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1. Press OK to start run Method Complete Press OK to continue 62 5 Making further runs 5 9 Running a stored method The KTAprime plus system can store up to 40 programmed methods a computer connected to the system can store up to 999 methods The methods are made either by using a method template or by programming line by line Programming a method is described in chapter 6 Method programming Go through the procedure below to run a stored method 5 9 1 Selecting a stored method 1 Perform the general preparation of the system according to the description in chapter 5 2 Preparing the system further 2 Select main menu Run Stored Method and press OK 3 Select the method number and press OK If a computer running PrimeView is connected to the system do as follows 1 Select main menu Run Stored Method and press OK 2 Select a method from either the system System or the computer PC Press OK 3 Select the method number and press OK 5 9 2 Starting the run 1 Press OK at the Press OK to start run prompt to start the run The progress of the method can be viewed on the computer monitor See section 5 7 During a run for a description of viewing and changing parameters during the run 5 9 3 Finishing the run 1 Press OK at the Method Complete prompt to finish the run To abort the run before it is finished press End Confirm the following message by selecting yes then press OK 2 When the
2. 4 6 2 Control keys end e Interrupt method operation before the run is completed e Stop manual operation hold cont e Hold method time or volume and the gradient at the current concentration Pump and fraction collector continue uninterrupted e Continue the normal method operation pause cont e Pause all operation without ending the method All functions including pump and fraction collector are stopped e Continue the normal method operation feed tube e Advance the fraction collector one position SSS AkTAprime plus User Manual 11 0026 44 Edition AA 39 System overview Templates Run Stored Method Manual Run Program Method Copy Method Set Parameters Check 40 4 6 3 Changing a parameter value Parameter Current value Set Flow Rate 15 0ml min 00 0 New value to be set To change a parameter value 1 2 4 7 Press OK to enter the set value mode Press A or V to change the set value A cursor below a text or numerical value shows what is affected when pressing the keys Press OK to verify the set value and exit the set value mode To cancel press Esc Main menu overview The main menu contains the following options Used to run pre made application templates and method templates This menu appears after the self test when turning on the system See sections 5 6 Starting a run and 5 8 Running a method template
3. no yes no eoi 58 Autozero on the recorder Select menu Autozero Press OK to set the recorder output signal to zero Setting an event mark Select menu Event Mark Press OK to set an event mark on the chart When running the system manually the options for changing the parameters during a run are different Refer to section 5 10 Running the system manually 5 7 3 Interrupting a run There are three ways to interrupt a run 5 7 4 1 Pressing the end button interrupts the run with the prompt End method Entering y and then pressing OK terminates the run Entering n and then pressing OK resumes the run Pressing the pause cont button stops the pump but the set flow rate and the gradient values are retained Press pause cont again to resume the run Pressing the hold cont button holds the gradient at the current value and the pump continues to run Press hold cont again to resume the gradient formation Completing a run When the run is finished the display shows Method Complete Press OK The post run printing display is shown If using a chart recorder press yes for making a print out see section 8 6 6 Printing curves directly after a run Otherwise select no The run is now completed If using PrimeView you can now evaluate the run and create a report KTAprime plus User Manual 11 0026 44 Edition AA Making further runs r 5 5
4. Note The UV lamp should be turned off overnight 5 12 3 Removal from cold room 1 Loosen all connections to prevent them sticking when the system returns to room temperature 2 Allow the separation unit to stabilize at room temperature for at least 12 hours 3 Tighten all connections and pump water through the system to check for leaks 4 Tighten any leaking connector 68 AKTAprime plus User Manual 11 0026 44 Edition AA Method programming 6 6 Method programming This section describes how to create customized methods using method templates or by programming line by line how to edit stored methods and to copy methods 6 1 General Fully customized methods for purification can be created either by using a method template or by programming line by line A method consists of a series of breakpoints which define changes of one or more parameter values The methods are programmed on a time or a volume base KTAprime plus can store up to 600 breakpoints in totally 40 user defined methods A computer connected to the system can store up to 999 methods To plan the method programming 1 Illustrate the run by the progress of the gradient the concentration of buffer B during the run 2 Define all breakpoints and the actions at the breakpoints that are required to achieve this progress 6 2 Programming using method templates In the method templates the breakpoints are predefined Only the length between the breakpoints
5. Used to run methods that are programmed by the user See section 5 9 Running a stored method Used to run the system manually without using methods See section 5 10 Running the system manually Used to program user specific methods See chapter 6 Method programming Used to copy methods between KTAprime plus and an external computer connected to the serial interface of the system See section 6 5 Copying a method Used to calibrate and set parameters for example for UV conductivity temperature pressure and flow rate See sections 9 20 Calibrations and 11 2 1 Set Parameters menus Used to check system parameters such as serial number pump run time and lamp intensity See section 11 2 2 Check menus KTAprime plus User Manual 11 0026 44 Edition AA Making further runs 5 Making further runs This chapter describes operations and procedures related to the daily work with AKTAprime plus It also gives further information to chapter 3 Making your first run The chapter also contains a list of other common topics and where to find more information about them 5 1 General This chapter contains information on the following topics Topic Page Preparing the system further 42 Purging the inle ttubingr 22888 4e RL Rte LA y 43 Scheduling calibrations 44 Applying sample of all sizes using a sample loop Superloop or
6. e Fixed volume fractionation e Peak fractionation Note Make sure that the fraction collector is properly installed and prepared before starting the run See section 8 4 Fraction collector 5 5 1 Fixed volume fractionation Fixed volume fractionation means that fixed fractions fixed volume time or number of drops are collected within a set interval of time or volume The fraction properties are preset in the application templates and the method templates In method templates and other stored methods they are set in the menus Set Fraction Base and Set Fraction Size 0 means no fractionation 5 5 2 Peak fractionation Peak fractionation allows you to collect peaks during the elution besides fixed volume fractions In this case the slope of the curve decides when the actual fractionation should start and end The properties for controlling the start and end points are set in the menu Set Peak Collect no means no peak fractionation Slope setting is described in section Setting peak collection on page 76 The illustration shows a UV curve where fixed volumes are collected in tube 1 6 until the peak is detected The peak is collected in tube 7 12 Fractionation and method start qe C C __ C KTAprime plus User Manual 11 0026 44 Edition AA Making further runs r 5 a 5 6 Starting a run 5 6 1 Final checks Before starting a method we recommend that you make certain checks to ensure that
7. Sample inject by 1 Select sample injection through the injection valve or through the system InjV Pump pump Set Pressure Limit 2 Set the pressure limit and press OK 1 00 MPa 1 00 Note The pressure limit should be set to the maximum backpressure limit of the column used 0 2 MPa the back pressure contribution from flow restrictor The maximum backpressure limit is found in the column instruction More information on using the flow restrictor is found on page 161 Set Flow Rate 3 Set the flow rate and press OK ml min 0 00 Set Fraction Size 4 Setthe fraction size 1 in the figure and press OK 0 0 ml 0 0 Set Equilibr Volume 5 Set the equilibration volume 2 and press OK 0 0 ml 0 0 Set Sample Inj Volume 6 Set the sample volume 3 to be injected and press OK 0 0 ml 0 0 Note The sample volume entered should include sample wash out volume if needed SSS 60 AKTAprime plus User Manual 11 0026 44 Edition AA Set Wash 1 Volume 0 0 ml 0 0 Set Elution Volume 0 0 ml 0 0 Set Wash 2 Volume 0 0 ml 0 0 Method ready yes yes no Save Method yes yes no Free Methods Sel Method Free Free Methods Sel Method Used Press OK to start run Method Complete Press OK to continue Making further runs 5 7 Set the wash 1 volume 4 and press OK This setting does NOT apply to the Gel filtration method template 8 S
8. capillaries from the plastic bag 10 Put the inlet tubing A1 and B in deionized water 11 Put the waste capillaries W1 W3 in waste 2 5 Connecting the mains cable WARNING AkTAprime plus must be connected to a grounded mains socket to prevent system parts from becoming live WARNING Only use mains cables delivered or approved by GE Healthcare 1 Turn AKTAprime plus to access the rear 2 Remove all tape holding the cables 3 Connect the system mains cable Der from the mains inlet to a properly 288822228 grounded mains socket Go ee D 4 Ensure that the other cables are E E E To properly connected to the rear con mains panel E outlet ei 5 Turn AKTAprime plus to access the front WARNING Do not block the rear panel of the system The mains power switch must always be easy to access 18 AKTAprime plus User Manual 11 0026 44 Edition AA Installation ass 2 6 Connecting a computer for PrimeView to the system AKTAprime plus can be delivered with PrimeView a software that allows data collection result evaluation and report generation on an external computer PrimeView package contains a software CD user manual and serial cable Note Before connecting the computer install PrimeView software on th
9. manual run AT application template MT method template running mode indication elapsed method volume or time current flow rate and pressure The available running modes are Run The system runs with the set flow rate End The system is not running Pause The pump is stopped but the set flow rate and the gradient values are retained Hold The pump continues to run but the gradient is held at the current value Running display 2 shows UV absorbance value pH concentration of buffer B and actual conductivity value in mS cm or uS cm Running display 3 shows the conductivity as a percentage of the maximum conductivity setting current temperature tube number and fraction size Running display 4 shows the position of the waste valve the buffer valve and the injection valve Printing progress The process parameters can be printed directly during the run on the recorder See section 8 6 Recorder REC 112 AKTAprime plus User Manual 11 0026 44 Edition AA Making further runs r 5 5 7 2 Changing parameters Some of the process parameters can be changed during the run They can be changed at any time during the run except the gradient The gradient can only be changed if no gradient is running or if the system is paused press pause cont or held press hold cont To change a parameter follow the instruction below The new setting takes effect immediately Changing the concentration of buffer B 1 Select menu Set Concent
10. off Set Length 2 Set the length volume or time for the target concentration of buffer B to be 0 00 ml 6 50 reached Press OK Set Target 3 Set the target concentration Press OK 00 B 50 The result will be a gradient starting with the concentration set in menu Set Concentration B and finishing with the target concentration AKTAprime plus User Manual 11 0026 44 Edition AA 63 5 Making further runs Set Flow Rate 0 1 ml min 0 8 Set Fraction Base ml min ml drp Set Fraction Size 00 0 ml 0 2 Set Pressure Limit 1 00 MPa 1 00 Set Buffer Valve Pos Pos 1 ies Set Inject Valve Pos Load Waste Load Inject 64 Setting the flow rate 1 Select menu Set Flow Rate The current setting is displayed Press OK 2 Set the flow rate and press OK Setting the fraction base 1 Select menu Set Fraction Base The current setting is displayed Press OK 2 Choose time min volume ml or drops drp Press OK Setting the fraction size 1 Select menu Set Fraction Size The current setting is displayed Press OK 2 Set the fraction size and press OK Setting the pressure limit 1 Select menu Set Pressure Limit The current setting is displayed Press OK 2 Set the pressure limit and press OK Note The pressure limit should be set to the maximum backpressure limit of the column used 0 2 MPa the back pressure contributi
11. recorder functions Autozero and Event mark are also available Set Concentration B 20 B Set Gradient off Set Flow Rate 1 0 ml min Set Fraction Size 0 05 ml Set Buffer Valve Pos Pos 1 Set Inject Valve Pos Load Autozero Event mark e To change a parameter see the instruction in the previous section 5 10 1 Preparing a manual run The new setting takes effect immediately e To autozero the recorder or to set an event mark on the chart select the desired option then press OK 5 10 5 Finishing the run Method Complete 1 Press OK at the Method Complete prompt to finish the run Press OK to continue To abort the run before it is finished press End Confirm the following message by selecting yes then press OK 2 When the run is finished the curves can be printed from the computer AKTAprime plus User Manual 11 0026 44 Edition AA 65 Making further runs 66 5 11 Cleaning after a run and storage All valves return to default position i e position 1 after a run CAUTION Do not allow solutions which contain dissolved salts proteins or other solid solutes to dry out in the UV flow cell Do not allow particles to enter the UV flow cell as damage to the flow cell may occur CAUTION Never leave the pH electrode in the flow cell for any period of time when the system is not used since th
