Home

ÄKTAxpress

1. Creating a method To create a method plan for performing metal ion stripping plan for perform ing metal ion l stripping Step Action Click the Method Wizard icon in the Method Editor Result The Method Wizard dialog box appears Strip Affinity Columns Main Selections Maintain Select Column Strip Affinity Column HiT rap_ Chelating HP_1_ml Global Last Page Select Number of Columns to Strip 1 Running Condition f Room Temperature i Cold Room 5 On the Strip Affinity Columns page e Select column type e Select the number of affinity columns e Select running conditions Click Next On Last page click Finish and save the metod plan e Run the method plan according to the standard procedure described in 6 6 1 Starting a run using a method plan on page 186 18 1178 00 AA ep 206 Operation 6 e Immerse the inlet tubing in flasks according to the check list on the Summary page Note The Metal Ion Stripping inlet tubing A8 have to be filled before starting the stripping run See 6 5 3 Purging the pump and inlet tubing on page 156 e p 207 7 Evaluation 7 Evaluation About this chapter This chapter contains descriptions of how results from a run can be evaluated In this chapter This chapter contains the following sections Topic Evaluation procedure overview Finding and opening results Viewing results Pooling fractions and adjusting the pools Creating pooling protocols Print
2. KTA design KTA xpress User Manual Amersham V Biosciences Table of Contents Table of Contents Mes RO el G arcuate tcataen asec a con mendeuumadneetiee weateuuoneaderteneesea tas 7 LL Important USSrrINTONMallONccsccsssecasuanarevernaci e 8 Lee roduc tN MaOs EE 9 IE E OLY A A E E A ETEA A N E E A A AE AE E T A E E 11 Zo WAVER O GUNG UO tress ce reset oie E E E 12 2 1 Introducing AKTAXDIe SS cccccceeceenceceececuececeeeeeeeeeaevecaevecaeeeneeteesetanternesenaeees 13 2 2 Purification Of recombinant PLOteINS ccececeeceeeeceeeeeteeeeteeeeeeateetseeateetsetaneanaees 18 Zoe Basle ODE aun ONNCIDIES asuneen a A O de iaasusan meena 19 2 4 AKTAxpress user GOCUMENtATION ccecccececeeccceececececcececuececeeeeaseecaeresereneness 20 3 KTAXpress OVGIVICW socciccos cctens ci cadccs cases dctecateeriaeceed actenetueidcd ented i nanenane nnan aanne 23 3 1 UNICORN OVGrVICW ccccccccccsceeeeeeeeeeeeeeateeeeeeteeeeeegteeesteteaeetegteeeateatsaeantatsaeass 24 3 2 Separation System OvVerviEWciu suiii Seca lau Gena udeidul Susur waste aad 27 S22 ols LOCATION OF the COMPONENTS ccuiduats tact aa candied a aa akexidie 28 2 oe c MICU TOW Dal aenema rrara 34 229 UDINE aNd CONMCCIOIS reirei absagvanas hagaad suaeiadacsauaseonadeus tastes 39 32 Ae FUNUN deS DEO anera a a E E nina eae 37 O22 e MACs and CONO arna a r A AE O naedeia vanes 43 Pe LADE oare N 46 3 3 Purification DrocessS Overvie W scis
3. To do this 1 Open a result file Place the mouse pointer in any corner of the area to be magnified Press and hold the left mouse button A magnifying glass icon will be added to the mouse pointer arrow on the screen Drag a box to cover the area to be magnified and release the mouse button Result The selected region is now displayed in the entire chromato gram window together with appropriate scales for the Y and X axes Use the arrow keys on the keyboard to move around in the chroma togram at the current zoom scale Possible undo actions when zooming e Undo zoom Right click in the window and select Undo zoom to undo the last Zoom step e Reset zoom Right click in the window and select Reset zoom to reset all zoom steps at once Further instruc Further instructions on how to change the viewing of the chromatogram can be moe found in How to view results in the UNICORN Online Manual 18 1178 00 AA ep 212 Evaluation 7 1 4 Pooling fractions and adjusting the pools Introduction After the last purification step of each protocol a fractionation is performed The resulting fractions are shown in the chromatogram A pooling suggestion of the fractions can be provided in UNICORN to calculate protein concentration and amounts This section describes the steps involved when creating the pooling suggestions and adjusting the pools Note When the fractionation of a peak is finished it will in the chro
4. Wetted materials The following wetted materials are used in AKTAxpress PEEK polyetheretherketone Pump UV monitor Valves Mixer Flow restrictor Online filter Tubing Unions connectors PTFE polytetrafluoroethylene Pump UV monitor Mixer Flow restrictor FEP fluorinatedethylenepropylene Tubing ETFE ethylenetetrafluoroethylene Flow restrictor Unions connectors PCTFE polychlorotrifluoroethylene Pump Conductivity cell PP polypropylene Online filter Inlet filter PVDF polyvinylidenefluoride Pump PE polyethylene Pump PFR fluororubber Flow restrictor e p 329 10 Reference information 10 2 Technical specifications 10 2 3 Wetted materials Titanium alloy Pump UV monitor Inlet filter Conductivity cell Quartz glass UV monitor Aluminium oxide Pump Stainless steel Elgiloy cobalt chromium nickel alloy Pump Ruby sapphire Pump Online filter 18 1178 00 AA ep 330 10 3 Introduction Import file content Example File specification Reference information 10 Import file format This section describes how to create import files and how they are used One or several import files can be used for storing information on one or several protein samples When starting a run using the wizard in System Control the import file can automatically be called upon and the information collected and inserted
