 Select the Column to be used in the drop down list Note Any HiTrap IEX means that any HiTrap ion exchange column can be used The same default values as for HiTrap Q
93. Maintenance 8 4 Maintenance when required 8 4 2 Cleaning check valves 8 4 2 Introduction Performing in place cleaning Performing offline cleaning 28 4090 22 AB p 320 Cleaning check valves Faulty operation of the check valves is usually indicated by e irregular flow e very low flow e unstable pressure traces Probable causes of this are air or dirt in a check valve preventing it from closing to seal and hold the pressure To solve the problem perform e Inplace cleaning If the problem remains continue with e Offline cleaning To perform inplace cleaning of the check valves Note It is recommended to use degassed buffers and solutions Step Action 1 Pump deionized water at a flow rate of 40 ml min for 2 minutes 2 Change solvent to 100 methanol and pump ata flow rate of 40 ml min for 4 minutes 3 Change solvent to deionized water and flush the system If this does not correct the problem follow the instructions for performing offline cleaning To perform offline cleaning of the check valves CAUTION Check valves have precision matched components and should only be disassembled by a trained person If the problem cannot be corrected the check valve should be replaced completely Required tools and material Maintenance 8 13 mm wrench 18 mm wrench Ultrasonic bath 100 methanol Step Action 1 Change solvent t
94. Note When performing a run using a method plan an initial system wash will automatically be included To purge the pump and or system Step Action 1 Carefully immerse the inlet tubing A1 in a flask containing the buffer to be used 2 Run a Pump Wash or a System Wash e Start UNICORN and select System Control Manual Pump e Select PumpWash and inlet A1 or SystemWash Za a AA C hiamidton her l we DT Ago upahe I has a chockas ha paramara faidi wall ba updated dang sahod an e Click Execute 6 4 8 Introduction Caution Purging proced ure Prerequisites Operation 6 Purging the pump with methanol Both pump heads must be purged with methanol if e The system has been left unused for a week or longer or e The pump has been run dry The purging will maintain the pumping capacity and protect the pump piston seals Note All inlet tubing that will be used in the next purification run should be filled manually before purging the pump CAUTION To protect the piston seals in the pump the pump must never be run with air in the inlet tubing The purging procedure in short 1 Flush out buffer using deionized water 2 Purge the pump using methanol 3 Flush out methanol using deionized water The following are required for automated customized system cleaning e one flask with at least 200 ml degassed methanol e one flask with a
95. Piston kit including piston spring seal 18 1112 13 and rinse membrane Monitor parts The following spare parts are available for the monitors UV monitor Item Code no Hg optics with 254 280 nm filters excl 18 1128 20 flow cell Hg lamp amp housing complete 18 1128 22 UV flow cell 2 mm 18 1128 25 Filter 254 nm 18 0620 01 Filter 280 nm 18 0621 01 UV test kit 2 mm flow cell 280 nm 18 1129 63 Conductivity monitor Item Code no Conductivity flow cell complete 18 1111 05 Air sensor Item Code no Air sensor complete 915N 11 0003 08 e p437 10 Reference information 10 6 Ordering information Flow restrictor Cables Fraction collector The following spare part is available for the fraction collector Item Code no Capillary loops Superloop 28 4090 22 AB p 438 The following spare part is available for the flow restrictor Item Code no Flow restrictor FR 902 18 1121 35 The following cables are available Item Code no UniNet 0 7 m 18 1109 74 UniNet 1 5 m 18 1117 75 UniNet 3 0 m 18 1109 75 UniNet 15 m 18 1117 74 UniNet Inline coupler 11 0003 79 Mains cable EU 220 V 19 2448 01 Mains cable US 115 V 19 2447 01 Drip box 11 0002 95 The following capillary loops are available Item Code no Capillary loop 11 0003 02 The following Superloops are available Item Code no Su
96. RN 5 2003 2014 General Electric Company 2006 2014 General Electric Company All rights reserved First published Aug 2006 All goods and services are sold subject to the terms and conditions of sale of the company within GE Healthcare which supplies them A copy of these terms and conditions is available on request Contact your local GE Healthcare representative for the most current information GE Healthcare Europe GmbH Munzinger Strasse 5 D 79111 Freiburg Germany GE Healthcare UK Ltd Amersham Place Little Chalfont Buckinghamshire HP7 9NA UK GE Healthcare Bio Sciences Corp 800 Centennial Avenue P O Box 1327 Piscataway NJ 08855 1327 USA GE Healthcare Japan Corporation Sanken Bldg 3 25 1 Hyakunincho Shinjuku ku Tokyo 169 0073 Japan 28 4090 22 AB 09 2014
97. RT 15 CR 12 28 4090 22 AB p 118 1 Recommended to pause overnight at RT Recommended solutions for ion exchange columns Methodology 4 The tables below list recommended solutions for the ion exchange columns supported by AKTAxpress See 4 5 1 Descriptions of columns on page 95 for a list of columns supported by AKTAxpress Column Solution Volume Flow rate ml min HiTrap Q SP HP 1and5 ml Step 1 2 M NaCl 4 CV RT 0 25 1 3 CR 0 20 1 Step 2 Deionized water 2 CV RT 0 25 1 3 CR 0 20 1 Step 3 1 M NaOH 4 CV RT 0 25 1 3 CR 0 20 1 Step 4 Deionized water 4 CV RT 0 25 1 3 CR 0 20 1 Step 5 30 isopropanol 2 CV RT 0 25 1 3 CR 0 20 1 Step 6 Deionized water 4 CV RT 0 25 1 3 CR 0 20 1 RESOURCE Q S 1and6 ml Step 1 1M NaCl 5 CV RT 4 6 CR 3 2 4 8 Step 2 Deionized water 2 CV RT 4 6 CR 3 2 4 8 Step 3 1M NaOH 5 CV RT 4 6 CR 3 2 4 8 Step 4 Deionized water 2CV RT 4 6 CR 3 2 4 8 Step 5 1M HCI 5 CV RT 4 6 CR 3 2 4 8 Step 6 Deionized water 2 CV RT 4 6 CR 3 2 4 8 Step 7 1M NaCl 5 CV RT 4 6 CR 3 2 4 8 Step 8 Deionized water 2CV RT 4 6 CR 3 2 4 8 Mono Q S Step 1 1M NaCl 4CV RT 0 5 CR 0 4 p119 4 Methodology 4 7 Solution alternatives 4 7 4 Customized CIP Solution Suggestions Columns Column Solution Volume Flow rate ml min Step 2 Deioniz
98. Remove the second screw and without allowing the pump head to twist sideways carefully pull it out 7 Place the pump head face down on the bench Pull out the piston togeth er with the return spring 8 Inspect the piston and return spring for sign of damage If damaged they should be replaced e p363 8 Maintenance 8 6 Replacement procedures 8 6 6 Replacing pump pis on seal Step Action 9 Wipe the piston with a clean cloth If salt solutions have been used the piston may be slightly corroded This corrosion can be removed with a rubber eraser If it cannot be wiped or rubbed clean scrape off any de posits with a scalpel or razor blade Inspect the piston with a magnifying glass for scratches Replace with a new piston if any scratches or cracks are found 10 Remove the two screws securing the drain plate and the rinse chamber Remove and discard the rinsing membrane Remove the rinse chamber Drain plate Rinsing membrane a Rinse chamber j 11 Gently withdraw the piston seal Discard the used seal Piston seal Le 28 4090 22 AB p364 Maintenance 8 Step Action 12 The pump head rinse chamber and drain plate should be carefully rinsed or cleaned in an ultrasonic bath if available If dirt can be seen on any surfaces the inlet and outlet check valves should be removed and cleaned separately see 8 4 2 Cleaning check valves on page 320 13 Slightly wet th
99. S or GF as last step in a multi step protocol 05 1 intermedicte DS GF Last step DS 1 Peak Collection DS GF Included Procedures DS 1 Included Procedures Fractionation Note The Peak Collection page and Included Procedures page show the same type of parameters independent of protocol step p 181 5 Method Wizard 5 5 Advanced Zone 5 5 5 Advanced Zone for Purify Desalting Gel Filtration DS 1 General and DS 1 Inter mediate pages parameters DS 1 Peak col lection paramet ers DS GF and DS GF Last Step pages parameters 28 4090 22 AB p 182 This page contains options and parameters related to flow rate pressure and volumes DS 1 Intunmudiato Fow Rate E 1 69 mn than Voke fo sysa93 cv Preme Lint os were Peak nectar Peak ingetan Push Veme w pio ot Peak lryection Mar Vokume 13 p o a Flow Rate Flow rate during the desalting step Elution Volume Volume used during the elution of the desalting column Pressure Limit Upper pressure limit during the desalting step Peak Injection only available on the DS 1 Intermediate parameters page e Peak Injection Flush Volume The extra volume besides the peak volume used to flush the loops when loading the sample onto the desalting column e Peak Injection Max Volume The maximum volume to be injected onto the desalting column Note The maximum injection volume is set to make sure that the peak volume flush volume do
100. System box and click OK twice Properties Run Daa Groups Pun Data Coir Curves Name V Ame Cleve Style and Color Logbook S miem apars type in shew Ret Mak Bloch start Eno dam ad wasters Metta a sinatra Result The system messages marks are displayed in the chroma togram e p269 6 Operation 6 7 During a run 28 4090 22 AB p 270 Step Action By looking in the chromatogram select the peak to continue with in the next purification step Information about in which loop the peak has been collected is displayed in the chromatogram Ending the run Status indicator colors Operation 6 Step Action 3 Select the loop in which the peak to continue with is collected e In System Control select Manual Alarms amp Mon Result The Alarms amp Mon Instructions dialog for the selected system is displayed e Select Man_Peak_Select in the list in the Instructions area Result The LoopPos drop down list is displayed to the right in the Parameters area e Select the loop containing the peak to be selected for next step in the LoopPos list and click Execute Fae G Aa update Il ihe chee te parameter halt i be updated Aero method nes Note If the peak is divided into several loops e g LP1 and LP2 choose any one of these loops in the LoopPos list e g LP2 to select the entire peak e Click Close to close the Alarms amp Mon Instructions dialog 4 Click Continue to pro
101. a run 6 4 11 Preparing the system when using a Superloop Connecting the Follow the instructions below to connect Superloop superloop CAUTION Make sure that no parts for example tubing or columns are positioned in front of the fraction collector hindering the ejection of the microplate The movement of the plate might destroy columns or tubing positioned in front of the fraction collector Step Action 1 Attach Superloop to the column holder on the right hand side 2 Disconnect the loop connected between port OUT 5 and IN 5 in the loop valve Use the fingertight key if necessary 3 Connect the tubing from the top of the superloop to the loop valve port OUT 5 4 Connect the tubing from the bottom of the superloop to the loop valve port IN 5 Filling the super Superloop should be manually filled with protease sample solution The Method Wizard loop contains an option for guided loading of Superloop in preparation and purification runs Dialog boxes will then automatically appear when starting the run giving instructions on how to fill the Superloop Note The capillary loops are filled with sample in the same way as the Superloop 28 4090 22 AB p234 Operation 6 Filling the Superloop using guided loading of Superloop interactive method Step Action 1 Connect a Luer fill port to the injection valve port MANUAL INJECTION 2 e Start the run When the system enters Pause
102. able within the limits of the Delta Plateau value during the Stable Time interval Select Largest Peak Based on either peak height or peak area to be transferred to the next column By default area is used to determine the largest peak More information For more information on peak handling and watch conditions see 4 2 Peak handling on peak handling on page 64 e p187 5 Method Wizard 5 5 Advanced Zone 5 5 8 Advanced Zone for Purify Fractionation 5 5 8 Introduction Fractionation 28 4090 22 AB p 188 Advanced Zone for Purify Fractionation This section describes the parameters used in the fractionation step during a purification run The peak fractionation options and parameters are described below The parameters define the final fractionation and how the final peaks are collected in the fraction collector Pesh Fistan Sizo 4 10 10 wt Fea echonaton Agcnthen Level AND Siope s Stat Level 2 16000 6060 mat Peak Fraction Size The maximum volume of each peak fraction Peak Fractionation Algorithm Choose to start and stop the peak fractionation based on the signal curve Level e Level_AND_Slope e Level_OR_Slope e Slope AND means that both conditions have to be met OR means that only one condition has to be met The fractionation start values are set by Start Level and Start Slope and the stop values by End Level and End Slope Different start and stop values for level and
103. acked with Super dex 75 prep grade For high resolution polishing step for proteins of sizes 3 70 kDa and for sample volumes of lt 13 ml Methodology 4 Supported GF columns and volumes Characteristics HiLoad 26 600 Superdex 200 prep grade 320 ml HiLoad column prepacked with Super dex 200 prep grade For high resolution polishing step for proteins of sizes 10 600 kDa and for sample volumes of lt 13 ml HiPrep 16 60 Sephacryl S 100 HR 120 ml HiPrep column prepacked with Sephacryl S 100 HR For high resolution polishing steps for proteins of sizes 1 100 kDa and for sample volumes lt 5 ml HiPrep 16 60 Sephacryl S 200 HR 120 ml HiPrep column prepacked with Sephacryl S 200 HR For high resolution polishing steps for proteins of sizes 5 250 kDa and for sample volumes lt 5 ml HiPrep 16 60 Sephacryl S 300 HR 120 ml HiPrep column prepacked with Sephacryl S 300 HR For high resolution polishing steps for proteins of sizes 10 1500 kDa and for sample volumes lt 5 ml HiPrep 26 60 Sephacryl S 100 HR 320 ml HiPrep column prepacked with Sephacryl S 100 HR For high resolution polishing steps for proteins of sizes 1 100 kDa and for sample volumes lt 13 ml HiPrep 26 60 Sephacryl S 200 HR 320 ml HiPrep column prepacked with Sephacryl S 200 HR For high resolution polishing steps for proteins of sizes 5 250 kDa and for sample volumes lt 13 ml HiPrep 26 60 Sephacryl S
104. action out 1500 1 ETFE let outlet valve port 7 Specification of piston rinsing tubing Maintenance 8 Label bold Description Length mm_ Inner Material or diameter designation mm F8 yellow Fraction out 1500 1 ETFE let outlet valve port 8 F9 blue Fraction out 1500 1 ETFE let outlet valve port 9 F10 green Fraction out 1500 1 ETFE let outlet valve port 10 F11 Fraction out 1500 1 ETFE let outlet valve port 11 Waste Injection 1500 1 ETFE Waste2 valve port 2 Waste Injection 1500 1 ETFE WasteF3 valve port 3 Waste Outlet valve 1500 1 ETFE WasteF 1 port 1 AKTAxpress has the following tubi Rinse Rinse In Rinse Out ng connections for the piston rinsing tubing e p349 8 Maintenance 8 6 Replacement procedures 8 6 2 Replacing tubing and connectors 28 4090 22 AB p 350 Designation Description Length mm Inner Material diameter mm Rinse In Rinsing solu 1350 1 2 PTFE tion inlet to pump head 2 lower port Rinse Pump head 2 200 3 Silicone upper portto including pump head 1 check valve lower port Rinse Out Pump head 1 1350 1 2 PTFE upper port to rinsing solu tion outlet 8 6 3 Maintenance inter val Reason for main tenance Required material Replacing flow re strictor Maintenance 8 Replacing flow restrictor When required for example when the restrictor creates too
105. add a small amount of salt for ex ample 20 mM to the desalting buffer However too high salt concentration might affect the binding ca pacity when using ion exchange in the following step e For manual desalting protocols it is only possible to load up to 3mlpersample loop if using two HiTrap Desalt ing columns in series 10 ml per sample loop if using a HiPrep 26 10 Desalting column Troubleshooting and corrective actions 9 No satisfactory elution Possible cause and action from IEX column e Check that the eluted peak from the previous step was collected in a capillary loop by inspecting the peak start and peak end markers in the chromato gram If not collected Open the used method plan and enter the Ad vanced Zone Change the watch parameters used in peak collection and run the sample again e Check that the appropriate size of desalting column was used in the previous step e If the separation is not good enough Open the used method plan and enter the Ad vanced Zone Change the length and or slope of the salt gradient or Decrease the flow rate Ifthe problem persists create a new method plan using a column that will give a higher resol ution result see 4 Methodology on page 58 e If the protein did not bind to the column it was dir ected to one of the fraction outlets F7 F10 To protein binding change the binding buffer or adjust buffer pH Check
106. aded onto the GF column the excess volume will be emptied from the loop to the collection tubes F9 F10 one for each sample The loops are then washed with buffer to waste 6 Elution of the AC column desalting ion exchange and gel filtration is repeated for each sample Tag cleavage The table below describes the tag cleavage procedure and includes the elution of the AC column Hence when tag cleavage is used this part replaces the Elution of the AC column part described previously in this section Step Description 1 An extra wash is performed with AC wash buffer before elution to wash out unbound sample The column is filled with cleavage buffer optional The protease in the superloop is loaded onto the columnis The system is paused for the set incubation time During the incubation the tags are cleaved from the protein LoopWash of all loops is performed with IEX binding buffer The AC column is eluted with AC binding buffer or the chosen concentra tion of AC elution buffer and the peak volume collected in a loop SystemWash is performed to fill the system with buffer After purification of all samples uncleaved protein and tags are flushed from the AC columns with buffer and collected in the fraction collector p57 4 Methodology 4 About this chapter In this chapter 28 4090 22 AB p58 Methodology This chapter gives an introduction to the meth
107. ake sure that the entire system has been flushed thoroughly with bacteriostatic solution for example NaOH and distilled water before service and maintenance This chapter contains the sections below Maintenance 8 Section See Maintenance program 8 1 Monthly maintenance 8 2 Six monthly maintenance 8 3 Maintenance when required 8 4 Calibration procedures 8 5 Replacement procedures 8 6 e p305 8 Maintenance 8 1 Maintenance program 8 1 Introduction Periodic mainten ance program 28 4090 22 AB p306 Maintenance program This section specifies the preventive maintenance to be performed on KTAxpress The maintenance is divided into e Monthly maintenance e Six monthly maintenance e When required For maintenance of columns see chapter 6 8 5 Cleaning of columns Overview on page 283 The following periodic maintenance should be made by the user of AKTAxpress Interval Maintenance action Monthly See 8 2 1 Checking flow restrictor on page 308 See 8 2 2 Changing pump rinsing solution on page 310 Six monthly See 8 3 1 Checking UV lamp run time on page 312 See 8 3 2 Cleaning UV cell on page 314 See 8 3 3 Cleaning conductivity cell on page 317 When required See 8 4 1 Externally cleaning the system on page 319 See 8 5 1 Calibration of zero pressure on page 329 See 8 5 2 Calibration of conductivity cell on page 3
108. al Loops lection e AC IEX 2 Peak Col lection e DS 1 Peak Collec tion Flush Volume Empty Miscellaneous Settings 20 ml Loops p69 4 Methodology 4 2 Peak handling 4 2 2 Peak collection parameters when using double loops 28 4090 22 AB p70 Parameter to change On the pages New Value Loop Wash Volume Miscellaneous Settings 40 ml Empty Loops Loop Wash Volume Miscellaneous Settings 40 ml 4 2 3 Introduction Overview Peak fractionation parameters Methodology 4 Peak fractionation parameters This section contains brief information about the parameters that control how peaks are collected in the last purification step The peak fractionation parameters determine how peaks are collected at the final fractionation during the last purification step These peaks containing the final product of the purification run are collected in the fraction collector The collection in the microplate is performed in a serpentine manner e For 96 well microplates that is first in A1 A2 A11 A12 and then in B12 B11 B2 B1 and so on e For 24 well microplates that is first in A1 A2 A5 A6 and then in B6 B5 B2 B1 and so on In Advanced Zone some of the parameters that control peak handling can be changed The final peak fractionation does not involve any watch instructions The fractionation simply starts when the signal level and
109. ample ID System 1 4 Syel Sanghi Eate ID Saton T Sanga Samp 21D System 1 Sy Sangin fompi 1 ID Syste Gewghe 2 Tampir 2 ID Spadam gt e p261 6 Operation 6 6 Starting a run 6 6 1 Starting a run using a method plan 28 4090 22 AB p262 Step Action 6 For each sample If desired enter the isoelectric point in pl the extinction coefficient for the protein in Ext Coeff and the molecular weight of the protein in MW The extinction coefficient is the absorbance of 1 mg ml solution of the purified protein at 280 nm in a 1 cm cuvette Note Note If using an import file containing this data for the pro teins the fields will automatically be filled when enter ing this page The path to the import file folder must then be specified in Miscellaneous Settings in Advanced Zone in the Method Wizard in Method Editor For more information on the import file see 10 3 Import file format on page 426 Data will be imported each time this page is entered If the fields have been edited manually and the page is entered again later the edited data will be overwrit ten See also 5 5 9 Advanced Zone for Purify Miscellaneous settings on page 190 Enter any optional text in the Free Text Sample box It will appear in the pool table and in the result file in Documentation Notes Meth od Notes in Evaluation Click Next Proceed to Final setup and starting the run below
110. an immobilized metal affinity chromatography column IMAC or a Glutathione affinity chromatography column When using a non tagged protease further purification is necessary to separate the target protein from the protease The following list describes a few proteases commonly used for affinity tag removal Protease Common Optimal Codenumber Comment recognition incubation or other sequence temperature supplier than marks the GE cleavage Healthcare site Thrombin LVPR GS 22 C 27 0846 01 non tagged Factor Xa IEGR 12 C 27 0849 01 Cuts with non tagged clean ends if an N terminal affinity tag is used PreScission LEVLFQ GP 4 C 27 0843 01 Highly specif GST tagged ic Low optim 3 al temperat o ure TEV protease ENLYFQ G 30 C Invitrogen Highly specif Histidine ic tagged p87 4 Methodology 4 4 Affinity tag removal 4 4 2 Description of proteases Note Low incubation temperature reduces risk of protein degradation 28 4090 22 AB e p88 4 4 3 Introduction Process overview for automatic on column tag remov al Methodology 4 Description of the affinity tag removal processes This section describes an on column and an off column affinity tag removal process The on column process is performed automatically in AKTAxpress whereas the off column process is performed in for example batch outside the system Automatic aff
111. and wash volume in Outlets or Fraction Collector Check the Summary page for information 5 5 9 Introduction Overview Method Wizard 5 Advanced Zone for Purify Desalting Gel Filtration This section describes the parameters used in the desalting gel filtration step Some of the default parameter values depend on the chosen column Note If two desalting steps are included in the protocol the parameter for the first desalting step is indicated by DS 1 Parameters for 1 step DS protocols and protocols with DS as last step are indicated by DS GF Note Make sure that the parameter values do not exceed the specification of the chosen column Depending on the choices made earlier in the Method Wizard different pages and parameters will appear in the Advanced Zone Select the pages for which to change parameter settings see Entering the Advanced Zone in 5 5 3 Advanced Zone for Purify on page 172 for more information The picture below shows an overview of the possible pages that may appear See the sections below for information about what parameters can be changed on the different pages Advanced settings for desalting gel filtration Protocols starting with a OS step except 1 step protocol 1 step protocol OS or GF with capillary loop or Superloop sample loading DS 1 Genero OS GF D5 11 Peak Collection DS GF Included Procedures DS 1 included Procedures Intermediate OS step D
112. are detected when all loops are occupied are collected in a separate outlet tube It is also possible to connect two loops in series in order to collect even larger peaks For information on parameters when using double loops see 4 2 2 Peak collection parameters when using double loops on page 68 The peak collection is controlled by a series of UV signal watch conditions included in the method When a watch condition is met a predefined action is issued for example start collecting a peak Another watch is then activated for detecting the peak maximum and so on The table below describes the basic watch instructions available in the Advanced Zone of the AKTAxpress Method Wizard Other instructions are combinations of these Note For more detailed information on watch instructions see UNICORN AKTAxpress User Reference Manual Option Explanation Greater_Than The signal exceeds a certain value Less_Than The signal falls below a certain value Slope_Greater_Than The slope of the signal curve exceeds a specified value Less_Than_Or_Valley The signal falls below a specified value or a valley is detected A valley is detec ted only after a Peak_Max has been detected and the valley is defined by a local minimum followed by an increase to 102 of the local minimum plus the Delta_Peak value p65 4 Methodology 4 2 Peak handling 4 2 1 Peak collection parameters Option Explanation Peak_Max
113. arge procedures See also the column instructions for column solution suggestions In this section This section contains the sub sections below Sub section See Standard CIP Solution Suggestions System 4 7 1 Standard CIP Solution Suggestions Columns 4 7 2 Customized CIP Solution Suggestions System 4 7 3 Customized CIP Solution Suggestions Columns 4 7 4 Metal lon Charge Solution Suggestions 4 7 5 Strip Solution Suggestions 4 7 6 28 4090 22 AB p 112 4 7 1 Introduction Solutions for CIP System within a purify method Solutions for CIP System and Loops within a prepare and maintain method Methodology 4 Standard CIP Solution Suggestions System Standard CIP system procedures contains three cleaning steps and can be run e included as a post run procedure within a purification run denoted CIP System or e separately within the Standard System and Column procedures method plan in Prepare and Maintain denoted CIP System and Loops with NaOH Water Buffer It is recommended to use the following solutions and in the order below e 0 5 M NaOH e deionized water affinity ion exchange binding buffer If cleaning sample inlets use the appropriate wash solution e g water or buffer from a separate bottle It is recommended to use the following solutions and in the order below 0 5 M NaOH e deionized water affinity ion exchange binding buffer
114. as been loaded from the Superloop or the capillary loops The report format xpressActiveChrom can also be selected if only the active zoomed window should be included in the report e Click the Edit button to modify the report format if needed Evaluation 7 Step Action 4 e Click the Print button Result The Print dialog box opens e Choose what pages and how many copies to print e Click OK Note Printers are set up in the File menu of the UNICORN Manager Customized re For instructions on how to create or edit customized reports see UNICORN AKTAxpress ports User Reference Manual e p303 8 Maintenance About this chapter Warning In this chapter 28 4090 22 AB p304 Maintenance This chapter describes the maintenance activities for KTAxpress WARNING The separation systems must not be opened by the user The systems contain high voltage circuits that can deliver a lethal electric shock WARNING NO SERVICEABLE PARTS INSIDE Do not open covers Service and planned maintenance should be performed by person nel authorized by GE Healthcare only WARNING When using hazardous chemicals take all suitable protective measures such as wearing protective glasses and gloves resistant to the chemicals used Follow local regulations and instructions for safe operation and maintenance of the system WARNING When using hazardous chemicals m
115. ase slide the housing firmly to the right into place A click will indicate that the housing is in position our at o w KO Wey ee oMa Maintenance 8 Step Action 10 Open the cap covering the filter wheel and check that the filter wheel is in the 280 nm position If this is not the case turn the wheel into the correct position A click will indicate that the wheel is in position 11 Fix the new lamp cable in the cable duct and connect it at the back of the system 12 Connect power to the system and set the unit in Idle mode Note If a low intensity warning is given even after a change of UV lamp the filter may need to be changed Contact a local GE Healthcare representative 13 Reset the UV lamp run time counter in UNICORN Note This step can only be made by an GE Healthcare service engineer p 357 8 Maintenance 8 6 Replacement procedures 8 6 5 Replacing check valves 8 6 5 Introduction Spare part and tools required Replacing the check valves 28 4090 22 AB p358 Replacing check valves A check valve should be replaced if for example dirt cannot be removed by cleaning the check valve The following spare parts and tools are required e Check valve kit 18 1128 66 containing one inlet check valve and one outlet check valve e 13 mm wrench e 18 mm wrench CAUTION The check valves should only be disassembled by a trained per
116. at a low flow rate and the columns can be attached one at a time The method will go from column position 1 to 5 by pressing Next Breakpoint See 6 4 9 Connecting columns and tubing on page 225 for information about the manual procedures to be performed Fill Inlets Manually Us filling inlets with buffer using a syringe attached to the ing Syringe pump head s This method is used when the inlets are filled with air The method will go from inlet S1 S4 A1 A8 B1 and B2 by pressing Next Breakpoint See 6 4 5 Filling inlets manually using a method plan on page 214 for information about the manual proced ures to be performed Automatic meth The table below describes the automatic method plans that can be created od options Select To Customized System clean the system loops and outlets using up to 5 differ Procedures ent buffers solutions 28 4090 22 AB p 140 Method Wizard 5 Select To Customized Column clean equilibrate the column s using up to 9 different Procedures solutions only one column type in each run Installation Test test the function of the pump switch valves and mixer See the nstallation Guide for more information Purge Pump with create a method plan for purging the pump with meth Methanol anol This should be performed if the pump has been run dry or the system has not been used for some days The procedure requires e about
117. ation run depends on preknown factors such as temperature the number of samples sample volumes column properties protocol if on column tag cleavage has been selected which and how many system and column procedures that have been selected e factors determined during a run such as sample viscosity flow control the length of the included wash steps The table below show estimations of the run times for d ifferent example protocols with maximum number of samples and the largest columns AC 5 ml IEX 6 ml DS HiPrep 26 10 Desalting GF HiPrep 26 60 Sephacryl at room temperature Protocol Estimated run times in Maximum number of RT with max number of samples samples Hours 1 step AC IEX 2 5 4 DS 0 5 4 GF 14 4 2 step AC IEX DS 45 4 Typical run times for included sys tem procedures Reference information 10 Protocol Estimated run times in Maximum number of RT with max number of samples samples Hours AC IEX GF 18 4 DS AC IEX 5 2 3 step AC IEX DS AC IEX 7 3 DS AC IEX DS 4 2 DS AC IEX GF 11 2 4 step AC IEX DS AC IEX DS 5 5 2 AC IEX DS AC IEX GF 12 5 2 1 Run times are approximate and with default settings Sample loading time on column tag cleavage system and column procedures are not included The table below show estimations of the run times for some differen
118. ays be performed and no elution or peak selection options can be selected for step 1 e If there is a second step following the AC IEX step and if gradient elution was selected options for How to select peak are displayed Select how to determine which peak to be transferred to next puri fication step Largest Manual Selection or Define B Interval only for gradient elution The options are described in the table below For protocols with an intermediate DS step By default Include Intermediate Desalting is selected This means that the intermediate desalting step in the protocol will be performed To remove the intermediate desalting step from the protocol uncheck this box Under Fractionation select fractionation technique type of microplate and if the method should pause if the last well of the microplate is reached to be able to change plate Click Next The table below describes the elution and fractionation options for a purification run Option Description related action Elution Step Elution 100 elution buffer during 5 CV set as default will be used for elution in AC IEX steps Note The default settings can be changed in the Advanced Zone ep 155 5 Method Wizard 5 4 Using the Purify option 5 4 2 Selecting Purify 2 2 page options Option Description related action Gradient Elution Up to four segments prior to regeneration can be used for el
119. being withdrawn from the pump chamber On the delivery stroke of the piston the inlet valve is sealed by the pressure developed and eluent is forced out through a similar check valve at the outlet Reference information 10 ci Outlet check valve Purge R gt valve i y Rinse chamber outlet LEO Rinse chamber Return spring Inlet check valve or Piston Piston seal Rinse membrane The pistons are actuated by cams eccentrics driven by the motors Force for the retraction of the pistons is provided by coil springs The length of stroke of the pistons is fixed and changes in the flow rate are made by varying the speed of the drive motor Piston seal rinsing system Leakage between the pump chamber and the drive mechanism is prevented by a piston seal The pistons and seals are continuously lubricated by the presence of liquid To prevent any deposition of salts from aqueous eluents on the piston and to prolong the life of the seals the low pressure chamber behind the pump head can be flushed continuously with a low flow of rinsing solution The flexible rinsing membrane sweeps the piston and seals the rinsing system Check valve f Optional path t i without recirculation Rinsing solution 20 ethanol p 405 10 Reference information 10 1 System description 10 1 1 Pump and related components Mixer The mixer is a static mixer with a volume of 0 37 ml Note Do not use organic solv
120. binding buffer should be used incubation Time Example on pro The protease volume and concentration can be automatically calculated by using the tease calculation Protease Calculator sheet found on the AKTAxpress strategy CD Whe vae of Anty comm wel be used 3 Mom miny vampirs at you nn on Pa system Som mich protein w be appted 1e each HeTrap aMhinty cotan mg Hon many Cy of protease wil be appted O7 s setai v ow Hom much protease do you need to coore mg of orones wen TM inounda a Cacwates requres concertranen of sreeonse SE in na Cae tates tone amenet of retenes required SEB eo ns The following example describes how to perform a manual calculation Conditions e Protein type Histidine tagged with TEV protease cleavage site e Number of samples 4 e Estimated amount of protein sample 20 mg e Affinity column HisTrap HP 5 ml e p239 6 Operation 6 4 Preparing the system for a run 6 4 12 Preparing for on column tag cleavage Using a column heater 28 4090 22 AB p 240 e Protease TEV protease 200 units mg target protein e Delay volumes 0 53 ml for the first column only loop valve to column valve plus delay volume for guided loading of Superloop 0 27 ml for all columns column valve to column Protease volume The default protease volume in the Method Wizard is 0 7 CV can be changed Volume needed per column 0 7 x 5 ml 3 5 ml Total volume needed for 4 samples 3 5 ml x 4 column
121. bing cutter and replace the ferrule e f air bubbles keep appearing in the sample loading tubing replace the tubing see 8 6 2 Replacing tubing and connectors on page 338 e If using a highly viscous sample dilute it to ease sample loading e Check that the flow restrictor generates a backpres sure of 0 2 0 05 MPa as follows Set the injection valve to Waste Run the pump manually at 10 ml min with water Note the backpressure in the Run Data window Connect the flow restrictor to the open end of the Waste tubing Note the IN marking Run the pump manually at 10 ml min with water Note the backpressure Calculate the backpressure difference If it is not within limit replace the flow restrictor See 8 6 3 Replacing flow restrictor on page 351 Troubleshooting and corrective actions 9 Elution problems Find the possible cause and action for a specific problem in the table below No satisfactory elution from Possible cause and action Affinity column If the protein did not bind to the column it will be found in the flowthrough flask as shown on the Summary page e Verify that the tagged target protein is present in the start sample e Check that the sample has been applied onto the affinity column e Check that appropriate wash and elution buffers were applied e HisTrap HP and HiTrap Chelating HP Ifthe target protein elutes during Wash 2 de crease the imidazol
122. bration N A time usage of the column before a puri fication run A method plan including equilibration blank run of the columns The illustration below gives an overview of how to include Equilibration Blank Run procedures in the purification method plan See 5 4 Using the Purify option on page 150 for detailed information Purify 2 2 the columns for which to include a conditioning procedure by checking the boxes the oppropricte conditioning procedure for eoch of the selected columns the columns for which to include a conditioning procedure by checking the boxes gt Select the appropriate conditioning procedure for eoch of the selected columns p 255 6 Operation 6 5 Column procedures before a purification run 6 5 2 Conditioning of columns CreateaStandard The illustration below gives an overview of how to perform Equilibration Blank Run System and procedures in the Standard System and Column Procedures method plan See 5 3 Column Proced Using the Prepare and Maintain option on page 136 for detailed information ures method plan including equilib ration blank run Select the columns for which to include a conditioning procedure Select Standard System and Column Procedures System Procedures oi o a Select column typ o B Select o e itioning procedure o a o a condi Select the positions occupied by the column type 1 5 Select