12. the buffer Valve ns cag Hee we nieuwe ves yeoeeta mena 45 Collecting fractions Asie ten Enr aaa 52 Starting a run using an application template 53 Viewing and changing parameters during a run 56 Performing a run using a method template 59 Performing a run using a stored method 62 Running the system manually 63 Cleaning after a run Storage 66 Operating the system in a cold room 68 Other topics Topic Page Connecting and using a recorder 110 Calibrating the flow pressure and Monitors 135 Injection valve positions and flow paths 158 Using the flow festrictOr 22422 ddtet id MA aa baba dane den aks 161 Chemical compatibilitus cs ceccais dee ceed wee eee nes tees 187 AKTAprime plus User Manual 11 0026 44 Edition AA 41 Making further runs 5 2 Preparing the system further To prepare AKTAprime plus for a run follow the instructions in chapter 3 Making your first run It covers the most commonly used applications This section contains additional instructions for example how to connect different types of columns purging the system flow path and different ways of injecting the samp
13. 1 150 ml onto the column Superloop is an accessory available in three sizes Volume Max allowed column pressure Code no 10 ml 4 MPa 18 1113 81 50 ml 4 MPa 18 1113 82 150 ml 2 MPa 18 1023 85 All the sample is applied which gives good reproducibility and high recovery The sample is not diluted as the buffer pushing the movable seal is kept separate The loaded sample can be injected all at once or in several smaller volumes down to 1 ml portions permitting automated repetition of sample injection The Superloop is filled manually with a syringe Preparation Prepare the injection valve and connect Superloop as follows 1 Connect the supplied luer Pos 1 LOAD female 1 16 male union Column connector to port 3 of the injection valve Pump Sample B syringe 2 Make sure that tubing for the Superloop waste is connected to port 4 of the injection valve Waste Waste 3 Make sure that Superloop is filled with liquid see separate Superloop instruction 4 Mount Superloop in a column holder as close to the injection valve as possible 5 Connect the bottom tubing to injection valve port 2 6 Connect the top tubing to injection valve port 6 7 Make sure that all connections are fingertight AKTAprime plus User Manual 11 0026 44 Edition AA 49 Making further runs Set Sample Inj Vol 0 0 ml 0 0 ee 50 Filling Superloop Fill the Superloop as follows 1 Set the inje
14. 1 2 AKTAprime plus user documentation mu 10 13 AKTAprime plus User Manual re 11 FA PSH OC sccicccteercesesssscescencesshessctescisssasesaaconcsssescaseessoveveeseetaststelsscaspeewvcwcesiasesbeesseasses 12 15 Typographical conventions asser 13 2 Installation Zils General semaine nue Ne RI 15 2 2 PRE REQUISITES sn nn tent inter 16 2 3 UNPACKING th SYSTEM aise 16 ZA Installing AHS syste Meesun nin 17 2 5 Connecting the mains cable waeeeeeceeccsssssseeesesssssccscsssssssssesesesseececssssssssssesess 18 2 6 Connecting a computer for PrimeView to the system uu 19 ZT Systemiself test cnrs tn 20 3 Making your first run 31 Generale ennines RN 21 3 2 Purification WOrKTOW nement 21 3 3 Pre requisites 3 4 Starting AKTAprime plus 3 5 Starting PAIM VIQW nn Rene i Ra E R HOE ABUTTEL Dr DArATION orana antenne 3 7 Sampl DEEPA GIN rest 3 8 Purification Setup 3 9 Selecting template and starting the run 3 10 Viewing the TUN LL Lt ess 311 Viewingthe result Les nn ea de a 3 12 Creating and printing a simple report ee cccssssssssseesssssccssssssssseeeees 3 13 Cleaning after a run 3 14 Making further runs amp System overview AV GENETTA fn en RM M Un nn LES Er AU An 4 2 System components scsrasuiusoinsiimenndn denin iii 4 3 SUSTSMAPIOW path naine nina 44 Functional description 45 Columns dnd CUBA sas minimum AG s Operator inter ace nn in tnt 47 MGM mMenu Over VIe eusian n EROR Ss AKTAprime plus User Manual 11 0026 44 Editi
15. 40 0 pos1 WASTE Re equilibration 115 S 0 1 0 pos 1 LOAD End method 120 S EEE AKTAprime plus User Manual 11 0026 44 Edition AA 87 Template description E 7 2 8 Albumin removal The Albumin removal application template is used for removing albumin Albumin Removal To access the template select Albumin Removal HiTrap Blue and HiTrap Blue press OK Column HiTrap Blue 1 ml Total run time approx 37 min sample application time B A 100 gt 25 50 75 100 Volume Action Volume Conc B Flow Fract BufferV Inject V Priming A1 0 0 40 0 pos 1 WASTE Equilibration 35 0 J 0 pos 1 LOAD Autozero 45 Sample application 45 0 1 1 pos 1 INJECT Elution 45 S 0 1 1 pos 1 LOAD End elution 55 S 0 1 0 pos 1 LOAD Priming B 55 1 S 100 40 0 pos 1 WASTE Albumin wash out 90 S 100 1 1 pos 1 LOAD End wash out 100 S 100 1 0 pos 1 LOAD Priming A1 100 1 S 0 40 0 pos 1 WASTE Re equilibration 1154S 0 1 0 pos 1 LOAD End method 120 S SSS 88 AKTAprime plus User Manual 11 0026 44 Edition AA Template description 7 2 9 Mab purification gradient elution The Mab purification gradient elution application template is used for purification of monoclonal antibodies using a pH gradient Mab Purification To access the template select Mab Purification Gradient elution and Gradient elution press OK Column HiTrap Protein A 1 ml alt HiTrap rProtein A or HiTrap Protein G 1 ml Total run time approx 63
16. 8 Running a method template AKTAprime plus contains four method templates based on the most common purification techniques When using a method template some parameters are set by the operator when preparing the run Before starting the run the operator has the option to save the settings in a method This allows the operator to edit the method later and to reuse it Go through the procedure below to run a method template 5 8 1 Selecting method template 1 Perform the general preparation of the system according to the description in chapter 5 2 Preparing the system further Templates 2 Select main menu Templates and press OK Method Template 3 Select sub menu Method Template and press OK 4 Select the desired template and press OK Gelfiltration Buffer Exchange lon Exchange Gradient elution HIC Gradient elution Affinity Step Gradient SSS AKTAprime plus User Manual 11 0026 44 Edition AA 59 Making further runs ey 5 8 2 Setting the parameters B A Gradient 6 100 gt Ser ts aie Fd Te The figure above shows the theoretical gradient concentration of B the UV curve and fractionation marks The numbers correspond to steps in the method to be defined before starting the run 1 Fraction size 2 Column equilibration volume 3 Sample application volume 4 Wash volume to remove unbound sample etc Wash 1 5 Elution volume 6 Wash volume to remove residues etc Wash 2
17. End Method menu yes yes no 2 Select yes and press OK Edit Breakpoint 3 Select a final breakpoint with the same parameters 0 00 ml 6 3 7 Saving the method When all breakpoints are set save the method as follows 1 Goto the Save Method menu Save Method 2 Select yes and press OK yes yes no SSS AKTAprime plus User Manual 11 0026 44 Edition AA 77 6 Method programming 6 4 Editing a stored method To edit an existing method follow the instruction below Refer to section 6 3 Programming line by line for more detailed information about setting the parameters 6 4 1 Selecting method Program Method 1 Select main menu Program Method and press OK Free Methods 25 2 Select the number of the method and press OK Sel Method Used 08 Method Occupied 3 Select yes and press OK edit edit clear Use the arrow buttons to go through the sub menus and change the parameters as desired see also section 6 3 Programming line by line 6 4 2 Editing an existing breakpoint Edit Breakpoint 1 Go to the Edit Breakpoint menu 0 00 ml 2 Use the down button to scroll through the existing breakpoints 3 Press OK at the desired breakpoint to enter the parameter menus 4 Edit the parameters as required All values are default the previously stored values Save Breakpoint 0 00 ml 5 Save the new parameter values by pressing OK 6 4 3 Editing time volume of a breakpoint Edit Breakpoint
18. The method templates are described in chapter 7 Template description 4 Go through the parameters using the arrow buttons and set the values as desired SSS 70 AKTAprime plus User Manual 11 0026 44 Edition AA Sample inject by InjV Pump Set Pressure Limit 1 00 MPa 1 00 Set Flow Rate ml min 0 00 Set Fraction Size 0 0 ml 0 0 Set Equilibr Volume 0 0 ml 0 0 Set Sample Inj Volume 0 0 ml 0 0 Set Wash 1 Volume 0 0 ml 0 0 Set Elution Volume 0 0 ml 0 0 Set Wash 2 Volume 0 0 ml 0 0 Method ready yes yes no AKTAprime plus User Manual 11 0026 44 Edition AA 6 2 2 1 Method programming 6 Setting the parameters Select sample injection through the injection valve or through the system pump Refer to section 5 4 Applying the sample for more information about sample application Note When using the system pump for sample application the sample tubing should be connected to port 8 on the Buffer valve Set the pressure limit and press OK Note The pressure limit should be set to the maximum backpressure limit of the column used 0 2 MPa the back pressure contribution from flow restrictor The maximum backpressure limit is found in the column instruction More information on using the flow restrictor is found on page 161 Set the flow rate and press OK Set the fraction size and press OK Set
19. a brief overview of AKTAprime plus including the system components a system flow scheme with functional description and a description of the operator interface 4 1 General AKTAprime plus is a single compact unit designed for simple protein purification at laboratory The unit includes the control system monitor pump fraction collector and valves for buffer selection sample injection gradient formation and flow diversion The system is operated from the push buttons and LCD display at the front panel 4 2 System components The location of the system components are shown below TE lt Column TE holder Flow Fraction diversion collector De valve DE Sample loop Monitor and controller D O KTA bie ug LCD display Column O0 Push buttons 00 p UV flow cell Pump Ve LA gt Nod 75 Flow gan restrictor 2 Conductivity cell Pressure Mixer Injection Buffer Switch valve Flow sensor valve valve restrictor 1 KTAprime plus User Manual 11 0026 44 Edition AA 35 System overview a 4 3 System flow path The illustration below shows the flow path of AKTAprime plus Buffer and sample Manual sample Monitoring Fractionation selection loading Pump Pressure Mixer sensor restr Conductivity z Switch valve Flow diversion Fraction collector Buffers Functions available in AKTAprime plus e Automatic buffer and
20. a software that allows real time monitoring evaluation and report generation on an external computer The system can also be delivered with Recorder 112 for simpler data presentation A brief system description of AKTAprime plus can be found in chapter 4 System overview 12 AKTAprime plus user documentation The following user documentation is included in AKTAprime plus User documentation Content AKTAprime plus User Manual System description How to use the system including safety instructions concepts operation maintenance and troubleshooting AKTAprime plus Cue Cards Short step by step instructions for selected applications using the preprogrammed application templates System preparation and value table for the method templates For related literature such as handbooks methods and principles see Ordering information AKTAprime plus User Manual 11 0026 44 Edition AA Introduction an 1 3 AKTAprime plus User Manual This manual has the following content Chapter Content Important user information Regulatory and safety information 1 Introduction Brief introduction to AKTAprime plus information about the user documentation and safety instructions 2 Installation Preparing the initial installation and performing the installation 3 Making your first run Short step by step instructions for preparing the system and performing a run using
21. applications after 15 min of lamp warm up but the full specifications are not obtained until after 1 hour 3 5 Starting PrimeView If the computer and PrimeView are not already started 1 Turn on the computer 2 Click PrimeView icon on the desktop of the Microsoft Windows operating system to open PrimeView module For customizing the view panes see PrimeView User Manual AKTAprime plus User Manual 11 0026 44 Edition AA Making your first run 3 6 Buffer preparation e Use high purity water and chemicals e Filter the buffers through a 0 45 um filter before use Prepare at least 500 ml of the following buffers Type of buffer Solution Binding buffer A1 20 mM sodium phosphate 0 5 M NaCl and 30 mM imidazole pH 7 4 Elution buffer B 20 mM sodium phosphate 0 5 M NaCl and 0 5 M imidazole pH 7 4 3 7 Sample preparation 1 Adjust the sample composition to the binding buffer by e diluting the sample in binding buffer or e buffer exchange using HiTrap Desalting or HiPrep 26 10 Desalting column 2 Filter the sample through a 0 45 um filter 3 8 Purification setup 3 8 1 Removing storage solution from the flow path At delivery and during storage the flow path is filled with 20 ethanol This should be removed before continuing the setup Note Do not use buffer with high salt concentration to flush out the ethanol It might cause too high backpressure To flush out the ethanol us
22. breakpoint must be at time volume 0 00 If several breakpoints have been set parameters can be changed in them by selecting the desired breakpoint with the arrow buttons In a new method the default value in the first breakpoint is 0 for all parameters B flow rate etc Buffer valve 1 Injection valve load and no peak collection All breakpoints after the first one will inherit the parameter values of the previous breakpoint Selecting a breakpoint Edit Breakpoint 1 Press OK to enter the breakpoint selection mode 0 00 ml 2 Select the desired time volume of the breakpoint with the arrow buttons and press OK Edit Breakpoint To create a new breakpoint go through all breakpoints After the last one the NEW breakpoint value changes to NEW Pressing OK here creates a new breakpoint with value 0 00 This value can be changed with the arrow buttons 74 AKTAprime plus User Manual 11 0026 44 Edition AA Method programming 6 Setting the concentration Set the concentration of buffer B as follows 1 Select menu Set Concentration B and press OK Set Concentration B 20 B 30 2 Set the desired concentration and press OK e To create a linear gradient set two breakpoints with different values for concentration of B This creates a linear gradient from the first to the second value over the interval between the breakpoints e Tocreateastep gradient set two breakpoints separated by 0 1