5. Maintenance 8 Click Close to finish the operation Setting the con If the conductivity cell has been replaced a cell constant has to be set ductivity cell con stanit The cell constant is shown on the packaging of the new cell If the packaging is missing perform a normal calibration according to 8 5 2 Calibrate current conductivity cell on page 245 To enter the cell constant of a new conductivity cell Read the cell constant from the packaging of the new cell In the UNICORN System Control module select the system with the new conductivity cell by clicking the system name System Control SYS 3 Method Result File View Manual System Help i arin m Expand gt gt a Calibration procedure Monitor Add cell constant value of a new cond cell See Help 25 0000 ee ay gt Read value 1 1 Reference value 1 Measured value fi 5 0 12cm Close Help Enter the cell constant in the Reference value 1 field Click Read value 1 Read value 1 After a few seconds a new cell constant is calculated and saved in the system 3 In the menu select System Calibrate and choose Cond_Cell under Monitor Click Close to finish the operation ep 245 8 Maintenance 8 6 Replacement procedures 8 6 Replacement procedures About this section This section describes replacement procedures of AKTAxpress In this section This section contains the follo
6. Method Wizard 5 Column equilibra To select column equilibration options tion options On the Column Equilibration page select the type of column s to be equilibrated Method Wizard Column Equilibration Main Selections Purify I Affinity Prepare System Column E quilibration I Desalting Last Page If lon Exchange W Gel Filtration During a run the affinity ion exchange and desalting columns will be equilibrated with 5 CV and the gel filtration columns with 2 CV of the appropriate buffer Note The chosen column s will be equilibrated before the sample is loaded on the affinity column Glick Next Finishing the creat When all selections are made continue with 5 2 5 Saving the method plan on page mi 106 e p 101 5 Method Wizard 5 2 Creating a method plan 5 2 4 Using the Maintain option 9 2 4 Using the Maintain option Introduction This section describes how to create a method plan for maintenance of the system and the columns The method plan can for example consist of cleaning instructions or an installation test Creating a method plan for mainten ance To create a method plan for maintenance On the Main Selections page select Maintain Click Next Method Wizard Hain Selections Maintain Last Page Main Selections i Prepare Purify Maintain Note When entering the Main Selections page the parameters values on the subsequent pages are set to d
7. Six monthly maintenance Maintenance when required Calibration procedures Replacement procedures 18 1178 00 AA ep 220 Maintenance 8 8 1 Maintenance program Introduction This section specifies the preventive maintenance to be performed on AKTAxpress The maintenance is divided into e Monthly maintenance e Six monthly maintenance e When required For maintenance of columns see chapter 6 8 3 Cleaning columns on page 202 Periodic mainten The following periodic maintenance should be made by the user of AKTAxpress ance program Monthly See 8 2 1 Checking flow restrictor on page 223 See 8 2 2 Changing pump rinsing solution on page 22S Six monthly See 8 3 1 Checking UV lamp run time on page 227 See 8 3 2 Cleaning UV cell on page 228 See 8 3 3 Cleaning conductivity cell on page 231 When required See 8 4 1 Externally cleaning the system on page 233 See 8 5 1 Calibration of zero pressure on page 242 See 8 5 2 Calibration of conductivity cell on page 243 See 8 6 1 Replacing capillary loops on page 247 See 8 6 2 Replacing tubing and connectors on page 249 See 8 6 3 Replacing flow restrictor on page 262 See 8 6 4 Replacing UV lamp on page 264 See 8 6 5 Replacing check valves on page 269 See 8 6 6 Replacing pump piston seal on page 272 See 8 6 7 Replacing a damaged pump piston on page 280 See 6 5 3 Purging the pump and inlet tubing on page 156 e p 221 8 Maintenan