123. ceed with the next purification step To stop the run on a system before it is finished Step Action 1 Check that the correct system is selected on the screen 2 Click End above the Run data view pane The status indicator is located at the bottom of System Control For information about how the indicator colors relate to the run status see 6 2 Connecting a system on page 197 p271 6 Operation 6 7 During arun Changing para meters Problems during arun 28 4090 22 AB p 272 The separation system can be controlled with manual instructions issued from the Manual menu in System Control These instructions can be used during the run to change system conditions in response to the results observed e When changing or loading a microplate during a run the system must first be set to Pause before pressing the Eject button on the system control panel If air is detected at buffer application the system enters pause mode The air should then be removed using a syringe see 6 4 16 Using air detection on page 247 for more information In case of other problems during the run see 9 1 Introduction to troubleshooting on page 372 6 8 About this section In this section Procedures after a run Operation 6 This section describes how to clean the system tubings and columns after a performed protein purification It also includes how to perform metal ion stripping of the affini
124. charge Blank run Strip CIP Re equilibrate Several of the procedures in the standard system and column procedures option can also be included within a purification run The lists below show which system and column procedures that are available in the standard system and column procedures option System Procedures Note The system procedures that can be included in a purification run are marked with a star e Remove Ethanol from System and Loops e Rinse All Outlets e CIP System and Loops with NaOH Water Buffer e Guided Loading of Superloop e Fi Sample Inlets with Buffer e Fill Buffer Inlets with Buffer e Fi System Loops Outlets with Ethanol System Procedure Description Remove Ethanol from In the beginning of the run the system is washed with System and Loops water The loops will also be washed Rinse All Outlets To rinse the outlets All outlet tubing F1 F11 including Frac F2 will be washed with solution from inlet A1 Note Insert a microplate in the fraction collector to collect solution from Frac F2 Methodology 4 System Procedure Description CIP System and Loops with NaOH Water Buffer Washes the system and loops with NaOH water and affinity ion exchange binding buffer Guided Loading of Su perloop In the beginning of the purification run the Superloop is filled with sample or protease solution When perform ing the filli
125. contents and the headings for the sections that the chapter contains The section begins with an introduction that summarizes the content Each section is divided into blocks of information with separating lines The blocks are identified by labels in the margins This makes it easier for you to quickly scan a page to find the exact topic you are looking for The table below describes the content in each chapter in this manual Chapter Contents 1 Preface Regulatory contact and safety informa tion 2 Introduction General system overview of AKTAxpress and basic operating principles back ground to protein purification and in formation about the user documenta tion 3 AKTAxpress overview Description of AKTAxpress and a purific ation process overview 4 Methodology A guide to selecting protocols buffers solutions and columns Peak handling is also described 5 Method Wizard Description of the Method Wizard Pre pare and Maintain and Purify 6 Operation Instructions for preparing the system for arun and performing a run on one or several systems 7 Evaluation Viewing and evaluating the results printing chromatograms and generat ing reports 8 Maintenance Maintenance schedules and instructions for preventive maintenance and repla cing spare parts 9 Troubleshooting and corrective ac tions Overview of error symptoms possible causes and corre
126. cording to the column instructions This will remove unspecific bound proteins and prevent column clogging WARNING system When using hazardous chemicals take all suitable protective measures such as wearing protective glasses and gloves resistant to the chemicals used Follow local regulations and instructions for safe operation and maintenance of the A There are two types of cleaning procedures available for column cleaning The table below briefly describes the cleaning procedures Cleaning procedure Description For more information see Standard column clean ing Automatic cleaning of the columns with two solu tions 6 8 6 Standard cleaning of columns on page 284 Customized column cleaning Automatic cleaning of the columns with up to nine solutions 6 8 7 Customized clean ing of columns on page 288 e p283 6 Operation 6 8 Procedures after a run 6 8 6 Standard cleaning of columns 6 8 6 Introduction Warnings and notes Prerequisites 28 4090 22 AB p284 Standard cleaning of columns Standard cleaning of the columns includes automatic cleaning of the columns with two solutions and can be run included as a post run procedure within a purification method denoted CIP Columns or within the Standard System and Column Procedures prepare and maintain method plan denoted CIP If cleaning columns using the Standard System
127. crease the the maximum sample loading volume from 10 ml to 20 ml 6 4 16 Introduction About air detec tion Air sensor sensitiv ity Air detection at buffer application Operation 6 Using air detection This section describes the automatic air detection in AKTAxpress The separation system is equipped with an air sensor which is located between the inlet valve and the pump Automatic air detection is used to e prevent air from entering the flow path during the purification and e detect when the sample supply is empty when applying sample e Check that the air sensor sensitivity on each system used is set to Low_Sensitivity default by selecting System Settings in System Control and then clicking Monitors The system must be in End mode Air detection during buffer application is used to prevent air from reaching the columns By default air detection is always enabled during buffer transport in a method plan made in the Method Wizard When air is detected for example due to empty buffer supply or a loose tubing end the system enters Pause mode To remove the air with a syringe Step Action 1 Check the buffer supply and the tubing 2 Fit a syringe at least 20 ml to a purge valve 3 Turn the purge valve half a turn to open it 4 Draw buffer with the syringe until the tubing is completely filled with buffer 5 Close the purge valve e p247 6 Operati
128. ctive actions Error codes ep25 2 Introduction 2 4 AKTAxpress user documentation Typographical conventions Prerequisites 28 4090 22 AB p26 Chapter Contents 10 Reference information Detailed hardware description technical and chemical specifications typical run times ordering information Menu commands field names and other text items from the software or on control buttons are quoted exactly as they appear in a bold typeface e Example Run Setup Search paths are shown in a bold typeface with a separating colon between each level e Example View Panes Customize i e the menu command Customize in the sub menu Panes from the View menu Text entries that UNICORN generates or that the user must type are represented by a monotype typeface e Example Connection change The following prerequisites must be fulfilled before you can use this system in the way itis intended e You need to have a general understanding of computers and Microsoft Windows operating system In most cases universal computer functions will not be explained e UNICORN must be installed and configured correctly on the computer e Youneed to understand the basic concepts of liquid chromatography Terminology and functionalities will be explained only when they differ from normal practice e Before you try to operate AKTAxpress you must study and understand AKTAxpress Operating Instructions About
129. ctors sensors mixer and column block Pump leakage Find the possible cause and action for a specific problem below Problem Possible cause and action Low buffer flow and disturbing noise Bad piston spring Disassemble the pump head and examine the piston spring If the spring is corroded check the piston seal and rinse membrane Make sure that the rinsing system is always used when working with aqueous buffers with salt concentration see 8 6 2 Replacing tubing and connectors on page 338 If the piston is damaged replace it see 8 6 6 Repla cing pump piston seal on page 361 Flow path leakage Find the possible cause and action for a specific problem below To perform a tubing leakage test see 8 4 4 Checking tubing leakage on page 325 Problem Possible cause and action Leakage around a pump head Piston seal or rinsing membrane incorrectly fitted or worn Replace the pump piston seals in the pump heads see 8 6 6 Replacing pump piston seal on page 361 Leaking connection or crystallized material around tubing connect ors Tubing connector leaking Unscrew the connector and check if it is worn or in correctly fitted If required replace the faulty connect ors see 8 6 2 Replacing tubing and connectors on page 338 e p389 9 Troubleshooting and corrective actions 9 4 Leakage problems 28 4090 22 AB p390 Problem Possible cause and action
130. ctors that are related to columns If leakage is found tighten the connectors or replace the tubings if ne cessary see 8 6 2 Replacing tubing and connectors on page 338 e p243 6 Operation 6 4 Preparing the system for a run 6 4 14 Checking the tubing Step Action 2 Air inspection Inspect the inlet tubings for air All tubings up to the pump heads should be inspected Samples Buffers Buffers 1 S4 Al A2 B1 A3 A8 B2 If air bubbles are found perform manual purging of the system see 6 4 4 Purging the pump and inlet tubing Overview on page 212 3 Buffer tubings Check that the tubings are fully immersed in the buffer containers Fix the tubings in the cap for example by e Fitting the inlet filter weights on the tubings e Running the tubings through the rubber cap included in the accessory kit 28 4090 22 AB e p244 6 4 15 Introduction Preparing samples and sample inlet tubings Setting the sample volume Operation 6 Preparing samples This section describes how to prepare the samples To prepare the samples and sample inlet tubings Step Action 1 For all columns except HisTrap FF Crude Clarify the samples using centrifugation and or filtration through a 0 45 um filter The tube holder is used for storing tubes with small sample volumes The optional flask holder can be used for sample flasks with larger volumes For Hi
131. d Maintain oe s a interactive Methods Last Por wet Column attachment or Fill inlets Manually Using Syringe or ami Purge Pump with Methanol or 0 instollation Test Customized System Procedures Customized System gt Lost Page Procedures Customized Column Procedures Customized Column gt Last Page Method Wizard 5 In this section This section contains the sub sections below Sub section See Selecting Prepare and Maintain option 5 3 1 The Prepare and Maintain page options 5 3 2 Selecting Standard System and Column Procedures options 5 3 3 Selecting Customized System Procedures options 5 3 4 Selecting Customized Column Procedures options 5 3 5 e p137 5 Method Wizard 5 3 Using the Prepare and Maintain option 5 3 1 Selecting Prepare and Maintain option 5 3 1 Introduction Selecting the pre pare and maintain option 28 4090 22 AB p 138 Selecting Prepare and Maintain option This section describes how to create a method plan for preparation and maintenance of the system and columns To select prepare and maintain option Step Action 1 On the Main Selection page select Prepare and Maintain Click Next Result The Prepare and Maintain page is displayed 2 Select the requested type of preparation maintenance procedure to be performed See 5 3 2 The Prepare and Maintain page options on page 140 for a description of the option
132. d by clicking the system name gt In the menu select System Calibrate and choose PumpPress under Monitor p 329 8 Maintenance 8 5 Calibration procedures 8 5 1 Calibration of zero pressure 28 4090 22 AB p 330 Step Action 3 Click Start calibrate and wait until it is enabled again 10 20 s Start calibrate Result The injection valve will automatically be switched to Waste and the pressure reading set to zero 4 Click Close to finish the operation 8 5 2 Introduction Calibrate current conductivity cell Maintenance 8 Calibration of conductivity cell Problems in the conductivity cell can cause abnormal conductivity readings in chromatograms There are two different procedures e Calibration of current conductivity cell e Setting the conductivity cell constant after replacement To calibrate the current conductivity cell Note Calibration of the conductivity cell is not normally required The cell constant is calibrated and set at the factory Note Perform the test at room temperature 20 30 C Step Action 1 Prepare 20 ml of calibration solution 1 00 M NaCl 58 44 g l and immerse the A1 tubing into the solution Note Make sure that the temperature of the calibration solution is the same as the room temperature 2 In the UNICORN System Control module select the system to be calib rated by clicking the system name IO 3 Start
133. d columns The procedures can be run e included within a purification method preferred if only the columns to be used in the run should be conditioned or e inthe Standard System and Column Procedures prepare and maintain method up to five columns of the same type can be conditioned simultaneously Conditioning of columns is performed by running e equilibration possible for all columns or e blank run possible for AC and IEX columns The columns will also be conditioned if the equilibration or blank run procedure is the last step of a combined procedure e g CIP Columns Re equilibrate The table below describes when conditioning of different types of columns should be performed Column type When to perform Use to equilibrate the conditioning columns with buffer and affinity columns e priortoafirst blank run N A time usage of the column e after metal ion charging affinity and ion Before a purifica equilibration N A exchange tion run columns ion exchange e priortoa first blank run provide the columns time usage of columns with ex the column changeable counter ions e after long term storage Prerequisites Create a purify method plan in cluding equilibra tion blank run Operation 6 Column type When to perform Use to equilibrate the conditioning columns with buffer and desalting and gel filtration columns prior to a first equili
134. d in the evaluation part of UNICORN For more ation actions information see UNICORN AKTAxpress User Reference Manual e p293 7 Evaluation 7 2 Finding and opening results 7 2 Introduction Recommended finding and open ing of files 28 4090 22 AB p294 Finding and opening results This section describes how to find the result files in UNICORN folders and open them In UNICORN it is recommended to find and open result files in the Evaluation module the following way to receive automatic pooling Step Action 1 Use the Recent Runs tab or the Find tab in the File Navigator on the left hand side of the Evaluation window to locate the result file Revert ure F s fod Arat tir name i Value of wanable Sample 0 Note See How to use the File Navigator in the UNICORN AKTAx press User Reference Manual for detailed instructions on how to locate files and set up File Navigator preferences Click the button to expand the list for the result file Spite 1008 for Hewne A Syoteent002 fw howe JOITA Evaluation 7 Step Action Double click a sample file for the purification for example Sample1 a 4 Syte fos Mameij Pan Sample APB 7 M Sydni lot Home Result The chromatogram of the sample is displayed and a pooling suggestion of the fractions is shown By default the chromatograms in a run are shown as opened windows T
135. d maintain method plans The table below describes for which procedures prepare and maintain methods are required If create the method plan For more information see column inlets are going to be connected to new columns in the column block using the drop to drop method Column Attachment 6 4 9 Connecting columns and tubing on page 225 there is a lot of air in all most inlet tubings Fill Inlets Using Syringe 6 4 5 Filling inlets manu ally using a method plan on page 214 e thesystem has been left unused for a week or longer or e the pump has been run dry Purge Pump with Meth anol 6 4 8 Purging the pump with methanol on page 223 new columns should be charged with metal ions Standard System and Column procedures and select the column proced ure Metal lon Charge Blank Run 6 5 1 Metal ion charging of chelating affinity columns on page 252 a customized cleaning of the system with up to five solutions should be per formed Customized System Pro cedures 6 8 4 Customized clean ing of the system on page 281 a customized cleaning of one column type with up to nine solutions should be performed Customized Column Procedures 6 8 7 Customized clean ing of columns on page 288 p 201 6 Operation 6 3 Creating method plans Procedures fre Several frequently used column procedures can be run separately in a prepare and
136. d of the purification run The image below shows the different stages on the affinity column The numbers indicate the steps described in the table above p89 4 Methodology 4 4 Affinity tag removal 4 4 3 Description of the affinity tag removal processes Performing auto matic affinity tag removal with AK TAxpress Recommended process overview for off column tag removal when us ing AKTAxpress 28 4090 22 AB p90 Stage 2 5 Load sample and wash rf Tag cleavage using AKTAxpress Stage 6 Stage 7 Stage 8 9 Stage 10 Inject protease Incubate Elution Affinity from Superloop cleaved protein regeneration i Affinity tag Cleaved tagged protease and protein non cleaved protein l Loop or Fraction fraction collector collector See 6 4 12 Preparing for on column tag cleavage on page 238 for instructions on how to perform on column affinity tag removal on AKTAxpress The table below describes the stages in an off column tag removal process using AKTAxpress Stage Description 1 Perform initial purification e Run an AC IEX protocol if the protease will be functional in AC IEX buffer or e Run an AC IEX DS protocol if a buffer exchange is preferred prior to cleavage Perform batch cleavage e Collect fractions e Add protease and incubate at preferred temperature and for required amount of time Methodology 4 Stage Description If an affin
137. de salting columns Solutions for ion exchange columns Solutions for gel filtration columns Methodology 4 The tables below list recommended solutions for the desalting columns supported by AKTAxpress See 4 5 1 Descriptions of columns on page 95 for a list of columns supported by AKTAxpress Column Step Solution All desalting columns Step 1 0 2 M NaOH Step 2 Deionized water The tables below list recom mended solutions for the ion exchange columns supported by AKTAxpress See 4 5 1 Descriptions of columns on page 95 for a list of columns supported by AKTAxpress Column Step Solution All ion exchange columns Step 1 2 M NaCl Step 2 IEX binding buffer The tables below list recommended solutions for the gel filtration columns supported by KTAxpress See 4 5 1 Descriptions of columns on page 95 for a list of columns supported by KTAxpress Column Step Solution All Gel filtration columns Step 1 0 5 M NaCl Step 2 Deionized water e p115 4 Methodology 4 7 Solution alternatives 4 7 3 Customized CIP Solution Suggestions System 4 7 3 Customized CIP Solution Suggestions System Introduction Customized CIP system procedures can be run only in the Customized System Procedures prepare and maintain method plan The procedure contains up to five cleaning steps Recommended It is recommended to use the following solution
138. drainage hole are facing downwards and the text UP OUT on the pump head is facing up wards Mount the complete pump head over the locating pins on the front panel Locate the metal end of the piston and the spring towards the drive cam e Hold the pump head firmly against the side panel of the housing with one hand Do not allow the assembly to twist under pressure from the return spring Using the hex key fit and tighten one of the hex screws se i e Fit and tighten the remaining screw 18 Refit the tubing e Reconnect the outlet tubing to the outlet check valves e Reconnect the inlet manifold e Refit the tubing of the piston seal rinsing system 19 Purge the pump and run in the new piston seal carefully following the instructions below The piston seal should be run in using 100 methanol CAUTION Before the start of each run ensure that there is an adequate supply of solution in the reservoirs Never allow the pump to run dry since this will affect the lifetime of the piston seals e p367 8 Maintenance 8 6 Replacement procedures 8 6 6 Replacing pump piston seal Step Action Fill a reservoir with 100 methanol and immerse the inlet tubing A1 Connect a male Luer syringe of at least 30 ml to the left purge valve Two syringes are included in the accessory kit supplied with the system Turn the left purge valve counter clockwise half a turn to open it Ma aD
139. e Any HiTrap means that any HiTrap affinity column can be used The same default values as for HisTrap HP are used These can be changed in the Advanced Zone The following AC columns are supported by AKTAxpress Supported AC columns Characteristics and volumes HisTrap HP HiTrap column prepacked with Ni Sepharose High eae Sn Performance a Ni precharged medium optimized for high resolution purification of histidine tagged proteins HisTrap FF HiTrap column prepacked with Ni Sepharose Fast Flow Tandil a Ni precharged medium which has high binding ca pacity for histidine tagged proteins at high flow rates p95 4 Methodology 4 5 Column alternatives 4 5 1 Descriptions of columns Desalting columns 28 4090 22 AB p96 Supported AC columns and volumes Characteristics HisTrap FF crude 1and5 ml HiTrap column prepacked with Ni Sepharose Fast Flow a Ni precharged medium which has high binding ca pacity for histidine tagged proteins at high flow rates The column enables direct loading of lysed unclarified samples without the need for any clarification that is centrifugation or filtration prior sample loading HiTrap Chelating HP 1and5 ml HiTrap column prepacked with Chelating Sepharose High Performance to be charged with the metal of choice for purification of histidine tagged proteins HiTrap IMAC HP 1and5 ml HiTrap column prepacked with IMAC Sepharose
140. e the tag has been cleaved off and the sample needs further purification p59 4 Methodology 4 1 Protocol descriptions and choice See 4 4 Affinity tag removal on page 84 for more information on affinity tags and removal of affinity tags Grouping of proto The protocols can be divided into four main groups based on protein starting material cols see the table below Protocol Description Group 1 Protocols suitable for purification of AC tagged proteins AC DS GF The protocols can be combined with on column tag cleavage in the first step AC DS IEX AC DS IEX DS GF Group 2 Protocols suitable for purification of AC DS AC double tagged proteins AC DS AC DS GF The protocols can be combined with on column tag cleavage in the first step Group 3 Protocols suitable for tagged proteins DS AC that should be purified using AC in the second or third step but first needs to DS AC DS GF IEX DS AC be desalted and or purified using ion IEX DS AC DS GF exchange chromatography Group 4 Protocols suitable for purification of a ACL DS JEX GF protein that has for example already ea been purified using AC and from which DS IEX IEX DS GF the tag has been removed on or off DS IEX DS GF IEX DS IEX column IEX DS IEX DS GF Protocols starting with an IEX step can be combined with on column tag cleavage in the first step Note On column tag cleavage on IEX is not recommen ded unless you know that
141. e 8 5 2 Calibration of conductivity cell on page 331 p317 8 Maintenance 8 4 Maintenance when required 8 4 Maintenance when required About this section This section describes the maintenance activities to be made when required In this section 28 4090 22 AB p 318 This section contains the sub sections below Sub section See Externally cleaning the system 8 4 1 Cleaning check valves 8 4 2 Checking pump pressure 8 4 3 Checking tubing leakage 8 4 4 Running installation test 8 4 5 Maintenance 8 8 4 1 Externally cleaning the system Maintenanceinter When required val Reason for main The system should be kept dry and clean to ensure proper functioning of the system tenance Chemical stains and dust should be removed Warning WARNING T Remove liquid or dirt from the system surface using a cloth and if necessary a mild cleaning agent Actions To clean the system follow the procedure below Step Action 1 Check that no active run is in progress on the system The run indicator should be off 2 Disconnect the mains power cable 3 Wipe the surface with a damp cloth Do not allow spilt liquid to dry on the instrument 4 To remove stains spray the system with 20 ethanol and wipe off the excess Note Be careful not to bend any tubing 5 Let the system dry completely before using it p319 8
142. e General check list below 2 Find the fault and corrective actions in one of the different sections de pending on the type of problem e Problems during a run Errors related to failure to load samples high back pressure and unsatisfactory elution from the columns see 9 2 Problems during a run on page 375 e Monitoring problems These are errors detected during a run or when evaluating the resulting chromatograms For example ghost peaks noisy signal signal drift and unstable readings see 9 3 Monitoring problems on page 382 e Leakage problems Problems related to physical problems with the flow path see 9 4 Leakage problems on page 389 e Alarms and connection problems Alarms in UNICORN and error codes on the separation systems see 9 5 Alarms and connection problems on page 391 3 Make the recommended corrective actions 4 If problem persists after corrective actions contact a local GE Healthcare representative General check list Check the following items before starting troubleshooting System checks e Is the correct system selected in UNICORN System Control For more details see 6 7 During a run on page 267 e Is the fan blowing at the back of the system 28 4090 22 AB p372 Troubleshooting and corrective actions 9 Monitor checks Is the UV monitor set to the correct wavelength For more details see 8 6 4 Replacing UV lamp on page 353 Is the UV cell locking nut properly tightened t
143. e changed the Back and Next buttons can always be used to return to the Advanced Zone page in question To enter the Advanced Zone Step Action 1 When all selections are made in the Method Wizard the Last Page ap pears e Click Next Lest Paye Allequred ieiechon we made Press Fireih to cave the selections as a Method Men Presi Next to continue to the Advanced Zone 2 Siwe Peete in a Man Foka Man Fakin hame AMan aiar ep165 5 Method Wizard 5 5 Advanced Zone 5 5 1 Advanced Zone introduction 28 4090 22 AB p 166 Step Action 2 On the first page in the Advanced Zone select the pages to be displayed in the Advanced Zone The following sections in this chapter describe the main procedures for changing parameter values e Advanced Zone for Prepare and Maintain e Advanced Zone for Purify chromatographic technique 5 5 2 Introduction Changing Purge Pump with Meth anol settings Method Wizard 5 Advanced Zone for Prepare and Maintain This section describes the Advanced Zone for a Prepare and Maintain method plan The following method plans allow parameter values to be changed e Purge Pump with Methanol e Customized System Procedures e Standard System and Column Procedures Note Make sure that the parameter values do not exceed the specification of the chosen column To change parameters in the Purge Pump with Methanol method pla
144. e concentration of wash 2 solution If the absorbance curve does not return com pletely to the base line after peak elution using step elution or if the absorbance curve increases during gradient elution the quality of the im idazole might be poor Change to imidazole with higher quality or increase the Peak_Max factor to be able to detect peak end e GSTrap HP and FF Many proteins bind better to GSTrap HP and FF at room temperature than in cold room whereas other protein shows the opposite Adding 1 mM DTT or other reducing agent to the buffer might increase the binding capacity e Tag cleavage High glycerol concentrations in the buffer might decrease the activity of some proteases For operation and optimization hints see 4 Methodology on page 58 and 6 4 12 Preparing for on column tag cleavage on page 238 p377 9 Troubleshooting and corrective actions 9 2 Problems during a run No satisfactory elution from Possible cause and action 28 4090 22 AB p378 Desalting column e Check that the eluted peak from the previous step was collected in a capillary loop by inspecting the peak start and peak end markers in the chromato gram If not collected Open the used method plan and enter the Ad vanced Zone Change the watch parameters on the affinity peak collection page Runthe sample again e Ifthe protein co elutes with the salt peak the recom mendation is to
145. e needed Buffer guidance is provided in the instructions for each column and in the purification handbooks see 4 5 1 Descriptions of columns on page 95 The table below lists the suggested buffers for AC runs with Histidine tagged proteins when using the columns HisTrap HP FF HisTrap FF crude HiTrap IMAC HP FF columns Note For information about suggested buffers when using the HiTrap Chelating column see the column instruction When performing suggested buffer binding 20 mM sodium phosphate 0 5 M NaCl 20 40 mM im idazole pH 7 4 Note The optimal imidazole concentration is protein dependent 20 40 mM is suitable for many proteins and for the IMAC columns if charged with Ni or Co extra wash before elu 20 mM sodium phosphate 0 5 M NaCl 50 mM imidazole tion pH 7 4 Note x of the extra wash buffer is added to the binding buffer This value can be changed in the Advanced Zone See 5 5 4 Advanced Zone for Purify Affinity lon Exchange on page 174 for details Example If using a binding buffer with 20 mM imidazole and 20 of extra wash buffer 100 mM imidazole the total concentration of imidazole during the extra wash will be 40 mM cleavage using TEV 20 mM sodium phosphate 0 5 M NaCl 50 mM imidazole protease pH 7 4 Note The imidazole concentration might need to be optimized especially if a different Histidine tagged protease than TEV is used p 109 4 Methodology
146. e new seal with water Place the seal in the hole on the pump head and press it down into position with a hard flat object 14 With the pump head still facing downwards on the bench place the rinse chamber onto the head with the rinse ports in line with the inlet and outlet check valves The conical depression in the rinse chamber should be facing upwards ready to accept the new rinsing membrane Fit the rinsing membrane with the conical face downwards D r Cc ER TT S Lee Se Ta ESZ S Bare G5 o IT ala a eh j y w JIL 15 Place the drain plate on top of the assembly Use the two screws to lock the complete assembly together Note Align the drainage hole in the drainage plate with the inlet check valve the opposite side of the pump head marked OUT UP e p365 8 Maintenance 8 6 Replacement procedures 8 6 6 Replacing pump pis on seal Step Action 16 Wipe clean the piston and remove all finger prints Wet the piston with water and then insert it into the return spring With the pump head facing downwards on the bench insert the piston into the pump head by pushing it gently but firmly vertically downwards into the seal Note Do not push the piston at an angle to the head and DO NOT twist the piston 28 4090 22 AB p366 Running in the new piston seal Maintenance 8 Step Action 17 e Turn the head so that the inlet valve and
147. e operator to start and pause a run The display indicates running status identity of the system error codes and so on UNICORN is a complete package for control and supervision of AKTAxpress It consists of software for interfacing the controlling computer to the separation system via the USB CAN converter box supplied The software runs under Microsoft Windows The Method Wizard in UNICORN provides easy creation of method plans for purification and for preparation and maintenance of the separation system and columns Before a run the operator chooses a purification method plan and the number of samples for each separation system The systems can then be started separately or simultaneously from UNICORN or from the control panel on each system The illustration below shows the System Control module in UNICORN when using four separation systems fee ana Henne B fod an oc A single separation system can handle one purification protocol at a time However itis possible to run several separation systems in parallel controlled by one single computer thus allowing several different purification protocols to be run Required installa tions Introduction 2 simultaneously UNICORN provides individual and simultaneous control of up to twelve separation systems by one single computer The individual systems can also be placed apart from each other thus allowing operation in cold room and room temperature in pa
148. e the values for these parameters Peak volumes for peaks eluted from affinity and ion exchange columns should be as small as possible It will increase the possibility to e load the entire peak volume onto any desalting or gel filtration column in the next step since their maximum loading volumes are limited e optimize the recovery in the ion exchange step by making sure that the entire peak volume is collected when eluted in the ion exchange gradient Only one loop is available for each peak so peak volumes larger than 7 5 ml will cause sample loss To minimize the peak volume e Choose a suitable column type and size e Adjust the gradient slope A steeper gradient will narrow the peaks Extra wash of af finity chelating column Flow rates and pressure limits Peak detection and collection Collecting large peaks Gradient elution Method Wizard 5 An extra wash of affinity chelating columns can be used to wash out loosely bound proteins The extra wash is an additional step before the elution of the target protein For a powerful wash the imidazole concentration B should be set to above 0 B If no imidazole is used B 0 the volume used in this step should be decreased to minimize the run time Note Do not raise the imidazole concentration too much as it might cause the target protein to elute No warning will be issued if the flow rate or pressure limit are set higher than the values recom
149. easures such as wearing protective glasses and gloves resistant to the chemicals used Follow local regulations and instructions for safe operation and maintenance of the system WARNING When using hazardous chemicals make sure that the entire system has been flushed thoroughly with bacteriostatic solution for example NaOH and distilled water before service and maintenance tis possible to include cleaning of the sample inlets after sample loading or after the purification run within a Purify method plan See 5 4 3 Selecting System Procedures page options on page 157 for information about how to include cleaning of sample inlets in the method plan See 4 3 1 System and column procedures within a purify method on page 74 for information about how to perform the procedure Cleaning proced ures using meth od plans Flushing the tubings manually Operation 6 There are two types of cleaning procedures available for system cleaning The table below briefly describes the cleaning procedures Cleaning procedure Description For more information see Standard system clean ing Customized system cleaning Automatic cleaning of the system with NaOH water and affinity ion exchange buffer Automatic cleaning of the system with up to five optional solutions 6 8 3 Standard cleaning of the system on page 278 6 8 4 Customized clean ing of the system on page 281
150. eavage parameters are described separately since they appear only when tag cleavage is selected Depending on the choices made earlier in the Method Wizard different pages and parameters will appear in the Advanced Zone Select the pages for which to change parameter settings see Entering the Advanced Zone in 5 5 3 Advanced Zone for Purify on page 172 for more information The picture below shows an overview of the possible pages that may appear See the sections below for information about what parameters can be changed on the different pages Advanced settings for AC IEX Advanced settings for AC IEX first step of a protocol intermediate or last step of a protocol On column tog cleavage ACHIEX 2 General AC IEX 2 Elution AC IEX 1 Tag Clevage General AC IEX 1 General AC IEX 1 Elution AC IEX 1 Tog Clevage Elution included Procedures Peak Collection y Fractionation ACHIEX 1 ACHIEX 1 included Procedures Peak Collection AC IEX 2 included Procedures AC TIEX 1 Fractionation Included Procedures Method Wizard 5 Note AC IEX 1 means that the step is the first one in a protocol AC IEX 2 means that the step is an intermediate step or last step in a protocol e g DS AC DS Note The Peak Collection page Included Procedures page and Fractionation page show the same type of parameters independent of protocol step AC IEX 1 Gener This page conta
151. ecifications This section specifies the general operating data of AKTAxpress For components data see 10 2 2 Components specifications on page 420 General performance data of AKTAxpress Parameter Data Flow rate range 0 1 65 ml min Pressure range 0 3 MPa 30 bar 435 psi Gradient formation data of AKTAxpress Step gradients quasi isocratic mixing Conditions e Water based buffers with different concentrations of salts e Mix proportions 5 95 Parameter Concentration accuracy Data 2 B Linear gradients Conditions e Water based buffers with different concentrations of salts Parameter Data Linearity 2 B within 20 80 B Physical data of AKTAxpress Parameter Data Supply voltage 100 240 V autorange 50 60 Hz Reference information 10 Parameter Data Power consumption Max 120 VA Degree of protection IP 20 Dimensions w250 x d490 x h660 mm Weight 30 kg Ambient temperature Temperature operation 4 to 40 C storage 25 to 60 C Relative humidity 20 95 non condensing Wetted material See 10 2 3 Wetted materials on page 424 CE conformity This product complies with the European directives listed in the table by fulfilling the corresponding harmonized standards A copy of the EC Declaration of Conformity is available on request Directive