23. for example by running the System Wash Method with all tubing inlets in water 2 Replace the column with a bypass capillary 3 Replace the pH electrode optional with a dummy pH electrode 4 Wash the system with 20 ethanol and store it in 20 ethanol The UV flow cell can also be stored dry by flushing as above with distilled water and then 20 ethanol through the flow cell Replace the red protective caps Never use compressed air as this may contain droplets of oil Storage of the pH electrode The pH electrode should always be stored in a 1 1 mixture of pH 4 buffer and 2 M KNO when not in use Electrode regeneration If the electrode has dried out immerse the lower end of the electrode overnight in a buffer with a 1 1 mixture of pH 4 buffer and 2 M KNO AKTAprime plus User Manual 11 0026 44 Edition AA 67 Making further runs 5 12 Cold room operation Cold room operation is sometimes necessary to keep the biomolecule s of interest stable 5 12 1 Preparation 1 Place the separation unit in the cold room 2 Turnon the system If the system already is turned on turn on the UV lamp 3 Allow the UV lamp to warm up for at least 15 minutes 4 Tighten all connections and pump water through the system to check for leaks 5 Tighten any leaking connector 5 12 2 Running 1 Ensure that the temperature of the buffers has reached the ambient temperature 2 Calibrate the pH electrode optional 3 Check the pH of the buffers
24. in bold S represents the sample volume B 100 10 20 30 40 50 60 valime Breakpoint ml Parameter Conc B Flow Fract BufferV Inject V 0 Equilibration 0 1 0 pos 1 LOAD 10 Sample applic 0 1 0 pos 1 INJECT 10 S Elution delay wash 0 1 0 pos 1 LOAD 20 S Elution start fraction 0 1 1 pos 1 LOAD start gradient 35 S Column wash 100 1 d pos 1 LOAD 49 9 S End column wash 100 1 0 pos 1 LOAD end fractions 50 S Priming 0 40 0 pos 1 WASTE 70 S Re equilibration 0 1 0 pos 1 LOAD 80 S End method 0 1 0 pos 1 LOAD 72 AKTAprime plus User Manual 11 0026 44 Edition AA Program Method Free Methods Sel Method Free Free Methods Sel Method Used 16 Method Occupied edit edit clear Clear Method 09 yes yes 25 no Method programming 6 To program a new method line by line 1 Select main menu Program Method and press OK The menu contains the sub menus shown below Move through the menus using the arrow buttons Free Methods 25 Sel Method Free 16 Set Method Base ml Set Fraction Base ml Set Pressure Limit 1 00 MPa Edit Breakpoint 0 00 ml Set Alarm at No alarm 10 0 Show B on Rec out 2 no yes no End Method no yes no Save Method no yes no 6 3 1 Selecting a method number 1 Select a number for the new method If t
25. min Delete Breakpoint 0 00 min 76 6 Method programming Setting peak collection The fraction size must be set gt 0 to activate peak collection If fraction size is set to zero fractions will not be collected at peaks The slope of the UV curve when a peak should be detected should be entered as mAU min The fraction collector will change tubes whenever the slope exceeds the set value The peak end is determined automatically by the system 1 Select menu Set Peak Collect and press OK 2 Inthe Set Slope menu set the slope and press OK Use a previous chromatogram from an identical run to determine the slope We recommend starting with a slope of about 10 of the peak height in mAu min with a time averaging of 2 6 s Perform a blank run with the chosen setting to check that tube changes do not occur as a result of baseline disturbances Setting UV signal autozero 1 Select menu Autozero and press OK 2 Select yes and press OK to set the output signal to zero At the next breakpoint the setting is reset to no Setting an event mark 1 Select menu Event Mark and press OK 2 Select yes and press OK to set an event mark on the chart Event Mark is reset to no at next breakpoint Editing time volume of a breakpoint 1 Select menu Edit time volume and press OK 2 Select Change or Replace and press OK to edit the time volume of the breakpoint Change will also change the time volume of all the followi
26. needs to be set for example the length of the elution in volume or time The illustration below shows an typical method for gradient elution with a linear gradient from 0 to 100 The sample is loaded manually through the injection valve The fraction collection starts at the beginning of the elution The numbers represent the parameters to be set The table shows the parameters at each interval S represents the sample volume Note The example only shows the general principles for programming using method templates Some applications might need additional parameters This is described in detail in chapter 7 Template description AKTAprime plus User Manual 11 0026 44 Edition AA 69 6 Method programming a B 100 10 20 30 40 50 60 us Parameter Volume Conc B Flow Fract BufferV Inject V 1 Equilibration 10 0 F 0 pos 1 LOAD volume 2 Sample volume Sample 0 F 0 pos 1 INJECT 3 Wash 1 volume 10 0 F 0 pos 1 LOAD 4 Elution volume 15 100 F 1 pos 1 LOAD 5 Wash 2 volume 15 100 F 1 pos 1 LOAD Re equilibration 20 0 F 0 pos 1 LOAD hidden in template 6 2 1 Selecting method template Templates 1 Select main menu Templates and press OK Method Template 2 Select sub menu Method Template and press OK 3 Select the desired template and press OK Gelfiltration Buffer Exchange lon Exchange Gradient elution HIC Gradient elution Affinity Step Gradient
27. problems are not encountered once the run has been started 1 Check that the inlet tubings are immersed in the correct bottles for the method you are selecting 2 Check that there is sufficient eluent available 3 Check that the waste bottle is not full and will accept the volume diverted to it during the run 4 Check that the pump has been purged i e no air in the inlet tubing If not purge the pump according to page 43 The application templates already include the tubing priming 5 Check that the correct wavelength is set on the optical unit and that the correct UV flow cell is installed For range setting refer to section 8 6 5 Setting analog outputs 6 Calibrate the pH electrode if required optional Refer to section 9 20 5 Calibrating the pH electrode optional 7 Check that the fraction collector has at least 40 tubes fitted 8 Check that the correct column has been fitted and equilibrated if not included in the method 9 Ifusing a chart recorder or a computer for monitoring the run make sure that it is set correctly SSS AKTAprime plus User Manual 11 0026 44 Edition AA 53 Making further runs 5 6 2 Selecting an application template AKTAprime plus is run by either using a pre made template or method or by running the system manually The following four running options are available 54 Application templates Templates for running the most frequent purifications These templates only require the sample vol
28. sample selection e Manual sample application e Fixed volume loops for applying samples from 100 ul to 5 ml e Superloop 10 ml Superloop 50 ml and Superloop 150 ml e Sample loading through the pump for large sample volumes e Online monitoring of UV and conductivity optional for pH e Peak detection and collection of up to 95 small fractions e Data presentation using PrimeView or Recorder 112 optional 36 AKTAprime plus User Manual 11 0026 44 Edition AA System overview 4 4 Functional description 4 4 1 System control AKTAprime controls all components within the system including programming of the fraction collector Application templates method templates for the most common chromatographic techniques or line by line programming can be used to create and run a purification Up to 40 user defined programs can be stored 4 4 2 Liquid delivery The single channel pump delivers liquid with high precision over a wide flow rate range which provides fast and reproducible purifications A 3 port switch valve and a mixer are used for gradient formation and a pressure sensor prevents damage to the column in case of too high pressure increase 4 4 3 Flow path control Two motorized rotary valves automatically control the flow path The 8 port valve is used for buffer or sample selection and the 7 port valve for sample injection 4 4 4 Monitoring The high precision online monitor makes it possible to measure UV absorbance
29. with the electrical components immediately switch off the system and contact an authorised service technician WARNING NaOH is injurious to health Avoid spillage WARNING When using hazardous chemicals ensure that the entire system has been flushed thoroughly with distilled water before service and maintenance WARNING Only spare parts that are approved or supplied by GE Healthcare may be used for maintaining or servicing the system WARNING Use ONLY tubings supplied by GE Healthcare to ensure that the pressure specifications of the tubings are fulfilled WARNING If the system is turned the external capillaries and other tubing may become entangled in nearby objects and be pulled from their connections causing leakage gt gt gt PP D 15 Typographical conventions Keyboard options menu selections and text on labels and panels are identified in the text by bold typeface Menu commands field names and dialog box prompts in PrimeView are also identified in the text by bold typeface A colon separates menu levels thus File Open refers to the Open command in the File menu AkTAprime plus User Manual 11 0026 44 Edition AA 13 Introduction 14 AKTAprime plus User Manual 11 0026 44 Edition AA Installation 2 Installation This chapter describes unpacking and installation of AKTAprime plus It also describes how to connect a computer to the system for using PrimeView for data col
30. 00 S 100 1 1 pos 1 LOAD End elution 110 S 100 1 0 pos 1 LOAD Priming A1 110 0 1 S 0 40 0 pos1 WASTE Re equilibration 125 S 0 1 0 pos 1 LOAD End method 130 S 93 AKTAprime plus User Manual 11 0026 44 Edition AA Template description D 7 2 15 Histidine tag purification The Histidine tag purification application template is used for purification of Histidine tagged proteins His Tag Purification To access the template select His Tag Purification HisTrap and press OK HisTrap Column HisTrap HP 1 ml Total run time approx 74 min sample application time B 100 gt 50 100 150 Volume Action Volume Conc B Flow Fract BufferV Inject V Priming B 0 100 40 0 pos 1 WASTE End priming B 25 100 40 0 pos 1 WASTE Priming A1 25 0 1 0 40 0 pos 1 WASTE Equilibration 60 0 1 0 pos 1 LOAD Autozero 70 Sample application 70 0 1 0 pos 1 INJECT Wash 3 70 S 0 1 0 pos 1 LOAD Elution 90 S 0 1 1 pos 1 LOAD Wash 4 110 S 100 1 Hb pos 1 LOAD End wash 4 127 S 100 1 0 pos 1 LOAD Priming A2 127 0 1 S 0 40 0 pos 1 WASTE Re equilibration 142 S 0 1 0 pos 1 LOAD End method 147 S i 94 AKTAprime plus User Manual 11 0026 44 Edition AA Template description eC _ 7 2 16 System wash The System wash template is used for rinsing and priming the tubings and the components in the system flow path System Wash Method 1 To access the template select System Wash Method and press OK Select Buffer V Pos 2 Select the buffer inlets
31. 1 Goto the Edit Breakpoint menu 0 00 mi 2 Use the down button to scroll through the existing breakpoints 3 Press OK at the desired breakpoint to enter the parameter menus Edit time volume 4 Select menu Edit time volume and press OK 12 7ml Edit time volume 5 Select Change or Replace and press OK to edit the time volume of the Change Replace breakpoint Change will also change the time volume of all the following breakpoints accordingly Replace will not change the time volume of the other breakpoints New time volume 6 Edit the time volume and press OK 12 7ml 15 0 78 AKTAprime plus User Manual 11 0026 44 Edition AA Edit Breakpoint 0 00 ml Edit Breakpoint NEW Save Breakpoint 0 00 ml Edit Breakpoint 0 00 ml Delete Breakpoint 0 00 ml Delete Breakpoint 0 00 ml yes Show B on Rec out 2 no yes no Save Method yes yes no Method programming 6 6 4 4 Inserting a new breakpoint 1 Goto the Edit Breakpoint menu 2 Use the down button to scroll through all existing breakpoints After the last breakpoint the breakpoint value changes to NEW 3 Press OK at breakpoint NEW to create a new breakpoint This breakpoint will have the value 0 00 4 Set the breakpoint value with the arrow buttons and press OK 5 Edit the parameters as required 6 Select the Save Breakpoint
32. 10_Pressure 05 AT 2004decO no003 10_Temp Peaktable H 06 AT 2004dec07n0003 10_Conc poser 17 AT2004dec07n0003 10_UV 01 BASEM Peak table name uven PEakt ee Peak window I Accept negative peaks Correlated baseline z Reject peaks J Peak skim fio ratio Baseline settings Correlated baseline 17 AT2004dec07n0003 10_UV 01 BASEM Column height 2 cm Save and Edit Peak Table Cancel Help Goe 30 KTAprime plus User Manual 11 0026 44 Edition AA 2 Making your first run Click OK A peak table is added at the bottom of the module Primeview Evaluation AT2004decO7n0003 10 _ C UNICORN Local Fil prime Result AT2004decO7n0003 RESI Bile Edit View Integrate Window Help lalx F AT2004de007n0008 10 UV ou AT2004decO7n0003 10 Cond AT2004dec07n0003 10 Fractions AT2004dec7n0003 10 Inject AT2004de007 no003 10_UV 01 BASEM AT jec07no003 10 Logbook mau 519 800 e00 400 H200 hooo 40 49 800 00 400 a a a 200 Break point Break point 7 Break point 8 Break point Cie Kee hes HHEH 0 Jar apal gpopip2papapsp p bo 5b 10 0 150 200 250 300 350 400 46 A AT2004dec07n0003 10_UV D1PEAK B AT2004dec07no003 10_UV 01 PEAKI This description describes how create and print a simple report However it is poss
33. A Template description DONE 7 2 5 IMAC purification The IMAC purification application template is used for purification of Histidine tagged proteins IMAC Purification To access the template select IMAC Purification Uncharged HiTrap and press Uncharged HiTrap OK Column HiTrap Chelating Total run time approx 87 min sample application time B 100 50 100 150 200 250 Volume Action Volume Conc B Flow Fract BufferV InjectV Priming B 0 100 40 0 pos2 WASTE End priming B 25 100 40 0 pos2 WASTE Priming A2 25 1 0 40 0 pos2 WASTE Water wash 60 0 1 0 pos 2 LOAD Priming A3 65 0 40 0 pos3 WASTE Metal ion application 100 0 1 0 pos 3 LOAD Priming A2 101 0 40 0 pos2 WASTE Water wash 116 0 1 0 pos 2 LOAD Priming A1 121 0 40 0 pos1 WASTE Equilibration 156 0 1 0 pos 1 LOAD Autozero 166 Sample application 166 0 1 0 pos 1 INJECT Buffer wash 166 S 0 1 0 pos 1 LOAD Elution 186 S 0 1 1 pos 1 LOAD Elution wash out 206 S 100 1 1 pos 1 LOAD End wash 223 S 100 1 0 pos 1 LOAD Priming A2 223 1 S 0 40 0 pos2 WASTE Re equilibration 238 S 0 1 0 pos 2 LOAD End method 243 S Ses AKTAprime plus User Manual 11 0026 44 Edition AA 85 Template description _ 7 2 6 GST tag purification The GST tag purification application template is used for purification of GST tagged proteins GST tag Purification To access the template select GST tag Purification GSTrap and GSTrap press OK Column GST