152. ed T agat Concennahon 9 maint inpor Fle Location C WIC ORIN ServeriMethodwigardimgor The options and parameters for emptying loops Flush Volume Empty Loops The volume used to flush the excess and discarded sample from the loops to the outlets F7 F10 Loop Wash Volume Empty Loops The volume used to wash the loops after emptying excess or discarded loops Loop Wash Volume Buffer volume used to wash the loops Note These values should be increased when using a double loop See 4 2 2 Peak collection parameters when using double loops on page 68 The options and parameters defining air sensor controlled sample loading Enable Air Sensor Controlled Sample Loading Used to provide loading of the entire sample volume The sample loading will stop when the sample supply is empty and air enters the air sensor See also 6 4 16 Using air detection on page 247 If not using this option the sample volume s has to be entered in the Wizard when starting the method plan in System Control Max Sample Volume The maximum allowed sample volume when air sensor controlled sample loading is enabled Method Wizard 5 Miscellaneous op Unit Molecular Weight The unit of the protein molecular weight that will be shown tions for example during the evaluation Preferred Target Concentration The target concentration of the protein When pooling the fractions in Evaluation the software calculates how each pool should be concentrated or di
153. ed Zone introduction 5 5 1 Advanced Zone for Prepare and Maintain 5 5 2 Advanced Zone for Purify 5 5 3 Advanced Zone for Purify Affinity lon Exchange 5 5 4 Advanced Zone for Purify Desalting Gel Filtration 5 5 5 Advanced Zone for Purify Included system procedures 5 5 6 Advanced Zone for Purify Peak Collection 5 5 7 Advanced Zone for Purify Fractionation 5 5 8 Advanced Zone for Purify Miscellaneous settings 5 5 9 Hints on optimizing run parameters 5 5 10 28 4090 22 AB p 164 5 5 1 Introduction The Advanced Zone Advanced Zone content Entering the Ad vanced Zone Method Wizard 5 Advanced Zone introduction This section describes parameters in Advanced Zone and how to change them The Advanced Zone in the Method Wizard is used for viewing and changing parameter values in method plans In new method plans the default values are shown They are preoptimized but can be changed to meet specific demands Values in new method plans as well as in previously made method plans can be changed Note Avoid changing default parameter values in a method plan unless the result is clearly understood Changing a parameter value might require adjustments of other parameter values as well The content in Advanced Zone depends on the options selected on the first page in Advanced Zone Only pages that are related to these parameters will be shown If a selection or parameter value in Advanced Zone needs to b
154. ed in front of the fraction collector hindering the ejection of the microplate The movement of the plate might destroy columns and tubings positioned in front of the fraction collector Attach the columns for a purification run to the column block or the column holder according to the table below A detailed specification can be found on the Summary page in the Method Wizard in System Control The illustration below shows the ports in the column block Upper ports Lower ports Position Column Column block ep225 6 Operation 6 4 Preparing the system for a run 6 4 9 Connecting columns and tubing Connecting the column tubing us ing a method plan 28 4090 22 AB p226 Position Column Port 1 Affinity ion exchange column for sample 1 Port 2 Affinity ion exchange column for sample 2 Port 3 e Affinity ion exchange column for sample 3 or e Affinity ion exchange column for the second affin ity ion exchange step or e 2x HiTrap Desalting column Port 4 e Affinity ion exchange column for sample 4 or e Affinity ion exchange column for the second affin ity ion exchange step or e 2x HiTrap Desalting column Port 5 2 x HiTrap Desalting column Column holder Left hand side Gel filtration column and or HiPrep Desalting column Right hand side Superloop see also 6 4 12 Preparing for on column tag cleavage on page 238 Tube for pump rinsing
155. ed to system preparation before and after the sample loading Included System Procedures incubation Teng alter LIP i 0 339 un Vohume Clean Pil Sampie inet Tuhinga 1 OSE mi Incubation Time after CIP The time the system is set to pause after the system has been filled with CIP solution Volume Clean Fill Sample Inlet Tubings Buffer volume used to clean fill the sample inlet tubings p 185 5 Method Wizard 5 5 Advanced Zone 5 5 7 Advanced Zone for Purify Peak Collection 5 9 7 Introduction Peak collection parameters 28 4090 22 AB p 186 Advanced Zone for Purify Peak Collection This section describes the parameters used in the peak collection step during a purification run The peak collection options and parameters are described below The parameters define how the peaks are collected in the loops ACAEX 1 Peak Collection Absa Lolot ot Suge Peaks n Sevend Loupe Die ONe Max Volume in Each Loop 75 105 65 m Max Number of Loco u Stat Colecnon Watch Level Greaear than bad 6000 6000 mu Watch Shope bietes har m EOOD SOOO trey Stop Colechun Pesk Max Facio os Bn Wateh avel i naa than w tanon snoa m i Wate Statie Pistas Shatin Teme us P9989 min Daba Pinani 5 P ANN matt Seint Laye Pesk Baud ur Dire OHadt Allow Collection of Single Peaks in Several Loops To collect single peaks with a volume larger than Max Volume in Each Loop in several loops Note When
156. ed water 2 CV RT 0 5 CR 0 4 Step 3 1M NaOH 4 CV RT 0 5 CR 0 4 Step 4 Deionized water 2 CV RT 0 5 CR 0 4 Step 5 1M HCI 4 CV RT 0 5 CR 0 4 Step 6 Deionized water 2 CV RT 0 5 CR 0 4 Step 7 1M NaCl 4 CV RT 0 5 CR 0 4 Step 8 Deionized water 2 CV RT 0 5 CR 0 4 Recommended The tables below list recommended solutions for the gel filtration columns supported solutions for gel by AKTAxpress filtration columns See 4 5 1 Descriptions of columns on page 95 for a list of columns supported by KTAxpress Column Solution Volume Flow rate ml min All HiLoad Super dex columns 16 600 and 26 600 Step 1 1M NaOH 4 CV RT 0 5 1 3 CR 0 4 1 Step 2 Deionized water 4 CV RT 0 5 1 3 CR 0 4 1 Step 3 30 isopropanol 0 5 CV RT 0 5 1 3 CR 0 4 1 Step 4 Deionized water 2 CV RT 0 5 1 3 CR 0 4 1 All HiPrep Sephacryl columns 16 60 and 26 60 28 4090 22 AB p 120 Methodology 4 Column Solution Volume Flow rate ml min Step 1 0 5 M NaOH 0 25 CV RT 0 3 0 8 CR 0 2 0 6 Step 2 Deionized water 4 CV RT 0 3 0 8 CR 0 2 0 6 Step 3 30 isopropanol 0 5 CV RT 0 3 0 8 CR 0 2 0 6 Step 4 Deionized water 4 CV RT 0 3 0 8 CR 0 2 0 6 p121 4 Methodology 4 7 Solution alternatives 4 7 5 Metal lon Charge Solution Suggestions 4 7 5 Metal lon Charge Solution Suggestions Introduction This sect
157. ee 5 5 7 Advanced Zone for Purify Peak Collection on page 186 for information Depending on the column preparation and post run procedures that was included in the purification run selected earlier in the Method Wizard different parameters will appear The same type of settings is however available for the different procedures For each procedure the flow rate and volume when performing the procedure can be changed For a list of possible procedures for the different columns see 4 3 1 System and column procedures within a purify method on page 74 ACAEX 1 ineluded Piowedures Flow Rate Eaudbeation a 10 69 mr Veduane taute atari ba S73 cv Flow Rate LIF M 0 bof tian Volume OP w M3339933 CV Fiw Nte Water ates PIP 1 PoS Vehsne Water ates P Pano Fim Nate Neen atiente 0 65 tmin Veame Raegitane s p a Cy ie Incubation Tene ater CIP 0 10 9999 min e Flow Rate Flow rate through the column during the procedure Examples Flow Rate Remove Ethanol Flow rate used when the ethanol is removed from the column Flow Rate Equilibration Flow rate used during the column equilibration e Volume Volume used to perform the procedure Examples Volume Remove Ethanol Volume used to remove ethanol from the column Volume Equilibration Volume used to equilibrate the column e Incubation Time after CIP The time the system is set to pause after the column s has been filled with CIP solution p179
158. eel Elgiloy cobalt chromium nickel alloy Pump Ruby sapphire Pump Online filter p425 10 Reference information 10 3 Import file format 10 3 Introduction Import file con tent Example File specification 28 4090 22 AB p426 Import file format This section describes how to create import files and how they are used One or several import files can be used for storing information on one or several protein samples The information can be added to the run data when starting a run in the wizard in System Control The import file must contain the following parameters for each sample ID e Sample volume e Isoelectric point e Extinction coefficient e Molecular weight e Freetext Note If the air sensor is used for applying the entire sample volume set the sample volume to for example 10 in the import file the sample volume parameter must always be gt 0 This illustration is an example of an import file containing information on three different samples IGFP His Volume 10 IGFP His pI 6 19 IGFP His Extcoeff 0 746 IGFP His Mw 28 IGFP His FreeText Green Fluorescent Protein APD 7 volume 10 APB pI 5 95 APD 7 ExtCoeff 0 4 APB MW 28 APB 7 FreeText Unstable at room temperature Arb 13 volume 10 APB 13 pI 5 56 APB 13 extcoeff 1 175 APB 13 Mw 70 APB 13 FreeText Avuid pH below 5 5 The import file can be created in a text editor for example Notepad Follo
159. elect File Instant Run Instant Run Result The Method Wizard in System Control appears 3 Select the requested method plan in the Method plan list Click Next Hun Method plan Method pisn Note yous De Ub 5 AL OS IEX G ra or Asty Get Hi stun placar Hie Lap HP eo Global al GE Suuperinne Hioed 16 00 Superdex 75 pep grade a Prepare and Martan Global Rowen Tempas Tost Step Sten Finan diane ia salnas nasd anna e p259 6 Operation 6 6 Starting a run 6 6 1 Starting a run using a method plan If selecting a Prepare and Maintain method plan Select on which System s the method plan shall be used It can be used on any or all selected systems Click Next Enter any optional text for example running data Click Next The text can be viewed later in the result file in Evaluation by selecting View Documentation Notes Method Notes The a wien by JP GE Hesthcae 6 Proceed to Final setup and starting the run below 28 4090 22 AB p260 Operation 6 If selecting a Purify method plan Step 4 Action e Select on which System s the method plan shall be used e Select the Number of Samples for each system s e Click Next Enter identification name s for the samplels max 74 characters A bar code reader can also be used An import file with sample information can be linked to this sample ID See 10 3 Import file format on page 426 Click Next S
160. elected earlier in the Method Wizard The flow rate when washing out weakly bound proteins before elution e Elution Cleaved Protein Flow rate used when eluting the cleaved protein after protease incubation Affinity Regeneration The flow rate used to regenerate the affinity column i e elution of tags tagged protease and uncleaved protein using affinity elution buffer p177 5 Method Wizard 5 5 Advanced Zone 5 5 4 Advanced Zone for Purify Affinity lon Exchange Tag Cleavage Elution paramet ers 28 4090 22 AB p 178 Pressure Limit Upper pressure limit during the affinity step Load same volume of protease on all columns To load the same protease volume on all columns as entered in Default Protease Injection see below To use separate volumes for different columns deselect this option The separate volumes have to be entered in the System Control wizard before starting the run Enable Watch Stable Baseline If Watch Stable Baseline is enabled the firstwash step will be completed when the baseline is stable or when the entire wash volume has been consumed The stability of the UV signal will be checked during the specified time interval The Stable Baseline condition is met when the signal fluctuates less than the set Delta Base value during the set Stable Time interval Note This page replaces the AC IEX 1 Elution page when on column tag cleavage is selected in the first part of the Method Wizard Volu
161. elow 96 well microplates Parameter Data Type Deep well plate 96 wells height 45 mm Well shape Square wells volume 2 ml 24 well microplates Parameter Data Type Deep well plate 24 wells height 45 mm Well shape Square wells volume 8 ml p 419 10 Reference information 10 2 Technical specifications 10 2 2 Components specifications 10 2 2 Components specifications Introduction This section specifies the operating data of the components in AKTAxpress For general data of the system see 10 2 1 System specifications on page 416 Pump data Pump data Parameter Data Flow rate range 0 1 65 ml min Flow rate accuracy 2 or 0 02 ml min whichever is greater Flow reproducibility RSD lt 0 5 Maintenance interval Sealings 2000 hours Mixer data Mixer data Parameter Data Internal volume 0 37 ml Flow restrictor Flow restrictor data data Parameter Data Back pressure 0 2 MPa nominal UV monitor data UV monitor data Parameter Data Wavelengths 280 and 254 nm set by lamp position and filter Optical path length 2mm Optical cell volume 2 ul 28 4090 22 AB p420 Conductivity mon itor data Reference information 10 Parameter Data Detector cell volume 30 ul Absorbance range 0 01 to 5 AU Autozero range 0 2 to 2 0 AU Linearity deviation lt 3 up to 2 AU
162. elow shows an example of this Stable Time Stadie Time condition met VA Delta Plateau Delta Plateau Condition not met p67 4 Methodology 4 2 Peak handling 4 2 2 Peak collection parameters when using double loops 4 2 2 Introduction Definition of double loop Why using a double loop Example of using two separate loops for collect ing peaks with volume gt 7 5 ml 28 4090 22 AB p68 Peak collection parameters when using double loops This section contains information about peak collection and which parameters that need to be modified when using double loops A double loop consists of two 10 ml capillary loops that are connected in series The protein eluted will be collected in one 20 ml loop instead of in two 10 ml loops For information about how to connect double loops see 6 4 10 Preparing the system when using double loops on page 231 During a purification run the intermediate peaks can be collected in five capillary loops The volume for these peaks is limited to 7 5 ml in each 10 ml loop due to band broadening effects If peak volumes larger than 7 5 ml is expected it is recommended to collect the eluate in a double loop instead of using two separate capillary loops The main advantage of using a double loop is that the double peak from the DS GF step obtained when using two separate capillary loops is avoided thus yielding a less diluted protein a better separatio
163. em and Column Procedures prepare and maintain method plan it is possible to include a procedure for rinsing all outlets see below Note Do not leave the system with salt buffer in the flow path It might damage the pump WARNING When using hazardous chemicals take all suitable protective measures such as wearing protective glasses and gloves resistant to the chemicals used Follow local regulations and instructions for safe operation and maintenance of the system WARNING When using hazardous chemicals make sure that the entire system has been flushed thoroughly with bacteriostatic solution for example NaOH and distilled water before service and maintenance Prerequisites Create a purify method plan in cluding CIP Sys tem Create a Standard System and Column Proced ures method plan including CIP Sys tem Operation 6 The following are required for automated standard system cleaning e Cleaning solutions The cleaning solutions use dedicated inlet tubings See 4 7 1 Standard CIP Solution Suggestions System on page 113 for information on recommended cleaning solutions e Amethod plan for cleaning the system The illustration below gives an overview of how to include CIP System in the purification method plan See 5 4 Using the Purify option on page 150 for detailed information Purify 2 21 if sample inlets should be cleoned after sample loading oppropric
164. ence information About this This chapter includes reference information for AKTAxpress chapter In this chapter This chapter contains the sections below Section See System description 10 1 Technical specifications 10 2 Import file format 10 3 Typical run times 10 4 Chemical resistance guide 10 5 Ordering information 10 6 e p401 10 Reference information 10 1 System description 10 1 About this section In this section 28 4090 22 AB p 402 System description This section describes the components of AKTAxpress For a system overview see 3 2 Separation system overview on page 31 This section contains the sub sections below Sub section See Pump and related components 10 1 1 Valves 10 1 2 Monitors 10 1 3 Fraction collector 10 1 4 10 1 1 Introduction Pump Reference information 10 Pump and related components This section describes the pump and mixer in AKTAxpress Mixer Pump heads General The pump in AKTAxpress is a high performance laboratory pump for applications where accurately controlled liquid flow is required It consists of two parallel pump heads with a cylinder piston in each pump head Each pump head also has two check valves and one purge valve The liquid is drawn into the inlet manifold by the action of the pump Twin reciprocating pump heads work in unison to deliver a low pulsation flow from the pump outlet
165. endent Unless otherwise List of chemicals List of chemicals and their compatibility to AKTAxpress Chemical Exposure lt 1 day Exposure up to 2 months Remarks Acetaldehyde OK OK Acetic acid lt 5 OK OK Acetic acid 70 OK OK Acetonitrile OK OK FFKM PP and PE swell Acetone 10 OK Avoid PVDF is affected by long term use Ammonia 30 OK OK Silicone is affected by long term use Ammonium chlor OK OK ide Ammonium bicar OK OK bonate Ammonium ni OK OK trate Ammoniumsulph OK OK ate 28 4090 22 AB p432 Reference information 10 Hydrochloric acid 0 1M OK OK Chemical Exposure lt 1 day Exposure upto2 Remarks months 1 Butanol OK OK 2 Butanol OK OK Citric acid OK OK Chloroform OK Avoid ECTFE CTFE PP and PE are af fected by long term use Cyclohexane OK OK Detergents OK OK Dimethyl sulphox Avoid Avoid PVDF is affected ide by long term use 1 4 Dioxane Avoid Avoid ETFE PP PE and PVDF are affected by long term use Ethanol OK OK Ethyl acetate OK Avoid Silicone not resist ant Pressure limit for PEEK decreases Ethylene glycol OK OK Formic acid OK OK Silicone not resist ant Glycerol OK OK Guanidinium hy OK OK drochloride Hexane OK Avoid Silicone not resist ant Pressure limit for PEEK decreases Silicone not resist ant e p43
166. ents in buffers The mixer is designed for use with water based solutions only If organic solvents are used incorrect gradients or incomplete mixing may be the result Flow restrictor The flow restrictor is positioned directly after the UV and conductivity monitors The restrictor generates a steady backpressure of 0 2 MPa to prevent air bubbles being formed in the monitors The flow restrictor is closed for pressures below 0 2 MPa 28 4090 22 AB p406 Reference information 10 10 1 2 Valves Introduction This section describes the valves in AKTAxpress Outlet valve Column valve Loop valve Injection valve Switch valves SW1 and SW2 Inlet valve Switch valves The switch valves SW1 and SW2 are 2 way 3 port valves used for buffer switching and gradient formation The valves have one port marked IN and two ports marked NC normally closed and NO normally open The valves have UNF 5 16 connectors The valves have no user replaceable parts NO NC i Z 2 i o Default position at NC NO startup inject The two positions of the valve gives the following flow paths A1 A2 position B1 82 position Buffer Buffer Buffer Buffer Al B2 Al B2 Inlet Inlet Inlet Inlet valve valve valve valve Buffer Buffer Buffer Buffer BI A2 Bl A2 Inlet valve The inlet valve is a 12 way 13 port valve For each 12 positions an inlet port is connected to the central outlet port The valve has M6 connectors The va
167. er and finally with 10 CV affinity ion exchange binding buffer General e Ablankrun should be performed before the first time the column is used and after long term storage e A blank run is often performed to make sure that the column is free of any bound proteins that may elute with the conditions that will be used during the run Affinity columns e By performing a blank run leakage of loosely bound metal ions can be avoided during the purification runs A blank run should always be performed after metal ion charging or stripping of a column lon exchange columns e Provides the ion exchanger with exchangeable counter ions Column post run procedures that can be included in a purification run Methodology 4 Column preparation Description procedure Remove Ethanol be Before the equilibration or blank run the columns are fore Preparation washed with water AC IEX columns Remove Ethanol be Before the equilibration the columns are washed with fore equilibration water DS GF columns A number of column post run procedures can be included in a purification run The following combinations of post run procedures are available e Fill with Ethanol all column types e CIP Columns Fill with Ethanol all column types Note If using chelating affinity columns the columns should be stripped before CIP to avoid formation of metal ion salts that can damage the column e CIP
168. ess you have already filled the Super loop with protease or sample before starting the run During purification run Clean Sample Inlets after Sample Loading only available for proto cols starting with After the samples have been loaded the system will pause and a message will appear on the screen Sample inlets should then carefully be immersed into a separate bottle containing the appropriate wash solution e g AC IEX water or buffer The sample inlet tubings are cleaned before the purification method continues Clean Pump with0 5M After each sample has been loaded via the pump the NaOH between Load ing of Different Samples only available for protocols starting with AC IEX pump is cleaned with 0 5 M NaOH followed by water and AC IEX binding buffer Wash Frac Tubing Between Samples Between fractionation of samples the fraction collector tubing will be washed with 2 ml buffer One microplate well will be used for each wash Post run After the purification run the procedures below can be included Fill System with Ethan ol After the purification has been completed the system is washed with water and filled with 20 ethanol The ca pillary loops used will also be filled CIP System then Fill with Ethanol After the purification run the system is cleaned with 0 5 M NaOH followed by water affinity binding buffer water and finally filled with 20 ethanol The capillary loops
169. f electrical and electronic equipment must not be disposed as unsorted municipal waste and must be collected separately Please contact an authorized representative of the manufacturer for information concerning the decommissioning of your equipment In this section This section contains the sub sections below Sub section See Replacing capillary loops 8 6 1 Replacing tubing and connectors 8 6 2 Replacing flow restrictor 8 6 3 Replacing UV lamp 8 6 4 Replacing check valves 8 6 5 Replacing pump piston seal 8 6 6 Replacing a damaged pump piston 8 6 7 p335 8 Maintenance 8 6 Replacement procedures 8 6 1 Replacing capillary loops 8 6 1 Replacing capillary loops Maintenanceinter When required for example when a capillary loop has clogged val Reason for main Clogged tubings may contaminate or cause high back pressure and hence preventing tenance a successful purification run Required material Capillary loop 11 0003 02 CAUTION Only spare parts approved or supplied by GE Healthcare may be used for maintaining and servicing the system Replacing capil To replace the capillary loops lary loops Step Action 1 Press the Eject button to put the collector plate in load inserted in system position 2 Set the system in Standby mode by pressing the On button This will disconnect the system from UNICORN RUN zz i 3 On the loop valve
170. ffer sugges tions IEX buffer sugges tions GF buffer sugges tions Further informa tion Methodology 4 Suggested buffers for DS runs If for example suggested buffer preparing for AC preparing for AIEX Use the AC binding buffer that will be used in the follow ing step 50 mM Tris HCI pH 8 0 preparing for CIEX 20 mM MES pH 6 0 preparing for storage include e g 10 glycerol in a suitable buffer e g 50 mM Tris HCl pH 7 5 150 mM NaCl Suggested buffers for IEX runs If for example suggested buffer binding to AIEX 50 mM Tris HCl pH 8 0 binding to CIEX 20 mM MES pH 6 0 elution from AIEX 50 mM Tris HCl pH 8 0 1 M NaCl elution from CIEX 20 mM MES pH 6 0 1 M NaCl Note In the examples above the pl of the target protein is 7 Suggested buffers for GF runs If for example suggested buffer preparing for further studies 50 mM Tris HCl pH 7 5 150 mM NaCl preparing for storage include e g 10 glycerol in a suitable buffer e g 50 mM Tris HCl pH 7 5 150 mM NaCl For further information see method handbooks in 4 8 Documents for further information on page 124 and product specifications p111 4 Methodology 4 7 Solution alternatives 4 7 Solution alternatives About this section This section contains suggestions on solutions for CIP strip and metal ion ch
171. ffinity lon Exchange 5 5 5 Advanced Zone for Purify Desalting Gel Filtration 5 5 6 Advanced Zone for Purify Included system procedures 5 5 7 Advanced Zone for Purify Peak Collection 5 5 8 Advanced Zone for Purify Fractionation ae cccssssssssessesssssccsssssssssusessesseccsssssssssssssssessccssssssssnssnessessees 5 5 9 Advanced Zone for Purify Miscellaneous SELLINGS ccccscccssssssssssssesssssesseccssssssssessssessecsssssssssssnsesess 190 5 5 10 Hints ON optimizing FUN POFAMETETS cssssescscccsssssseccscssssusccessssssessccsssussscsesssssscecesssssssccesssssusesessessee 192 6 OP OFAC OM ees csccsscncesctesduvsaccecsastovcacunvecnsnecsesbavearensbenavsbsunsusuestondesstesvunedossoevbebantesaeveveddteteebsveduansbentsteesateaveen 195 6 1 OPEV ALON OVERVIOWe siini a n a A A A N 196 6 2 Connecting A Syste n n A ET AE T O A N ees 197 Ga Creating MET ODI Scie Ra E a e A E AA Revue nae UN 200 6 4 Preparing the system Tord C asneineneniniiii ie ae n E A eco 203 64d Printing OEA SUMMA eene a E EEEE EA R 205 6 4 2 Preparing b ffers and Sol tigN Szira E 207 6 4 3 Preparing outlet and waste tUDING cceccscccssssssssussessssssssssssssssusssessessessessssssssssssessessecsssssssssnusessssees 210 6 4 4 Purging the pump and inlet tubing OVEFVICW eecccscssscssssssssseessssesscsssssssssusssesssssecssssssssuasseesesee 212 6 4 5 Filling inlets manually using a method PION seceessesssssscssssssssssesessessccssssssss
172. fication preparation and maintenance of the separation systems and columns The Method Wizard also allows editing of stored method plans Before a run the operator chooses method plan and the number of samples for each separatio n system The systems can then be started separately or simultaneously from UNICORN For more information about the Method Wizard see 5 Method Wizard on page 125 Operation over view AKTAxpress overview 3 The step by step operation of the separation system is described below f Had alta anon at Di k ABA AA Run preparations A new method plan is created using the Method Wizard in the Method Editor module If required a traditional method can also be created in the Method Editor Starting a run Method plans are started on single or multiple systems using a wizard in the System Control module Traditional methods can only be started on a single system and not by using the System Control wizard p29 3 AKTAxpress overview 3 1 UNICORN overview Evaluating the results Run data is collected continuously and when the run has finished the result files can be processed using standard run procedures or own developed run procedures Suggestions for automatic pooling of fractions will be made when opening a single chromatogram Help functions The table below describes how to access the on line help utility included in UNICORN software If you want to access then
173. fication run 6 5 Starting a run 6 6 During a run 6 7 Procedures after a run 6 8 e p195 6 Operation 6 1 Operation overview 6 1 Operation overview Introduction This section gives an overview of the operation of AKTAxpress Work flow The typical work flow in AKTAxpress after turning on a system and connecting it can be divided into a number of steps Step 1 6 are described in separate sections in this chapter Step 7 and 8 are described in chapter 7 Evaluation on page 292 1 Creating method plans l 2 Preparing the system for a run l 3 Procedures before a purification run conditioning of columns metal ion charging of uncharged chelating affinity columns l 4 Storting o run using a method pian t 5 During o run viewing and changing parameters l 6 Procedures after a run cleaning the system and columns 1 8 Create a report 28 4090 22 AB p196 Operation 6 6 2 Connecting a system Introduction This section describes how to connect to a separation system from UNICORN Up to twelve separation systems can be connected in the System Control module Connectingasep The separation systems that are available in UNICORN are shown in the vertical bar aration system at the left hand side of System Control Disconnected systems are identified by a blue connector symbol Note To add more systems in UNICORN see AKTAxpress Installation Guide Left c
174. flushed A detailed description of the process steps can be found in the section 3 3 2 Description of the process steps on page 54 p53 3 AKTAxpress overview 3 3 Purification process overview 3 3 2 Description of the process steps 3 3 2 Description of the process steps Introduction This section contains a detailed description of the main process steps during the purification process described in the flow chart The description is generalized to cover up to four samples A separate description of the tag cleavage procedure is also included Note The description covers the most important process steps only Manual system The table below briefly describes how the operator prepares AKTAxpress for a run setup before starting the run Note The method plan to be used should be created before commencing the system setup Note Print out the Summary page from the System Control Method Wizard before starting setting up the system The Summary page provides information on for example buffer and solution volumes inlets outlets column positions and so on Step Description 1 The buffer containers and sample tube s are filled and sealed See the Summary page for information on which buffers to be used and the buffer volumes 2 All inlet outlet and waste tubing is immersed in the correct flasks See the Summary page for information on inlet outlet and waste tubing positions 3 An empty microplate is
175. ge samples DS GF protocols or for loading of protease when performing on column tag cleavage This section describes how to pre fill the Superloop with buffer connect the Superloop to the system and fill the Superloop with protease sample For information regarding how to create a method plan including on column tag cleavage and how to calculate protease volume and concentration see 6 4 12 Preparing for on column tag cleavage on page 238 Automatic affinity tag removal can be combined with all AKTAxpress protocols that start with an AC IEX step There are two protocols including Superloop sample loading e DS multiple sample injections from Superloop e GF multiple sample injections from Superloop To avoid pressing air into the system pre fill the Superloop with buffer manually before connecting it to the system The table below describes how to pre fill the Superloop with buffer For more detailed information see the the Superloop instructions Step Action 1 Remove the upper end pieces 2 Fill the upper chamber with buffer 3 Replace the end pieces and ensure there is no air trapped 4 Turn the Superloop upside down and fill the lower chamber in the same way 5 Reinsert the inner end piece and turn it until the slot lines up with the countersink in the glass tube Replace the other end pieces and ensure there is no air trapped e p233 6 Operation 6 4 Preparing the system for
176. gged proteins and protocols that can be combined with on column and off line batch tag cleavage are provided All protocols starting with an affinity ion exchange chromatography step include tag cleavage as an option AKTAxpress provides the following features e AMethod Wizard to make it easy to create method plans e Automatic purification of up to four samples in a single run e Purification protocols containing up to four chromatography steps e Automated on column affinity tag removal in the first step of purification protocols starting with an affinity ion exchange chromatography step epi5 2 Introduction 2 1 Introducing AKTAxpress The separation system 28 4090 22 AB p16 e Purification protocols supporting purification of both well expressed and low expressed proteins double tagged proteins and proteins that have been cleaved off line e Up to 50 mg of protein can be purified per sample e Up to twelve AKTAxpress systems can be run in parallel controlled from one computer Different purification protocols can be run simultaneously e The systems can be placed next to each other or they can stand separately if preferred AKTAxpress comprises a number of separation systems and a computer with monitor running the control and analysis software The separation system is a compact chromatography unit containing the components required for automated multi step purification processes The illustration on nex
177. ging the columns 28 4090 22 AB p 252 Create a method plan including Metal lon Charge Blank Run Charge the columns Operation 6 The illustration below gives an overview of how to include Metal lon Charge Blank Run in the Standard System and Column Procedures method plan See 5 3 Using the Prepare and Maintain option on page 136 for detailed information 000060 OO Z eo e o 080 0 000 0 0 5 00008000 8 8 a g a column tupe 1 5 i 0 l lz 0 l Note The Metal lon Charging inlet tubing have to be filled before starting the charging run See 6 4 4 Purging the pump and inlet tubing Overview on page 212 Note The metal ion waste that exits the system through tubing F11 should be collected in a separate waste bottle To charge the columns and perform a blank run Step Action Summary page 1 Immerse the inlet tubing in flasks according to the check list on the 2 Run the method plan according to the standard procedure described in 6 6 1 Starting a run using a method plan on page 259 e p253 6 Operation 6 5 Column procedures before a purification run 6 5 2 Conditioning of columns 6 5 2 Introduction Conditioning pro cedures When should con ditioning of columns be per formed 28 4090 22 AB p 254 Conditioning of columns Conditioning of columns should be performed to obtain well conditioned and equilibrate
178. give the protein the required sample characteristics needed for the next application Is it important to have a charge and or size homogenous sample after the purification To obtain a charge homogenous sample include an IEX or DS IEX step in the purification protocol To obtain a size homogenous sample include a GF step in the purification protocol To obtain both include an IEX GF or DS IEX GF step in the purification protocol Guidance for When choosing protocol start by considering the questions in the previous section choice of purifica to get an idea of what steps should be included in the protocol Choosing protocol tion protocol can be divided into two steps 1 Choose a subset of protocols based on the starting material Depending on the starting material for example if the protein is tagged double tagged or if the tag has already been removed from the protein different subsets of protocols are suitable for the purification The table below gives guidance on which protocols that are appropriate to select among based on the starting material To refine the selection further see step 2 below ep61 4 Methodology 4 1 Protocol descriptions and choice 28 4090 22 AB p62 If you have Select among the protocols a tagged recombinant protein that start with an AC step Group 1 protocols a double tagged recombinant protein that include two AC steps Group 2 protocols a tagged protein
179. gn you must reinstall the driver Continue to step 4 Right click the Amersham URJ45 icon and choose Properties Result The Amersham URJ45 Properties dialog is opened Click the Driver tab Troubleshooting and corrective actions 9 Step Action 5 e Click the Update Driver button Result The Hardware Update Wizard is opened e Click Next 6 e Select the option Install from a list or Wolcome to the Hardware Update Wizard That vrawd helps you inital sottrae tor Somer dhan UR JSS M your hardware case with an mstaltohun CD S w Happy disk insert it now What do you ward the mead tv du rata the yultewe automate shy Hecunmnended inta bom abt u pete oson Advanced Oki Newt to conte e __ Sone _ e Click Next 7 e Select the option Include this location browse to the folder C Progam Files KVASER Drivers and click OK Baa ma anovai neds Theten aka Anana BE Maaa S Senet immovable madia Hope CDADM I tech thes cadan intha saach C Dant seach Iwil chonse the drie to instal he dve you chonze wd be mach for your hadware ie O cra i e Click Next e p399 9 Troubleshooting and corrective actions 9 7 Checking USB CAN driver Step Action 8 The wizard should find the correct driver e Click Next and then Finish 9 Close the Device Manager and the System Properties dialog 28 4090 22 AB p400 Reference information 10 10 Refer
180. graphy is performed in three steps protein binding column wash and protein elution In addition to these steps affinity tag removal can also be performed on the AC column Binding buffer The buffer for binding has to match the binding properties of the used affinity column Extra wash buffer The extra wash is used to wash out non specifically bound impurities that do not come off with the normal binding buffer wash Use a buffer with slightly higher amount of imidazole than the binding buffer but not too close to the concentration in the elution buffer because it might cause pre elution of the target protein Note The extra wash is recommended when running HisTrap or HiTrap Chelating IMAC columns Cleavage buffer If affinity tag cleavage is performed the binding buffer can often be used If preferred an alternative cleavage buffer can be used to condition the affinity column before injection of the protease Elution buffer The elution buffer should be chosen to elute the target protein efficiently For AKTAxpress step or gradient elution of the bound protein from the AC column is used to achieve an elution volume or purity suitable for the next purification step Desalting can be performed in preparation for an ion exchange affinity chromatography step or as a last buffer exchange step DS before IEX AC If IEX AC is to be performed after DS use the IEX AC binding buffer in the desalting step ep 107 4 Methodol
181. gy 4 8 Documents for further information 4 8 Introduction Further informa tion 28 4090 22 AB ep 124 Documents for further information This section includes a list of the different documents that can be used to find more information on protein separation and related topics Further information on protein separation can be found in the following documents Handbook Documents Code number Affinity Chromatography Handbook 18 1022 29 Principles and methods Gel Filtration Principles and methods 18 1022 18 GST Gene Fusion System Handbook 18 1157 58 lon Exchange Chromatography Prin 18 1114 21 ciples and methods Strategies for Protein Purification 28 9833 31 Recombinant Protein Purification 18 1142 75 For details about column characteristics buffer choices cleaning procedures and so on see each column instruction Method Wizard 5 5 Method Wizard About this This chapter describes the Method Wizard in UNICORN chapter In this chapter This chapter contains the sections below Section See Method Wizard introduction 5 4 Working with method plans 5 2 Using the Prepare and Maintain option 53 Using the Purify option 5 4 Advanced Zone 5 5 ep125 5 Method Wizard 5 1 Method Wizard introduction 5 1 Introduction The Method Wiz ard The method plan Using a method plan 28 4090 22 AB p 126 Method Wizard introduction
182. he connector NOTE Insert the tubing fully to the bottom of the port Ferrule NOTE Insert the tubing fully to the bottom of the port e Tighten the connector properly For areas difficult to access use the fingertight key available in the accessory kit Fingertight connectors should be tighten by fingers only but for areas difficult to access use the special key available in the ac cessory kit M6 and 5 16 should be tighten using the special key for M6 4 Ig D gt x e Press the On button to reconnect the system to UNICORN p341 8 Maintenance 8 6 Replacement procedures 8 6 2 Replacing tubing and connectors Step Action 9 Verify the new tubing by e Running a flow through the replaced capillary tubing and check the connectors for leakage e Running the tubing leakage test see 8 4 4 Checking tubing leakage on page 325 Adjusting the The fraction collector tubing should be adjusted in height when replaced fraction collector tubing Step Action 1 Adjust the capillary tubing end to match the height of the microplate The capillary end should be positioned about 1 mm above the microplate 1 mm Specification of KTAxpress has the following tubing connections for the system flow path system tubing For information about the piston rinsing tubing see 8 6 2 Replacing tubing and connectors on page 338 28
183. he chromatogram window on top is the active window There is also a minimized temporary chroma togram window See Basic presentation of chromatograms in UNICORN KTAxpress User Reference Manual for further information about chro matograms Alternative find See How to open a result file in UNICORN AKTAxpress User Reference Manual for ing and opening alternative ways to open result files Automatic pooling will not be performed if result of files files are opened in any of the alternative ways e p295 7 Evaluation 7 3 Viewing results 7 3 Introduction Zooming in the chromatogram Further instruc tions 28 4090 22 AB p296 Viewing results This section describes how to view the results In the active chromatogram window zooming in on a designated area of the chromatogram is the easiest and quickest way to enlarge different parts of a curve To do this Step Action 1 Open a result file 2 e Place the mouse pointer in any corner of the area to be magnified e Press and hold the left mouse button A magnifying glass icon will be added to the mouse pointer arrow on the screen e Drag aboxto cover the area to be magnified and release the mouse button Result The selected region is now displayed in the entire chromatogram window together with appropriate scales for the Y and X axes 3 Use the arrow keys on the keyboard to move around in the chromato gram at the current zoom scale
184. he lamp 417 418 System error Internal error Restart system If problem remains contact service e p395 9 Troubleshooting and corrective actions 9 6 Error code list 28 4090 22 AB p396 Code Description 420 421 System error Internal error Restart system If problem remains contact service 422 Filter size error The filter size in the UV lamp is out of range Check that the method is made for AKTAxpress 423 47 System error Internal error Restart system If problem remains contact service 428 Measurement warning The UV lamp has been switched off or the conductivity cell disconnected when measuring the absorbance or the conductivity Try to switch on the UV lamp If it does not help restart the system If the problem still remains contact service 429 Lamp intensity warning The intensity of the UV lamp is too low Replace the lamp 430 Dark current warning The dark current in the UV lamp is higher than the allowed limit Check if the seal is broken 431 UV auto zero warning The UV value was out of the allowed range 0 2 to 2 AU when trying to set the relative absorbance signal to zero 480 No peak found No peak was found by the Peak_Select instruction 481 No peak selected No peak has been selected Use the Peak_Select instruction to select a peak 500 501 System error Internal error Restart system If problem remains contact service 9 7 Int
185. hione S trans Glutathione 26 kDa ferase depending on the number of histidines and the used linker between the tag and the target protein p85 4 Methodology 4 4 Affinity tag removal 4 4 1 Description of affinity tags Purification prop erties 28 4090 22 AB p 86 Histidine tags Histidine tags are the most widely used affinity tags They are normally composed of a sequence of 6 histidines that can bind with high affinity to metal ions The tags are small and therefore often considered to have little effect on the target protein This tag is also beneficial to use if purification under denatured conditions is required because no specific native fold of the tag itself is needed for binding GST tags GST tags often improve the expression and solubility of the target protein The interaction with glutathione is highly specific and a one step purification therefore often results in a reasonably pure material Purification of Histidine or GST fusion proteins is simple and uses mild elution conditions that minimize the risk of damage to the functionality of the target protein 4 4 2 Introduction About proteases Protease types Description of proteases Methodology 4 This section describes different types of proteases that can be used for affinity tag removal When using a Histidine or GST tagged protease the cut off target protein can be eluted while the protease is still bound to
186. ht connectors The valve has no user replaceable parts Fraction collector Waste_ F3 Injection valve N ON F4 Fio oj Ffs Flow restrictor F10 Default position at startup The valve is used for redirecting samples from a column to a capillary loop passing samples to the fraction collector and passing liquids to flowthrough containers and waste For flow path details see 3 2 2 Liquid flow path on page 38 28 4090 22 AB p410 10 1 3 Introduction UV monitor Reference information 10 Monitors This section describes the monitors and flow restrictor in AKTAxpress Flow restrictor UV monitor Pressure sensor Conductivity monitor Air sensor The UV optical unit houses the Hg lamp the wavelength filter and the UV flow cell The light beam is directed through a double conical or straight flowthrough cuvette of 2 ul illuminated volume to a photodetector The photodetector current is fed to the signal processing circuitry in the system Optical unit Outlet Flow Photo Lamp Filter Lens Beam splitter cell detector AN ee A Di 3 254 nm 280 nm Photo detector Vs System Lamp voltage Microprocessor The reference signal comes from the same point in the lamp as the signal measuring the sample thus assuring a stable baseline by eliminating the effects of variations in lamp intensity The Hg lamp emits light only at certain wavelengths It does no
187. idering the new delay volume 6 4 13 Introduction Microplate re quirements Installing a micro plate Operation 6 Preparing the fraction collector This section describes how to load a microplate in the fraction collector The microplates for the fraction collector in AKTAxpress must fulfill the following requirements e Deep well plate with 96 or 24 wells height 45 mm e Square well design not cylindrical wells e Volume 2 ml 96 wells or 8 ml 24 wells The following manufacturer s microplates are tested and approved by GE Healthcare to be used with AKTAxpress 96 well microplates e Greiner no 780270 780280 780285 e Eppendorf no 2605662 e Nunc no P7616 e Corning no A9347 24 well microplates e Thomson no 931565 WARNING T Do not put any body parts in front of the fraction collector The microplate can be ejected both manually and automat ically by the system e p241 6 Operation 6 4 Preparing the system for a run 6 4 13 Preparing the fraction collector WARNING Ensure that no parts for example tubing or columns are positioned in front of the fraction collector hindering the ejection of the microplate The movement of the plate might destroy columns or tubing positioned in front of the fraction collector Follow the instruction in the table below to install a microplate Step Action 1 Press the Eject button to eject the fraction collector
188. in another folder under the Home folder enter the name of the folder in the field If the name of the folder does not exist the folder is created automatically The current date folder will then be created in this folder instead click Finish to complete the method plan go to step 2 or click Next to enter the Advanced Zone for changing parameter val ues See 5 5 Advanced Zone on page 164 Method Wizard 5 Step Action 2 On the Save As page select destination folder in the Method plan tree e Click Create Folder to create a new folder if preferred In the dialog that appears enter the name Click OK e f preferred additional information can be typed in the Notes field Nee NFA Aray Deining lon Tar hange HisTiap HPI miji RERI an le meget Bowes Tempar er tat Stap Crap Fiann Hens tn srie peah Monet JradStro DS Ireid Pd Seep Fokger velect a folder in Methoc plan nesl 3 Enter a name of the method plan and click OK Result The method plan is saved It can now be started from the System Control module on a single or on several separation systems simultan eously e p133 5 Method Wizard 5 2 Working with method plans 5 2 3 Editing a method plan 5 2 3 Editing a method plan Introduction This section describes how to edit an existing method plan Editing a method To edit a method plan plan Step Action 1 In the Method plan list in the Method Wizard select the me
189. ing Protocol and send the list to a printer or export the list to a file Step Action 1 e Open a result file in the Evaluation module e Make sure the pools are satisfactorily adjusted See 7 4 Pooling fractions and adjusting the pools on page 297 e Click the Add to Pooling Protocol button Result The pooled fractions from the active result file is added to the Pooling Protocol 2 e Repeat step 1 to add pooled fractions from other result files 3 e Click the View Pooling Protocol button Result The Pooling Protocol dialog box opens recieve Pretec eee zi hartere to do ancem p fend Seyid Pod Vd Core tet Endod ean tae 7 L asmpie Hermt Aa d i ee osmo i 6 0000 ecm bee swo wW o ww test 0700 00000 6 w00 F Shon hatos M She dotare Demre ot we Boca Coe Wer Evaluation 7 Step Action 4 e Click Show all fractions to display the individual fractions instead of fraction ranges for the pools e Click Show all columns to display all the information columns from the Pool table Possible actions in the Pooling Protocol Note The information in the Pooling Protocol is saved for the individual UNICORN user The Pooling Protocol can only be cleared by clicking the Delete or Delete all button e To delete a single pool select a pool and click the Delete button e To clear the whole protocol click the Delete all button e To print the protocol click the Print butto
190. ing e g System Wash If required replace the tubing and connectors see 8 6 2 Replacing tubing and connectors on page 338 Pump piston assembly leaking Replace the piston assembly in the pump head see 8 6 7 Replacing a damaged pump piston on page 370 Noisy baseline signal See Irregular flow above e p387 9 Troubleshooting and corrective actions 9 3 Monitoring problems Fault Possible cause and action Waves onthe pressure See Irregular flow above curve Irregular pressure See Irregular flow above trace Faulty air sensor Find the possible cause and action for a specific air sensor fault in the table below If the problem remains contact the local GE Healthcare representative Fault Possible cause and action Too high sensitivity e Wrong sensitivity setting Change to low sensitivity in System Settings in Sys tem Control by selecting Monitors Airsensor No response e The air sensor is not connected Check the air sensor cable e The air sensor is disabled Check that the air sensor has not been disabled Select System Settings in System Control and then Alarms Alarm_AirSensor 28 4090 22 AB p388 9 4 Leakage problems Troubleshooting and corrective actions 9 Introduction This section specifies troubleshooting for when there are leakage problems The problems can be related to e Pump leakage e Other leakage in valves tubing conne
191. ing procedure in 6 4 7 Purging the pump and system on page 222 e Locking nut in optical unit not properly tightened Turn the locking nut to the stop position See also 8 6 4 Replacing UV lamp on page 353 e Air in buffers Check that there is no air in the buffers Degas the buffers if necessary e The buffers might be impure Make sure the buffers have been filtered e Air in the UV flow cell There may be air in the flow cell Check that the flow restrictor gives a back pressure of 0 2 MPa see 8 2 1 Checking flow restrictor on page 308 e Dirty UV cell Clean the UV cell see 8 3 2 Cleaning UV cell on page 314 Signal drift See Noisy signal above Instability See Noisy signal above e p 383 9 Troubleshooting and corrective actions 9 3 Monitoring problems 28 4090 22 AB p384 Fault Possible cause and action Low sensitivity e Aging UV lamp Check the lamp run time see 8 3 1 Checking UV lamp run time on page 312 and replace it if neces sary see 8 6 4 Replacing UV lamp on page 353 e UV lamp in wrong position Check that the lamp position and the filter position both are set to the wavelength to be used 280 nm or 254 nm see 8 6 4 Replacing UV lamp on page 353 e The theoretical extinction coefficient too low Calculate the theoretical extinction coefficient of the protein If it is zero or very low at 280 nm the protein cannot be detected Troubleshooting a
192. inity tag removal can be combined with any of the AKTAxpress protocols starting with AC IEX The table below describes the stages in an AKTAxpress affinity tag removal method Stage Description 1 The software assists the user to manually fill the superloop with the re quired protease solution at the beginning of the automated affinity tag removal method Affinity tagged target protein binds to an AC IEX column Unbound protein and other material is washed off with binding buffer Optional An extra wash of the AC IEX column is performed Optional The AC IEX column is washed with cleavage buffer Protease is loaded onto the column from the superloop NIL oO Om EEI aN N The system is set to pause in order to allow on column cleavage incub ation The protease cleaves the target protein from the tag The free target protein is washed out with AC IEX binding buffer or extra wash buffer from the AC IEX column and is collected in the capillary loop s Tagged protease tags and non cleaved protein stays on the column Note If non tagged protease has been used the protease will also be released with the target protein The target protein is further purified 10 Substances still bound to the column i e the affinity tags tagged pro tease and non cleaved target protein are eluted with elution buffer during the post run procedures and collected in the fraction collector at the en
193. injection of selected peaks can not be per formed The delay volume the LoopParameter instruction volume 0 44 ml between the UV lamp and the loop valve has not passed Wait until the volume has passed and try again 28 4090 22 AB p 380 Troubleshooting and corrective actions 9 Problem Possible cause and action When finishing fraction ation using the instruc tions Fractionation Stop and or Peak_FracStop it is not possible to turn the outlet valve to another position than WasteF1 The default delay volume 0 764 ml between the UV lamp and the fraction collector has not passed Wait until the volume has passed and try again Miscellaneous The table below describes miscellaneous problems that may occur during a run Problem Possible cause and action During elution if the maximum number of loops is reached the warning Instruction Ig nored is issued Peaks detected during regeneration of affinity columns if on column tag cleavage was selec ted are not collected in the fraction collector This is due to a delay in the UV watch instruction but the UV watch instruction will still be executed The fraction collector is full and the option Change plate if out of fractions was not selected when creating the method plan in the Method Wizard The flow will be dir ected to a predefined outlet instead Note Always make sure that Change plate if out
194. ins options and parameters related to flow rate volumes and pressures al parameters as described below It is displayed for protocols starting with AC IEX Note If tag cleavage was selected in the first step the Tag Cleavage General page will appear instead See Tag Cleavage General page parameters below for information on which parameters that can be changed Flow rate e Sample Loading Sample loading flow rate e Wash Out Compl Sample loading Flow rate when washing out unbound sample from the column after sample loading e Extra Wash Before Elution only appears if this was selected earlier in the Method Wizard The flow rate when washing out weakly bound proteins before elution e Elution Flow rate during elution of affinity columns Volumes e Complete Sample Loading An extra volume of buffer used to get the entire sample onto the column by pushing the last part of the sample from the injection valve to the column valve p175 5 Method Wizard 5 5 Advanced Zone 5 5 4 Advanced Zone for Purify Affinity lon Exchange AC IEX 1 Elution and AC IEX 2 Elution paramet ers 28 4090 22 AB p176 e Wash Out Unbound Sample Affinity ion exchange buffer volume CV used for washing out sample that did not bind to the column e Extra Wash Before Elution only appears if this was selected earlier in the Method Wizard The wash volume used to wash out weakly bound proteins before elution Pressure Limit Upper
195. interval 10 000 positions 1 year 28 4090 22 AB p422 Capillary loops Fraction collector Capillary loop data Reference information 10 Parameter Data Volume 10 ml Fraction collector data Parameter Data Collector type X Y collector for 96 or 24 deep well mi croplates e p423 10 Reference information 10 2 Technical specifications 10 2 3 Wetted materials 10 2 3 Wetted materials Introduction This section specifies the wetted materials of AKTAxpress Wetted materials The following wetted materials are used in AKTAxpress e PEEK polyetheretherketone Pump Pressure sensor UV monitor Valves Mixer Flow restrictor Online filter Tubing Unions connectors e PTFE polytetrafluoroethylene Pump UV monitor Mixer Flow restrictor e FEP fluorinatedethylenepropylene Tubing e ETFE ethylenetetrafluoroethylene Flow restrictor Unions connectors e PCTFE polychlorotrifluoroethylene Pump Conductivity cell e PP polypropylene Online filter Inlet filter 28 4090 22 AB p424 Reference information 10 PVDF polyvinylidenefluoride Pump PE polyethylene Pump PFR fluororubber Flow restrictor FFKM perfluoroelastomer Pressure sensor Titanium alloy Pump Pressure sensor UV monitor Inlet filter Conductivity cell Quartz glass UV monitor Aluminium oxide Pump Stainless st
196. ion contains metal ion charge solution suggestions that can be used as a starting point for the metal ion charge recharging procedures Metal ion solution guidance is also provided in the instructions for each column and in the purification handbooks Important Metal ion waste shall be collected in outlet F11 Recommended The table below lists the recommended solutions for metal ion charge recharge of solutions chelating affinity columns supported by AKTAxpress See 4 5 1 Descriptions of columns on page 95 for a list of columns supported by AKTAxpress Step Solution 1 Deionized water 2 0 1 M metal ion solution of choice 3 Deionized water 28 4090 22 AB p 122 4 7 6 Introduction Important Recommended solutions Methodology 4 Strip Solution Suggestions This section contains strip solution suggestions that can be used as a starting point for the chelating affinity column strip procedures Strip solution guidance is also provided in the instructions for each column and in the purification handbooks Metal ion waste shall be collected in F11 The table below lists the recommended solutions for strip of chelating affinity columns supported by AKTAxpress See 4 5 1 Descriptions of columns on page 95 for a list of columns supported by AKTAxpress Step Solution 1 Histidine affinity A buffer with 50 mM EDTA 2 Deionized water e p123 4 Methodolo
197. ionation pauses al lowing the microplate to be replaced Note If this box is left unchecked the flow will be directed to a predefined outlet 28 4090 22 AB p 156 Method Wizard 5 5 4 3 Selecting System Procedures page options Introduction The options on the System Procedures page depend on the choice of purification protocol and if on column tag cleavage was included Selecting options The table below describes how to select System Procedures page options Step Action 1 Select Remove Ethanol to remove ethanol from the system before starting the run Result The system flow path will be rinsed with water including the loops e p157 5 Method Wizard 5 4 Using the Purify option 5 4 3 Selecting System Procedures page options Step Action 28 4090 22 AB p 158 For protocols starting with an AC IEX step Select procedures to be included in the purification run If on column tag cleavage was selected the option Guided loading of Superloop will appear See 4 3 1 System and column procedures within a purify method on page 74 for a description of the available system procedures System Procedures Prevarshor T Hemewe Lihanot itom System FE Campin inier Tubes vath Bitte Rayma Manes interacted L2 fustad aning ot Supetiong Papani Mami iarann Upton oveitatle f on column 1g cleavoge wos included haina ey C Oman Sample Indete ahe S amaha Losiny Reges Marna
198. itable protective measures such as wearing protective glasses and gloves resistant to the chemicals used Follow local regulations and instructions for safe operation and maintenance of the system Prepare the required volumes of the buffers and solutions needed in the run see Summary page For best purification results use deionized water and high purity chemicals Filtering of liquids through a 0 45 um filter and degassing the liquids is recommended Note Do not use organic solvents in buffers The mixer is designed for use with water based solutions only If organic solvents are used incorrect gradients or incomplete mixing might occur e Prepare required buffers according to the chosen purification protocol and fill the containers e Place the containers on a suitable shelf under the bench If the containers have caps each cap must have a vent opening to prevent a vacuum from forming as buffer or solution is extracted Equilibration binding elution and washing buffers as well as cleaning solutions are introduced into the system through the inlet tubing A1 A8 B1 and B2 Depending on the type of protocol to be run the buffers and solutions use different inlet tubings The required number of buffer and solutions varies and depends on the type of e p207 6 Operation 6 4 Preparing the system for a run 6 4 2 Preparing buffers and solutions purification protocol and which system and or column pr
199. ity tagged protease was used Run an AC protocol and collect the flowthrough fraction which will con tain the cleaved protein Note If a non tagged protease was used further purification steps will most likely purify away protease cleaved off af finity tag and non cleaved protein from your wanted cleavage protein Perform further purification e If an AC IEX protocol was run in step 1 you want to continue with DS in the further purification Choose for example between DS DS IEX DS IEX GF protocols e If an AC IEX DS protocol was run in step 1 you can continue with for example IEX DS or IEX GF to achieve higher purity of your cleaved target protein p9l 4 Methodology 4 4 Affinity tag removal 4 4 4 Conditions for affinity tag cleavage 4 4 4 Introduction Conditions affect ing cleavage Cleavage time and conditions for on column cleav age using AKTAx press Raising cleavage temperature Buffer sugges tions 28 4090 22 AB p92 Conditions for affinity tag cleavage This section describes conditions important for affinity tag cleavage Protease protein ratio buffer composition time and temperature are all important factors for efficient cleavage All these conditions can be optimized to fit the specific protein Different proteases need different amounts of time for cleavage If cleavage is not performed at the protease s optimal temperature the cleavage time will be
200. ke sure that the column is free of any bound proteins that may elute with the conditions that will be used during the run Affinity columns e By performing a blank run leakage of loosely bound metal ions can be avoided during the purification runs A blank run should always be performed after metal ion charging or stripping of a column lon exchange columns e Provides the ion exchanger with exchangeable counter ions Customized Sys tem Procedures Customized Column Proced ures Methodology 4 Column procedure Description Strip columns Strips metal ions off the first step chelating affinity column using 5 CV strip buffer e g affinity binding buffer with 50 mM EDTA followed by 10 CV water Note Outlet F11 will always be used to collect the metal ion waste Metal lon Charge Re Charges new uncharged or stripped first step chelating charge affinity columns with the appropriate metal ion in three steps 5 CV water 1 CV 0 1 M metal ion solution of choice and finally 5 CV water Note Outlet F11 will always be used to collect the metal ion waste The Customized System Procedures method plan provides cleaning preparation of the chosen tubing loops and valves with up to 5 cleaning solutions one cleaning solution at a time When running the method the system will pause and a message appear when the inlets should be inserted into a new cleaning solution See 4 7 3 Customized CIP Soluti
201. l Volume used to remove ethanol from the column Volume Equilibration Volume used to equilibrate the column e Incubation Time after CIP The time the system is set to pause after the column s has been filled with CIP solution 3 Click Next to go through the pages and change the required values Method Wizard 5 Step Action When the Last page is displayed continue with 5 2 2 Saving the method plan on page 132 ep171 5 Method Wizard 5 5 Advanced Zone 5 5 3 Advanced Zone for Purify 5 5 3 Introduction Entering the Ad vanced Zone 28 4090 22 AB p 172 Advanced Zone for Purify This section gives an introduction to the Advanced Zone for a Purify method plan Note Avoid changing default parameter values in a method plan unless the result is clearly understood Changing a parameter value might require adjustments of other parameter values as well The first page that appears in the Advanced Zone contains a list of parameters that can be changed in the current method plan The method plan is based on the selections made in the first part of the Method Wizard To select parameters 2 Click Next to go through the subsequent pages Changing Purify parameters Method Wizard 5 Step Action 3 On Last Page click Next to save the method plan See 5 2 2 Saving the method plan on page 132 The pages appearing in the Advanced Zone corresponds to the
202. l The life time is 4000 x1000 that is 4 000 000 strokes 28 4090 22 AB p324 8 4 4 Introduction Checking the tubing leakage Maintenance 8 Checking tubing leakage Problems with leaking connectors can be found running a tubing leakage test To check for leakage in the system tubing Step Action 1 Check that the inlet tubing to be tested is properly immersed in a flask with deionized water Start the pump e Select System Control Manual Pump e Select a flow rate of for example 20 ml min e Click Execute to start the flow Select a flow path matching the tubing to be checked for example e InletValve and A1 e OutletValve and F12 e LoopSelection and LP1 e Click Execute to set the valves into position Inspect the UV curve for abnormal peaks see below e 1p 325 8 Maintenance 8 4 Maintenance when required 8 4 4 Checking tubing leakage Abnormal UV Below is an examples of abnormal UV curve due to leaking connectors causing air curve bubbles in the tubing may po 180 o 100 20 0 300 wo ihi 28 4090 22 AB p326 8 4 5 Introduction Procedure Maintenance 8 Running installation test To check the function of the separation system an installation test can be performed on all separation systems for example after a prolonged stop The test can be started to run simultaneously on all systems if required The installation test
203. l chelating columns 1and5 ml Step 1 1 M NaOH 1 CV RT 1 5 CR 0 8 4 Step 2 Deionized water 10 CV RT 1 5 CR 0 8 4 Step 3 30 isopropanol 10 CV RT 0 5 2 5 CR 0 4 2 Step 4 Deionized water 10 CV RT 1 5 CR 0 8 4 Step 5 2 M Nacl 1 CV RT 1 5 CR 0 8 4 Step 6 Deionized water 10 CV RT 1 5 CR 0 8 4 GSTrap columns 1 and 5 ml e p117 4 Methodology 4 7 Solution alternatives 4 7 4 Customized CIP Solution Suggestions Columns Recommended Column Solution Volume Flow rate ml min Step 1 6 M GuaHCl 2 CV RT 1 5 CR 0 8 4 Step 2 GST binding buffer 5 CV RT 1 5 CR 0 8 4 Step 3 1 Triton X 1002 2 CV RT 1 5 CR 0 8 4 Step 4 GST binding buffer 5 CV RT 1 5 CR 0 8 4 1 Recommended to pause 1 hour 2 Alternatively 3 4 CV 70 ethanol The tables below list recommended solutions for the desalting columns supported by solutions for de KTAxpress salting columns See 4 5 1 Descriptions of columns on page 95 for a list of columns supported by KTAxpress Column Solution Volume Flow rate ml min HiTrap Desalting Step 1 1mgpepsin mlin 1 CV RT 5 CR 4 0 1 M acetic acid 0 5 M NaCl Step 2 0 2 M NaOH 2 CV RT 5 CR 4 Step 3 Deionized water 5 CV RT 7 5 CR 6 HiPrep 26 10 De salting Step 1 1 mg pepsin mlin 1 CV RT 10 CR 8 0 1 M acetic acid 0 5 M NaCl Step 2 0 2 M NaOH 2 CV RT 10 CR 8 Step 3 Deionized water 5 CV
204. l ion stripping and recharging of affinity columns 290 create a prepare and maintain method plan including strip 291 create a purify method plan including strip 290 prerequisites 290 strip procedures 290 Method introducing 126 Methodology 59 Method plan create a new 130 deleting 135 editing 134 introducing 126 main selections 131 saving 132 starting 259 ypes 200 using the Prepare and Maintain option 136 using the Purify option 150 Method Wizard buttons 128 introducing 126 croplate recommended 241 Molecular weight 262 Monitors description 411 0 On column tag removal 238 Online help 30 Operation work flow 196 Ordering information 436 Outlet tubing 210 Outlet valve 410 P Peak collection parameters 65 Peak collection parameters when using double loops 69 Peak fractionation parameters 71 Peak handling 64 peak collection parameters 65 peak fractionation parameters 71 Peak max factor 67 Pooling fractions 297 Pooling protocol add pools 300 exporting 301 printing 301 Pools 297 adjusting 299 Pre filling the Superloop with buffer 233 Preparing for on column tag cleavage 238 Preparing the system when using a Superloop 233 Pressure calibration 329 Mi Index checking 324 curve 388 Printing pooling protocol 301 Print report 302 Process description 54 Process flow chart 53 Product numbers 436 Protease calculation 239 descriptions 87 types
205. lamp has a life time of typically Wavelength 254 nm Wavelength 280 nm In room temperature 7000 h 3500 h In cold room 2000 h 2000 h The lamp run time can be checked from UNICORN see 8 3 1 Checking UV lamp run time on page 312 Required material Hg lamp in housing including cable 18 1128 22 e Cross head screwdriver CAUTION Only spare parts approved or supplied by GE Healthcare may be used for maintaining and servicing the system Replacing the UV When replacing the lamp the complete lamp housing including UV lamp should be lamp replaced Lamp housing UV lamp Filter housing 4 Detector housing Flow cell To change the UV lamp including housing e p353 8 Maintenance 8 6 Replacement procedures 8 6 4 Replacing UV lamp Note It is recommended that an GE Healthcare service engineer changes the UV lamp The user can change the lamp but cannot reset the lamp run time in UNICORN WARNING The UV monitor uses high intensity ultra violet light To pre vent injury to eyes do not remove the optical unit while the lamp is ON Step Action 1 Disconnect the power to the system for example by unplugging the mains power cable from the system CAUTION To prevent equipment damage the mains power to the separation system must be disconnected before any cable is plugged or un plugged at the rear of the system 2 Remove the connector UV monitor lamp
206. lan Create os Change Method plan Note Hse New te create a Method plan Set Daaa p 127 5 Method Wizard 5 1 Method Wizard introduction The Method Wiz The table below describes the Method Wizard buttons ard buttons Click To go back to the previous page lt Back go to the next page Next gt finish creating the method plan and save it Finish cancel the settings and close the wizard Nothing is Cancel saved open help texts for the active page Help restore all settings to the default values Enabled on the Set Default first page only delete a selected method plan or folder Delete PRE REE Changing selec When changing a selection made on the Purify pages in the Method Wizard all tions parameter values belonging to column type in the Advanced Zone will be set to default Any changes made earlier in the Advanced Zone have to be made again Note Avoid changing default parameter values in the advanced zone unless the result is clearly understood Changing a parameter value might require adjustments of other parameter values as well 28 4090 22 AB p 128 Method Wizard 5 5 2 Working with method plans About this section This section provides instructions for how to work with method plans In this section This section contains the sub sections below Sub section See Creating a new method plan 5 2 1 Saving the method plan 5
207. leaned prepared Cuttomized System Procedures Sameie ir nta Os Ose Pidter inlets Oa Das Oas On Oe Inket 1 in aaye enabled and tie options hainw utes Al ne rint Duets Inmet Microplate in the Fraction Colector D Cokan Vaive Al Cohen Valve pasione need typsrs tubing 2 C Hotel The system wil Pane arei a message wal appee uinn A rew skann a needed Note Buffer inlet A1 is always used for cleaning of the outlets column valve and loops For each solution step move the A1 and other selected inlet tubings to the appropriate flask containing the next cleaning solution Select Outlets to clean all outlets A microplate must be placed in the fraction collector Select Column Valve to clean all column positions All positions require bypass tubing Method Wizard 5 Step Action 4 Select which loops to clean in the Loops drop down list If Loop 5 is se lected it must be a capillary loop If no loops should be cleaned select OFF 5 Select Number of Cleaning Solutions to use 6 Proceed to 5 2 2 Saving the method plan on page 132 e p147 5 Method Wizard 5 3 Using the Prepare and Maintain option 5 3 5 Selecting Customized Column Procedures options 5 3 5 Selecting Customized Column Procedures options Customized To select Customized Column Procedures options Column Proced ures options Step Action 1 On the Customized Column Procedures page select
208. let click Next Breakpoint instead of filling it with liquid 8 Repeat step 1 7 to fill the other inlet tubings 28 4090 22 AB p 216 6 4 6 Introduction Operation 6 Filling inlets manually using UNICORN If there is a lot of air in most all inlet tubings and only a few inlets should be filled with liquid fill the inlets manually using UNICORN Note If all most inlet tubings should be filled with liquid consider filling the inlets using a method plan see 6 4 5 Filling inlets manually using a method plan on page 214 Filling inlettubing To fill the inlet tubing A1 A8 and 1 S4 A1 A8 and S1 S4 Step Action 1 Check that the inlet tubings to be filled is properly immersed in the correct containers flasks tubes 2 In System Control select the system which tubings should be filled 3 To fill the A1 inlet tubing or any other inlet tubing first switch the valves e Select System Control Manual Flowpath e Select InletValve and A1 or whichever inlet tubing to be filled e Click Execute to set the valves to the correct positions reli e TO Ache agate P tha u chast et the panamanie hait will he ipint dang nating mat p 217 6 Operation 6 4 Preparing the system for a run 6 4 6 Filling inlets manual 28 4090 22 AB p 218 ly using UNICORN Step Action 4 Connect a male Luer syringe of at least 20 ml to a purge valve Tw
209. lick the symbol of the system to be connected Result The system is connected and the symbol changes to a white status indicator The run data curves and logbook for the system is shown Tile Yiew Manual System lelp Hold Pause Continue End ay Instant Run l Status indicator The table below shows how the indicator colors relate to the run status colors Indicator color Run status Example of events White End sys1 Q p 197 6 Operation 6 2 Connecting a system Indicator color Run status Example of events yellow flashing Green Run or Manual sys1 Q Yellow Hold Sample loading sys1 Q Red Pause Pump wash sys1 Red Background color Error Air detected e g running out of buffer Changing system The currently selected separation system is indicated by orange bars above and below display the system symbol to the left To display the status for another separation system than the currently selected left click the appropriate system symbol The run status will be displayed in the right pane Disconnectinga Right click the symbol of the system to be disconnected and select Disconnect separation sys tem 28 4090 22 AB p 198 Result The system is disconnected and the symbol changes Operation 6 p 199 6 Operation 6 3 Creating method plans 6 3 Introduction Method plan types Method plans when performi