34. Desalting HiPrep Desalting IMAC Purification Uncharged HiTrap Albumin Removal HiTrap Blue GST tag Purification GSTrap Mab Purification Gradient Elution HisTrap On column Refolding IgM Purification Anion Exchange HiTrap Q Cation Exchange HiTran SP Affinity Purification His tag Purification HisTrap System Wash Method HiTrap IgM Purification Sample appl volume 0 0 4 Inthe Sample appl volume menu set the sample volume with the up and down buttons Press OK Press OK to start run 5 Tostart the run press OK at the Press OK to start run prompt AKTAprime plus User Manual 11 0026 44 Edition AA For more information about application templates see chapter 7 Template description 55 i M RUN 10 0 ml 20 0 ml min 1 10 MPa 0 00002AU 20 B pH 8 50 22 90mS cm Cond 78 8 Tc 22 4 C Tube 01 Frac 5 0 ml Waste V waste BV 1 IV waste ae 56 Making further runs 5 7 During a run 5 7 1 Viewing the run Besides viewing the run in PrimeView the process parameters can be viewed directly on the front panel display Running display Four display alternatives with run data are available Select the desired running display by pressing or V Running display 1 shows method number or type M
35. GE Healthcare AKTAprime plus User Manual 0 KTA Important user information All users must read this entire manual to fully understand the safe use of AKTAprime plus WARNING The WARNING sign highlights instructions that must be followed to avoid personal injury It is important not to proceed until all stated conditions are met and clearly understood CAUTION The Caution sign highlights instructions that must be followed to avoid damage to the product or other equipment It is important not to proceed until all stated conditions are met and clearly understood Note The Note sign is used to indicate information important for trouble free and optimal use of the product Declaration of conformity This product meets the requirements of applicable CE directives A copy of the corresponding Declaration of Conformity is available on request The CE symbol and corresponding declaration of conformity is valid for the instrument when it is used as a laboratory device It is not intended for clinical or in vitro use of for diagnostic purposes used as a stand alone 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 Contents Contents 1 Introduction ET EGen rdl isa et En 9
36. NG The system must not be opened by the user It contains high voltage circuits that can give a lethal electric shock WARNING Always disconnect the power supply before attempting to replace any item during maintenance WARNING When the lamp power is on the lamp socket carries a dangerous voltage Do not connect disconnect with the system switched on WARNING The system uses high intensity ultra violet light Do not remove the UV lamp while the system is running Before replacing a UV lamp ensure that the lamp cable is disconnected from the rear of the system to prevent injury to eyes If the mercury lamp is broken make sure that all mercury is removed and disposed according to national and local environmental regulations 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 There must always be a sample loop connected to ports 2 and 6 of the injection valve This is to prevent liquid spraying out of the ports when switching the valve This is especially dangerous if hazardous chemicals are used AKTAprime plus User Manual 11 0026 44 Edition AA Introduction WARNING If there is a risk that large volumes of spilt liquid have penetrated the casing of the system and come into contact
37. ON Do not allow solutions which contain dissolved salts proteins or other solid solutes to dry out in the UV flow cell Do not allow particles to enter the UV flow cell as damage to the flow cell might occur Buffers not containing any salt can be left in the system for a short time after a run even overnight not in the pH electrode see instructions below If a buffer containing salt has been used flush the flow path with deionized water This is especially important if an organic solvent will be used in the next run To flush the flow path 1 Filla syringe with five times the sample loop volume of deionized water 2 Rinse the sample loop by injecting the water through the fill port on the injection valve 3 Put all used inlet tubings in water 4 Inthe Templates menu select Application Template and then System Wash Method 5 Select the used inlet ports Inlets A1 and B will always be washed 6 Press OK to start the method The system flow path is now automatically flushed For information on cleaning and long term storage see section 5 11 Cleaning after a run and storage AKTAprime plus User Manual 11 0026 44 Edition AA 33 Making your first run 3 14 Making further runs This chapter contains instructions for performing a specific purification run More information on using AKTAprime plus further is found in chapter 5 Making further runs Chapter 5 Making further runs contains information on the
38. ONS sercon 143 10 2 System 10 53 HPRESSUCS CUIVE wiaicscssecesteccecssvsayvssesscncastezseeverseanitinneanstecedsceaveivtnasantaaesctitieconee 144 104 UV CUVE nn nn an E Conti 145 10 5 Conductivity CULVER saipe namai 146 10 6 pH c rveloptional issues nine 147 TOZ MIXE opeope e 148 10 8 PUM D ennea E n S 148 10 9 Fraction Coletor ersinnen 149 10 10 Buffer valve and injection valve weeccccccsssssssssssssessssccssssssssssssseeesseesee 149 10 11 Error MESSAGES nos asenn iii ein r EA RA 150 10 12 Adjusting the spring tension of the delivery arm 154 10 13 Removing trapped air bubbles wo cccccssssssssseessssscsccsssssssssseessssessssen 154 10 14 Restart after power failure oe ccccssccsssssssssssseessseccsssssssssseeessssessessssns 154 11 Reference information 11 1 SUSteMIAeSCTIDHONE ne AM nee 11 27 MENUS e ne Ne Re SR NE rt de 113 Technical specifications 11 4 Chemical resistance guide and chemical compatibility 187 115 Ordering MPO TION scsi ine k ei EEA EE 188 Index rr AKTAprime plus User Manual 11 0026 44 Edition AA 7 Contents About this manual This manual describes how to run AKTAprime plus chromatography system using preprogrammed application templates and method templates It also describes how to create methods and running the system manually System description and instructions for installation maintenance and troubleshooting are also included in this manual 8 AKTAprime plus User Manual 11 0026 44 E
39. Recorder REC V2 has ban teen EME Mn Maintenance 9 1 P riodic GIST CO nn nn nine na 119 9 2 cleaning the System naiara e 121 9 3 Cleaning the system flow path cssssssesssssscccssssssssssseesessscsssssssssssseees 122 9 4 Moving the system 9 5 Checking the UV monitor nnsssiessiinnimiiiniinineniiis 124 96 Checking the pump is 125 9 7 Checking the fraction collector csssssssessssssscscssssssssssssessssccssssssssssssees 126 9 8 Checking the rotary valves wicccccccccsssssssssssssesssessccssssssssssseesessscsssssssssssssees 9 9 Cleaning the UV flow cell in place 9 10 Cleaning the UV flow cell off line oo ccccsssssscssssssssseneessscescssssssssseees 126 9 11 Cleaning the conductivity flow cell off line oo ccccscccssssssssseeeees 127 AKTAprime plus User Manual 11 0026 44 Edition AA Contents 9 12 Replacing the conductivity cell ccesseeeeeeesssssccccsssssssessesesseccsssssssssseeees 9 13 Replacing plates in the rotary valves 9 14 Removing and assembling the pump 9 15 Replacing O rings in the PUMP ou eesssssssseeesssssscccsssssssssseessssseccesssssssseeees 9 16 Cleaning and replacing check valves in the PUMP o sssssssseseees 130 9 17 Replacing mixer CHAMBER sise 9 18 Cleaning the pH electrode Optional occecccccccccssssssseesscccccsssssssseeenees 9 19 Replacing the pH electrode optional 9 20 COND RMON S sn st unt ne tn 10 Troubleshooting 10 1 FaultS and OCtl
40. STE Wash 2 buffer if Wash 2 is used Wash 2 optional 5 0 F V pos 2 LOAD Priming 20 ml of C3 0 40 0 pos 1 WASTE buffer A Re equilibration D 0 F 0 pos 1 LOAD equilibr volume 1 If using the pump for sample application Buffer V pos 8 and Inject V LOAD 2 If using the pump for sample application 15 ml is automatically added to Wash 1 3 These steps are hidden in the template and cannot be changed AKTAprime plus User Manual 11 0026 44 Edition AA Installing and modifying components 8 Installing and modifying components This chapter describes how to install and modify components in AKTAprime plus It also describes how to install and use optional components 8 1 General Some applications might require that some of the components in AKTAprime plus have to be modified or changed For example the UV sensitivity can be changed by using another UV flow cell the wavelength of the optical filter can be changed or the rack in the fraction collector changed according to the tube size used The optional pH electrode allows for real time monitoring of the pH and if a recorder is used a print out The chapter covers the following components e The optical unit including flow cells and filters e The conductivity cell e The fraction collector e The pH flow cell and electrode optional e The recorder REC 112 optional AKTAprime plus User Manual 11 0026 44 Edition AA 99
41. V Priming B 0 100 40 0 pos3 WASTE End priming B 25 100 40 0 pos3 WASTE Priming A3 25 1 0 40 0 pos3 WASTE Priming A1 50 0 40 0 pos1 WASTE Equilibration 85 0 1 0 pos 1 LOAD Autozero 95 Sample application 95 0 1 0 pos 1 INJECT Buffer wash 1 95 S 0 1 0 pos 1 LOAD Priming A2 115 S 0 40 0 pos2 WASTE Buffer wash 2 150 S 0 1 0 pos 2 LOAD Refolding 160 S 0 0 5 0 pos 2 LOAD End refolding 190 S 100 0 5 0 pos 2 LOAD Priming A3 200 S 100 40 0 pos 3 WASTE Buffer wash 3 200 1 S 0 40 0 pos3 WASTE Priming B 205 S 0 40 0 pos3 WASTE Buffer wash B 205 1 S 100 40 0 pos3 WASTE Elution 220 S 100 T 1 pos 3 LOAD Re equilibration 240 S 0 1 1 pos 3 LOAD End method 257 S l amisi 92 KTAprime plus User Manual 11 0026 44 Edition AA Affinity Purification any HiTrap 7 2 14 Affinity purification The Affinity purification application template is used for purification of for example benzamidine Template description To access the template select Affinity Purification any HiTrap and press OK Column any HiTrap column Total run time approx 47 min sample application time B 100 50 100 130 Volume Action Volume Conc B Flow Fract BufferV Inject V Priming A1 0 0 40 0 pos1 WASTE Equilibration 35 0 1 pos 1 LOAD Autozero 45 Sample application 45 0 1 1 pos 1 INJECT Wash incl frac 45 S 0 1 1 pos 1 LOAD Wash excl frac 52 S 0 1 0 pos 1 LOAD End wash 65 S 0 1 0 pos 1 LOAD Priming B 65 0 1 S 100 40 0 pos 1 WASTE Elution 1
42. an application template 4 System overview Description of AKTAprime plus 5 Making further runs Detailed instructions for performing a run for example for sample application fraction collection cold room operation and storage 6 Method programming Programming a method using method templates or by editing line by line 7 Template description Detailed description of the application templates and method templates 8 Installing and modifying Instructions for installing and assembling components parts in the system Instructions for recorder REC 112 9 Maintenance Maintenance schedules and instructions for preventive maintenance replacing spare parts and calibration 10 Troubleshooting Overview of error symptoms possible causes and corrective actions Error messages 11 Reference information Detailed hardware description technical and chemical specifications ordering information ft AKTAprime plus User Manual 11 0026 44 Edition AA 11 Introduction 12 gt gt gt BRP 14 Safety IMPORTANT AkTAprime plus is intended for laboratory use only not for clinical or in vitro use or for diagnostic purposes e The system is designed for indoor use only e Donot use in dusty atmosphere or close to spraying water e Operate in accordance with local safety instructions e Do not block the air inlet or outlet of the system WARNING The system must be connected to a grounded mains socket WARNI
43. and 5 on the INJECTION VALVE and port NO on the fraction collector valve in waste Connect the HisTrap HP 1 ml column between port 1 on the INJECTION VALVE and the upper port of the UV flow cell Use a suitable length of PEEK tubing and 1 16 male connectors Note Other unions and connectors might be required for other columns 3 8 3 Preparing the fraction collector Fill the fraction collector rack with 18 mm ll tockknob tubes minimum 40 pcs Adjust the height of the delivery arm using the lock so that the bottom of the tube sensor is about 5 mm below the top of the tubes The tubes should always be below the horizontal mark on the tube sensor AKTAprime plus User Manual 11 0026 44 Edition AA Making your first run a 3 If necessary adjust the length of the tubing exposed according to the sequence shown below Tubing holder 4 Check that the sensor is in the correct position for the tube size The eluent tubing should be over the center of the collection tube Use the red sensor control to position the tube holder 5 Rotate the rack by hand until the rear half of the tube sensor rests against tube 1 6 Press feed tube on the front panel The bowl moves to the correct position to collect the first fraction in tube 1 Set Parameters 7 Make sure that drop synchronization is turned on Note Drop synchronization can NOT be used at flowrates above 3 ml m
44. cations are not obtained until after 1 hour Note At delivery the flow path is filled with 20 ethanol as protection The ethanol should be removed before a purification run See section 3 8 1 Removing storage solution from the flow path AKTAprime plus User Manual 11 0026 44 Edition AA Making your first run 3 Making your first run This chapter contains step by step instructions for performing a typical purification run after the installation using an application template Note It can also be used as a guide for running the system on a daily basis 3 1 General The run described in this section is an affinity purification of Histidine tagged proteins using a HisTrap HP column The sample is injected from the sample loop PrimeView is used for monitoring result evaluation and report generation Histidine tag is mostly used for facilitating purification of expressed recombinant proteins The Histidine tag is small and does not usually interfere with the function activity or structure of the protein HisTrap HP column is designed for this purpose and has for example high binding capacity and compatibility with several different additives For more information on performing a run for example sample application fraction collection running method templates and cold room operation see chapter 5 Making further runs WARNING When using hazardous chemicals take all suitable protective measures such as wearing protective gla