210. lifesciences com contact GE Healthcare Bio Sciences AB Bj rkgatan 30 751 84 Uppsala Sweden www gelifesciences com GE and GE monogram are trademarks of General Electric Company Amersham GSTrap HiLoad HiPrep HisTrap HiTrap Mono Q Mono S MonoBeads PreScission RESOURCE Sephacryl Sephadex Sepharose SOURCE Superdex Superloop UNICORN are trademarks of General Electric Company or one of its subsidiaries IMAC Sepharose products Ni Sepharose products and Fe Sepharose products These products are sold under a license from Sigma Aldrich under patent number EP 1276716 Metal chelating compositions and equivalent patents and patent applications in other countries GST Gene Fusion Vectors A license for commercial use of GST Gene Fusion Vectors under US patent 5 654 176 and equivalent patents and patent applications in other countries must be obtained from Millipore Corp formerly Chemicon International Inc AcTEV is a trademark of Life Technologies Corporation Excel Microsoft and Windows are registered trademarks of Microsoft Corporation TALON is a registered trademark of Clontech Laboratories Inc All other third party trademarks are the property of their respective owner Any use of UNICORN and AkTAxpress software is subject to GE Healthcare Standard Software End User License Agreement for Life Sciences Software Products A copy of this Standard Software End User License Agreement is available on request UNICO
211. ludes purification with affinity chromatography followed by desalting and finally ion exchange chromatography Abbreviations found in this manual Abbreviation Chromatography technique AC Affinity chromatography DS Desalting IEX lon exchange chromatography GF Gel filtration KTAxpress provides protocols for purification in up to four steps The protocols are fully automated and can be combined with on column tag cleavage When selecting options for a purification run the options are based on the protocol AC DS IEX GF but it is still possible to run any kind of protocol See 5 1 Method Wizard introduction on page 126 for more information Samples are loaded automatically via the system pump for protocols starting with an AC or IEX step For protocols starting with a DS or GF step the sample s is pre loaded manually with a syringe into the capillary loops Superloop in the beginning of the run Protocols where on column procedure is included contain manual filling of the Superloop with protease solution before or in the beginning of the run Protocols starting with AC or IEX can be combined with on column tag cleavage in the first step It is however recommended to perform on column tag cleavage only for protocols that start with an AC step unless you know that your tag specifically binds to the IEX column Protocols starting with an IEX DS or GF step are suitable for proteins where for exampl
212. lumns e Strip solutions EDTA and water See also the column instructions and or 4 7 6 Strip Solution Suggestions on page 123 e Amethod plan for cleaning the columns The illustration below gives an overview of how to include Strip in the purification method plan See 5 4 Using the Purify option on page 150 for detailed information Select the first step affinity column for wich to include a Strip procedure by checking the box Select the appropriate Strip procedure Operation 6 Create a Standard The illustration below gives an overview of how to include Strip in the Standard System System and and Column Procedures method plan See 5 3 Using the Prepare and Maintain option Column Proced on page 136 for detailed information ures method plan including Strip I matography Step box Select o a e Cheloting ond column type o D Select o a Strip procedure o e T oy fe Select x the positions occupied by the column type 1 5 i a a a Jo ogaoga Strip the columns Note using the method plan Note The Metal lon Stripping inlet tubing A8 have to be filled before starting the stripping run See 6 4 6 Filling inlets manually using UNICORN on page 217 The metal ion waste that exits the system through tubing F11 should be collected in a separate waste bottle To strip the columns Step 1 Action
213. luted in order to get the set target concentration Import File Location The path to the folder where import file is stored The import file might contain information about for example extinction coefficient and molecular weight of the proteins to be used For more information on the import file see 10 3 Import file format on page 426 p191 5 Method Wizard 5 5 Advanced Zone 5 5 10 Hints on optimizing run parameters 5 5 10 Introduction Low protein amounts Peak volume 28 4090 22 AB p 192 Hints on optimizing run parameters This section contains hints and directions for how to optimize method plans for troublefree operation of KTAxpress Most of the adjustments are made in Advanced Zone in the Method Wizard To obtain as good recovery as possible when running samples with low protein amount it is recommended to change the settings on the following pages in the Advanced Zone as described in the table below Pagels in Advanced Parameter to be Recommended setting Zone changed change Fractionation Peak Fractionation Al Use Level if the baseline gorithm is stable at the final puri fication step Fractionation Start Level Start Slope Decrease the values for Peak Max Factor Minim um Peak Width End Level End Slope these parameters Peak Collection only for intermediate steps Watch Level Greater than Watch Slope Great er than Watch Level Less than Decreas
214. lve has no user replaceable parts C3in C4in Bypass Injection valve Clout C2out C3ou C2in Clin UV monitor Bypass C5out C4out at startup The six positions gives the following flow paths Column 1 position C4in C3in C2in C5in Clin Clout C5out C4out C2out C3out ou Column 4 position C4in C3in C2in C5in Clin Clout C5out Gout C2out C3out C4ou Column 2 position C4in C3in C2in C5in Clin Clout C5out C4out C2out C3out ou Column 5 position C4in C3in C2in C5in Clin Clout C5out C4out C2out C3out u Default position Column 3 position C4in C3in C2in C5in Clin Clout C5out C4out C2out C3out ou Bypass position default C4in C3in C2in C5in Clin Bypass Bypass Clout C2out c C5out C4out Sout Loop valve The loop valve is a 2x6 way 14 port valve used for directing a flow to any of up to five sample capillary loops and one bypass route The loop valve is identical to the column valve The valve has fingertight connectors The valve has no user replaceable parts e p409 10 Reference information 10 1 System description 10 1 2 Valves L2 L3 L4 Ll L5 Injection valve Bypass Bypass Injection valve L5 L1 L4 L2 Default position L3 at startup For flow path see 10 1 2 Valves on page 407 Outlet valve The outlet valve is a 12 way 13 port valve For each 12 positions an outlet port is connected to the central inlet port The valve has fingertig
215. lve has no user replaceable parts e p407 10 Reference information 10 1 System description 10 1 2 Valves Injection valve Default position at startup The valve is used for buffer selection and sample loading For flow path details see 3 2 2 Liquid flow path on page 38 The injection valve is a 4 way 8 port valve used for main flow direction in the system The valve is also used for manual injection of samples and filling Superloop through a syringe The valve has fingertight connectors The valve has no user replaceable parts Center j e Y 6 5 startup inject oe Default position at The four positions of the valve gives the following flow paths Inject position Reinject position Manual Manual inject Waste inject Waste Loop valve Loop valve upper port Waste upper port Waste Outlet Loop valve Outlet Loop valve valve lower port valve lower port Column Inlet from Column Inlet from valve pump valve pump Load position Waste position Manual Manual inject Waste inject Waste Loop valve Loop valve upper port Waste upper port Waste Outlet Loop valve Outlet Loop valve valve lower port valve lower port Column Inlet from valve pump Column Inlet from valve pump 28 4090 22 AB p 408 Column valve five columns and one bypass route Reference information 10 The column valve is a 2x6 way 14 port valve used for directing a flow to any of up to The valve has fingertight connectors The va
216. m and column procedures within a purify method on page 74 Customized System Column Procedures prepare and maintain methods Run system and column procedures within a customized prepare and maintain run if any of the options below should be performed e Many columns of the same type should be prepared cleaned e Customized preparation cleaning of the system with up to five solutions should be performed Customized System Procedures method plan e Customized preparation equilibration of the columns with up to nine solutions should be performed Customized Column Procedures method plan Standard System and Column Procedures prepare and maintain method Run system and column procedures within the standard system and column procedures prepare and maintain method if e The columns to be prepared are going to be used in a purification run or should be maintained after a purification run and e The procedures to be run are only available within the Standard System and Column Procedures method plan cannot be included within a purification run These are System Procedures Rinse outlets p79 4 Methodology 4 3 System and Column Procedures 4 3 2 System and column procedures in prepare and maintain methods Standard System and Column Pro cedures 28 4090 22 AB p 80 Wash System and Loops Fill Buffer Inlets with Buffer automatically Column Procedures Strip Metal lon Charge Blank Run Strip CIP Re
217. me parameters can be changed as described below ACAEX 1 Tag Cleavage Elubor Viae Congiote Sam ie onting ib P eg i Wash fhd I inbesun Sampie p awa cy F matbenten Cevcvnge Rudter Sf sansa oy Detak Protesoe Iree ion or p aan ry Protease Inemtiun Dulay OS 0 990088 mi Ehdur Laves Froter id 0 saas Cv Airey Koga tron fe s3333 CY Ema Wash Beto kor bs jo S933353 Cv Volumes e Complete Sample Loading An extra volume of buffer used to get the entire sample onto the column by pushing the last part of the sample from the injection valve to the column valve e Wash Out Unbound Sample Affinity ion exchange buffer volume CV used for washing out sample that did nor bind to the column e Default Protease Injection The protease volume for each column e Protease Injection Delay The volume between the loop valve and the column valve e Elution Cleaved Protein The affinity ion exchange binding buffer volume used for elution of cleaved protein e Affinity Regeneration The volume of affinity elution buffer used to regenerate the affinity column i e elution of tags tagged protease and uncleaved protein AC IEX 1 Peak Collection para meters AC IEX 1 In cluded Proced ures parameters Method Wizard 5 e Extra Wash Before Elution The wash volume used to wash out weakly bound proteins before elution For more information on tag cleavage see 4 4 Affinity tag removal on page 84 S
218. mended for the columns used Note Changing the flow rate will change the slope of an eluting peak The default values for detecting peak start and peak end are set to match typical peaks eluting from the supported columns The slope and levels values though might need to be adjusted if other parameters are changed The slope of a peak is changed if e the flow rate is changed e the gradient slope is changed The default start and stop level values might need adjustment if e small peaks are expected e large peaks with peak volumes gt 7 5 ml unless using a double loop e the peaks are broadened due to for example a more flat gradient Note If the level values are decreased for very large or broad peaks the peak volume collected might be too big for the loop available or exceed the loading volume of the next column A double loop allows collection of larger peaks A large peak collected in two single loops might result in a double peak eluted in the next purification step By using a double loop instead the entire peak can be collected in one loop thus eliminating the double peak in the next step See 4 2 2 Peak collection parameters when using double loops on page 68 The following recommendations apply to protocols that include gradient elution e Make sure that the baseline is stable This can be obtained by using pure imidazole in the AC elution buffer e p193 5 Method Wizard 5 5 Advanced Zone 5 5 10 Hints
219. mode press Continue Result A dialog box appears requesting the syringe to be fitted e Filla syringe with at least 5 ml of affinity binding buffer or the buffer used in the protease sample solution e Fit the syringe in the fill port and click Continue Note Do not inject the buffer before clicking Continue fsvetem tonal srs i mera a a inses a nytinge with gt Senl butter n the inection Vaive Press CONTINUE R coe ee y GE a 3 A new dialog box appears requesting the buffer to be injected e Inject the buffer and click Continue Do not remove the syringe before clicking Continue Jeqect a loast 5 mi butter Do not remove the seinge Pres CONTINUE R _ ae eee E 1p 235 6 Operation 6 4 Preparing the system for a run 6 4 11 Preparing the system when using a Superloop Step Action 4 A new dialog box appears requesting a syringe with protease sample solution to be fitted e Remove the syringe from the fill port e Fill the syringe with the requested amount of protease sample solution re fit it in the fill port and click Continue Do not inject the protease sample before clicking Continue insent syge wath prosepie colhon Prest CONTINUE R PME ums ey E 5 A new dialog box appears requesting the protease sample solution to be injected e Inject the protease sample solution and click Continue to proceed the run Do not remove the syringe The protease sample will automatically be loaded onto
220. munication are located on the rear of the system ors UniNet network Conductivity flowcell UV monitor optical unit Air sensor AC power inlet UV monitor lamp 28 4090 22 AB ep 32 AKTAxpress overview 3 Location of The columns can be attached to AKTAxpress in different places depending on the columns size of the column Column type Location Small columns Small columns are connected to the column block either directly or by using different unions depending on the type of column See 6 4 9 Connecting columns and tubing on page 225 for information about the unions The block has five ports In some applications two columns are connected in series p33 3 AKTAxpress overview 3 2 Separation system overview 3 2 1 Location of the com ponents 28 4090 22 AB p34 Column type Location Large columns 30 and 50 mm diameter Columns with o d 30 mm are attached by using the left hand column holder with snap locks see figure below The holder can hold two columns Note When using one of the HiPrep 16 60 Sephacryl S 100 S 200 S 300 HR gel filtration columns 0 d lt 30 mm insert the HiPrep 16 xx Column Wrap into the column holder before attaching the column Columns with o d 50 mm can be attached using an op tional column holder made for these columns This column holder is ordered separately and should be mounted in place of the standard column holder
221. n Step Action 1 Change the required values on the Advanced Settings Purge Pump with Methanol page Advanced Seltmys Purge Pump with Methanol Ware Wash Voha Step 1 T Wia 10 989 st Methanol Volume Step gt 10 M SITA mi Methanol Volume Step 2 2 UW SIIIN mi Water Wash Volume Step 2 wo W SIITA mi Water Wash Volume Step 1 Volume used to clean the pump with water before it is purged with methanol e Methanol Volume Step 1 Volume used to purge the pump with methanol e Methanol Volume Step 2 Volume used for the second methanol step e Water Wash Volume Step 2 Volume used to clean the pump with water after the methanol wash 2 Click Next The Last Page is displayed Continue with 5 2 2 Saving the method plan on page 132 p 167 5 Method Wizard 5 5 Advanced Zone 5 5 2 Advanced Zone for Prepare and Maintain Changing Custom ized System Pro cedures settings Changing Stand ard System and Column Proced ures settings 28 4090 22 AB p 168 To change parameters in the Customized System Procedures Step Action 1 Change the required values on the Advanced Settings Customized System Procedures page Advanced Setting Lustumszed Sprlem Procedures Sangle indet FA Vane E W 9999991 mi Pairs trdet FA Vodne ie po E mi Lanp Wash vaime w Pain sa a Sample Inlet Fill Volume Volume used for filling the sample inlet tubing Buffer Inlet Fill Volume Volume u
222. n and a smaller risk for salt contamination The examples below illustrates the advantage of collecting large peaks in double loops instead of in separate loops If collecting the peak in two separate loops re injection of the complete eluate from two loops onto desalting or gel filtration columns will give a characteristic double peak This peak is caused by wash out and delay volumes between emptying the two loops not from sample impurity mAU Peak from the AC step Double peak from the DS GF step min Methodology 4 Example of using If collecting the peak in a double loop re injection of the eluate onto desalting or gel a double loop for _ filtration columns will give a characteristic single peak collecting peaks with volume gt 7 5 mAU ml Peak from the AC step Peak from the DS GF step min Modifying the Depending on the protocol and included procedures the parameters for peak collection parametersinthe need to be changed when using double loops The table below describes which the method plan parameters and pages that are affected The parameters are changed in the Advanced Zone See also 5 5 3 Advanced Zone for Purify on page 172 Parameter to change On the pages New Value Max Volume in Each Loop AC IEX 1 Peak Col 15 ml lection e AC IEX 2 Peak Col lection e DS 1 Peak Collec tion Allow Collection of Single e AC IEX 1 Peak Col No Peaks in Sever
223. n clarified by centrifugation and or filtration prior to sample loading Are the correct buffers used for the chosen columns and proteins Are the chosen columns suitable for the chosen target proteins The pH of some buffers changes with the temperature p373 9 Troubleshooting and corrective actions 9 1 Introduction to troubleshooting Note The mixer is designed for use with water based solutions only If organic solvents are used incorrect gradients or incomplete mixing may be the result 28 4090 22 AB p374 9 2 Introduction Pressure prob lems Troubleshooting and corrective actions 9 Problems during a run This section specifies troubleshooting for problems related to sample loading and elution It also describes some problems that may arise when running methods that have been created in the Method Editor in UNICORN Find the possible cause and action for a specific problem in the table below Problem Possible cause and action High back pressure e Check the calibration of zero pressure see 8 5 1 Calibration of zero pressure on page 329 e Check the flow restrictor see 8 2 1 Checking flow restrictor on page 308 e Make sure that the samples have been centrifuged and or filtered through a 0 45 um filter and that no precipitation has occurred prior to sample loading e A5mlaffinity column instead of a 1 ml column is recommended for large sample volumes more than 20 30 ml e Clean
224. n page 259 Operation 6 6 8 4 Customized cleaning of the system Introduction Customized cleaning of the system includes cleaning of the system with up to five solutions It is performed by running the Customized System Procedures prepare and maintain method plan The system will be automatically cleaned It is possible to select to clean the sample inlets buffer inlets outlets column valve and loops in the method plan Warnings and Note Do not leave the system with salt buffer in the flow path It might damage notes the pump WARNING When using hazardous chemicals take all suitable protective measures such as wearing protective glasses and gloves resistant to the chemicals used Follow local regulations and instructions for safe operation and maintenance of the system WARNING T When using hazardous chemicals make sure thot the entire system has been flushed thoroughly with bacteriostatic solution for example NaOH and distilled water before service and maintenance Prerequisites The following are required for automated customized system cleaning Cleaning solutions The operator moves the inlet tubing from solution to solution See 4 7 3 Customized CIP Solution Suggestions System on page 116 for information on recommended cleaning solutions A Customized System Procedures method plan for cleaning the system including selected sample and buffer inlets loops and outlet
225. n parameter values and click OK p 265 6 Operation 6 6 Starting a run 6 6 2 Starting a manual run 28 4090 22 AB p266 Note Note Before starting a method run created in the Method Editor using a 24 well microplate for fractionation select the Specials radio button the SetPlateType instruction and choose the 24_wells radio button in the SetPlateType Parameters area See the UNICORN Reference Manual for AKTAxpress for information about method runs that have been created in the Method Editor Avoid changing other default system settings parameter values unless the result is clearly understood 6 7 Overview Viewing the run Operation 6 During a run This section describes how to view the progress of the run how to manually select a peak for the next purification step and how to change parameters during a run The progress of the run can be viewed in detail in System Control By clicking the separation system symbol on the left the current status for the particular system can be displayed Up to three view panes Run Data Curves and Logbook can be displayed showing different aspects of the run in real time e The Run Data view pane displays the current values for selected run parameters e The Curves view pane displays the monitor signal values graphically e The Logbook view pane shows the actions as the run proceeds All actions and unexpected conditions are logged with date time
226. n seal p 361 8 Maintenance 8 6 Replacement procedures 8 6 6 Replacing pump piston seal Replacing piston seal and mem brane 28 4090 22 AB p362 To replace the piston seal Step Action 1 Change solvent to deionized water and flush out all salt Move all input buffers bottles below the level of the pump heads to prevent siphoning CAUTION REPLACING SPARE PARTS Read the instructions carefully Check the orientation of each part before continuing with the next instruc tion Beware that for example some individual parts of the pump head can be assembled incorrectly 2 Disconnect the electrical power to the system to be maintained 3 Remove the tubing e Disconnect and remove the tubing from the pump head outlets upper ports e Remove the tubing of the piston seal rinsing system plug in fittings 4 Unscrew the two white knurled screws under the pump to release the support bracket that locks the inlet manifold into the inlet valves M 5 Gently lower and remove the complete manifold Maintenance 8 Step Action 6 e Using the hex key unscrew and completely remove one of the two hex screws locking the pump head in position e When unscrewing the second locking screw push firmly on the front face of the pump head to compensate for the pressure of the piston return spring Hold the pump head firmly to prevent it from twisting gd is f P UY Z Me e
227. n some cases however the choice may be affected by the column choices in the surrounding steps Therefore the procedure of choosing columns below is described per chromatography step ending with an example of how to choose column combinations for a four step protocol Questions to be considered before choosing columns How much purified protein is needed after the final purification step Protein loss increases with each successive purification step e How much sample is to be loaded e What purity and resolution is required Recommended column choice for the AC step Amount of target protein Which target protein tog Which target protein tag Use in AC step Histrap HP Gstrap 48 HisTrap HP GSTrap 48 1ml imi Sml Sml Comments on alternative column choices p101 4 Methodology 4 5 Column alternatives 4 5 2 Choosing columns Choosing column for the DS step 28 4090 22 AB p 102 Step Comments on alternative column choices AC Overloading loading more sample on a column than specified of an affinity column can be performed to minimize non specific binding It often results in a purer end product AC If there is a low expressed target protein in a large sample volume consider using a 5 ml HiTrap column to reduce the risk of column clog ging or protein degradation due to time consuming sample loading AC To save time HisTrap FF crude can be used Then no clarification
228. n the containers according to the table below Summary page Inlet tubing Buffer or solution Al See the Summary page for the specific run A2 lon exchange binding buffer 28 4090 22 AB p 208 Operation 6 Inlet Buffer or solution tubing A3 0 2 M NaOH A4 See the Summary page for the specific run A5 Water A6 0 5 M NaOH A7 e 1 0 M NaOH for CIP of chelating columns or e 6M Guanidine HCI or Triton X 100 for CIP of GSTrap columns A8 20 Ethanol B1 Affinity ion exchange elution buffer B2 See the Summary page for the specific run ST S2 S3 Metal ion charging solution e g 0 1 M NiSO S4 Metal stripping solution e g histidine affinity A buffer with 50 mM EDTA Note Inlet tubing S3 metal ion charging solution must be manually filled before starting the charging run Note The waste from the metal ion charging and stripping runs is collected through outlet tubing F11 e p209 6 Operation 6 4 Preparing the system for a run 6 4 3 Preparing outlet and waste tubing 6 4 3 Preparing outlet and waste tubing Introduction This section describes where the outlet and waste tubings should be immersed The outlet and in The illustration below shows the location of the outlet and injection valves on the jection valves instrument as well as detailed views of the tubing from the valve ports Outlet valve utlet valve Injection valve
229. n to print the protocol on the default Windows printer e To save the protocol as a file click the Export button to save the protocol in one of the following formats text txt Excel xIs HTML htm XML xml 5 e Click the Close button to close the Pooling Protocol dialog box Result If the protocol was exported or only edited the dialog box will close If the protocol was printed a dialog box will open asking if the list is to be deleted and a new one started e p301 7 Evaluation 7 6 Printing report 7 6 Introduction How to print a standard report 28 4090 22 AB p302 Printing report Reports can be printed after a completed run To find information on how to create reports see UNICORN KTAxpress User Reference Manual This section describes how to print reports from runs with AKTAxpress The table below describes how to print a report in the Evaluation module using report formats Step Action 1 Open a result file 2 e Select File Report or e Click the Report icon Result The Generate Report dialog box opens Genes ate Kaper Glebe spana dramagile L nop Pa or paanan ory 3 e Select xpress 1sample xpress 2samples xpress 3samples or xpress 4samples report format depending on how many samples are included in the result file Report formats ending with Loop e g xpress 1sample Loop should be selected for runs where the sample s h
230. nal is out of range See section 9 3 Monitoring problems on page 382 4 UV lamp has been on for less time than specified in WarmUpTime The recommended warm up time is 60 min The parameter WarmUpTime is found in System Settings Alarms in UNICORN 5 The flow is lower than specified in MinFlow MinFlow might be too low The parameter MinFlow is found in Manu al Pump Flow in UNICORN 6 The pressure is out of range Related to PumpWash SystemWash or LoopWash If the pressure is too high press Continue aborts the wash and repeat the wash If problem remains check system loops for blockage 51 57 System error nternal error Restart system If problem remains contact service 60 61 System error nternal error Restart system If problem remains contact service 62 Illegal mode exchange t could be caused by a WATCH instruction with action CONTINUE that occurred when the system was in RUN state Check the method Other wise there could be an internal error Restart system If the problem re mains call service 100 105 System error Internal error Restart system If problem remains contact service 106 Pressure calibration error Check that the system is at zero pressure 107 Pump synchronization error By pressing END the pump will be synchronized If this doesn t help re start the system If problem remains contact service 200 218 System error Internal error Restart system If problem remains contact service 219 Instruc
231. ncluding the system procedure Fill Sample Inlet Tubings with Buffer Other inlets are automatically filled during run by System Wash 5 4 3 Selecting System Procedures page options on page 157 Operation 6 If then For instructions see air is present in the sys tem remove the air manually by purging the pump and system using Pump Wash or System Wash from UNICORN 6 4 7 Purging the pump and system on page 222 e the system has been left unused for a week or longer or e the pump has been run dry both pump heads must be purged with methanol 6 4 8 Purging the pump with methanol on page 223 When performing a run using a method plan an initial system wash will automatically be included e p213 6 Operation 6 4 Preparing the system for a run 6 4 5 Filling inlets manually using a method plan 6 4 5 Introduction Create the meth od plan Fill the inlets 28 4090 22 AB p 214 Filling inlets manually using a method plan If there is a lot of air in most all inlet tubings fill the inlets with liquid using a method plan This procedure is divided into two steps e Create the method plan Fill Inlets Using Syringe e Fill the inlets using a syringe Note If only a few inlet tubings should be filled with liquid consider filling the inlets one at a time from UNICORN see 6 4 6 Filling inlets manually using UNICORN on page 217 The illus
232. nd collected in a flowthrough collection flask 4 When using several samples during the run step 1 3 are repeated serially for all samples before proceeding to the next step if selected in the Method The table below describes the elution procedure of the AC columnis Step Description 1 A LoopWash of all loops is performed with IEX binding buffer 2 An extra wash optional is performed with AC wash buffer before elution to wash out unbound sample If peaks are detected they will be collected in F5 F6 3 The AC column is eluted with a step gradient using AC elution buffer and the peaks collected in loops The largest peak is selected p55 3 AKTAxpress overview 3 3 Purification process overview 3 3 2 Description of the process steps Step Description 4 The nonselected peaks are emptied from the loops to the collection tubes in outlet F7 F8 one for each sample The loops are then washed with IEX binding buffer to waste Desalting The table below describes the desalting procedure of the collected peak volume Step Description 1 A SystemWash is performed to fill the system with IEX binding buffer 2 The AC peak collected in the loop is loaded onto the DS column peak volume flush volume 3 The DS column is eluted with IEX binding buffer and the peak volume collected in a loop 4 If the whole peak volume cannot be loaded onto the DS column the exce
233. nd corrective actions 9 Faulty conductiv Find the possible cause and action for a specific conductivity curve fault in the table ity curve below If the problem remains contact the local GE Healthcare representative Problem Possible cause and action Baseline drift or noisy signal e Leaking tubing connections Check for leaking tubing connections and correct see 6 4 14 Checking the tubing on page 243 e Air in the conductivity flow cell There may be air in the flow cell Check that the flow restrictor gives a back pressure of 0 2 MPa see 8 2 1 Checking flow restrictor on page 308 e Column not equilibrated Equilibrate the column If necessary clean the columns using a method plan for Column cleaning see 6 8 5 Cleaning of columns Overview on page 283 e Air might be trapped in the pump Purge the pump according to the procedure in 6 4 7 Purging the pump and system on page 222 e Dirty conductivity cell Clean the conductivity cell see 8 3 3 Cleaning con ductivity cell on page 317 Waves on the conduct ivity curve e Air might be trapped in the pump Purge the pump according to the procedure in 6 4 7 Purging the pump and system on page 222 Conductivity measure ment with the same buffer appears to change over time e Dirty conductivity cell Clean the conductivity cell see 8 3 3 Cleaning con ductivity cell on page 317 e The ambient temperature might have decreased or increa
234. ng purification Purify method plans 28 4090 22 AB p200 Creating method plans This section gives a brief description of which settings and method plans are frequently used when operating AKTAxpress There are two main types of method plans available e Prepare and Maintain Preparation and maintenance of the system and columns e Purify Purification of protein When performing purification using AKTAxpress several system and column procedures can be performed before and after the purification These procedures may e require separate prepare and maintain method plans to be run before and or after purification or e be included as preparation or post run procedures within a purification run To perform a purification create a purify method plan according to the instructions in 5 4 Using the Purify option on page 150 When creating the purify method plan e include the appropriate system and column procedures to be run See 4 3 1 System and column procedures within a purify method on page 74 for information about which system and column procedures that can be included inarun e if double loops will be used in the run make sure to change the appropriate loop parameters in the Advanced Zone See 4 2 2 Peak collection parameters when using double loops on page 68 Procedures fre quently used re quiring prepare and maintain method plans Operation 6 Some procedures can only be run in prepare an
235. ng the system will pause and messages ap pear to guide you through the operation Fill Sample Inlet Tubings Fills the sample inlets with buffer Fill Buffer Inlet Tubings Fills the buffer inlets with buffer Fill System Loops Outlets with Ethanol After the run has been completed the system is washed with water and filled with 20 ethanol The capillary loops used and the fraction collector tubing will also be filled Column Procedures The column procedure combinations that can be run are listed below See the table below for a description of each possible procedure separately Note The column procedure combinations that also can be included in a purification run are marked with a star e Remove Ethanol all column types e Fill Columns with Ethanol all column types e Equilibrate all column types e CIP Re equilibrate all column types except chelating columns e Blank run AC IEX columns only e S rip first step chelating affinity columns only e Metal lon Charge Blank Run first step chelating affinity columns only e Strip Recharge Blank Run first step chelating affinity columns only e Strip CIP Recharge Blank run first step chelating affinity columns only e Strip CIP Re equilibrate first step chelating affinity columns only p8l 4 Methodology 4 3 System and Column Procedures 4 3 2 System and column procedures in prepare
236. nnectors included in the separation system All tubing located before the pump in the flow path sample and inlet tubing has inner diameter i d 1 6 mm and outer diameter o d 1 8 This tubing is connected with M6 fittings except for tubings connected to the pump air sensor and switch valves where 5 16 fittings are used The tubing material is FEP All tubing located after the pump in the flow path system and outlet tubing has i d 1 0 mm and o d 1 16 This tubing is connected with narrow headed fingertight connectors The tubing material is ETFE except the outlet tubing to the fraction collector which is made of PEEK The system also includes stop plugs to be used in non used ports in the valves and the column adapter The illustration shows the three types of tubing fittings used in AKTAxpress Ferrule Se O o LA nI 5 16 fitting M6 fitting Narrow headed fingertight connector e When connecting an M6 or a 5 16 fitting the orientation of the ferrule must be correct see the illustration above e When connecting a fitting the tubing must be inserted fully into the connection port before tightening the fitting The inlet and outlet tubing has the following length Tubing Tubing designation Length Buffer tubing A1 A8 B1 B2 1 5 m Fraction outlet tubing F3 F11 1 5m p39 3 AKTAxpress overview 3 2 Separation system overview 3 2 3 Tubing and connectors Tubing labels 28
237. not exceed the maximum sample loading volume of the column See 5 5 7 Advanced Zone for Purify Peak Collection on page 186 for information on settings These page contains options and parameters related to flow rate pressure and volumes Flow Rate Flow rate during the gel filtration Pressure Limit Upper pressure limit during the desalting gel filtration step Included Proced ures parameters Method Wizard 5 Elution Volume before Fractionation Volume used for elution of the gel filtration desalting column before the peak fractionation starts Elution Volume with Fractionation Volume used for elution of the gel filtration desalting column after the peak fractionation has started Peak injection only available on the DS GF Last Step page e Peak Injection Flush Volume The extra volume besides the peak volume used to flush the loops when loading the sample onto the desalting gel filtration column e Peak Injection Max Volume The maximum volume to be injected onto the desalting gel filtration column Note The maximum volume is set up to make sure that the peak volume flush volume do not exceed the maximum sample loading volume of the column Depending on the column preparation and post run procedures that was included in the purification run selected earlier in the Method Wizard different parameters will appear The same type of settings is however available for the different procedures For each proced
238. ns 13 CE certification 12 Cell constant 334 Changing parameters 272 Checking USB CAN driver 397 Check valve replace 358 Check valves cleaning 320 Chemical resistance 432 Choosing buffer 109 Choosing CIP solution 113 114 116 117 Choosing columns 101 Choosing metal ion charge solution 122 Choosing protocol considerations 61 Choosing strip solution 123 Chromatography abbreviations 59 CIP columns 284 288 system 278 281 CIP solution choosing 113 114 116 117 Cleaning columns 283 system 276 319 Cleavage conditions 92 Cleavage time 92 Column choice AC DS IEX GF 105 AC step 101 DS step 102 GF step 103 IEX step 103 Column heater 92 240 Column procedures before a purification run blank run 254 conditioning 254 equilibration 254 Columns alternatives 95 choosing 101 connecting 225 e p445 Index descriptions 95 for affinity chromatography 95 for desalting chromatography 96 for gel filtration chromatography 98 for ion exchange chromatograpy 97 unions 229 Column types 95 Column valve 409 Component specifications 420 Conductivity curve 385 Conductivity cell calibration 331 Connect a separation system 197 Connecting double loops 231 Connecting the Superloop 234 Connection guide 229 Connectors fingertight 341 M6 341 Content description 25 Cutting tool 340 D Description of affinity tag removal processes 89 affinity tags 85 buffers 107 columns