45. conductivity and pH optional A temperature sensor inside the conductivity cell provides automatic temperature compensation for pH and conductivity measurements UV absorbance UV detection wavelengths 254 and 280 nm are supplied with the system Other wavelengths for special applications are available including 214 nm if higher sensitivity is required Conductivity The conductivity monitor gives reliable measurements over the range of values typically seen during purification of biomolecules pH optional The true pH conditions during purification can be monitored and recorded by placing a flow cell containing the pH electrode into the flow path after the conductivity flow cell 4 4 5 Fraction collection Fractionation is performed by fixed volume collection or automatic peak fractionation Peak fractionation can be based on peak detection using slope sensing Fraction marks and fraction numbers allow easy identification of fractions and peaks AKTAprime plus User Manual 11 0026 44 Edition AA 37 System overview 38 A 3 port flow diversion valve allows automatic diversion to waste so that only required fractions are collected A selection of rack sizes is available 4 4 6 Data presentation AKTAprime plus can be delivered with either PrimeView software for monitoring evaluation and report generation or with Recorder 112 for simpler data presentation of runs PrimeView PrimeView offers real time monitoring of the chromato
46. ction valve to position INJECT Start the pump and let it run until the movable seal has reached the bottom of Superloop Stop the pump and set the injection valve to position LOAD Pos 2 INJECT 1 Column Sample syringe Load a large volume syringe with sample Gently load the syringe contents into the Superloop through port 3 Leave the syringe in position The loaded sample can be injected all at once or in several smaller volumes down to 1 ml portions The volume to inject is set in templates or in programmed methods in menu Set Sample Inj Vol The sample is applied to the column when the injection valve is set to position INJECT When the required volume has been injected set the valve to LOAD When using method templates this is performed automatically AKTAprime plus User Manual 11 0026 44 Edition AA Making further runs 5 4 3 Applying 10 ml or more of sample using the buffer valve Larger sample volumes can be applied directly from a sample vessel through the buffer valve Note In isocratic techniques e g size exclusion chromatography band broadening will be large when applying sample through the buffer valve Preparation 1 The sample must be particle free and filtered through a 0 45 um filter Otherwise the inlet filter might get clogged quickly 2 Connect an inlet tubing to port 8 on the buffer valve 3 Place the other end of the inlet tubing in
47. d to copy it to the computer To copy a method to another method number Note Displays including the PC option only appear when a computer running PrimeView is connected to the system 1 Select main menu Copy Method and press OK 2 Atthe Copy Method From sub menu select from which unit you want to copy the method System AKTAprime plus system PC computer Press OK 3 Enter the number of the method and press OK 4 At the Copy Method To sub menu select to which unit you want to copy the method Press OK 5 Enter the new number of the method and press OK Free means that the selected number is free for storing a new method Used means that the number is already occupied 6 If the Method occupied display appears select to clear the number or not yes or no Press OK 7 Confirm the selected procedure by pressing OK Copy Method Press OK PC 274 gt System 29 Copy Method Press OK System 25 gt PC 285 Copy Method Press OK PC 274 gt PC 285 Copy Method Press OK System 25 gt System 29 The method is copied to the new method number AKTAprime plus User Manual 11 0026 44 Edition AA Template description 7 Template description 7 1 General AKTAprime plus contains a collection of templates for common chromatographic applications The templates are grouped into two categories e Application templates Only the sample volume needs to be entered before
48. dition AA Introduction 1 Introduction This section is an introduction to AKTAprime plus the documentation included and the content in this manual It also contains an overview of the safety instructions 11 General AKTAprime plus is a compact liquid chromatography system designed for one step purification of proteins at laboratory scale When compared to traditional operations AKTAprime plus offers significant advantages in terms of speed capacity and fraction collection RAT AKTAprime plus features e Easy unpacking and installation e Pre programmed application templates for specific common purification steps e Method templates for all common chromatography techniques e Cue cards for simple and quick operation e Compatible with a range of prepacked columns such as HiTrap HiPrep and RESOURCE e High accuracy and reproducibility e Flow rates up to 50 ml min and pressures up to 1 MPa AKTAprime plus User Manual 11 0026 44 Edition AA 9 Introduction 10 The control system monitor pump and fraction collector together with valves for buffer selection sample injection gradient formation and flow diversion form a single compact unit The high precision on line monitor includes the possibility to measure UV conductivity and pH optional The system is operated using the push buttons and LCD display at the front panel AKTAprime plus can be delivered with PrimeView
49. e computer according to PrimeView User Manual WARNING The computer should be installed and used according to the instructions provided by the manufacturer of the computer To connect a computer to the system 1 Make sure the mains power to a AKTAprime plus and the computer is turned off dm dis 2 Connect the serial signal cable to socket RS 232 on the system 3 Connect the other end of the cable to the serial interface on the computer Note Data is sent through pin 3 and received through pin 2 Ground is on pin 5 computer 4 Switch on the mains power to AKTAprime plus and the computer 5 Start PrimeView according to PrimeView User Manual SSS AKTAprime plus User Manual 11 0026 44 Edition AA 19 Installation Selftest Please wait AKTAprime plus V3 00 Templates 20 2 7 System self test Start the system and run the system self test as follows 1 Switch on the system at the mains switch on the rear panel The system now performs a self test 2 First the system name and software version number is shown for a few seconds Several messages are then shown during the self test If an error is detected an error message is shown on the display 3 The self test takes about 30 40 seconds When the start up is completed with no errors the display shows the Templates menu and is ready for use Note The system can be used for most applications after 15 min of lamp warm up but the full specifi
50. e View Help MAU UV Cond pH Pressure Temp Cone Curves pane 424 404 38 364 344 324 3 0 28 264 men e E ET min 00 01 02 03 04 05 06 OF 08 09 10 11 12 13 14 15 16 1 7 18 19 20 Logbook pane For Help press F1 OEnd I No watch Controlled By prime i e The Curves pane displays monitor signal values graphically e The Logbook pane displays all actions e g method start and end base instructions and method instructions and unexpected conditions e g warnings and alarms The log is saved in the result file Selecting curves to be displayed 1 In PrimeView module select View Properties 2 Inthe Properties dialog click the Curves tab 3 Inthe Display curves list select the curves you want to display 4 Click OK For more information on customizing the view panes see PrimeView User Manual Goe 28 KTAprime plus User Manual 11 0026 44 Edition AA Making your first run 3 10 2 Finishing the run Method Complete Press OK to continue 1 Press OK at the Method Complete prompt This will cause all valves to return to their default positions 3 10 3 Aborting a run To abort a run before it is completed 1 Press the end button End method 2 Select yes and press OK yes yes no 3 11 Viewing the result PrimeView Evaluation module provides facilities for the presentation and evaluation of separation results si 1 Tostar
51. e run The selected values depend on the choice of column see the Method Templates value table cue card for recommended values e Sample injection using the sample pump or via the sample port Any volume changes due to the selection are handled automatically within the templates Note When using the pump and buffer valve sample application the sample is always applied through port 8 on the buffer valve In the following sections the method templates are illustrated by the buffer gradient during the run The table below the gradient shows how the parameters to be entered correspond to the steps during the run F in the tables represents the recommended flow rate of the column that is used see the Method Template value table cue card V represents the fractionation volume For more information on how to run a method template refer to section 5 8 Running a method template AKTAprime plus User Manual 11 0026 44 Edition AA Template description S 7 3 2 Gelfiltration buffer exchange Gelfiltration To access the template select Gelfiltration Buffer Exchange and press OK Buffer Exchange B A 100 rA 1 3 Volume Action Step Conc B Flow Fract BufferV InjectV Equilibration 1 0 F 0 pos 1 LOAD Sample application 2 0 F V posi INJECT Elution 3 0 F V pos 1 LOAD 1 If using the pump for sample application Buffer V pos 8 and Inject V LOAD 7 3 3 lon exchange lon Exchange To access th
52. e template select lon Exchange Gradient elution and press OK Gradient elution B 100 A g Volume Action Step Conc B Flow Fract BufferV Inject V Equilibration 1 0 F 0 pos 1 LOAD Sample application 2 0 F 0 pos 1 NJECT Wash 12 3 0 F 0 pos1 LOAD Elution 4 0 F V pos LOAD Wash 2 12 ml 5 100 F V pos 1 LOAD Priming 20 ml of A 0 40 0 pos 1 WASTE buffer A Re equilibration B3 0 F 0 pos 1 LOAD equilibr volume 1 If using the pump for sample application Buffer V pos 8 and Inject V LOAD 2 If using the pump for sample application 15 ml is automatically added to Wash 1 3 These steps are hidden in the template and cannot be changed AKTAprime plus User Manual 11 0026 44 Edition AA 97 Template description HIC Gradient elution Affinity Step Gradient 98 7 3 4 HIC hydrophobic interaction chromatography To access the template select HIC Gradient elution and press OK For buffer gradient and parameter settings table see the lon exchange method template at the previous page 7 3 5 Affinity To access the template select Affinity Step Gradient and press OK B 100 Volume Action Step Conc B Flow Fract BufferV Inject V Equilibration 1 0 F 0 pos 1 LOAD Sample application 2 0 F 0 posi INJECT Wash 12 3 0 F 0 pos1 LOAD Priming 15 ml of A 100 40 0 pos 1 WASTE elution buffer B Elution 4 100 F V pos 1 LOAD Priming 15 ml of B 0 40 0 pos 2 WA
53. et the elution volume 5 and press OK 9 Set the wash 2 volume 6 and press OK This setting does NOT apply to the Gel filtration method template 10 Select yes at the Method ready prompt and press OK 5 8 3 Storing the method 1 Select yes to store the method then press OK Otherwise select no and press OK 2 To store the method select a method number and press OK Free means that the selected number is free for storing a new method Used means that the number is already used Select a free method number and press OK Alternatively press OK to clear the number in the Clear Method menu 5 8 4 Starting the run 1 Press OK at the Press OK to start run prompt to start the run 2 See section 5 7 During a run for a description of viewing and printing run data and changing parameters during the run 5 8 5 Finishing the run 1 Press OK at the Method Complete prompt to finish the run To abort the run before it is finished press End Confirm the following message by selecting yes then press OK 2 When the run is finished the curves obtained can be printed on the chart recorder or from the computer This is described in section 8 6 6 Printing curves directly after a run AKTAprime plus User Manual 11 0026 44 Edition AA 61 Run Stored Method Run Stored Method Number 13 Run Stored Method From System PC Run Stored Method From PC No 73 or Run Stored Method From System No 13
54. following topics Topic Page Preparing the system for a run 42 Purging the inlet tubing 43 Scheduling calibrations 44 Applying sample of all sizes using a sample loop Superloop or thebuiler vale int RE a dead Rene EP 45 Collecting fractions AR M nn ere 52 Starting a run using an application template 53 Viewing and changing parameters during a run 56 Performing a run using a method template 59 Performing a run using a stored method 62 Running the system manually 63 Cleaning after a run Storage 66 Operating the system in a cold room 68 Other topics Topic Page Connecting and using a recorder 110 Calibrating the flow pressure and monitors 135 Injection valve positions and flow paths 158 Using the flow restrictor 161 Chemical compatibility 187 SEE 34 AKTAprime plus User Manual 11 0026 44 Edition AA System overview 4 System overview This chapter contains
55. g in the appropriate buffers Purge kit 4 Runthe pump at 0 1 ml min I Set Flow Rate Press OK 0 0ml min 00 1 to start run Filling inlet tubing A1 A8 1 Go to Set Buffer Valve using the arrow buttons 2 Set the chosen A inlet and click OK The valve switches to the selected port 3 Draw buffer with the purge syringe until liquid enters the syringe 4 Repeat step 1 3 until all chosen A inlet tubing is filled Filling inlet tubing B 1 Goto Set Concentration B and set the concentration to 100 2 Click OK The switch valve turns to the inlet B port 3 Draw buffer with the purge syringe until liquid enters the syringe 4 Replace the purge tubing with the stop plug 5 Stop the pump by pressing end and then OK EE AKTAprime plus User Manual 11 0026 44 Edition AA 43 Making further runs to start run 4 Press pause cont run the pump for at least 30 s and press pause cont The table below lists the type and frequency of calibrations that can be done on AKTAprime plus Refer to section 9 20 Calibrations for descriptions of how to Only necessary after replacing wear parts Only necessary if specific conductivity with high Must be done when changing the flow cell Must be done when changing the flow cell _ Flushing the pump with 100 methanol 1 Put inlet tubing A1 in 100 methanol 2 Runthe pump at 40 ml min for 10 20 s and press pause cont I Set Flow Rate Press OK 0 0ml min 00 1 3 Set the fl