239. ns prepacked with Sepha dex G 25 Superfine For fast buffer ex change and desalting of samples with a volume of lt 3 ml 1 If you expect peaks larger than 7 5 ml from the affinity step we recommend using a double loop See 4 2 2 Peak collection parameters when using double loops on page 68 for more information lon exchange IEX columns separate proteins based on differences in charge The separation is mediated by a reversible interaction between a charged protein and an oppositely charged chromatographic media Note Any HiTrap IEX means that any HiTrap ion exchange column can be used The same default values as for HiTrap Q HP are used These can be changed in the Advanced Zone Anion exchange columns Anion exchange columns are used to separate negatively charged proteins The following anion exchange columns are supported by AKTAxpress Supported anion IEX columns and volumes Characteristics HiTrap Q HP Columns prepacked with Q Sepharose Lana Stil High Performance for high resolution purification RESOURCE Q Columns prepacked with SOURCE iandeml 15Q Fast purification with high resolu tion Mono Q 5 50 GL 1ml Column prepacked with MonoBeads for purification with the highest resolu tion p97 4 Methodology 4 5 Column alternatives 4 5 1 Descriptions of columns Gel filtration columns 28 4090 22 AB p98 Cation exchange columns Cation exchange
240. nt wash steps are executed serially for all samples before elution of the first protein starts Sample application from the capillary loops Guided loading of sample into capillary loop one sample at a time Sample application from the Superloop Guided loading of sample into Superloop One sample is then injected onto columns up to four times The buffer is selected by the inlet valve If a gradient is required it is created using a switch valve The pump transports the buffer through the column to elute the captured protein Introduction 2 Intermediate frac The eluted protein peak is detected by the UV cell If the protein is to be further purified tion collection in a subsequent step the peak volume will be directed to one of the loops where intermediate fractions are stored Large peaks can be collected in double loops The selected peak is then loaded directly from the loop onto the next column for the next purification step Peaks for the next purification step can be selected based on area or height largest peak the peak volume for the set B interval or by manually selecting a peak Final fraction col After the last purification step the eluted volume of the final protein peak is directed lection by the outlet valve to the deep well microplate in the fraction collector p23 2 Introduction 2 4 AKTAxpress user documentation 2 4 Introduction User documenta tion package 28 4090 22 AB p24
241. o RESOURCES 1 x 1 ml 17 1178 01 RESOURCE S 1 x 6 ml 17 1180 01 HiTrap SPHP 5 x 1 ml 17 1151 01 HiTrap SP HP 5 x 5 ml 17 1152 01 Mono S 5 50GL 1x1ml 17 5168 01 Gel filtration Item Code no HiLoad 16 600 Superdex 75 prep grade 17 1068 01 1x 120m HiLoad 16 600 Superdex 200 prep grade 17 1069 01 1x 120m HiLoad 26 600 Superdex 75 prep grade 17 1070 01 1x 320m HiLoad 26 600 Superdex 200 prep grade 17 1071 01 1x 320m HiPrep 16 60 Sephacryl S 100 HR 1 x 17 1165 01 120m HiPrep 16 60 Sephacryl S 200 HR 1 x 17 1166 01 120m HiPrep 16 60 Sephacryl S 300 HR 1 x 17 1167 01 120m HiPrep 26 60 Sephacryl S 100 HR 1 x 17 1194 01 320m HiPrep 26 60 Sephacryl S 200 HR 1 x 17 1195 01 320m HiPrep 26 60 Sephacryl S 300 HR 1 x 17 1196 01 320m e p443 10 Reference information 10 6 Ordering information Documents The following documents are available Item Code no UNICORN User Manual 11 0003 68 AKTAxpress User Manual 28 4090 22 AKTAxpress Cue Cards 28 4090 23 AKTAxpress Operating Instructions 28 9579 08 AKTAxpress Installation Guide 28 4090 29 Superloop Instructions 56 3015 99 Affinity Chromatography Handbook 18 1022 29 Gel Filtration Handbook 18 1022 18 Strategies for Protein Purification 28 9833 31 Handbook Recombinant Protein Purification 18 1142 75 Handbook Antibody Purification Handbook 18 1037 46 lon Exchange Chromatography amp Chro 11 0004
242. o syringes are included in the accessory kit supplied with the system 5 Turn the purge valve counter clockwise half a turn to open it O SS he LAY heey OREN oN 5 Io j N 5 y Ha e J f an 7 6 Slowly draw buffer with the syringe When fluid starts to enter the syringe close the purge valve Check that there is no visible air left in the A1 or whichever chosen tubing Note If air in the other pump head is suspected draw buffer through that pump head as well Filling the inlet tubing B1 and B2 Operation 6 Step Action 7 If required remove the syringe empty it and insert it in the purge valve again before continuing 8 Repeat step 1 6 to fill other inlet tubings To fill the inlet tubing B1 and B2 Step Action 1 Check that the inlet tubings B1 and B2 are properly immersed in the correct flasks 2 Start the pump at a low flow rate e Start UNICORN and select System Control Manual Pump e Select Flow and FlowRate 0 1 ml min Melon Dt Fiwd cote te r ve Ade apdate il the a checked the parameter haidi mil be parod Sarg setot anl e Click Execute to start the flow p 219 6 Operation 6 4 Preparing the system for a run 6 4 6 Filling inlets manually using UNICORN 28 4090 22 AB p 220 Step Action 3 To fill the B1 inlet tubing the inlet valve first has to be set e Select System Control Manual Pump
243. o deionized water and flush out all used buffers solu tions Move all input buffers bottles below the level of the pump heads to prevent siphoning 2 Disconnect electrical power from the system 3 Remove the tubing from the pump heads 4 Loosen the upper valve from the pump head using the 18 mm wrench a ee m p FA A 8 E 4 z f 5 Gently remove the check valve CAUTION Handle the check valves with care when they have been removed from the pump heads to prevent loss of any internal components ep 321 8 Maintenance 8 4 Maintenance when required 8 4 2 Cleaning check valves Step Action 6 Unscrew the two white knurled screws under the pump to release the support bracket locking the inlet manifold into the inlet valve Gently lower and remove the complete manifold ae Loosen the lower valve from the pump head using the 13 mm wrench CAUTION Handle the check valves with care when they have been removed from the pump heads to prevent loss of any internal components Gently remove the check valve Immerse the complete valves in methanol and place them in an ultra sonic bath for some minutes Then repeat the ultrasonic bath with deionized water 28 4090 22 AB p322 Maintenance 8 Step Action 10 Refit the check valves The inlet check valve with a lip for the manifold and a larger diameter opening is fitted to the side marked
244. o the stop position For more details see 8 6 4 Replacing UV lamp on page 353 Is the air sensor sensitivity set to low in UNICORN System Control to avoid unnecessary stops due to minor air bubbles Flow path checks Is all tubing connected correctly Is there leakage at any of the connections Is any tubing folded or twisted Is the inlet tubing immersed in correct buffer solutions Is the gel filtration column located in the left hand column holder If located in the right hand holder it will hinder the movement of the fraction collector Is any tubing interfering with the ejection and loading of the fraction collector Is there a 2 ml deep 96 well or an 8 ml deep 24 well microplate in the fraction collector For more details see 6 4 13 Preparing the fraction collector on page 241 Is there a risk that the number of wells in the microplate will not be sufficient for the run If this is the case make sure that Change plate if out of fractions is selected when creating the method otherwise the flow will be directed to a predefined outlet For more details see 5 4 2 Selecting Purify 2 2 page options on page 154 Purification checks Does the positioning of the columns correspond to the selections made in the Method Wizard Refer to the Summary page Have all columns been cleaned and prepared according to the column recommendations Have the samples been adjusted to binding buffer conditions Have the samples bee
245. ocedures that are included in the method Note Sometimes the inlet tubings A1 A8 B1 and B2 are not enough for all solutions needed because the chosen protocol and the selected system and column procedures require more inlets A message will then appear in the wizard suggesting a suitable action e g using one or several sample inlets as inlet tubing s for the additional solution s or if this is not possible making new selections requiring less buffer inlets The maximum number of samples that can be run is decreased if using sample inlets as buffer inlets The liquid supply required for each inlet for a specific purification method plan is shown on the Summary page in the Method Wizard in System Control For information on suitable buffers for different columns see 4 6 2 Buffer suggestions on page 109 e Immerse the inlet tubing in the containers according to the table below Summary page Inlet Buffer or solutions tubing A1 A4 See the Summary page for the specific run A5 Water A6 0 5 M NaOH A7 See the Summary page for the specific run A8 20 ethanol B1 B2 See the Summary page for the specific run S1 S4 See the Summary page for the specific run Inlet supply for The liquid supply for prepare and maintain methods differ from the purification preparationand methods Also different prepare and maintain methods differ in inlet tubing use maintenance e Immerse the inlet tubing i
246. odology for AKTAxpress It contains information and guidelines for purification runs such as How to choose a protocol How peaks are handled in the different steps of a purification run Which system and column procedures that can be included in a run or run separately in a prepare and maintain run How to perform affinity tag removal How to choose columns How to choose buffers How to choose CIP strip and metal ion solutions The perfect scheme for purifying a protein remains an empirical process and further optimization might be necessary This chapter contains the sections below Section See Protocol descriptions and choice 41 Peak handling 42 System and Column Procedures 43 Affinity tag removal 44 Column alternatives 4 5 Buffer alternatives 4 6 Solution alternatives 4 7 Documents for further information 4 8 4 1 Introduction Protocol definition Protocols avail able Sample loading techniques for dif ferent protocols On column tag cleavage and off column tag cleav age combinations with protocols Methodology 4 Protocol descriptions and choice This section describes the different protocols that can be used with AKTAxpress and gives guidance on how to choose protocol for a purification In this manual the name of a protocol is an abbreviation of the combination of chromatography techniques used in the purification Example The protocol AC DS IEX inc
247. of fractions is selected if there is a risk that the fraction collector may be filled during a run For more details see 5 4 2 Selecting Purify 2 2 page options on page 154 e p381 9 Troubleshooting and corrective actions 9 3 Monitoring problems 9 3 Introduction Faulty UV curve 28 4090 22 AB p382 Monitoring problems This section specifies troubleshooting for monitoring problems during a run The monitoring problems can be related to e UV curve e Conductivity curve e Pressure curve e Air sensor Find the possible cause and action for a specific UV curve fault in the table below If the problem remains contact the local GE Healthcare representative Fault Possible cause and action Ghost peaks e Air in buffers Check that there is no air in the buffers Degas the buffers if necessary It is recommended to use de gassed buffers for best performance e Dirtor residues in the flow path from previous run Clean the system using a method plan for System Cleaning see 6 8 2 Cleaning system Overview on page 276 e Residues in the columns from previous run Replace or clean the columns using a method plan for Column cleaning see 6 8 5 Cleaning of columns Overview on page 283 Troubleshooting and corrective actions 9 Fault Possible cause and action Noisy signal e Air might be trapped in the pump Purge the pump using methanol according to the methanol purg
248. of the sample prior purification is needed Use in DS step Recommended column choices for the DS step Which sample volume 2 x HiTrap Desalting Comments on column choices Step Comments on column choices DS We recommend to use the HiPrep 26 10 Desalting column when the DS step is part of a multi step protocol because the system will dilute inter mediate peaks during peak handling DS 1 ml HiTrap affinity columns can be combined with 2 x HiTrap desalting columns Band broadening effects in the system combined with a limited sample loading volume on the 2 x HiTrap desalting column might how ever reduce protein recovery DS 5 ml HiTrap affinity columns should not be combined with 2 x HiTrap desalting columns since the eluted affinity peak might be larger than the volume possible to load on the 2 x HiTrap desalting column Methodology 4 Choosing column Recommended column choices for the IEX step for the IEX step Amount of target protein Which pl has the target protein Which pi has the target protein Use in IEX step eecourceg RESOURCES RESOURCE Q RESOURCE S imi iml 6ml Emi Step Comments on alternative column choices IEX Resource Q S gives higher resolution than HiTrap Q SP but Mono Q S gives the highest resolution Preferably use columns with higher resolution in late purification steps as a polishing step
249. ogy 4 6 Buffer alternatives 4 6 1 Description of buffers lon exchange chromatography buffers Gel filtration chro matography buf fers 28 4090 22 AB p 108 e DSas last step If DS is used as a final purification step the buffer should be chosen to match the activity or application that will follow e g crystallization NMR studies functional studies or storage The choice of buffer and its pH for IEX chromatography depends on the pl of the target protein and if an anion or a cation exchange column should be used Normally the pH of the buffer is selected to be at least 1 pH unit below or above the pl of the target protein e If using an anion exchange column denoted Q e g Resource Q use a buffer with one pH unit above pl of target protein e Ifusing a cation exchange column denoted S e g Resource S use a buffer with one pH unit below pl of target protein Gel filtration is used as a final purification step to perform a high resolution purification of the protein The buffer should be chosen to match the application that will follow e g crystallization NMR studies functional studies or storage 4 6 2 Introduction AC buffer sugges tions for Histid ine tagged pro teins Methodology 4 Buffer suggestions This section contains buffer suggestions that can be used as a starting point for purification For optimal behaviour in a specific protein purification optimization might b
250. on 6 4 Preparing the system for a run 6 4 16 Using air detection Air detection at sample loading 28 4090 22 AB p248 After removing the air the run can proceed by clicking Continue Note If running PumpWash SystemWash or LoopWash instead to remove the air the system will automatically go back to Pause mode after the wash Air detection during sample loading makes it possible to apply the entire sample volume and still prevent air from reaching the column By default air detection is enabled during sample application in a method plan made in the Method Wizard When air is detected during sample loading the inlet valve switches to port A1 The pump then uses a few milliliter of affinity binding buffer to apply sample that is left in the flowpath onto the column After that the pump flushes the air out with affinity binding buffer at 20 ml min The flush volume can be changed on the Miscellaneous Settings page in Advanced Zone in the Method Wizard The sample loss when using air detection is approximately 0 5 ml Setting a fixed sample volume It is also possible to set a fixed sample loading volume e To set a fixed volume the air sensor option must first be cleared in the method plan This is done on the Miscellaneous Settings page in Advanced Zone by unchecking Enable AirSensor Controlled Sample Loading Miscellaneous Settings Flush Voksen Empty Loops a 0 100 mt Loop Wath Vohame Empty Loop 2 Pa 10 999 mt Lo
251. on Suggestions System on page 116 for a list of recommended cleaning solutions for the columns supported by AKTAxpress The Customized Column Procedures method plan provides e Cleaning of the chosen column type with up to 9 cleaning solutions or e Equilibration of the column with an optional number of solutions Recommended for removing ethanol and equilibrate new columns with buffer and for adding ethanol to columns prior to storage this can also be performed within a purify method or in the standard column and procedure prepare and maintain method For the required amounts of solutions see the Summary page in the wizard when starting a run Note Only one column type can be cleaned equilibrated in each run For instructions on how to clean a specific column see the column instructions See also 4 7 4 Customized CIP Solution Suggestions Columns on page 117 for a list of recommended cleaning equilibrating solutions for the columns supported by AKTAxpress p83 4 Methodology 4 4 Affinity tag removal 4 4 Affinity tag removal About this section This section describes the affinity tag removal processes on and off column that can be performed in combination with the supported AKTAxpress protocols In this section This section contains the sub sections below Sub section See Description of affinity tags 44 1 Description of proteases 44 2 Description of the affinity tag removal processes 4 43 Condi
252. on optimizing run parameters e For 1 step protocols Use Level as Peak Fractionation Algorithm on the Fractionation page e For multi step protocols Decrease the Watch Slope Greater than value on the Peak Collection page s Proteaseinjection This applies to protocols that include tag cleavage volume The default value for the protease injection is 0 7 CV If using a larger value the target protein might be lost since it will pass through the column before peak collection is activated 28 4090 22 AB p 194 About this chapter Prerequisites In this chapter Operation Operation 6 This chapter describes the normal work flow when operating AKTAxpress from starting the system creating a method plan and preparing the system to cleaning the system and columns after the run It is assumed that the system and computer have been started according to the instructions in AKTAxpress Installation Guide WARNING In case of an emergency situation the system mains power connector must always be easy to disconnect A WARNING The computer must be used in a dry location and according to the environmental requirements stated in the manufac turer s instructions A This chapter contains the sections below Section See Operation overview 6 1 Connecting a system 6 2 Creating method plans 6 3 Preparing the system for a run 6 4 Column procedures before a puri
253. ools are required Piston kit 18 1112 13 containing piston spring seal and rinse membrane e 1 4 inch wrench e 3mm hex key e Screwdriver e 100 methanol for running in the new piston seal To replace the piston and the seal follow the instructions in 8 6 6 Replacing pump piston seal on page 361 About this chapter Warning In this chapter Troubleshooting and corrective actions 9 Troubleshooting and corrective actions This chapter describes the troubleshooting and corrective actions of AKTAxpress WARNING T When using hazardous chemicals take all suitable protective measures such as wearing protective glasses and gloves resistant to the chemicals used Follow local regulations and instructions for safe operation and maintenance of the system This chapter contains the sections below Section See Introduction to troubleshooting 9 1 Problems during a run 9 2 onitoring problems 9 3 Leakage problems 9 4 Alarms and connection problems 9 5 Error code list 9 6 Checking USB CAN driver 9 7 p371 9 Troubleshooting and corrective actions 9 1 Introduction to troubleshooting 9 1 Introduction to troubleshooting Introduction This section describes the troubleshooting procedure and includes a general check list of common errors to be checked Troubleshooting To troubleshoot AKTAxpress procedure Step Action 1 Always start checking th
254. oops See 4 2 2 Peak collection parameters when using double loops on page 68 for more information Loop extension Kit 28 9044 38 containing e Scapillary loops e 51 16 female 1 16 female unions e double stick tape The extra capillary loops can be mounted on the right hand side of AKTAxpress Use for example double stick tape to fasten the capillary loops The illustration below shows where it is recommended to mount the capillary loops indicated by the shaded area on the right hand side of the instrument 4 capillary loops here the loop ends should face the front of the instrument p 231 6 Operation 6 4 Preparing the system for a run 6 4 10 Preparing the system when using double loops Connecting a double loop 28 4090 22 AB p 232 The illustration shows how to connect an extra capillary loop to one of the existing loops The loops are joined using a 1 16 female 1 16 female union Note It is a good idea to use double loops for all five loops because it is not possible to predict in which loops the peaks will be collected F 1 16 female 1 16 female union Loop 1 No Extra copillary loop Loop valve OUT 6 4 11 Introduction Protocols for affin ity tag removal Protocols for Su perloop sample loading Pre filling the Su perloop with buf fer Operation 6 Preparing the system when using a Superloop The Superloop can be used for loading of lar
255. op Wath Voke 2 Pa 10 999 mt Lv Franio aa Sanace larmade Sangin aartng Max Sample Volume S 3988 E e When the method plan is started in System Control the sample volume has to be entered on the Settings page in the Method Wizard Operation 6 Note Make sure that the sample supply is sufficient for the given sample volume Otherwise the sample loading will be stopped when air enters the air sensor The flow path is then automatically flushed to remove the air e p249 6 Operation 6 4 Preparing the system for a run 6 4 17 Using flow control 6 4 17 Introduction About flow control 28 4090 22 AB p 250 Using flow control This section describes the flow control function in AKTAxpress The flow control is used to avoid exceeding the maximum pressure limit of the column When flow control is used the flow is automatically decreased when the pressure approaches the set maximum limit If the pressure decreases the flow slowly increases towards the set flow rate and so on If the flow rate falls below the minimum limit 0 1 ml min the system will issue an Alarm and enter Pause mode 6 5 Introduction In this section Operation 6 Column procedures before a purification run Before performing a purification run e uncharged chelating affinity columns should be charged with metal ions e columns should be conditioned to obtain well equilibrated columns This section contains the
256. or exchange columns e Check the online filter if used It can become clogged if unfiltered buffers are used e There might be dirt or residues in the flow path Clean the system using a method plan for system cleaning see 6 8 2 Cleaning system Overview on page 276 e If using a highly viscous sample dilute it to ease sample loading p 375 9 Troubleshooting and corrective actions 9 2 Problems during a run Sample loading problems 28 4090 22 AB p376 Find the possible cause and action for a specific problem in the table below Problem Possible cause and action Sample loading failure e Make sure that no air has entered the inlet tubing when moving the tubing from buffer solution to the sample If air enters the tubing and the system is set to pause it is possible to fill the required inlet manually from UNICORN see 6 4 6 Filling inlets manually using UNICORN on page 217 Remember to switch back to the original inlet valve position in UNICORN BEFORE pressing Continue e Donot place the sample inlet tubing too close to the bottom of the sample tube Air bubbles might other wise be created causing the sample loading to stop due to the air sensor e Purge the pump to remove trapped air bubbles see 6 4 7 Purging the pump and system on page 222 e Check the sample inlet tubing connectors A ferrule could be distorted or a connector tightened too hard Cut the tubing end using a tu
257. or the slope of the signal curve exceeds a defined value During the fractionation a specified maximum volume is collected in each well in the fraction collector The fractionation continues at least until the Minimum Peak Width time has elapsed The fractionation stops when the signal level and or the slope of the signal curve falls below a defined value The Stop Slope condition cannot be met unless Peak_Max has been detected The illustration below describes this Start Siope OR Start Level UV max x Peak Max Factor 0 5 Stop Level OR Stop Slope Note If the flow rate is changed the shape of the peak will change as well Hence the slope values might need to be changed accordingly ep7l 4 Methodology 4 2 Peak handling 4 2 3 Peak fractionation parameters Note When the fractionation of a peak is finished the subsequent fraction will appear to be very large in the chromatogram This is because the well position of the fraction collector is shown in the chromatogram If a new peak is detected only the delay volume will be collected in this well and the new peak will be collected in the next well 28 4090 22 AB p72 Methodology 4 4 3 System and Column Procedures About this section This section describes the different system and column procedures that can be e included in a purification run e performed by running a separate prepare and maintain method In this section
258. ormation about how to pre fill the Superloop with buffer connect the Superloop to the system and fill the Superloop with protease see 6 4 11 Preparing the system when using a Superloop on page 233 Automatic affinity tag removal can be combined with all AKTAxpress protocols that start with an AC IEX step The table below shows the main steps to be performed when performing on column tag cleavage in AKTAxpress Step Action 1 Pre fill the Superloop with buffer and connect it to the system See 6 4 11 Preparing the system when using a Superloop on page 233 2 Create a Purify method plan including on column tag cleavage see below 3 Calculate the volume and concentration of protease needed see below 4 During the run the software will give guidance to filling Superloop The filling is performed manually with a syringe in the injection valve See 6 4 11 Preparing the system when using a Superloop on page 233 The rest of the tag cleavage procedure is performed automatically Operation 6 Create a method The illustration below gives an overview of how to include on column tag cleavage plan including on jn the purification method plan See 5 4 Using the Purify option on page 150 for detailed column tagcleav information age Select protocol starting with AC IEX Select On Column Tog Cleavage Fill Colurnns with Cleavoge Buffer if o specific Cleavage buffer other than the ordinary affinity
259. ouble free and optimal use of the product WARNING This is a Class A product In a domestic environment this product may cause radio interference in which case the user may be required to take adequate measures Recycling 28 4090 22 AB p10 Preface 1 This symbol indicates that the waste of electrical and electronic equipment must not be disposed as unsorted municipal waste and must be collected separately Please contact an authorized representative of the manufacturer for information concerning the decommissioning of your equipment epil 1 Preface 1 2 Product information 1 2 CE certification 28 4090 22 AB p 12 Product information This product meets all requirements of applicable CE directives A copy of the corresponding Declaration of Conformity is available on request The CE mark and corresponding declaration of conformity is valid for the instrument when it is used as a standalone unit or connected to other CE marked GE Healthcare instruments or connected to other products recommended or described in this manual and used in the same state as it was delivered from GE Healthcare except for alterations described in this manual Preface 1 1 3 Safety Read the Operating Read the Operating Instructions before starting the installation Instructions Available in several languages e g English German Spanish French Italian and Swedish WARNING T The KTAxpress system is in
260. ough the flow cell WARNING CORROSIVE CHEMICALS NaOH is corrosive and therefore dangerous to health Avoid spillage and wear protective glasses The standard recommendation is to pump 1 M NaOH at a flow rate of 1 ml min for 2 minutes 2 Stop the flow and leave the cell filled with liquid for 30 minutes 3 Rinse with buffer or deionized water 4 If this does not correct the problem follow the instructions for making offline cleaning 28 4090 22 AB p 316 8 3 3 Maintenance inter val Reason for main tenance Performing in place cleaning Maintenance 8 Cleaning conductivity cell Every 6 months or when required A clean flow cell is essential for ensuring correct measurements If the conductivity measurements are not comparable to previous results the electrodes in the flow cell may be contaminated and requires cleaning See also 9 3 Monitoring problems on page 382 To make an inplace cleaning of the conductivity flow cell Step Action 1 Pump a cleaning or sanitizing agent through the flow cell WARNING CORROSIVE CHEMICALS NaOH is corrosive and therefore dangerous to health Avoid spillage and wear protective glasses The standard recommendation is to pump 1 M NaOH at a flow rate of 1 ml min for 2 minutes 2 Stop the flow and leave the cell filled with liquid for 15 minutes 3 Rinse with buffer or deionized water 4 Make a calibration of the conductivity monitor se
261. pending on the chosen type When all required selections are made the method plan is saved with a user specified name Note When selecting options for a purification run the options are based on the protocol AC DS IEX GF but it is still possible to run any kind of protocol Before using a method plan in a run some additional information must be added for example selecting the separation systems to be used number of samples name of the result file and so on This information will be requested in a wizard in the System Control module where the final preparation before starting the run is made When the wizard is finished a unique method for each of the systems is created and started See 6 6 1 Starting a run using a method plan on page 259 Opening the Method Wizard The first page in the Method Wiz ard Method Wizard 5 To open the Method Wizard Step Action 1 Click the Method Editor button located in UNICORN task bar Result The Method Editor module appears 2 Click the Method Wizard icon located in the tool bar Alternatively choose File Method Wizard lt Result The Method Wizard appears The first page shown below contains a list of previously created and stored method plans and a New option for creating a new method plan The first time the Method Wizard is used the list only contains the Installation test The Note pane to the right gives a short summary of the chosen method p
262. perloop 1 x 10 ml 18 1113 81 Proteases Filters Connectors and unions Reference information 10 Item Code no Superloop 1 x 50 ml 18 1113 82 The following proteases are available Item Code no PreScission Protease 500 units 27 0843 01 Thrombin 500 units 27 0846 01 Factor Xa 400 units 27 0849 01 The following filters are available Item Code no Filter holder kit 11 0004 07 Online filter holder 18 1112 44 Screw lid GL45 incl cap membrane 11 0004 10 The following connectors and unions are available Item Code no Fingertight connectors 1 16 18 1172 63 Fingertight stop plug 11 0003 55 Tubing connector M6 18 1172 64 Ferrule yellow for 1 8 OD tubing 18 1121 18 Tubing connector for pump inlet 18 1121 17 Wrench M6 key 19 7481 01 Nipple wrench 11 0003 56 Tubing cutter 18 1112 46 Union M6 male to 1 16 female 18 1112 57 e p439 10 Reference information 10 6 Ordering information Item Code no Union M6 female to 1 16 male 18 1112 58 Union M6 female to 1 16 female PEEK 18 1123 94 Union 1 16 male to 1 16 male 18 1120 93 Union 1 16 female to 1 16 female 11 0003 39 Union Luer female to 1 16 male 18 1112 51 Red fingertights 28 4010 81 Tubing The following tubing is available Item Code no ETFE tubing i d 1 mm o d 1
263. placed in the fraction collector 4 The inlet tubing is filled if not included in the method plan and the pump heads purged 5 The correct columns are fitted and equilibrated if not included in the method plan Starting the run The table below briefly describes the procedure for starting a purification process Step Description 1 A wizard dedicated for initializing the run is started in the System Control module 28 4090 22 AB p54 Loading the sample onto the AC column Elution of the AC column AKTAxpress overview 3 Step Description 2 The requested information in the wizard is filled in See also 6 6 1 Starting arun using a method plan on page 259 3 The last page in the wizard Summary specifies the method requirements totally and for each of the systems It is recommended to print this list since it will not be saved When clicking Run on the Summary page all systems enter Run mode The table below describes how the sample is loaded onto the AC column Note Individual affinity columns are always used for each sample Step Description 1 The first sample is loaded onto the first AC column The inlet valve switches to the inlet with AC binding buffer when the sample tube is empty air sensor detection 2 Air is removed through a Waste port in the injection valve 3 Unbound sample is flushed from the column with buffer AC binding buffer a
264. plan 6 4 5 Filling inlets manually using UNICORN 6 4 6 Purging the pump and system 6 4 7 Purging the pump with methanol 6 4 8 Connecting columns and tubing 6 4 9 Preparing the system when using double loops 6 4 10 Preparing the system when using a Superloop 6 4 11 Preparing for on column tag cleavage 6 4 12 Preparing the fraction collector 6 4 13 Checking the tubing 6 4 14 Preparing samples 6 4 15 Using air detection 6 4 16 e p 203 6 Operation 6 4 Preparing the system for a run 28 4090 22 AB p204 Sub section See Using flow control 6 4 17 Operation 6 6 4 1 Printing out a summary Introduction Before starting preparing the system asummary can be printed out from the method Wizard in System Control The Summary is useful during the preparation of the system columns and all solutions This section contains brief information about the Summary page and how to print it For detailed information see 6 6 1 Starting a run using a method plan on page 259 The Summary The Summary page specifies the method requirements The tabs for each system page specifies the required columns loops buffers solutions and so on and where they should be connected The total requirement of buffers and solutions is listed on the Summary tab AP Barty Radies Strip or CIP ACAEX Fest Step AF AEX Wash Buster botase Finan DS GF Baster HieTrop_ HPI Hitap HPI m HiLood_16 00_Sup p 20
265. r check that the local UNICORN workstation iS running 9 6 Introduction Error codes Troubleshooting and corrective actions 9 Error code list This section contains a list of error codes and their meaning The error codes are presented on the display of the separation system as a flashing code Exxx To enable identification of the system unit number the display will alternate between flashing the error code and the system unit number Error The following error codes can occur Code Description 1 Either of the following two error messages appears in UNICORN e The pressure is out of range If the pressure is too high decrease the flow rate check column back pressure or check system for blockage If problem remains contact service e Changing the inlet valve position is not allowed when B gt 0 Changing the inlet valve position will affect the gradient and is therefore not allowed when B gt 0 2 Either of the following two error messages appears in UNICORN e The conductivity is out of range Make sure that the buffers and concentrations used are correct Calibrate conductivity If problem remains contact service e Air detected by the air sensor See section 9 3 Monitoring problems on page 382 e p393 9 Troubleshooting and corrective actions 9 6 Error code list 28 4090 22 AB p394 Code Description 3 The UV sig
266. rallel AKTAxpress separation system and the computer with UNICORN control software should be installed and tested by GE Healthcare personnel ep19 2 Introduction 2 2 Purification of recombinant proteins 2 2 Introduction Protein purifica tion Multi step purifica tion of recombin ant proteins AKTAxpress for high throughput multi step purifica tion 28 4090 22 AB p 20 Purification of recombinant proteins This section introduces the reader to purification of recombinant proteins and thus gives a background to the development of the system Success in isolation and purification of a target protein is highly based on the knowledge of the protein characteristics Protein purification serves to deliver a suitable amount of pure target protein in its native biological state for use in further studies The requirements on the end product may vary considerably For instance a certain enzyme may be considered pure enough for activity studies at a relatively moderate mass purity provided that the end product is free of substances interfering with the activity of the enzyme Pure enough for structure analysis of a protein on the other hand normally means a rather high mass purity since interfering substances may affect the techniques used in structure determinations The purification itself is almost never a final goal but rather a necessary step on the way to performing protein research in many differen
267. ration system from UNICORN is found in 6 Operation on page 195 p49 3 AKTAxpress overview 3 2 Separation system overview 3 2 5 Indicators and controls Power on off To make it possible to turn on off the power to an AKTAxpress separation system whenever required e Make sure that at least one of the two connectors on the system mains cable is easy to access after the system installation or e Connect the system mains cable to a separate mains power switch which is easy to access 28 4090 22 AB p50 AKTAxpress overview 3 3 2 6 Labels Introduction This section describes the labels on the separation systems Labels Labels for safety product no CE waste disposal C tick and rating are located on the rear panel of the separation systems A system no label is located on the left front rail The safety label warns the user of risk for personal injury Do not proceed until the instructions are clearly understood and all stated conditions are met BE cast ep51 3 AKTAxpress overview 3 3 Purification process overview 3 3 Purification process overview Aboutthis section This section gives an overview of the steps during a typical purification process In this section This section contains the sub sections below Sub section See Process flow chart 3 3 1 Description of the process steps 3 3 2 28 4090 22 AB p52 AKTAxpress overview 3 3 3 1 Proces