56. graphy run for documentation and evaluation Documentation is simplified since result files contain a complete record of a run including method curve data and a run log PrimeView allows rapid preparation of customized reports Recorder 112 Recorder 112 can be used to display data during the run for example the UV trace and conductivity gradient and other run data that has been stored during the run such as flow pressure pH and theoretical gradient 4 5 Columns and tubing A wide range of pre packed columns for the most commonly used techniques such as ion exchange gel filtration hydrophobic interaction and affinity chromatography can be used with AKTAprime plus A list of the recommended pre packed columns is given in section 11 5 1 Recommended columns On delivery the system is equipped with i d 1 6 mm inlet tubing i d 0 75 mm tubing from the pump to the outlet and i d 1 0 mm waste tubings When running columns with low maximum pressure limits at high flow rates PEEK tubing with a larger inner diameter can be used instead to prevent increased backpressure which might cause damage to the column Note lftubings are changed the delay volume to the fraction collctor must be changed AKTAprime plus User Manual 11 0026 44 Edition AA System overview as 4 6 Operator interface 4 6 1 Menu navigation A or V Find a specific menu option e OK Enter a menu e Esc Return one menu level lt ESC AorV i AoV
57. he display shows Free the selected number is free for storing a new method Press OK and continue with the parameter settings If the display shows Used a method already occupies that number Select a free number and press OK To clear a stored method press OK only 2 Toclear the stored method in the Method Occupied menu select clear and press OK 3 Select yes in the Clear Method menu and press OK AKTAprime plus User Manual 11 0026 44 Edition AA 73 6 Method programming 6 3 2 General parameter set up Setting the method base 1 Select menu Set Method Base and press OK Set Method Base ml min ml 2 Select time min or volume ml and press OK Setting the fraction base 1 Select menu Set Fraction Base and press OK Set Fraction Base ml min ml drp 2 Choose time min volume ml or drops drp Press OK Setting the pressure limit Set Pressure Limit 1 Select menu Set Pressure Limit and press OK 1 00 MPa 1 00 2 Set the pressure limit and press OK Note The pressure limit should be set to the maximum backpressure limit of the column used 0 2 MPa the back pressure contribution from flow restrictor The maximum backpressure limit is found in the column instruction More information on using the flow restrictor is found on page 161 6 3 3 Setting breakpoints The breakpoints are set on a time or volume base depending on the Method Base setting in the previous section The first
58. ible choose from a variety of objects to include in a report including chromatograms methods documentation free text and more in the customized No Retention min Area mAutmin Height m u 1 11 50 0 3267 0 433 Peak table za a a osez Ga Ex D 32131 3410 301 113 3 12 Creating and printing a simple report report interface To create and print a report 1 Select File Report to open the Generate Report dialog Format Preview SSS Generate Report AKTAprime plus User Manual 11 0026 44 Edition AA xi Contents Customized Report Format 31 Making your first run 2 Click Preview to open the Customize Report and see the entire report layout PRES fe Dew Berg pene ew Neag ely Ted ARE deles TT oi EE imwa Tei pee PLAT LES U Bomp ada Tine Dur yon 1 as Pa PrimeView Brport mn ua a w animant ones he arena PIS CEE dene as S nsnuause Bass as ELEREEETL ES clea ia pia Tha Pose 12cFs Preview Made 3 Click Exit to return to the Generate Report dialog 4 Click Print to open the Print dialog r Printer Name seupp ps3 6 L52_4_ 4050 r Print Range Copies Lo Number of copies fi 2 Pages E Enter page numbers and or page ranges separeted by commas For example 1 3 5 23 LK cena 5 Click OK to print the report 32 AKTAprime plus User Manual 11 0026 44 Edition AA Making your first run 3 13 Cleaning after a run CAUTI
59. ilation inlet The system can be operated at normal ambient temperatures in the range 4 to 40 C It should be located in a place of low temperature variations away from heat sources draughts and direct sunlight AKTAprime plus requires 100 120 220 240 V 50 60 Hz electrical supply with safety grounding Flasks for buffers and waste are needed Unpacking the system Begin by creating a dry and clean working area of 120 x 80 cm that allows easy access Then follow the step by step instructions below Note Save all the original packing material If the system has to be repacked for transportation or otherwise it is important that the system can be safely packed using the original packing material Remove the cardboard hood the red strap that secures the system to the pallet and other packing material Check the contents against the enclosed packing list Also check enclosed packages Store all boxes and plastic bags in a convenient nearby place CAUTION Take care not to damage any capillaries or components when lifting the instrument or when opening the plastic cover CAUTION Do not lift the system by the pillar 4 Grip the instrument between the cushions and gently lift it onto the work area Take care not to damage any capillaries or components when doing this AKTAprime plus User Manual 11 0026 44 Edition AA Installation 5 Open the plastic cover from the top and fold down to uncover the s
60. in Set Drop Sync Active wes 3 8 4 Preparing the monitors 1 Check the UV lamp filter position and the lamp position See section 8 2 Optical unit 2 Calibrate the pH electrode optional See section 9 20 5 Calibrating the pH electrode optional AKTAprime plus User Manual 11 0026 44 Edition AA 25 Making your first run 3 8 5 Filling the buffer inlet tubing When running an application templates the buffer inlet tubing will automatically be filled with buffer The procedure below can then be ignored For other applications fill the inlet tubing manually with buffer as described in the following procedure 1 If there are large amounts of air in the tubing fill the tubing using the Purge kit See section 5 2 2 Purging pump and inlet tubing Templates 2 Select Templates in the main menu using the A and V buttons and press OK Application template 3 Select Application Template and press OK System Wash Method 4 Select System Wash Method and press OK Select Buffer V Pos 5 Select the used tubing By default A1 and B will always be washed B A pak pan ean E a En OK 6 Move the cursor to OK and press OK 7 Press OK to start the method Press OK to start run Ms ie 8 When the method is finished empty the first collection tube It will contain a small amount of liquid after the system wash SSS 26 AKTAprime plus User Manual 11 0026 44 Edition AA Luer u
61. ing deionized water 1 Put the inlet tubing A1 A8 that is used and B in deionized water Note At delivery only A1 and B are installed 2 Put all waste capillaries W1 W3 in waste Templates 3 Select Templates in the main menu using the A and V buttons and press OK Application template 4 Select Application Template and press OK AKTAprime plus User Manual 11 0026 44 Edition AA 23 Making your first run Select System Wash Method and press OK Choose to wash the A2 A8 inlet tubing that is used by pressing OK at those cursor positions A1 and B will always be washed Note At delivery only A1 and B are installed Scroll to OK and press the OK button Press OK to start the method When the method is finished replace the first collection tube It will contain a small amount of water after the system wash Removing large amounts of air from the inlet tubing If there are large amounts of air in the inlet tubing use the Purge kit to remove it as described in section 5 2 2 Purging pump and inlet tubing System Wash Method 5 6 Select Buffer V Pos B A 2 3 4 5 _ _ _ OK 7 Press OK to start run 8 9 From injection valve 1 2 1 16 male connector 3 4 HisTrap column 24 3 8 2 Preparing the tubing and column Put inlet tubing A1 in the binding buffer Put inlet tubing B in the elution buffer Put the three waste capillaries brown color from port 4
62. is may cause the glass membrane of the electrode to dry out Dismount the pH electrode from the flow cell and fit the end cover filled with a 1 1 mixture of pH 4 buffer and 2 M KNO3 Do NOT store in water only 5 11 1 Between runs Buffers not containing any salt can be left in the system for a short time after a run even overnight not in the pH electrode see instructions below If a buffer containing salt has been used the flow path should be flushed with deionized water This is especially important if an organic solvent will be used in the next run To flush the flow path 1 Filla syringe with five times the sample loop volume of deionized water 2 Rinse the sample loop by injecting the water through the fill port on the injection valve 3 Put all used inlet tubings in water 4 Inthe Templates menu select Application Template and then System Wash Method 5 Select the used inlet ports Inlets A1 and B will always be washed 6 Press OK to start the method The system flow path is automatically flushed 5 11 2 Storage overnight The system except the pH electrode if used can be left filled with buffer overnight For storage of the pH electrode see the separate instruction below AKTAprime plus User Manual 11 0026 44 Edition AA Making further runs r 5 5 113 Weekend and long term storage If you are not using the system for a few days or longer 1 Wash all tubing and flow paths used with deionized water
63. le 5 2 1 Connecting different types of columns 1 Ifthe column is large use a column holder to secure it 2 Connect the column between port 1 of the injection valve and the upper port of the UV flow cell Use a suitable length of PEEK tubing in combination with unions and connectors supplied with the system The illustration shows two columns one with 1 16 fingertight fittings HiTrap and one with M6 fittings HiPrep HiTrap column HiPrep column injection valve From U n HiTrap column D 1 16 Male Ci O To UV cell 1 16 Male Union 1 16 Female M6 Male HiPrep column Union 1 16 Female M6 Male 1 16 Male Note More information regarding columns buffer solutions etc for the provided applications is found on the cue cards supplied 42 KTAprime plus User Manual 11 0026 44 Edition AA Making further runs SESE 5 2 2 Purging pump and inlet tubing If there are large amounts of air in the tubing or if you suspect air in the pump use the Purge kit to purge the flow path Air bubbles that still are trapped in the pump causing increased pulsation can be removed by flushing 100 methanol through the pump These two procedures are described in the following two sections Purging the flow path using the Purge kit 1 Remove the stop plug from the mixer 2 Connect the Purge kit to the mixer 3 Put the used inlet tubin
64. lection 2 1 General CAUTION Read the following information carefully to ensure that the system is installed correctly AKTAprime plus is assembled calibrated and fully tested before shipping For safe transportation some components have been secured and need to be released from strappings Accessories such as fittings tubing column holders etc are enclosed in separate packages After the installation procedure has been performed AKTAprime plus is ready for purification work 2 1 1 Installation procedure overview e Preparing for installation 16 e Unpacking AKTAprime plus 16 s Installing the SyStem 15555 trance hante ie needa uit wa nt 17 e Connecting the mains power cabling 18 e Connecting a computer for using PrimeView 19 e Running the system self test 20 AKTAprime plus User Manual 11 0026 44 Edition AA 15 ED oio 16 Pre requisites AKTAprime plus requires a working area of about 120 x 80 cm width x depth The mains power switch on the rear panel must always be easy to access The system should be installed on a stable laboratory bench To ensure correct ventilation the system requires a free space of 0 1 m behind and in front of it Do not place soft material under the system It might block the vent
65. lus User Manual 11 0026 44 Edition AA Template description a 7 2 12 IgM purification The IgM purification application template is used for purification of monoclonal antibody IgM IgM Purification To access the template select IgM Purification HiTrap IgM Purification and HiTrap IgM Purification press OK Column HiTrap IgM Purification 1 ml Total run time approx 48 min sample application time B 100 50 100 150 Volume Action Volume Conc B Flow Fract BufferV Inject V Priming A1 0 0 40 0 pos1 WASTE Equilibration 35 0 1 0 pos 1 LOAD Autozero 45 Sample application 45 0 1 0 pos 1 INJECT Wash 45 S 0 1 0 pos 1 LOAD End wash 55 S 0 1 0 pos 1 LOAD Priming B 55 1 S 100 40 0 pos1 WASTE Elution 1 90 S 100 1 1 pos 1 LOAD End elution 1 100 S 100 1 0 pos 1 LOAD Priming A2 100 1 S 0 40 0 pos2 WASTE Elution 2 135 S 0 1 1 pos 2 LOAD Priming A1 145 S 0 40 0 pos1 WASTE Re equilibration 160 S 0 1 0 pos 1 LOAD End Method 165 S AKTAprime plus User Manual 11 0026 44 Edition AA 91 Template description D 7 2 13 On column refolding The On column Refolding application template is used for protein renaturation on a column On column Refolding To access the template select On column Refolding HisTrap and press OK HisTrap Column HisTrap 1 ml Total run time approx 160 min sample application time B A 100 50 100 150 200 250 Volume Action Volume Conc B Flow Fract BufferV Inject
66. menu Save the new breakpoint by pressing OK 6 4 5 Deleting a breakpoint 1 Goto the Edit Breakpoint menu 2 Use the down button to scroll through the existing breakpoints 3 Press OK at the desired breakpoint to enter the parameter menus 4 Select the Delete Breakpoint menu and press OK 5 Select yes and press OK to delete the breakpoint 6 4 6 Printing the method Print out the modified method gradient curve on recorder channel 2 as follows 1 Goto the Show B on Rec out 2 menu 2 Select yes and press OK The recorder now prints out the gradient curve 6 4 7 Saving the method When all breakpoints are set save the method as follows 1 Goto the Save Method menu 2 Select yes and press OK AKTAprime plus User Manual 11 0026 44 Edition AA 79 Copy Method Copy Method From System PC Copy Method From System No 25 Copy Method To System PC Copy Method To System free No 29 Copy Method To PC used No 285 Method occupied Clear it yes no Copy Method Please wait 80 6 Method programming 6 5 Copying a method An existing method can be copied to another method number in the system The system totally has 40 method numbers By connecting a computer running PrimeView to the system an extra 999 methods can be stored for future use However a method always has to be stored on the system first The Copy Method function can then be use