268. re increases the membrane bulges which is detected by the strain gauge The pressure sensor housing is made of PEEK other wetted parts are made of titanium and FFKM perfluororubber Air sensor Reference information 10 The air sensor is a high precision monitor designed for continuous monitoring of air bubbles in the inlet flow path When air is detected the system is either paused or performs an action that is set in the method plan The flow path has i d 1 5 mm The air sensor is made of PEEK e p413 10 Reference information 10 1 System description 10 1 4 Fraction collector 10 1 4 Fraction collector Introduction This section describes the fraction collector in AKTAxpress Fraction collector The fraction collector is an X Y collector with a free arm The design gives a compact design unit with efficient operation 4 28 4090 22 AB p414 Reference information 10 10 2 Technical specifications About this section This section contains specifications of AKTAxpress and its components In this section This section contains the sub sections below Sub section See System specifications 10 2 1 Components specifications 10 2 2 Wetted materials 10 2 3 ep 415 10 Reference information 10 2 Technical specifications 10 2 1 System specifications 10 2 1 Introduction Performance data Gradient forma tion Physical data 28 4090 22 AB p 416 System sp
269. re the outlet valve and fraction collector located at the lection top of the system Outlet valve ay Fraction collector Waste Fraction outlets F3 F11 Outlet valve During the last purification step the outlet valve directs the peak volume to the fraction collector The outlet valve also provides up to nine outlets for collecting flowthrough volumes and nonselected peaks and a waste outlet The outlet valve is also a part of the intermediate fraction collection where it is used to direct the peak volume to the loop valve WARNING Never place flasks on the top of the separation system If they become full and overflow liquid might penetrate the system causing a short circuit Fraction collector The fraction collector is a X Y collector made for 96 square well 8x12 well positions or 24 square well 4x6 well positions microplates The default volume of a well is 2 ml for 96 well microplates or 8 ml for 24 well microplates TT uninglene 28 4090 22 AB p 46 Indicators and controls AKTAxpress overview 3 This section describes the indicators and controls on AKTAxpress The separation systems are equipped with the following indicators and controls H T Run indicator lt Pa BESA 5 x fa 4 i Pause indicator ata 4 Van Pile 2 H ad X jipa Fakir OS lj lt gt Status display 0 Ga B5505 S Pause Continue O RUN Next breakpoint
270. rify option the same type of pages will always be displayed but the options on the pages may differ depending on the protocol to be run The illustration below shows the pages that will be displayed for the Purify option in the first part of the Method Wizard and what type of settings that can be selected For information about the Advanced zone see 5 5 3 Advanced Zone for Purify on page 172 Select protocol running condition columns On column tog cleavage loptionall elution how to select peak for next step froctionotion the next run or storoge Prepore the columns for the run Prepare the columns for the next run or storoge On last poge finish or enter Advanced Zone Method Wizard 5 Selecting the Puri To select the purify option fy option Step Action 1 On the Main Selection page select Purify 2 Click Next In this section This section contains the sub sections below Sub section See Selecting Purify 1 2 page options 5 4 1 Selecting Purify 2 2 page options 5 4 2 Selecting System Procedures page options 5 43 Selecting Column Preparation page options 5 4 4 Selecting Column Post Run page options 5 4 5 p151 5 Method Wizard 5 4 Using the Purify option 5 4 1 Selecting Purify 1 2 page options 5 4 1 Selecting Purify 1 2 page options Selecting options To select options on
271. roduction Checking the driver installation Troubleshooting and corrective actions 9 Checking USB CAN driver If there are connection problems between the computer and the separation systems the USB CAN driver installation can be checked To verify that the CAN drivers have been correctly installed Step Action 1 Open the System Properties dialog box e Open the Control Panel and choose System or e press down the Windows key and the Pause Break key simultan eously 2 In the System Properties dialog box click the Hardware tab I System Nestore Automatic Updates Nemote Genwi Compus None Hardware Advanced Add Hardware Wizard The Add Hardware Wizard helps you install hardware Add Haidware Wizard are AT ists al the hardware devices invlated prieg a the Device Manager to change the Hardware profiles prowde a way for you to set up and store agh aimant hardware contig aations Hardware Prufites n Pe e Click the Device Manager button Result The Device Manager is opened e p397 9 Troubleshooting and corrective actions 9 7 Checking USB CAN driver 28 4090 22 AB p398 Step Action In the Device Manager click the plus sign to expand the CAN Hardware KVASER item e Ifthe Amersham URJ45 icon looks like the image above the install ation was successful If so go to step 10 e Ifthe Amersham URJ45 icon has an exclamation or warning si
272. s Trichloroacetic OK OK acid 1 Trifluoroacetic OK OK acid 1 Urea OK OK o Xylene OK OK PP and PE are af p xylene fected by long term use e p435 10 Reference information 10 6 Ordering information 10 6 Ordering information Introduction This section lists the systems accessories and user replaceable spare parts available for AKTAxpress If not stated otherwise the code number contains a pack of one 1 unit Systems The following systems are available Item Code no AKTAxpress 18 6645 01 USB CAN device 28 9692 01 AKTAxpress software package 28 9053 45 includes UNICORN 5 31 strategies user manuals on CD and printed cue cards Holders The following holders are available for the system Item Code no Tube holder 18 1177 80 Tubing holder 18 1177 81 Flask holder optional 18 1177 79 Large column holder clip 0 d 30 mm 2 11 0002 92 pcs Large column holder AKTAxpress o d 28 4007 37 50 mm HiPrep 16 xx Column Wrap 28 9021 50 Pump parts The following spare parts are available for the pump Item Code no seals and 2 rinse membranes Pump piston seal kit including 2 piston 18 1112 04 28 4090 22 AB p436 Reference information 10 Item Code no Check valve kit including 1 inletand1 18 1128 66 outlet check valve
273. s Method Wizard 5 Step Action 3 If selecting Standard System and Column Procedures select the columns to be prepared by checking the appropriate boxes in the Column list Depending ont the DS column shred be usni chaning the fnet emcond o uit chenmnatioguanhy ten direnn hiin rinto vail he rand 3 Click Next e ifthe Last Page is displayed continue with 5 2 2 Saving the method plan on page 132 e Otherwise continue with the instructions in the appropriate section below 5 3 3 Selecting Standard System and Column Procedures op tions on page 142 5 3 4 Selecting Customized System Procedures options on page 146 5 3 5 Selecting Customized Column Procedures options on page 148 p 139 5 Method Wizard 5 3 Using the Prepare and Maintain option 5 3 2 The Prepare and Maintain page options 5 3 2 The Prepare and Maintain page options Introduction There are two categories of prepare and maintain page methods e Interactive methods When running these methods manual procedures are included e Automatic methods These method are fully automated no manual procedures are included Interactive meth The table below describes the interactive method plans that can be created od options Select to create a method plan for Column Attachment attaching the column tubing to the columns without trapping air bubbles in the columns When running the method plan the flow is started
274. s e p281 6 Operation 6 8 Procedures after a run 6 8 4 Customized cleaning of the system Create the Cus The illustration below gives an overview of how to create the Customized System tomized System Procedures method plan See 5 3 Using the Prepare and Maintain option on page Procedures meth 136 for detailed information od plan Cleaning the sys To run the method plan for cleaning the system tem Step Action 1 Wash the outside of the inlet tubings with water and or ethanol 2 Immerse the ends of the tubings to be used in the container with cleaning solution 1 see also the Summary page Note Inlet tubing A1 is used for all cleaning solutions The oper ator moves the inlet tubing from solution to solution If the system will not be used for a longer time use a bacteriostatic solution e g 20 ethanol as the final wash solution If the column valve is to be cleaned remove the columns and reconnect the tubings to the column block If the outlets are to be cleaned insert a microplate Run the cleaning method plan as described in 6 6 1 Starting a run using a method plan on page 259 28 4090 22 AB p282 6 8 5 Introduction Warning Cleaning proced ures Cleaning of columns Overview Operation 6 When running different types of purification methods and different samples after each other the columns should be cleaned between the runs ac
275. s 0 27 ml x 4 columns 0 53 ml 15 6 ml Protease concentration Amount of protease per column 20 mg x 200 units 4000 units Protease concentration 4000 units 3 5 ml 1143 units ml Conclusion For this example fill Superloop with 15 8 ml of 1143 units ml TEV protease Note When injecting the protease solution into Superloop the protease will be diluted with buffer corresponding to the volume in the capillaries between the injection valve and Superloop In this calculation example and in the Protease Calculator sheet this dilution is overlooked When using small protease volumes e g one sample with 1 ml affinity column the dilution might have to be considered when calculating the protease concentration Note Try to keep the glycerol concentration low in the protease solution since high glycerol concentration affects TEV activity negatively If a system is used in a cold room it is possible to raise the temperature in the affinity column by using a column heater The efficiency of some proteases increases with increased temperature thus decreasing the incubation time Note Carefully raise the temperature if the protein is temperature sensitive The column heater is not controlled from AKTAxpress but can be physically placed on top of the system Extra long capillaries might be needed between the column valve and the affinity column used The protease volume then must be changed in the Advanced Zone in the Method Wizard cons
276. s Interaction O Oean Pump veh 05 M NaOH beween Loading of Ditevent Samphss O Wash Fran Tubang Betwoweny Sampler Post Run x tnchade Post Aun inateuction FA System vath F hanal CIP System then F wth Fihanal CO CIP System then Fil with Butter T Cean Sample iniets Regares Marusa interaction Note If the Superloop should be filled with protease solution during the run if performing on column tag cleavage make sure that the Guided Loading of Superloop box is checked Note Only one of the options Clean Sample Inlets After Sample Loading or Clean Sample Inlets Post Run Instruction can be selected Method Wizard 5 Step Action For protocols starting with a DS GF step Select procedures to be included in the purification run See 4 3 1 System and column procedures within a purify method on page 74 for a description of the available system procedures L Wash Fiat Tubing Betwnnn Samples Post Run Z brechade Pust Run inemuactron FA System vih Ethan T OIP System theory FA vath Ethanoi A CIP System then Fi wth Butter To perform any post run procedures check the Post Run box and select the appropriate option See 4 3 1 System and column procedures within a purify method on page 74 for a description of the system post run procedures Click Next p 159 5 Method Wizard 5 4 Using the Purify option 5 4 4 Selecting Column Preparation page options 5 4 4 Selecting Col
277. s and in the order below solutions for Cus tomized System Procedures e deionized water 0 5 M NaOH e When the system shall not be used for a longer period of time use 20 ethanol as the last cleaning step 28 4090 22 AB p116 4 7 4 Introduction Prerequisites Recommended solutions for affin ity columns Methodology 4 Customized CIP Solution Suggestions Columns Customized CIP column procedures can be run only in the Customized Column Procedures prepare and maintain method plan The procedure contains up to nine eaning steps c The suggested solutions can be used as a starting point for the cleaning preparation equilibration procedures For optimal cleaning preparation equilibration of the columns optimization might be needed C IP column solution guidance is also provided in the instructions for each column and n the purification handbooks The columns must be filled with water prior to CIP It is possible to choose Wash Columns with Water before CIP in the beginning of the method The tables below list recommended solutions for the affinity columns supported by KTAxpress Note If using chelating affinity columns the columns should be stripped before CIP to avoid formation of metal ion salts that can damage the column See 4 5 1 Descriptions of columns on page 95 for a list of columns supported by AKTAxpress Column Solution Volume Flow rate ml min Al
278. s and materials e Syringe e Surface active detergent solution like Decon 90 Deconex 11 RBS 25 or equivalent e Deionized water Step Action 1 Set the system in Standby mode by pressing the On Standby button This will disconnect the system from UNICORN RUN i 28 4090 22 AB p314 Maintenance 8 Step Action 2 Fill a syringe with deionized water 3 Remove the UV monitor capillary tubing from the conductivity cell and the column valve 4 Connect a syringe to the upper capillary tubing using the connecting pieces A and B B A I Ss os 3 Ve rr A Luer female to 1 16 male 18 1112 51 B Union 1 16 female to 1 16 female 11 0003 39 5 Inject water through the cell in small amounts while holding the lower capillary tubing in a flask 6 Fill the syringe with a 10 surface active detergent solution like Decon 90 Deconex 11 RBS 25 or equivalent and inject through the cell 5 times 7 After the last injection leave the detergent solution in the flow cell for at least 20 minutes p 315 8 Maintenance 8 3 Six monthly maintenance 8 3 2 Cleaning UV cell Step Action 8 Rinse the syringe and then flush the flow cell with water 10 ml 9 Reconnect the capillary tubing Performing in To make an inplace cleaning of the UV flow cell place cleaning Step Action 1 Pump a cleaning or sanitizing agent thr
279. s flow chart Introduction This section contains a flow chart of the process steps during a typical run with AKTAxpress Process flowchart The example below shows the main steps during a four step standard purification of a single sample of a protein Affinity AC Desalting DS lon exchange IEX Gel filtration GF Loading sample on AC column lon exchange Sample loaded onto Pump wash AC column Peak in loop loaded onto IEX column e Unbound sample flushed from Loop for the loaded peak emptied and column flushed Unbound sample flush e Gradient elution of IEX IEX binding buffer IEX elution buffer gradient Peaks collected in loops and largest peak selected Nonselected peaks emptied and loops flushed AC oe DS IEX GF elution fractionation Sample protein solt 7 _ loading f Flow through Non selected peoks microplate Step elution of AC Desalting Gel filtration Loop wash System wash e System wash e 2 nd wash to wash out Peak in loop loaded onto DS Peak in loop loaded onto GF unbound sample column column e Elution of AC e Elution of DS e Elution of GF e Peaks collected in loops and Peaks collected in loops and e Peaks collected in fraction largest peck selected largest peak selected collector e Nonselected peaks emptied Excess of selected peak from e Excess of selected peak and loops flushed AC column emptied and loops from injection emptied and flushed loops
280. s then set to PAUSE mode The pump related items are located at the lower left hand side of the system Pressure sensor Mixer Pump heads Pump The pump is located at the lower left hand side of the separation system It is a low pulsation pump equipped with two pump heads which can deliver up to 65 ml min at pressures up to 3 MPa Mixer The buffers used for creating gradients are mixed in a static mixer which is located directly after the pump The volume of the mixer chamber is 0 25 ml Column handling AKTAxpress overview 3 Pressure sensor A pressure sensor is connected directly after the mixer for continuous detection of the system pressure The column handling items are the column valve and two types of column supports a column block for small columns and column holder for large columns Column block Column valve Column holder Holder for Superloop and rinsing solution tube Column valve The column valve is located at the upper left hand side of the front panel The valve makes it possible to select between up to five columns and a by pass tubing Column block The column block located directly above the column valve can take up to five small columns with fingertight fittings Column holder The rail on the left hand side is equipped with snap locks for fastening up to 2 large columns o d 30 mm while the right hand side snap locks are used for Superloop and tubes for piston seal rin
281. sTrap FF Crude columns Stir the samples before sample loading Note No clarification that is centrifugation and or filtration is needed Immerse the sample inlet tubing in affinity binding buffer Fillthe sample inlet tubing with affinity binding buffer in order to prevent air from entering the flow path To perform it manually see 6 4 6 Filling inlets manually using UNICORN on page 217 It can also be performed automatically during the system preparation if selected in the method plan Gently move the sample inlet tubing to the sample flasks or tubes tubing S1 to sample 1 tubing S2 to sample 2 and so on Make sure that no air enters the tubing If automatic filling is selected in the method plan a dialog will appear requesting the sample inlet tubing to be moved Tip If air enters the tubing set the system to pause if not already paused It is then possible to fill the required inlet manually from UNI CORN see 6 4 6 Filling inlets manually using UNICORN on page 217 Remember to switch back to the original inlet valve position in UNICORN BEFORE pressing Continue All purification protocols have air detection enabled by default during the sample application The sample application stops automatically when the sample supply is empty and air detected See 6 4 16 Using air detection on page 247 The sample e p245 6 Operation 6 4 Preparing the system 6 4 15 Preparing samples Purification pro
282. sed The conductivity of the solution changes with tem perature Since there is no temperature compensa tion the measured conductivity will be different at different temperatures e The buffer might loose its characteristics over time Change buffer e p385 9 Troubleshooting and corrective actions 9 3 Monitoring problems 28 4090 22 AB p386 Problem Possible cause and action Ghost peaks in gradi ent profile Non linear gradients e Air bubbles are passing through the flow cell Check for leaking tubing connections and correct see 6 4 14 Checking the tubing on page 243 e Air might be trapped in the pump Purge the pump according to the procedure in 6 4 7 Purging the pump and system on page 222 e Air might be trapped in the pump Purge the pump according to the procedure in 6 4 7 Purging the pump and system on page 222 e Dirt or residues in the flow path from previous run Clean the system using a method plan for system cleaning see 6 8 2 Cleaning system Overview on page 276 e Organic solutions have been used The mixer is designed for non organic solutions only e Faulty switch valve Flush through to clear any blockage by running e g System Wash Slow response to B changes See Non linear gradient above Note A delay of 3 ml plus the column volume is normal Incorrect absolute conductivity value e Bad calibration Calibrate the conductivity cell see 8
283. sed for filling the buffer inlet tubing Loop Wash Volume Volume of cleaning solution to be used for washing each selected loop Incubation Time after CIP The time the system is set to pause after the chosen tubing loops and valves have been filled with CIP solution Click Next The Last Page is displayed Continue with 5 2 2 Saving the method plan on page 132 Depending on the selections made in the first part of the method wizard the corresponding pages appear in the advanced zone When settings have been made for one page click Next to display the next page Advanced Settings System Procedures Method Wizard 5 Step Action 1 Change the required values on the Advanced Settings System Proced ures page Advanced Seltngs System Fruceduies Voke Loup Wash w Mo 10 999 wt Incubabon lene afer LIP 0 W men Volume Loop Wash Volume of cleaning solution to be used for washing each selected loop Incubation Time after CIP The time the system is set to pause after the system has been filled with CIP solution 2 Click Next Result The next selected page appears Advanced Settings Column Preparations Step Action 1 Depending on the columns to be prepared selected earlier in the Method Wizard one or two pages per column will be included in the advanced zone p 169 5 Method Wizard 5 5 Advanced Zone 5 5 2 Advanced Zone for 28 4090 22 AB p 170
284. set to zero see 8 5 1 Calibration of zero pressure on page 329 Start a flow from A1 to injection valve waste by e Start UNICORN and select System Control Manual Flowpath e Select Injection Valve and Waste Click Execute e Select System Control Manual Pump e Select a flow rate of 5 ml min Click Execute 28 4090 22 AB p308 Maintenance 8 Step Action 4 Start a flow from A1 to outlet valve waste by e Select System Control Manual Flowpath e Select Injection Valve and Inject Click Execute 5 Inspect the pressure curve in the UNICORN System Control window MPa Pressure 05 0 4 02 SS 01 j 0 0 a serene 00 of O02 O03 of O05 O06 oF 08 og 10 Check that the pressure difference is within the range 0 1 to 0 3 MPa If this is not the case the flow restrictor should be replaced see 8 6 3 Replacing flow restrictor on page 351 e p309 8 Maintenance 8 2 Monthly maintenance 8 2 2 Changing pump rinsing solution 8 2 2 Maintenance inter val Reason for main tenance Changing solution 28 4090 22 AB p 310 Changing pump rinsing solution Every month The pump rinsing solution should be changed to avoid bacteriological growth The rinsing solution can be placed in a 50 ml test tube mounted in the right hand column holder The pump piston seal rinsing system Check valve Optional path without i Rinsing solution recirculation _
285. shown in the table below Immerse injection valve port tubing into the flask container for 2 Waste 2 3 Waste 3 used for e g system wash and intermediate loop collection p211 6 Operation 6 4 Preparing the system for a run 6 4 4 Purging the pump and inlet tubing Overview 6 4 4 Caution Purging the pump and inlet tubing Overview CAUTION To protect the piston seals in the pump the pump must never be run with air in the inlet tubing Purging the pump The table below describes when different purging procedures should be performed and inlet tubing 28 4090 22 AB p212 and references to instructions for each procedure If then For instructions see e there is a lot of air in the inlet tubings and e most all inlet tubings should be filled with liquid fill the inlet tubings with liquid using the interact ive Prepare and Maintain Fill Inlets Using Syringe method plan 6 4 5 Filling inlets manu ally using a method plan on page 214 e there is a lot of air in the inlet tubings and e only a few inlets tubings should be filled with liquid manually fill the inlets by choosing the inlets to be filled from UNICORN 6 4 6 Filling inlets manu ally using UNICORN on page 217 there is only a small amount of air in the tubings automatically fill the inlet tubings within a purifica tion run by i
286. sing solution Note When using one of the HiPrep 16 60 Sephacryl S 100 S 200 S 300 HR gel filtration columns 0 d lt 30 mm insert the HiPrep 16 xx Column Wrap into the column holder on the left side before attaching the column Note Columns with o d 50 mm can be attached using an optional column holder made for these columns This column holder is ordered separately and should be mounted in place of the standard column holder left side The online filter supplied can be connected to the top of the gel filtration columns using a 1 16 male M6 female connector p43 3 AKTAxpress overview 3 2 Separation system overview 3 2 4 Functional description UV andconductiv The UV cell and the conductivity cell are located at the middle part of the system ity detection Flow restrictor UV cell Conductivity cell UV monitor The UV cell is connected in the flow path directly after the column valve It provides online detection of the absorbance of the eluted protein The optical unit is equipped with a Hg lamp a detector and a filter with a wavelength of 280 nm If required a wavelength of 254 nm can be used by turning the filter wheel and adjusting the housing oe o Wow Ww D T TETA UV cell WA p Optical unit 4 ii a dih oh The UV lamp is turned on by default The warm up time is 60 minutes If not using the system for a few days or longer turn off the lamp in System Settings in System Control by selecting Moni
287. solution 1 If an individual desalting column is used for each sample the column for sample 1 is connected to port 5 and the column for sample 2 to port 4 in the column block Note Insert stop plugs or non used connectors into empty ports in the column block to prevent dirt from entering the flow path Note Some applications require two columns of the same type connected in series This is marked x2 in the column list in the Method Wizard To avoid introducing air into the column connect the fitting drop to drop to the column also when connecting an online filter to the gel filtration inlet tubing This can be performed by using the method plan Column Attachment This procedure is divided into two steps e Create the method plan Create a method plan for attaching the columns Connecting the in let tubing fitting Operation 6 e Connect the inlet tubing fitting The illustration below gives an overview of how to create the method plan See 5 3 Using the Prepare and Maintain option on page 136 for detailed information O OO00008 Prepare and Maintain Lost Poge To connect the fitting Step Action 1 Run the method plan as described in 6 6 1 Starting a run using a method plan on page 259 Note The method will go from position 1 to 5 Result The column valve is set to position 1 and the pump starts the flow at a low flow rate Fix the tubing drop
288. son To replace the check valves Step Action 1 Change solvent to deionized water and flush out all salt Move all input buffers bottles below the level of the pump heads to prevent siphoning 2 Disconnect electrical power from the system 3 Remove the tubing from the pump heads 4 Loosen the upper valve from the pump head using the 18 mm wrench E A ZA Maintenance 8 Step Action 5 Gently remove the check valve and replace it with a new one CAUTION Handle the check valves with care when they have been removed from the pump heads to prevent loss of any internal components cy Od 6 Unscrew the two white knurled screws under the pump to release the support bracket that locks the inlet manifold into the inlet valve eS z gt 7 Gently lower and remove the complete manifold e p359 8 Maintenance 8 6 Replacement procedures 8 6 5 Replacing check valves 28 4090 22 AB p 360 Step Action 8 Loosen the lower valve from the pump head using the 13 mm wrench CAUTION Handle the check valves with care when they have been removed from the pump heads to prevent loss of any internal components Gently remove the check valve 9 Install the new check valves The inlet check valve with a lip for the manifold and a larger diameter opening is fitted to the side marked IN of the pump
289. ss 8 2 Monthly MAINTENANCE Eui enaere tia K A EE N EE Ae 8 2 1 Checking flow restrictor 8 2 2 CHANGING PUMP riNSiINg SOUTO Nassua e A EEEE 310 3 3 SIX MONAY MAINENANCE monnieri o E OAE EE AOE ER 311 8 3 1 Checking UVlamp TUN tiM E ienen a ii E e EA EE 312 83 2 Cl gning UV Cellera nn aa E AN AN 314 8 3 3 Cleaning Conductivity Cell assastea dati witiid a RE RE T 317 8 4 MGINTEMAMCE When reg iredniisieneaianmenikanaiiiarmi ii ina eii AE 318 8 41 Externally cleaning the Syste Miniset a TERURE 319 gA CISGIAIMNG CHECK VIVES aee AE E AE A UE ER 320 8 4 3 Checking p mp PLESSU LS ninnreeneiseeeii e A OE RARA 324 9 44 Checking tubing l 10 lt 0 6 Enirar nien a LE E UE OERE 325 8 4 5 Running installation test 8 5 CAIDA OM procedur Sanmi aa a E A a oa 8 5 Calibration OF zero presSSUre geninin EA a A 329 825 2 CAlIbratlon OF conductivity C l niaii e t e E T E N a 331 28 4090 22 AB ep6 Table Of Contents B56 RE placement PrOCSAULSS Es cdevecshis votes aocssessesssosassvvtovesoncoonvsssl cebstateasvntvatrdedlcetssbovvavinnns E E 335 8 6 Replacing capillary loo pS vecavvecsasdassssssssosaseestevsvovtsaveedeessstinesccssvecvenseeenaesaassscoossavnebiatdgoundassisovvonseuvseetgantassses 336 8 6 2 Replacing tubing and CONNECHOSS cssseessesssssccsssssssssusssessessesssssssssussuessesseseessssssssssesssssssecessssssssusssesseseees 338 8 63 Replacing TOW TESTIT siie i a B ANRE RE 351 8 64 Peplacino UV a Dae a A O
290. ss volume will be emptied from the loop to the collection tubes F9 F10 one for each sample The loop is then washed with IEX binding buffer to waste lon exchange The table below describes the ion exchange procedure of the collected peak volume Step Action 1 A PumpWash is performed to fill the pump with IEX binding buffer 2 The DS peak collected in the loop is loaded onto the IEX column peak volume flush volume 3 The loop with the loaded peak is flushed with IEX binding buffer to waste 4 Unbound sample is flushed from the IEX column to waste with IEX binding buffer Peaks are collected in outlet F9 F10 5 The IEX column is eluted with an IEX binding buffer IEX elution buffer gradient and the peak volumes are collected in loops 6 The nonselected peaks are emptied from the loops to the collection tubes F9 F10 one for each sample The loops are then washed with buffer to waste 28 4090 22 AB p 56 AKTAxpress overview 3 Gel filtration The table below describes the gel filtration procedure of the collected peak volumes Step Description 1 SystemWash is performed to fill the system with GF buffer 2 The largest IEX peak collected in the loops is loaded onto the GF column The maximum volume is 5 ml 3 The GF column is eluted with GF buffer 4 The eluted peaks are collected in the fraction collector 5 If the whole peak volume cannot be lo
291. sssccsssssssssssesssesecsssssssssusessssssessssssssussseees 138 5 3 2 The Prepare and Maintain PAGE OPTIONS eeeccccccssssssssssssessssessccssssssssssssssessecsssssssssusecsssseessssssssnuseesees 140 5 3 3 Selecting Standard System and Column Procedures OPtiONS cccccscsssssssssssseessesssecsssesssseeees 142 5 3 4 Selecting Customized System Procedures OPtiONS cacecccssccssssssssessesssssccsssssssssusessessecsssssssssssseees 146 5 3 5 Selecting Customized Column Procedures OPtiONS cceccccccccssssssssssseessessecssssssssssssessssesssecssssasseeeees 148 54 Using the PUTI ORTON inion dsescascseeeseasesaaahsasasceinad axvsssteatatcasa UE OAOE 150 541 Selecting PUrity T 2 pdoe Option enrian a a 152 542 S lecting Purify 227 PAGELOPLIONS issscssssstccccccssesshSovexesacsasesuesoooibostevseessassuusostessiecces tabi ausatacosstessovsstessies 154 5 4 3 Selecting System Procedures page OPtiONS ccsssssssesssssscssecssssssssssssssessecsssssssssssessesscesssssssssunseeses 157 5 4 4 Selecting Column Preparation PAGE OPtIONS ccesssssesssscssccsssssssssssessessccsssssssssusesssssccssssssssuasseesses 160 5 4 5 Selecting Column Post RUN page OPtiONS eeccecccccsssssssssssesssssscsecsssssssssssessessessssssssssnsessessecsessssssnusseesees 162 BS AAV COG E740 p RAAE ET E OE 164 551 AG VENCEd ZOMEIMELOC UCT OM i E AAA 165 28 4090 22 AB ep4 Table Of Contents 5 5 SeAAVENCEE ZONE LOR PUN Vesian A AA 5 5 4 Advanced Zone for Purify A
292. sub sections below Sub section See Metal ion charging of chelating affinity columns 6 5 1 Conditioning of columns 6 5 2 ep 251 6 Operation 6 5 Column procedures before a purification run 6 5 1 Metal ion charging of chelating affinity columns 6 5 1 Metal ion charging of chelating affinity columns Introduction New uncharged or stripped chelating affinity columns must be charged with metal ions before they can be used in a purification run Note If columns should be stripped and recharged with the same type or another type of metal ions this can be included within a purification run after purification or within a prepare and maintain run See 6 8 8 Metal ion stripping and recharging of affinity columns on page 290 for information Metal ion charge Metal ion charging of chelating affinity columns is performed by including the Metal procedure lon Charge Blank Run procedure in the Standard System and Column Procedures method plan By including the procedure Blank Run as the last step the columns are both charged and equilibrated and will be ready for use in a purification run Up to five affinity columns of the same type can be charged simultaneously Prerequisites The following are required for metal ion charging of the chelating affinity columns e Metal ion charge solutions See the column instructions and or 4 7 5 Metal lon Charge Solution Suggestions on page 122 e Amethod plan for char
293. suppliers p93 4 Methodology 4 5 Column alternatives 4 5 About this section In this section 28 4090 22 AB p94 Column alternatives This section contains general descriptions of the columns currently supported by KTAxpress and guidelines for selection of appropriate column combinations for different applications This section contains the sub sections below Sub section See Descriptions of columns 4 5 1 Choosing columns 4 5 2 4 5 1 Introduction Column types Affinity chromato graphy columns Methodology 4 Descriptions of columns This section describes the different types of columns currently supported by AKTAxpress Four main column types are used for chromatography in AKTAxpress Column type abbreviation Separation property Affinity chromatography AC column Function Desalting DS column N A used for buffer exchange lon exchange IEX column Charge Gel filtration GF column Size More information on the main column types is found below Affinity chromatography AC columns separate proteins on the basis of a reversible interaction between a protein and a specific ligand attached to a chromatographic matrix Affinity chromatography can be used whenever a suitable ligand is available The AC column binds specific molecules very efficiently even from large sample volumes with a low concentration of target molecules Not
294. susessessecessssssssnssseseeseees 214 6 4 6 Filling inlets mMan lly sing UNICORN seseina naaa eni 217 6 4 7 P rging the pump and SYSTEM suiii aA EA AE A EAE 6 4 8 Purging the pump with methanol 6 49 Connecting columnas ANd TUBING siheso e a Aak 6 4 10 Preparing the system when using double loops 6 4 11 Preparing the system when using a SUPCTMOOPL vcccccsssssssssssssessessesecsssssssssusssesssssecssssssssuassneseeeess 6 4 12 Preparing for on column tag cleavage aiii e A ER iA 6 4 13 Preparing the fraction CONSCtOL csssssssssccssssssssssssssessssccssssssssssssssssssessecsessssssssusscsessesesssnsssssssesesseess Gi TAS CHECKING HS LUBLIN seis ssvccicesseibosceccessns sass casovtassotscosssslaccossdavoatentvestunassessssbeovnawvalvosstausseslosbonveneecebuveetes 6 4 15 Preparing Sarpe Sirera E NO ONAE REOOO GAG USING Ar dJEtECUON naea E OEE O AOA 64 17 USIN TOW COMO liceeni oraiiio a Aa EOE ROE ERER 6 5 Column procedures before a purification FUN cc cccccsssssssssesesssssccsssssssssssesssesccsssssssssusssessessesssssssssssssesseseees 6 5 1 Metal ion charging of chelating affinity columns 6 5 2 Conditioning OF COlUMINS seats ass Se a aE R ian EN ERAO 6 6 Starting d TUN aseeseen ineei E EEA EEE EEE AEE 6 6 1 Starting G r m using d method Plasenn E RER eps Table Of Contents 6 6 2 Staring a TAG GLMCA as e racceeit scsi e Closes cand EONO HE EEREN 265 6 77 DUTING A TUN peregi A EA EN Ea RA E E
295. t emit light at 280 nm so for this wavelength the light is converted at a fluorescent surface before it passes p411 10 Reference information 10 1 System description 10 1 3 Monitors Conductivity mon itor Pressure sensor 28 4090 22 AB p412 the filter On the lamp housing there is a special exit for 280 nm light which means that the lamp position needs to be changed when working with this wavelength The conductivity monitor flow cell is positioned below the UV housing The flow cell has two cylindrical titanium electrodes positioned in the flow path of the cell An alternating voltage is applied between the electrodes and the resulting current is measured and used to calculate the conductivity of the eluent The monitor controls the AC frequency and increases it with increasing conductivity between 50 Hz and 50 kHz giving maximum linearity and true conductivity values The conductivity is automatically calculated by multiplying the measured conductance by the flow cell s cell constant The cell constant is precalibrated on delivery but can be measured with a separate calibration procedure see 8 5 2 Calibration of conductivity cell on page 331 Liquid flow gt The pressure in the flow path is continuously monitored by a pressure sensor The liquid chamber in the pressure sensor housing is equipped with a titanium membrane A strain gauge is attached to the rear side of the membrane When the liquid pressu
296. t least 300 ml degassed and deionized water e amethod plan e p223 6 Operation 6 4 Preparing the system 6 4 8 Purging the pump with methanol Create the meth od plan fora run The illustration below gives an overview of how to create the method plan See 5 3 Using the Prepare and Maintain option on page 136 for detailed information f Prepare and Maintain Purging the pump To purge the pump with methanol with methanol 28 4090 22 AB p224 Step Action 1 Prepare one flask with at least 200 ml of degassed methanol inlet A1 2 Prepare one flask with at least 300 ml of degassed and deionized water inlet A5 3 Carefully immerse the inlet tubings that will be filled during the procedure in the respective flasks 4 IMPORTANT Put both Waste tubings from the injection valve into a separate waste flask to collect the methanol separately 5 Run the method plan as described in 6 6 1 Starting a run using a method plan on page 259 6 4 9 Introduction Cautions Attaching the columns for a purification run Operation 6 Connecting columns and tubing This section describes how to connect columns and tubings that are used in a purification run CAUTION Tighten the columns properly in the column block to avoid leakage Over tightening might though rupture the column connectors CAUTION Make sure that no parts for example tubings or columns are position