67. min sample application time B A 100 25 50 75 100 125 Volume Action Volume Conc B Flow Fract BufferV Inject V Priming B 0 100 40 0 pos1 WASTE End priming B 25 100 40 0 pos1 WASTE Priming A1 25 1 0 40 0 pos 1 WASTE Equilibration 60 0 1 0 pos 1 LOAD Autozero 70 Sample application 70 0 1 0 pos 1 INJECT Elution delay 70 S 0 1 0 pos 1 LOAD Elution 80 S 0 1 1 pos 1 LOAD Elution wash out 100 S 100 1 1 pos 1 LOAD End wash out 117 S 100 1 0 pos 1 LOAD Priming A1 117 1 S 0 40 0 pos 1 WASTE Re equilibration 132 S 0 1 0 pos 1 LOAD End method 137 S EEE AKTAprime plus User Manual 11 0026 44 Edition AA 89 Template description Anion Exchange HiTrap Q Cation Exchange HiTrap SP 90 7 2 10 Anion exchange The Anion exchange application template is used for separation of molecules that have negative charge To access the template select Anion Exchange HiTrap Q and press OK Column HiTrap Q 1 ml Total run time approx 63 min sample application time For buffer gradient and parameter table see Mab purification gradient elution 7 2 11 Cation exchange The Cation exchange application template is used for separation of molecules that have positive charge To access the template select Cation Exchange HiTrap SP and press OK Column HiTrap SP 1 ml Total run time approx 63 min sample application time For buffer gradient and parameter table see Mab purification gradient elution AKTAprime p
68. ml or min with different values for the buffer B concentration This creates an immediate change in the B concentration between the breakpoints a step gradient Setting the flow rate 1 Select menu Set Flow Rate and press OK Set Flow Rate 0 1 ml min 0 8 2 Set the flow rate cannot be 0 0 and press OK Setting the fraction size 1 Select menu Set Fraction Size and press OK Set Fraction Size 00 0 ml 02 2 Set the fraction size and press OK Setting the buffer valve position 1 Select menu Set Buffer Valve Pos and press OK Set Buffer Valve Pos Pos 1 1 2 Setthe position and press OK Refer to the number printed on the buffer valve Setting the injection valve position 1 Select menu Set Inject Valve Pos and press OK Set Inject Valve Pos Load Waste Load Inject 2 Set the position and press OK Waste the flow is diverted to waste ports 4 and 5 Load the sample loop is loaded between ports 2 and 6 when injecting the sample through port 3 Inject the sample loop is emptied through port 1 and the flow directed to the column AKTAprime plus User Manual 11 0026 44 Edition AA 75 Set Peak Collect no Set Slope 0 00 mAU min Autozero no yes Event Mark no yes n Edit time volume 12 7ml Edit time volume Change Replace New time volume 12 7ml 15 0 Save Breakpoint 0 00
69. ng breakpoints accordingly Replace will not change the time volume of the other breakpoints 3 Edit the time volume and press OK Saving the breakpoint To save the breakpoint select menu Save Breakpoint and press OK Deleting a breakpoint To delete an existing breakpoint select menu Delete Breakpoint and press OK AKTAprime plus User Manual 11 0026 44 Edition AA Method programming 6 ee 6 3 4 Setting an alarm If an alarm should sound during or after the run SR 1 Goto the Set Alarm at menu No alarm 26 00 2 Enter the desired time or volume elapsed from the method start then press OK For example entering 26 ml will sound an alarm when 26 ml has been pumped from the method start Entering zero deactivates the alarm 6 3 5 Printing the method If using a chart recorder print the programmed method concentration of B curve on recorder channel 2 as follows Show B onRecout2 1 Goto the Show B on Rec out 2 menu no yes no 2 Select yes and press OK The recorder now prints out the theoretical B curve If using a computer it also possible to print the method from the computer Refer to the PrimeView User Manual for more information 6 3 6 Ending the method The method ends at the last breakpoint If a period of constant parameters is required at the end of the method enter a final breakpoint with the same parameters as the last one End Method 1 Go the
70. nion Templates Application Template His Tag Purification HisTrap Sample appl volume 0 0 Press OK to start run aes Making your first run 3 8 6 Filling the sample loop Using an injection fill port 1 Connect a sample loop between port 2 and 6 Luer union on the INJECTION VALVE Make sure that the sample loop is large enough for your sample 2 Connect a luer female 1 16 male union to port 3 3 Fill a syringe with five loop volumes of deionized water or binding buffer 4 Fitthe syringe in the luer union and carefully inject the buffer 5 Remove the syringe and fill it with at least two loop volumes of the sample 6 Carefully inject the sample into the sample loop Do NOT remove the syringe after the injection because the loop might otherwise be emptied due to self drainage 3 9 Selecting template and starting the run 1 Select Templates in the main menu and press OK 2 Select Application Template and press OK 3 Select His Tag Purification HisTrap and press OK 4 Set the sample volume and press OK 5 Press OK to start the purification run AKTAprime plus User Manual 11 0026 44 Edition AA 27 Making your first run 3 10 Viewing the run 3 10 1 Viewing the run in PrimeView When the pump starts running the progress of the run can be viewed in the two panes in PrimeView Prime View prime Result C prime Manual Runs prime Manual Run 7 RES 18 x Fil
71. not waste sample The sample loop must be completely filled with buffer before the sample can be loaded Complete filling is used when reproducible sample volumes are required An excess of sample is used to ensure that the sample loop is filled completely In preparative applications the sample volume should be at least two times the volume of the sample loop For analytical reproducibility use five times the volume of the sample loop as sample volume About 2 3 loop volumes of sample are required to achieve 95 of maximum loop volume Five loop volumes gives better precision Preparation Prepare the injection valve as follows 1 Connect the supplied luer female 1 16 male union connector to valve port 3 2 Make sure that a waste tubing is 7 TS l ni connected to port 4 of the injection valve AkTAprime plus User Manual 11 0026 44 Edition AA 45 Making further runs 46 3 Mount the sample loop between ports 2 and 6 of the injection valve Five sizes of sample loops are available Sample loop Code no Loop 100 ul 25 MPa 18 1113 98 Loop 500 ul 10 MPa 18 1113 99 Loop 1 ml 10 MPa 18 1114 01 Loop 2 ml 10 MPa 18 1114 02 Loop 5 ml 1 MPa 18 1140 53 An injection fill port can be used instead of the luer union connector Then prepare the injection valve as follows 1 Loosely thread the injection fill port screw into valve port 3 Insert an injection needle 0 7 mm o d into the injection fill po
72. on AA 5 Contents Making further runs 5T GOMOD sise attente nie 41 5 2 Preparing the system further oi ccessssesssssssscssssssssssesseessssecsesssssssnseeesess 42 5 3 Calibrations schedules 44 5 4 Applying the sample 45 5 5 Collecting fractions 52 5 6 Starting a run 53 5 7 Dring d TUN aan A nn rer 56 5 8 Running a method template oac eccessssseesesssssscccsssssssssssesessecsecssssssssseesess 59 5 9 Running a stored method 62 5 10 Running the system manually 63 5 11 Cleaning after a run and storage 66 D 12 Cold FO0m op ration ss nds 68 Method programming 6 1 GOR SRC leas i e e hA NE 6 2 Programming using method templates z 6 3 Programming line by line s ss sssssssssssssssssssssissrrssisssrssssssrssssssrssnsrsnesnernes 6 4 Editing a Stored method weeecccccccsssssssssssseesssssesscssssssssssussesssssecsssssssssssseesees 65 COpuing A MENO nina nn Rea Template description AL G n ral san annee anni 81 7 2 Application templates ou sssscssscsssssssscessssssssessscesssssessssssssssesssssesssssseessss 81 1 3 M tho d t mbplates aseisccsscissieessstisivasascteadectstesnisaissssataitagenlerecobissssitiaatialieabsien 96 Installing and modifying components 8 1 General 8 2 Optical unit 8 3 Co du tivity cell essas a E BA FractionColl ctor sn nn ti 8 5 pH flow cell and electrode optional 8 6
73. on from flow restrictor The maximum backpressure limit is found in the column instruction More information on using the flow restrictor is found on page 161 Setting the buffer valve position 1 Select menu Set Buffer Valve Pos The current setting is displayed Press OK 2 Set the position and press OK Refer to the number printed on the buffer valve Setting the injection valve position 1 Select menu Set Inject Valve Pos The current setting is displayed Press OK N Set the position and press OK Waste the flow is diverted to waste ports 4 and 5 Load the sample loop is loaded between ports 2 and 6 when injecting the sample through port 3 Inject the sample loop is emptied through port 1 and the flow directed to the column AKTAprime plus User Manual 11 0026 44 Edition AA Making further runs r 5 Sy 5 10 3 Starting the run Press OK to 1 Press OK at the Press OK to start run prompt to start the run start run 5 10 4 During the run Viewing parameters The progress of the method can be viewed on the computer monitor See section 5 7 During arun for a description of viewing and changing parameters during the run Changing parameters Most parameter values can be changed at any time during a manual run The gradient can only be changed if no gradient is running or if the system is paused press pause or held press hold The following parameters can be changed during the run In addition the
74. ow rate to 5 ml min using the arrow buttons 5 Move inlet tubing A1 to deionized water 6 Press pause cont and run the pump for 1 min 7 Finish by pressing end and then OK 5 3 Calibrations schedule perform these calibrations Component How often Pump Conductivity flow cell Cell constant accuracy is measured Temperature Entering a new cell constant Pressure offset When required pH electrode optional Every day Ool 44 KTAprime plus User Manual 11 0026 44 Edition AA Making further runs 5 4 Applying the sample In AKTAprime plus the sample can be applied in three ways depending on the volume to inject The table below lists the options together with the recommended sample volume ranges Volume to inject Sample application technique 25 ul 5 ml Sample loop manual filling 1 150 ml Superloop manual filling gt 10ml Pump together with buffer valve The different sample techniques are described in the following sections 5 4 1 Applying 25 ul 5 ml of sample using a sample loop Partial or complete filling The sample loop can be filled partially or completely depending on whether high recovery or reproducible sample volumes are most important Partial filling is used when high recovery is required The sample volume loaded should be at maximum 50 of the loop volume The volumetric accuracy is that of the syringe Partial filling allows the injected volume to be changed without changing the loop and does
75. rap 1 ml Total run time approx 37 min sample application time B A 100 gt 25 50 75 100 Volume Action Volume Conc B Flow Fract BufferV Inject V Priming A1 0 0 40 0 pos 1 WASTE Equilibration 35 0 1 0 pos 1 LOAD Autozero 45 Sample application 45 0 0 3 0 pos 1 INJECT Wash 45 S 0 1 0 pos 1 LOAD End wash 55 S 0 1 0 pos 1 LOAD Priming B 55 1 S 100 40 0 pos 1 WASTE Elution 90 S 100 1 1 pos 1 LOAD End elution 100 S 100 1 0 pos 1 LOAD Priming A1 100 1 S 0 40 0 pos 1 WASTE Re equilibration 115 S 0 1 0 pos 1 LOAD End method 120 S a 86 AKTAprime plus User Manual 11 0026 44 Edition AA Template description 7 2 7 Mab purification step elution The Mab purification step elution application template is used for purification of monoclonal antibodies by step elution Mab Purification To access the template select Mab Purification Step elution and Step elution press OK Column HiTrap Protein G 1 ml alt HiTrap Protein A or HiTrap rProtein A 1 ml Total run time approx 37 min sample application time B A 100 gt 25 50 75 100 Volume Action Volume Conc B Flow Fract BufferV Inject V Priming A1 0 0 40 0 pos1 WASTE Equilibration 35 0 1 0 pos 1 LOAD Autozero 45 Sample application 45 0 1 0 pos 1 INJECT Wash 45 S 0 1 0 pos 1 LOAD End wash 55 S 0 1 0 pos 1 LOAD Priming B 55 1 S 100 40 0 pos 1 WASTE Elution 90 S 100 1 1 pos 1 LOAD End elution 100 S 100 1 0 pos 1 LOAD Priming A1 100 1 S 0
76. ration B The current setting is displayed Press OK Set Concentration B 20 B 30 2 Set the new concentration and press OK Changing the flow rate 1 Select menu Set Flow Rate The current setting is displayed Press OK Set Flow Rate 0 1 ml min 0 8 2 Set the new flow rate and press OK Changing the fraction size 1 Select menu Set Fraction Size The current setting is displayed Press OK Set Fraction Size 00 0 ml 02 2 Set the new fraction size and press OK Setting the buffer valve position 1 Select menu Set Buffer Valve Pos The current setting is displayed Press OK Set Buffer Valve Pos Pos 1 1 2 Setthe new position and press OK Refer to the number printed on the buffer valve Setting the injection valve position 1 Select menu Set Inject Valve Pos The current setting is displayed Press OK Set Inject Valve Pos Load Waste Load Inject 2 Set the new position and press OK Waste the flow is diverted to waste ports 4 and 5 Load the sample loop is loaded between ports 2 and 6 when injecting the sample through port 3 Inject the sample loop is emptied through port 1 and the flow directed to the column AKTAprime plus User Manual 11 0026 44 Edition AA 57 Making further runs E Autozero Event Mark End method yes yes no Method Complete Press OK to continue Memory Print Out
77. rt Note Use a needle with a round tip Tighten the fill port until the nozzle has formed a seal around the needle s tip i e when it feels as if you are penetrating a septum at the end of the injection fill port The seal should allow easy insertion and removal of the needle Mount the syringe holder in the fill port Check the waste tubing and mount the sample loop as described for using a luer union connector AKTAprime plus User Manual 11 0026 44 Edition AA Making further runs EEE Filling the sample loop Two techniques can be used for filling the sample loop partial or complete filling Type of filling Volume to load Partial filling max 50 of the sample loop volume Complete filling 2 5 times the sample loop volume Partial filling Partial loop filling is achieved as follows Note The flow must be off For example when running the system manually press Pause 1 Set the injection valve to position pos 1 LOAD LOAD Column 2 Load the syringe with a large volume of deionized water or Sample ae syringe binding buffer at least 5 times the loop volume 3 Fill the sample loop carefully Waste 4 Setthe injection valve to position Pos 2 INJECT INJECT Column 1 Note Ifthe syringe is taken out when s Ra a ample the injection valve is in position syringe LOAD self drainage will occur and air enter the sample loop 5 Load the syringe with the required volume of sample maximum 50 of the sample loop vol
78. run is finished the curves obtained can be printed from the computer AKTAprime plus User Manual 11 0026 44 Edition AA Making further runs r 5 5 10 Running the system manually To run the AKTAprime plus system manually without using a pre programmed method or a template follow the procedure described in the sections below 5 10 1 Preparing a manual run 1 Perform the general system preparation refer to section 5 2 Preparing the system further Manual Run 2 Inthe main menu select menu Manual Run and press OK 5 10 2 Setting the parameters Use the arrow keys to go through the menu options and set the parameters as required The settings take effect as soon as the instruction is confirmed by pressing OK Setting the method base 1 Select menu Set Method Base The current setting is displayed Press OK Set Method Base ml min ml 2 Select time min or volume ml and press OK Setting the concentration Set the start concentration of buffer B as follows 1 Select menu Set Concentration B The current setting is displayed Press OK Set Concentration B 20 B 30 2 Set the desired concentration and press OK Setting a gradient To create a gradient from the start enter the target concentration of buffer B and the duration of the gradient in volume or time depending on the method base Set Gradient 1 Select menu Set Gradient default setting off Press OK
79. sses and gloves resistant to the chemicals used Follow local regulations and instructions for safe operation and maintenance of the system 3 2 Purification work flow A typical purification run consists of the following main steps e Buffer preparation e Sample preparation e Purification setup e Selecting template and starting the run e Viewing the run e Viewing the result e Creating and printing a report AkTAprime plus User Manual 11 0026 44 Edition AA 21 Making your first run Selftest Please wait AKTAprime plus V3 00 Templates td Ci PrimeView 22 3 3 Pre requisites The system and computer must be installed and functioning as described in chapter 2 Installation IMPORTANT Before using AKTAprime plus read all the safety information in section 1 4 Safety 3 4 Starting AKTAprime plus If the system is not already turned on 1 Turn on the system using the mains switch at the rear panel The system now performs a self test 2 First the system name and software version number is displayed Several messages are then shown during the self test If an error is detected during the self test an error message is shown 3 All parameters are automatically set to factory default values during the self test 4 The self test takes about 30 40 seconds When the test is completed the display shows the Templates menu Note The system can be used for most
80. starting a run e Method templates Require more information to be entered e g flow rate elution volume and equilibration volume Methods that are made from the method templates can also be stored for future use 7 2 Application templates 7 2 1 General The following application templates are included e Desalting e Affinity purification e Purification of Histidine tagged proteins e Purification of GST tagged proteins e Purification of monoclonal antibodies e IgM purification e Removal of albumin e Protein on column refolding e Anion exchange e Cation exchange e System Wash Method AKTAprime plus User Manual 11 0026 44 Edition AA 81 Template description Templates Application template 82 The process parameters in the application templates can not be changed Only the sample volume needs to be entererd e Sample application is always made by using a syringe and a sample loop e The buffer solutions to use are described in the application cue card For more information on how to run an application template refer to section 5 6 Starting a run The application templates are described below The templates are illustrated by the buffer gradient during the run The table shows how the parameter values change accordingly Parameter S represents the sample volume 7 2 2 Selecting an application template 1 In the main menu choose menu Templates and press OK 2 Choose menu Application Templa
81. t PrimeView Evaluation module click PrimeView Evaluation icon on eu i ae the Windows desktop Evaluation 3 11 1 Opening a result file 1 Select File Open to open the Open Result dialog 2 Select the result file example AT2005feb12n0001 res and click OK All contents of the opened result file are transferred to the Evaluation module and the chromatogram is automatically opened Prane Vire Evaluation LAT 004der ODEI C UNICORN ocal FE prime Result ATID Ader ToD EST Luigi x uh ria Est Va Vesgus TA 21812 amp 458 0 ji Er ees N a Curves FE pane isoo e hono ae h _ an D ae He Dreak pou ah SSS AKTAprime plus User Manual 1 1 0026 44 Edition AA 29 Making your first run a ey 3 11 2 Changing the chromatogram layout The chromatogram includes a number of curves that have been created during the method run such as UV conductivity pH and fraction marks To change the layout of the chromatogram 1 Right click in the chromatogram window and select Properties The Chromatogram Layout dialog is opened 2 Carry out the changes on the different tabs to get the desired layout for header curves and peak table 3 Click OK 3 11 3 Integrating peaks To make a simple integration of the UV curve peaks 1 Select Integrate Peak integrate to open the Integrate dialog x Chromatogram Target peak table g 01 T 2004dec07n0003 10 UV 02 AT 2004decO no003 10_Cond 03 AT2004dec0 no003 10_pH 04 AT2004decO no003
82. te and press OK 3 Select the desired template with the up and down buttons AKTAprime plus User Manual 11 0026 44 Edition AA Template description ee 7 2 3 HiTrap desalting The HiTrap desalting application template is used for desalting a sample solution Desalting To access the template select Desalting HiTrap Desalting and press OK HiTrap Desalting Column HiTrap 5 ml Desalting Total run time approx 9 min sample application time B A 100 20 40 60 80 Volume Action Volume Conc B Flow Fract BufferV Inject V Priming A1 0 0 40 0 pos 1 WASTE Equilibration 35 0 5 0 pos 1 LOAD Autozero 60 Sample application 60 0 5 0 pos 1 INJECT Elution 60 S 0 5 1 pos 1 LOAD End method 75 S AKTAprime plus User Manual 11 0026 44 Edition AA 83 Template description _ lt lt lt 7 2 4 HiPrep desalting The HiPrep desalting application template is used for desalting a sample solution Desalting To access the template select Desalting HiPrep Desalting and press OK HiPrep Desalting Column HiPrep 26 10 Desalting Total run time approx 18 min sample application time B 100 100 200 300 400 pre Action Volume Conc B Flow Fract Buffer V Inject V Priming A1 0 0 40 0 pos 1 WASTE Equilibration 35 0 20 0 pos 1 LOAD Autozero 300 Sample application 300 0 20 0 pos 1 INJECT Elution 300 S 0 20 5 pos 1 LOAD End method 400 S 84 KTAprime plus User Manual 11 0026 44 Edition A
83. the bottle with the sample 4 Fill the inlet tubing with sample See section 5 2 2 Purging pump and inlet tubing Applying the sample 1 Select a template in sub menu Method Template in menu Templates or select a stored method where the buffer valve is used for sample application In a stored method the buffer valve must be set to position 8 and the injection valve to position LOAD when applying the sample 2 In method templates select sample application with pump 3 Set the required parameters and the sample volume 4 Startthe run Note Whenusing the buffer valve for sample application an extra 15 ml of buffer is used for washing after the sample application Cleaning the pump WARNING NaOH is injurious to health Avoid spillage When the pump has been used for sample application cleaning the pump might be required If so pump a cleaning or sanitizing agent through the pump by running the System Wash method The standard recommendation is to pump 1 M NaOH for 30 minutes and then wash out immediately with buffer AkTAprime plus User Manual 11 0026 44 Edition AA 51 Making further runs Set Fraction Base ml min ml drp Set Fraction Size 1 00 ml Set Peak Collect no Set Slope 0 00 mAU min 0 00 mAU SEE 52 5 5 Collecting fractions Fractions are collected in tubes in the fraction collector It is possible to collect fractions in two different ways
84. the equilibration volume 1 in the figure and press OK Set the sample volume 2 to be injected and press OK Set the wash 1 volume 3 and press OK This setting does NOT apply to the Gel filtration method template Note 15 ml of buffer is automatically added to Wash 1 when using the system pump for the sample application Set the elution volume 4 and press OK Set the wash 2 volume 5 and press OK This setting does NOT apply to the Gel filtration method template 10 Select yes at the Method ready prompt and press OK 71 6 Method programming 6 2 3 Storing the method Save Method 1 To store the method select yes and press OK yes yes no 2 Select a method number and press OK Free Methods 25 Free means that the selected number is free for storing a new method Sel Method Free 16 Used means that the number is already occupied Free Methods 25 Select a free method number and press OK Alternatively press OK to clear Sel Method Used 16 the number in the Clear Method menu The programming is now finished and the method ready for use 6 3 Programming line by line The example below illustrates a simple method for a gradient elution with a linear gradient from 0 to 100 The sample is loaded manually through the injection valve Fraction collecting starts at the beginning of the elution The table shows the breakpoints in method Values actively entered for each breakpoint are shown
85. to be washed and press OK B A 2 3 St tiation at OK Note A1 and B are pre selected and will always be washed Total run time depends on the number of buffer inlets selected If all are selected the approximate run time is 9 min The table below shows how the tubings are washed Action Volume Conc B Flow Fract BufferV Inject V Wash B 0 100 50 0 pos 8 WASTE End wash B 24 9 100 50 0 pos 8 WASTE Wash A8 25 0 50 0 pos 8 WASTE Wash A7 50 0 50 0 pos7 WASTE Wash A6 75 0 50 0 pos6 WASTE Wash A5 100 0 50 0 pos5 WASTE Wash A4 125 0 50 0 pos4 WASTE Wash A3 150 0 50 0 pos 3 WASTE Wash A2 175 0 50 0 pos2 WASTE Wash A1 200 0 50 0 pos1 WASTE Air wash out 225 0 50 0 pos1 WASTE Wash Inject v load 250 0 0 5 0 pos 1 LOAD Wash Frac tubing 251 0 0 5 2 pos 1 LOAD End Method 252 AKTAprime plus User Manual 11 0026 44 Edition AA 95 Template description Templates Method template 96 7 3 Method templates 7 3 1 General The system is supplied with templates for the four most common purification techniques e Gel filtration buffer exchange e lon exchange e Hydrophobic interaction chromatography e Affinity Find a method template as follows 1 In the main menu choose menu Templates and press OK 2 Choose menu Method Template and press OK The following selections must be made before starting the run e Pressure limit flow rate fraction size and the volumes during the main steps of th
86. ume 6 Insert the syringe into the luer union on the injection valve 7 Set the injection valve to position LOAD 8 Carefully inject the sample into the sample loop Do NOT remove the syringe after the injection Otherwise the loop might be emptied due to self drainage 9 The sample will be injected onto the column when the valve is switched to INJECT in the method EE AKTAprime plus User Manual 11 0026 44 Edition AA 47 Making further runs Say Complete filling With complete loop filling the sample volume can only be changed by changing the loop size Complete filling is achieved as follows Note The flow must be off For example when running the system manually press Pause 1 Set the injection valve to position pgs 1 LOAD LOAD Column 2 Load the syringe with sample 2 5 times the loop volume Sample syringe 3 Carefully inject the sample into the sample loop Do NOT remove the syringe after the injection Otherwise the loop might be emptied due to self drainage 4 The sample will be injected onto the column when the valve is switched to INJECT in the method When emptying the sample loop a buffer volume of at least five times the sample loop volume should be used to flush the loop and ensure that all sample is injected onto the column es 48 AKTAprime plus User Manual 11 0026 44 Edition AA Making further runs 5 4 2 Applying 1 150 ml of sample using a Superloop Superloop allows introducing larger volumes of sample
87. ume as input all other process parameters are preset This section describes how to select and run an application template Method templates Templates for common purification techniques ion exchange hydrophobic interaction affinity and gel filtration These templates require more input from the operator such as flow rate and elution volume See section 5 8 Running a method template Stored methods These methods are programmed line by line and stored by the operator When creating a stored method all process parameters must be programmed A stored method can also be based on a method template See section 5 9 Running a stored method Manual run By running the system manually the operator chooses not to use a pre programmed template or method The process parameters are set before the run but they can not be stored for future use See section 5 10 Running the system manually AKTAprime plus User Manual 11 0026 44 Edition AA Making further runs r 5 This section describes how to use an application template for performing a run This information can also be found on the cue cards supplied To use an application template Templates 1 Inthe main menu choose menu Templates and press OK Application Template 2 Choose menu Application Template and press OK 3 Choose an application template and press OK Desalting Mab Purification HiTrap Desalting Step Elution
88. ystem 6 Remove the plastic cover by gently tilting the system back and forth while pulling out the plastic cover 7 Remove the protection pad placed under the fraction collector bowl 2 4 Installing the system 1 Raise the column holder to the top Column position holder 2 Putatleast 20 collection tubes in the bowl starting at the first position Lock knob 3 Loosen the lock knob holding the delivery arm and raise the arm 4 Adjust the delivery arm so that the tube sensor touches the Sy First position collection tubes of the outer 7 l track 5 Adjust the arm bracket so that the bottom of the tube sensor is about 5 mm below the top of the tubes The tubes should always be below the horizontal mark on the tube sensor Lock knob 6 Lock the arm bracket at this height with the lock knob 7 Check that the sensor is in the correct position for Sensor control the tube size The eluent tubing should be over the center of the collection tube Use the red sensor control to position the tube holder AKTAprime plus User Manual 11 0026 44 Edition AA 17 Installation 8 Rotate the rack counter clockwise by hand until the rear half of the tube sensor rests against tube 1 When the fraction collector is started the bowl moves to the correct position to collect the first fraction in tube 1 9 Remove the inlet tubings and the brown waste

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