297. t page is a schematic drawing showing the components in the flow path outlet Introduction 2 ale 24 or 96 well microplate Us aa En valve waste 0 2 metal ion i MPs ig loops tiow not usad i 1 5 with through peaks Paim alternative sample 1 4 sample 1 4 TSN double Kops a a wn a UV cell a Lr f ip lt i ZY column fa sae vate columns 7 A 15 alterative superioop sensor valve meet 6 waste inlet valve bump gradient ava SESI oa a om BOH pumo rinse buffers butters butters Samples Al 61 A2 82 A38 14 solution The main components are as follows Combined sample and buffer pump Switch valves for buffer selection and gradient formation Separate rotary valves for sample and buffer selection manual injection column selection loop selection and outlet flow direction UV and conductivity cells Loops for intermediate fraction collection Fraction collector with deep well microplate The columns required for the chosen purification can be attached to the system either by threading them into the column block small columns or by securing them with snap locks in the column holder large columns ep17 2 Introduction 2 1 Introducing AKTAxpress The UNICORN control system Several purifica tion protocols in parallel 28 4090 22 AB p18 The separation system is equipped with a control panel with push buttons and a display The push buttons allow th
298. t scientific areas The need to produce pure target proteins quickly and easily has increased the usage of recombinant proteins greatly in recent years So has also the wealth of techniques and products used for their amplification and purification The advantage of using a fusion protein to facilitate purification of a recombinant protein is widely recognized Fusion proteins are simple and convenient to work with and for many applications a single step purification using affinity chromatography is sufficient to achieve the desired level of purity However if there is no suitable fusion system or when a higher degree of purity is required a multi step purification process will be necessary AKTAxpress has been developed to meet the growing demand for quick and easy purification of recombinant proteins with high purity The system is based on purification strategies that involve combinations of protein capture on affinity columns buffer exchange in desalting columns intermediate purification in ion exchange columns and polishing in gel filtration or ion exchange columns AKTAxpress supports different purification protocols containing up to four chromatography steps Column and system preparation as well as cleaning in place and maintenance can be included in the purification protocols The intermediate protein peaks between the steps are collected in loops The purification protocols in AKTAxpress are primarily designed for purifica
299. t system procedures that can be included in a purification run using the largest columns AC 5 ml IEX 6 ml DS HiPrep 26 10 Desalting GF HiPrep 26 60 Sephacryl at room temperature RT System procedure Typical run time in RT min Preparation Remove Ethanol from System 10 During run Clean sample inlets 4 samples 5 Wash Frac Tubing 4 samples 5 Post run Fill system with ethanol 10 CIP system then fill with ethanol 35 CIP system then fill with buffer 25 e p429 10 Reference information 10 4 Typical run times Typical run times for included column proced ures 28 4090 22 AB p430 1 Run times are approximate and with default settings Sample loading time is not included The table below show estimations of the run times for some different column procedures that can be included in a purification run using the largest columns AC 5 ml IEX 6 ml DS HiPrep 26 10 Desalting GF HiPrep 26 60 Sephacryl at room temperature RT Column procedure Typical run timet RT CR min Preparation AC Equilibration 5 Blank Run 25 Remove Ethanol before preparation 5 DS Equilibration 10 Remove Ethanol before preparation 5 IEX Equilibration 5 Blank Run 25 Remove Ethanol before preparation 5 GF Equilibration 325 Remove Ethanol before preparation 155 Post run AC Fill with Ethanol 5 CIP
300. te CIP procedure if sample inlets should be cleaned after the run The illustration below gives an overview of how to include CIP System and Loops with NaOH Water Buffer in the Standard System and Column Procedures method plan e p279 6 Operation 6 8 Procedures after a run 6 8 3 Standard cleaning of the system Cleaning the sys tem within a puri fy method plan Cleaning the sys tem using the Standard System and Column Pro cedures method plan 28 4090 22 AB p280 See 5 3 3 Selecting Standard System and Column Procedures options on page 142 for detailed information Select Stondard System and Column Prox System Procedures aee 0000 M 0 CIP System and Loops with NoOH Woter Buffe the loops to be woshed Run the purify method plan as described in 6 6 1 Starting a run using a method plan on page 259 CIP System is selected as Post Run and cleaning of the system will be performed after purification If cleaning sample inlets the system will pause and the sample inlet tubings should be moved to a separate flask with the appropriate solution To run the prepare and maintain method plan for cleaning the system Step Action 1 Wash the outside of the inlet tubings with water and or ethanol 2 If the outlets are to be cleaned insert a microplate 3 Run the cleaning method plan as described in 6 6 1 Starting a run using a method plan o
301. tended for research use only and should not be used in any clinical or in vitro procedures for diagnostic purposes epi13 2 Introduction About this chapter In this chapter 28 4090 22 AB ep 14 Introduction This chapter contains an introduction to AKTAxpress and the basic operating principles It also gives a background to multi step purification of recombinant proteins and general information about the user documentation This chapter contains the sections below Section See Introducing AKTAxpress 21 Purification of recombinant proteins 2 2 Basic operating principles 2 3 AKTAxpress user documentation 2 4 2 1 Introduction General Features Introduction 2 Introducing AKTAxpress This section gives a general overview of AKTAxpress AKTAxpress is a liquid chromatography system intended for automated multi step purification processes The system has been developed and optimized for purification of Histidine tagged and Glutathione S Transferase GST tagged recombinant proteins from clarified or crude cell lysates AKTAxpress supports a number of purification protocols containing up to four chromatography steps for example affinity chromatography followed by desalting ion exchange and gel filtration Protocols for purification of both low expressed proteins and well expressed proteins are included Also protocols supporting purification of double ta
302. that the appropriate anion cation ex change column is used e To collect all IEX peaks run the sample again using the AC DS IEX protocol All peaks will then be collec ted in the microplate p379 9 Troubleshooting and corrective actions 9 2 Problems during a run No satisfactory elution from Possible cause and action Gel filtration column e Check that the eluted peak from the previous step was collected in a capillary loop by inspecting the peak start and peak end markers in the chromato gram If not collected Open the used method plan and enter the Ad vanced Zone Change the watch parameters used in the peak collection Run the sample again e Ifthe separation is not good enough change to a more suitable gel filtration column or divide the start sample into aliquots Perform repetitive or parallel purifications where less protein is applied in each run e For manual gel filtration protocols it is only recom mended to load up to 5 ml sample per sample loop Problems when The table below describes some problems that may occur when running methods running methods that have been created in the Method Editor in UNICORN For information regarding created in the parameters and how to create methods in the Method Editor see UNICORN Reference method editor Manual for AKTAxpress Problem Possible cause and action When using timer func tions e g Timer_Stop re
303. the Purify 1 2 page Step Action 1 On the Purify 1 2 page select Number of Purification Steps and Puri fication Protocol For guidance on how to select the number of steps and type of protocol see 4 1 Protocol descriptions and choice on page 59 J O2 Prsiieanon Protocol Attray Desahing lon Exchange Gel Fitraton Alr y Hutia HP d Glub Decahing HPrep_26 10_Decating Global lon fuchange FESOURCE Q10 Global Gel Finanen Hiosd 1550_5wmdm_ 7 ep gate akbal Rumney lantan C Room Temperanae Cold Room O Dre Coksnn Tay Cleavage O Esma Wash of tat Stag Cohamne baiara Fhian 2 Choose the column s to be used in the drop down list s For information about appropriate columns see 4 5 2 Choosing columns on page 101 28 4090 22 AB p 152 Method Wizard 5 Step Action Protocols starting with an AC IEX step Check the On Column Tag Cleavage box to perform on column tag cleavage in the first step Additional options for filling the columns with cleavage buffer and changing the protease incubation time are displayed See 4 4 3 Description of the affinity tag removal processes on page 89 for more information about on column tag cleavage Ww One Coker Tag Clerenge ly FE Cakera weh Cheavage Butter tneubomion Tose Pots o Select Fill Columns with Cleavage Buffer to fill the affinity ion ex change columns with cleavage buffer if a specific cleavage buffer other than the ordinar
304. the column s during the purification run leqect ceoteate solvice Do not remove the nenge Pret CONTINUE ty o am a ol Filling Superloop manually The protease can also be injected directly into Superloop with a syringe before starting the run by connecting the syringe to the lower port of the Superloop Itis also possible to connect Superloop to the loop valve as usual and manually setting the injection valve and loop valve to the correct positions To set the valve positions manually Step Action 1 Select Manual Flowpath in System Control 2 Set LoopSelection to LP5 and click Execute 28 4090 22 AB p236 Operation 6 Step Action e Before fitting or removing the syringe set InjectionValve to Inject and click Execute e To inject liquid into the superloop Set InjectionValve to Load and click Execute Inject the protease solution e p237 6 Operation 6 4 Preparing the system for a run 6 4 12 Preparing for on column tag cleavage 6 4 12 Introduction Protocols for affin ity tag removal Main steps 28 4090 22 AB p238 Preparing for on column tag cleavage This section describes how to create a method plan including on column tag cleavage and how to calculate protease volume and concentration It also describes how to use a column heater with KTAxpress in case the temperature needs to be increased during cleavage For inf
305. the general help utility open the Help menu in any of the software modules context specific help e click the Help button in the dialog box or topics e press the F1 key on the keyboard 28 4090 22 AB p 30 3 2 About this section In this section AKTAxpress overview 3 Separation system overview This section shows the location of the components in AKTAxpress as well as the controls indicators and rating labels It also describes the liquid flow path with a functional description of the components This section contains the sub sections below Sub section See Location of the components 3 2 1 Liquid flow path 3 2 2 Tubing and connectors 3 2 3 Functional description 3 2 4 Indicators and controls 3 2 5 Labels 3 2 6 ep3l 3 AKTAxpress overview 3 2 Separation system overview 3 2 1 Location of the components 3 2 1 Location of the components Introduction This section contains an overview of the components in the separation system Fluid handling All fluid handling components are located on the front of the separation system components Outlet valve ___ Fraction collector Column block ____ Capillary ae T Control panel Column valve SNe Ta Loop valve UV cell iry Conductivity cell A Pressure sensor ii Switch valves Mixer n ya nl poe Inlet valve Air sensor Electrical connect Connectors for power and com
306. this chapter In this chapter AKTAxpress overview 3 AKTAxpress overview This chapter contains an overview of AKTAxpress including UNICORN control software and the components in the separation system It also contains an overview of the purification process steps This chapter contains the sections below Section See UNICORN overview 3 1 Separation system overview 3 2 Purification process overview 3 3 ep27 3 AKTAxpress overview 3 1 UNICORN overview 3 1 Introduction UNICORN control system Software modules Method Wizard 28 4090 22 AB p28 UNICO RN overview This secti on is an overview of UNICORN UNICOR Ho UNICOR is a complete software package for control and supervision of AKTAxpress he software also enables the computer to communicate with the separation systems via the USB CAN converter box software runs under Microsoft Windows UNICORN consists of four integrated modules which are described in the table below Module Function UNICORN Manager File handling and administration Method Editor To create and edit methods and method plans System Control To start method plans and to control and monitor the separations processes on line Evaluation To evaluate and present stored results from the separa tion processes The Method Wizard in UNICORN provides easy creation of method plans for puri
307. thod plan to be edited Lreate w Change Method plan Method pian Note a New Ae gt a pac os Lp AC OS x oF aah atiae Dar Hu lep HP a Gioball DIAC GF Hioad 160 Superdex 75 pep aade A GF Supertoop Global aad Papae and Martan Fiocen Tempra ge bat Stan Click Next Change the values as desired on the subsequent pages Note When changing a selection made on the Purify pages in the Method Wizard all parameter values belonging to column type in the Advanced Zone will be set to default Any changes made earlier in the Advanced Zone have to be made again If further parameter values need to be changed go to Advanced Zone by clicking Next repeatedly Note Avoid changing default parameter values in the advanced zone unless the result is clearly understood Changing a parameter value might require adjustments of other parameter values as well Make the appropriate selections and edit the desired parameter values as described in 5 5 1 Advanced Zone introduction on page 165 and the subsequent sections Save the method plan as described in 5 2 2 Saving the method plan on page 132 Note The changes can be written in the Notes field in the Save As dialog 28 4090 22 AB p 134 5 2 4 Introduction Method Wizard 5 Deleting a method plan This section describes how to delete an existing method plan Deletinga method To delete a method plan plan Step Action 1
308. tion ignored The method instruction or manual command could not be executed Troubleshooting and corrective actions 9 Code Description 220 Set last tube error An attempt was made to set the last tube to a position already used Set the last tube to a non used position 221 222 System error Internal error Restart system If problem remains contact service 223 Bad combination of valve positions The Injection valve is in position reinject and the Outlet valve is in position LoopFracF12 224 CAN bus error The communication with the instrument is broken Restart system If problem remains contact service 300 308 System error Internal error Restart system If problem remains contact service 400 401 System error Internal error Restart system If problem remains contact service 402 Cell constant out of range The calculated conductivity cell constant is outside the accepted range Calibrate with a liquid of known conductivity in the range 0 1 300 mS cm 403 Cond sine table not calibrated The conductivity sine table has not been calibrated Contact service 404 Conductivity cell error The cond cell is either not connected or broken 405 408 System error Internal error Restart system If problem remains contact service 410 415 System error Internal error Restart system If problem remains contact service 416 The UV lamp is broken The UV lamp is broken Replace t
309. tion of the most common fusion proteins Histidine tagged and Glutathione S Transferase GST tagged proteins Tag removal is optional in all protocols starting with an affinity ion exchange step Introduction 2 Up to twelve AKTAxpress systems can work in parallel where each system can handle up to four different samples through the whole purification procedure independently of the other systems ep2l 2 Introduction 2 3 Basic operating principles 2 3 Introduction Sample applica tion and wash Elution 28 4090 22 AB ep 22 Basic operating principles This section gives a brief introduction to the function of AKTAxpress It describes the main steps during a purification run as shown in the table below Step Action 1 Sample application and wash 2 Elution 3 Intermediate fraction collection 4 Final fraction collection A functional description of AKTAxpress and a detailed description of the steps during the purification process can be found in section 3 3 Purification process overview on page 52 The sample can be loaded via the system pump from the capillary loops or the Superloop Sample application via the system pump The sample is selected by the inlet valve and transported by the pump to the affinity ion exchange column which captures the protein When using several samples during the run each sample is loaded and captured onto its own affinity column Then the subseque
310. tions for affinity tag cleavage 444 28 4090 22 AB p84 4 4 1 Introduction About affinity tags Affinity tag types Methodology 4 Description of affinity tags This section gives a general description of affinity tags and their use as well as a more detailed description of the Histidine and GST affinity tags Affinity tags are often used to facilitate the purification of recombinant proteins An affinity tag is included on DNA level for the recombinant protein normally at the N or C terminus The affinity tag can bind to an affinity chromatography medium with a suitable ligand allowing specific purification For several applications the affinity tag has to be removed from the target protein If a protease recognition sequence has been introduced between the affinity tag and the protein the affinity tag can be removed during the purification process of the protein When the tagged protein is subjected to its specific protease cleavage occurs at the recognition site Cleavage can be performed in batch or on column All AKTAxpress purification protocols starting with an affinity ion exchange chromatography step can be combined with automatic on column affinity tag cleavage d Protease Cleavage site Tags supported by KTAxpress protocols Affinity tag type Binds to Size Histidine polyhistidine Metal ions 1 3 kDa tag with n histidines Example Ni2 Zn2 Cu2 Co2 GST Glutat
311. to cols with manual sample loading 28 4090 22 AB p246 for a run volumes can also be set manually in the Wizard in System Control when starting the run Purification protocols starting with a DS or GF step in the Method Wizard require manual sample loading into the capillary loops or a Superloop Protocols using Superloop sample loading can be used for samples with large volumes One sample can be loaded from the Superloop a little at a time instead of distributing the sample into several loops When running these protocols the sample is injected with a syringe through the fill port MANUAL INJECTION in the injection valve into the loops Superloop in the loop valve When performing sample loading from capillary loops up to four loops can be filled Dialog boxes will automatically appear during the run with instructions for filling the loop superloop See also 6 4 11 Preparing the system when using a Superloop on page 233 for a description of the procedure Note The sample volume must be lower than the maximum sample loading volume of the desalting or gel filtration column used In addition the sample volume must always be lower than the loop volume which is 10 ml unless a double loop is used then it is 20 ml Observe that the sample volume should be entered when starting the method plan in System Control as well Example The HiPrep Desalting column alone can take up to 15 ml When used in AKTAxpress use a double loop to in
312. to drop See the table below for information about the connection of tubings from the column valve ports to the columns e p227 6 Operation 6 4 Preparing the system for a run 6 4 9 Connecting columns and tubing Step Action 3 Click Next Breakpoint to proceed to the next column position Note To skip a column position click Next Breakpoint instead of attaching a column 4 Repeat steps 2 3 until all tubings have been attached The table and illustration below describe how the tubing should be connected from the column valve ports to the columns See also the connection guide below for how to use unions to connect the tubing to the columns Column valve port Connect to column in Bypass Bypass 1 Column block upper port 1 2 Column block upper port 2 3 Column block upper port 3 or to DS GF column 4 Column block upper port 4 5 Column block upper port 5 or to DS GF column OUT 1 Column block lower port 1 OUT 2 Column block lower port 2 OUT 3 Column block lower port 3 or to DS GF column OUT 4 Column block lower port 4 OUT 5 Column block lower port 5 or to DS GF column 1 Connect the IN ports to the tubing on top of the desalting or gel filtration column s and the OUT ports to the tubing at the bottom of the columnis The columns are fitted to the column holder Note Do not connect any columns to the bypass ports 28 4090 22 AB
313. tors UV lamp Conductivity monitor The conductivity cell connected directly after the UV cell is primarily used for verifying gradients during the elution The measurement range is 1 uS cm to 999 9 mS cm Flow restrictor A flow restrictor is connected in the flow path directly after the conductivity cell to prevent air from precipitating and getting trapped in the UV cell The flow restrictor creates a backpressure of 0 2 MPa tg Flow restrictor 28 4090 22 AB p44 Intermediate frac tion collection AKTAxpress overview 3 The intermediate fraction collection is handled by a loop valve and five capillary loops Capillary loops Loop valve Loop valve The loop valve is located at the upper right hand side It is used to direct an intermediate peak volume from the UV cell and the outlet valve into a suitable loop where it is stored for the next purification step Capillary loops Five loops each one with a volume of 10 ml are connected to the loop valve The loops have i d 1 0 mm and are made of ETFE tubing It is possible to connect an extra capillary loop to each of the existing loops to get double loops with a volume of 20 ml The loops are joined using a 1 16 female 1 16 female union See also 6 4 10 Preparing the system when using double loops on page 231 p45 3 AKTAxpress overview 3 2 Separation system overview 3 2 4 Functional description Final fraction col The final fractionation parts a
314. tration below gives an overview of how to create the method plan See 5 3 Using the Prepare and Maintain option on page 136 for detailed information To fill the inlets using a syringe Step Action 1 Check that the inlet tubings to be filled are properly immersed in the correct containers flasks tubes 2 Run the method plan as described in 6 6 1 Starting a run using a method plan on page 259 Result The method will start with inlet 1 It will then continue with S2 S4 A1 A8 B1 and B2 when pressing next breakpoint Operation 6 Step Action 3 Connect a male Luer syringe of at least 20 ml to a purge valve Two syringes are included in the accessory kit supplied with the system 4 Turn the purge valve counter clockwise half a turn to open it hoa a a Il OFN EN NG 5 es gt D f f 5 Slowly draw buffer with the syringe When fluid starts to enter the syringe close the purge valve Check that there is no visible air left in the 1 tubing Note If air in the other pump head is suspected draw buffer through that pump head as well p 215 6 Operation 6 4 Preparing the system for a run 6 4 5 Filling inlets manually using a method plan Step Action 6 If required remove the syringe empty it and insert it in the purge valve again before continuing 7 To proceed with the next inlet press the Next Breakpoint button on the instrument Note To skip an in
315. ty columns This section contains the sub sections below Sub section See Emptying drip plate 6 8 1 Cleaning system Overview 6 8 2 Standard cleaning of the system 6 8 3 Customized cleaning of the system 6 8 4 Cleaning of columns Overview 6 8 5 Standard cleaning of columns 6 8 6 Customized cleaning of columns 6 8 7 etal ion stripping and recharging of affinity columns 6 8 8 e p273 6 Operation 6 8 Procedures after a run 6 8 1 Emptying drip plate 6 8 1 Introduction Emptying drip plate 28 4090 22 AB p274 Emptying drip plate The drip plate located below the microplate in the fraction collector should be emptied when necessary The purpose of the drip plate is to collect any overflow from the microplate Overflow might indicate an error see 9 1 Introduction to troubleshooting on page 372 If the drip plate becomes full there is a risk of damage to the system To empty the drip plate located under the fraction collector follow the procedure below WARNING Do not put any body parts in front of or close to the fraction collector The microplate can be ejected and retracted both manually and automatically by the system CAUTION Ensure that no parts such as tubings or columns are positioned in front of the fraction collector hindering the ejection of the microplate The movement of the plate might destroy columns or tubings positioned
316. ulated in mg ml or M and Amount is calculated in mg or mole Evaluation 7 Step Action 3 The pooled fractions can be adjusted manually e To include or exclude adjacent fractions in a pool Click the numbered marker under the pool and drag the sideline e Toadd more pools Click between the droplines under a fraction to create a new pool and drag the sidelines to include more adjacent fractions e To delete pools Click the numbered marker to select the pool and click the Delete button Click the Delete All button to clear all pools e To restore the pools created by UNICORN Click the Default Pool button Note To calculate concentration and amount for peaks that have not been fractionated perform a simulated peak fractionation See UNICORN AKTAxpress User Reference Manual for more information e p299 7 Evaluation 7 5 Creating pooling protocols 7 5 Introduction Creating pooling protocol 28 4090 22 AB p 300 Creating pooling protocols Information about pooled fractions from different result files can be assembled in the pooling protocol The pooling protocol can be printed for use when pooling the samples physically This section describes how to add pools to the pooling protocol and how to print or export the pooling protocol Once the pools have been satisfactorily adjusted they can be added to the pooling protocol The table below describes how to add pools to the Pool
317. umn Preparation page options Introduction The options on the Column Preparation page depend on the choice of purification protocol i e the number and type of steps included and the order of the steps Selecting options To select options on the Column Preparation page Step Action 1 Select the requested columns to be prepared by checking the boxes for the columns in the different steps First Chromatography Step Second Chromatography Step etc If you do not wish to prepare any columns leave all boxes on this page unchecked and click Next 28 4090 22 AB p 160 Method Wizard 5 Step Action Select the appropriate column preparation s to be included in the puri fication See 4 3 1 System and column procedures within a purify method on page 74 for a description of the options Depending on the selected protocol the number of columns vary and different types of columns may occupy the different column positions M Remgve Lthanot betore L auabeaticn Note The above example shows options for an AC DS IEX GF protocol Click Next p 161 5 Method Wizard 5 4 Using the Purify option 5 4 5 Selecting Column Post Run page options 5 4 5 Selecting Column Post Run page options Introduction The options on the Column Post Run page depend on the choice of purification protocol i e the number and type of steps included and the order of the steps Selecting options To
318. understood Option Use to create a method plan for Prepare and Maintain Preparation and maintenance of the system and or the columns Example of actions Filling the buffer and sample inlets with buffer Equilibrating the columns Performing differ ent types of cleaning operations and customized equi librations Running installation test Instructions for the Prepare and Maintain option are given in 5 3 Using the Prepare and Maintain option on page 136 Purify Purification operations Example of actions Performing different types of protein purification runs Instructions for the Purify option are given in 5 4 Using the Purify option on page 150 Note Some of the prepare and maintain options can also be included in a purify method plan For more information on the options available on the current page click Help ep 131 5 Method Wizard 5 2 Working with method plans 5 2 2 Saving the method plan 5 2 2 Introduction Saving the meth od plan 28 4090 22 AB p 132 Saving the method plan This section describes how to save the method plan To save the method plan Step Action 1 On the Last Page page click Store Results in a Main Folder to define where the results should be stored By default the results are stored in a folder directly under the Home folder The default name of the folder is the current date To store the results
319. ure the flow rate and volume when performing the procedure can be changed For a list of possible procedures for the different columns see 4 3 1 System and column procedures within a purify method on page 74 e Flow Rate Flow rate through the column during the procedure Examples Flow Rate Remove Ethanol Flow rate used when the ethanol is removed from the column Flow Rate Equilibration Flow rate used during the column equilibration e Equilibration Volume Volume used to perform the procedure Examples e p183 5 Method Wizard 5 5 Advanced Zone 5 5 5 Advanced Zone for Purify Desalting Gel Filtration Volume Remove Ethanol Volume used to remove ethanol from the column Volume Equilibration Volume used to equilibrate the column e Incubation Time after CIP The time the system is set to pause after the column s has been filled with CIP solution Fractionation See 5 5 8 Advanced Zone for Purify Fractionation on page 188 for information on settings 28 4090 22 AB p 184 5 5 6 Introduction Included System Procedures para meters Method Wizard 5 Advanced Zone for Purify Included system procedures This section describes the parameters used in the system preparation steps before and after a purification run The settings on this page are independent of the purification steps included in the protocol The Included System Procedures page contains options and parameters relat
320. ution in AC IEX steps The default gradient is 0 50 elution buffer during 20 CV followed by 50 100 elution buffer during 2 CV prior to clean after elution Note The default settings can be changed in the Advanced Zone How to select peak Largest The largest peak will be transferred to the next column Based on Area set as default or Height Define B Interval only available for gradient elution Prior to start a B interval will be set and the eluate during this interval is collected in loops and will be transferred to the next step Note The B interval is set and can be changed in the Advanced Zone or in System Con trol Manual selection only available for gradient elution After elution the system will pause Manually select which peak to transfer to the next purification step See Manually selecting a peak for the next purification step in 6 7 During a run on page 267 for more informa tion Fractionation Peaks Peaks and Fixed Select to only fractionate peaks Select to fractionate with a fixed volume When peaks Volume are detected they are collected using peak fractionation Note Check the Change plate if out of fractions box when using this option 24 well plate Select to collect fractions in a 24 well microplate 96 well plate Select to collect fractions in a 96 well microplate Change plate if out of fractions If the last tube is reached the fract
321. w the format description below e The file name is optional but the extension must be txt e Column 1 Sample ID Column 2 Parameters Column 3 Parameter values e The import file must contain all five parameters Each parameter must have a value gt 0 Using the import file Reference information 10 e The parameter names must be written according to the example above e Separate the columns by pressing Tab once e Press Enter to create a new line e The default storage location of the import file is C UNICORN Server MethodWizardImport The path can be changed in each method plan see 5 5 9 Advanced Zone for Purify Miscellaneous settings on page 190 When starting a run using the wizard in System Control the import file s can automatically be called upon e Enter the Sample ID s on the Sample ID System X Y page for example GFP His Note The Sample ID s from the import file on the previous page are used e Click Next The data in the file s is collected and inserted on the Settings page in the wizard e p427 10 Reference information 10 4 Typical run times 10 4 Introduction Factors affecting the run time for a purification run Typical run times for the different protocols at room temperature 28 4090 22 AB p 428 Typical run times This section lists typical run times for protocols and system and column procedures that have been included in the run The run time for a purific
322. with 5 CV desalting buffer and the gel filtration column with 2 CV gel filtration buffer Strip columns After purification strips metal ions off the first step affin ity column using 5 CV strip buffer e g affinity binding buffer with 50 mM EDTA followed by 10 CV water Note Outlet F11 will always be used to collect the metal ion waste Recharge After purification and after stripping of the first step af finity column the columns are recharged with the appro priate metal ion in three steps 5 CV water 1 CV 0 1 M metal ion solution of choice and finally 5 CV water Note Outlet F11 will always be used to collect the metal ion waste 4 3 2 Introduction When to run sys tem and column procedures within a prepare and maintain method Methodology 4 System and column procedures in prepare and maintain methods This section describes the different system and column procedures that can be performed by running the prepare and maintain method plans e Standard System and Column Procedures e Customized System Procedures e Customized Column Procedures Note Other types of prepare and maintain method plans are also available These are described in 5 3 2 The Prepare and Maintain page options on page 140 System and column procedures for preparing and maintaining the system and columns can be run separately in prepare and maintain methods or within a purification method see 4 3 1 Syste
323. x x x AC DS IEX x x IEX DS IEX x DS IEX GF x x x AC DS IEX x x x x GF p63 4 Methodology 4 2 Peak handling 4 2 Introduction Overview In this section 28 4090 22 AB p64 Peak handling This section contains information about how the peaks are handled in the different purification steps All purification steps in the standard protocols in the Method Wizard include peak handling In Advanced Zone some of the parameters that control peak handling can be changed The parameters available for each chromatography technique are described in the following sections This section contains the sub sections below Sub section See Peak collection parameters 4 2 1 Peak collection parameters when using double loops 4 2 2 Peak fractionation parameters 4 2 3 4 2 1 Introduction Overview Peak collection and watch condi tions Methodology 4 Peak collection parameters This section contains brief information about the parameters that control how peaks are collected in the different purification steps The peak collection parameters are used in watch conditions to determine how peaks are collected between the purification steps These peaks are collected in one or more of the five loops One peak can also be divided into several loops depending upon the volume The selected peak is then used in the next purification step Peaks that
324. y affinity binding buffer should be used before adding protease Select the Incubation Time the time required for the added protease to cleave the tag from the protein Information on factors affecting the cleavage time can be found in 4 4 4 Conditions for affinity tag cleavage on page 92 Protocols starting with an AC IEX step Select Extra wash of 1 st Step Columns before Elution to wash the af finity ion exchange column with affinity ion exchange wash buffer in order to remove weakly bound contaminants Click Next e p153 5 Method Wizard 5 4 Using the Purify option 5 4 2 Selecting Purify 2 2 page options 5 4 2 Selecting Purify 2 2 page options Introduction The options on the Purify 2 2 page depend on the choice of purification protocol in the previous step and if on column tag cleavage was included Selecting options To select options on the Purify 2 2 page Action Depending on the chromatography step different options appear on the Purify 2 2 page See steps 2 4 and the table below for information about how to select options for the different chromatography steps 28 4090 22 AB ep 154 Purify 2 2 page options Method Wizard 5 Step Action For AC IEX steps e Select how to elute the protein by choosing Step Elution or Gradient Elution The options are described in the table below Note If on column tag cleavage is selected step elution will alw
325. ypass Column valve 160 1 ETFE port 1 to port 7 UV Column valve 200 1 ETFE port OUT to UV cell Cond UV cell to 200 1 ETFE conductivity cell Restr Conductivity 350 1 ETFE cell to restrict or OutV Restrictor to 160 1 ETFE outlet valve port IN Loop Loop Outlet valve 500 1 ETFE FracF12 port 12 to in jection valve port 7 LoopV1 Injection 260 1 ETFE valve center port to loop valve port IN Bypass Loop valve 160 1 ETFE port 1 to port 7 e p347 8 Maintenance 8 6 Replacement procedures 8 6 2 Replacing tubing and connectors 28 4090 22 AB p348 Label bold Description Length mm_ Inner Material or diameter designation mm LoopV2 Loop valve 260 1 ETFE port OUT to injection valve port 4 L1 capillary Loop valve 12800 ETFE oop port 2 to 8 10 ml L2 capillary Loop valve 12800 1 ETFE oop port 3 to 9 10 ml L3 capillary Loop valve 12800 1 ETFE oop port 4 to 10 10 ml L4 capillary Loop valve 12800 1 ETFE oop port 5 to 11 10 ml L5 capillary Loop valve 12800 1 ETFE oop port 6 to 12 10 ml Frac Frac Outlet valve 260 1 PEEK CollF2 port 2 to frac tion collector F3 red Fraction out 1500 1 ETFE let outlet valve port 3 F4 yellow Fraction out 1500 1 ETFE let outlet valve port 4 F5 blue Fraction out 1500 1 ETFE let outlet valve port 5 F6 green Fraction out 1500 1 ETFE let outlet valve port 6 F7 red Fr

ÄKTAxpress User Manual - GE Healthcare Life Sciences

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ÄKTAxpress User Manual - GE Healthcare Life Sciences