8. The standard delivery of AKTAxpress includes four separation systems as shown in the illustration above The flask holder is optional ep13 2 Introduction 2 1 Introducing KTAxpress KTAxpress fea tures 18 1178 00 AA p14 AKTAxpress provides the following features A Method Wizard to make it easy to create method plans Automatic purification of up to four samples in a single run Purification protocols containing up to four chromatography steps Automated affinity tag removal in combination with any multi step purification protocol supported by KTAxpress Up to 50 mg of protein can be purified per sample Up to twelve KTAxpress system can be run in parallel controlled from one computer Different purification protocols can be run simultaneously The systems can be placed next to each other or they can be separated if preferred The separation system Introduction 2 AKTAxpress comprises a number of separation systems and a computer with monitor running the control and analysis software The separation system is a compact chromatography unit containing the components required for automated multi step purification processes The illustration below is a schematic drawing showing the components in the flow path outlet Lo valve 96 well plate waste A ANNY 0 2 metal ion 7 y waste 7 J flow not used through peaks Cond sample 1 4 sample 1 4 UV c
9. 18 1178 00 AA ep 2 0 Maintenance 8 Loosen the lower valve from the pump head using the 13 mm wrench CAUTION Handle the check valves with care when they have been re moved from the pump heads to prevent loss of any internal components Gently remove the check valve manifold and a larger diameter opening is fitted to the side marked IN of the pump head Tighten the valves until fully finger tight and then use the wrench to tighten a further 1 3rd 110 of a turn CAUTION Do not over tighten the valves since damage to the internal components can occur Refit the outlet tubing and the inlet manifold Reconnect the mains power cable Purge the pump carefully and check that the pumping action has been corrected see 6 5 3 Purging the pump and inlet tubing on page 156 lo j egm S Install the new check valves The inlet check valve with a lip for the e p271 8 Maintenance 8 6 Replacement procedures 8 6 6 Replacing pump piston seal 8 6 6 Replacing pump piston seal Introduction The pump piston seals should be replaced if e There are signs of liquid leakage between a pump head and the panel e The volume of the rinsing solution has increased or decreased CAUTION The pump head should only be disassembled by a trained person CAUTION Do not disassemble the pump head unless there is good reason to believe that there is an internal leakage Always make sure that sufficient spare compon
10. 600 sgg 400 300 200 100 0 100 200 300 400 Status indicator The table below shows how the indicator colors relate to the run status a colors a 18 1178 00 AA ep 146 Disconnecting a separation system Operation 6 Error indication When a warning or an alarm is issued from a system the background of the system symbol starts flashing and the background color turns yellow e Right click the symbol of the system to be disconnected and select Disconnect Result The system is disconnected and the symbol changes System Control S 5 1 Method Result File Yiew Manual System Help Hold Continue End SYS 1 3 Pressure gt gt UY gt gt Expand v Remaining time v Step in Method iv Acc Yolume ra Acc Time W Logbook Color Settings Customize Disconnect Changing system To display the status for the another separation system left click the system symbol display of the system on the left e p 147 6 Operation 6 4 Creating a method plan 6 4 Creating a method plan Introduction This section gives a brief description on how to use the Method Wizard to create a new method plan More information on creating a method plan can be found in 5 2 1 Creating a new method plan on page 89 Method plan types There are three main types of method plan available e Preparation of the system and columns e Purification of protein e Maintenance of the sys
11. Code 200 218 219 220 221 222 223 224 300 303 L H 402 403 af A 405 408 i T p N Troubleshooting and corrective actions 9 System error Internal error Restart system If problem remains contact service Instruction ignored The method instruction or manual command could not be executed Set last tube error An attempt was made to set the last tube to a position already used Set the last tube to a non used position System error Internal error Restart system If problem remains contact service Bad combination of valve positions The Injection valve is in position reinject and the Outlet valve is in position LoopFracF12 CAN bus error The communication with the instrument is broken Restart system If problem remains contact service System error Internal error Restart system If problem remains contact service System error Internal error Restart system If problem remains contact service Cell constant out of range The calculated conductivity cell constant is outside the accepted range Calibrate with a liquid of known conductivity in the range 0 1 300 mS cm Cond sine table not calibrated The conductivity sine table has not been calibrated Calibration of the conductivity sine table will start now and takes approximately 3 minutes to complete Conductivity cell error The cond cell is either not connected or broken System erro
12. Flushing the tubing manually Operation 6 Enter a Method plan name and click OK to save the method plan Method plar af ds iex prep col 2003 12 12 af ds prep ds Clean System af ds ad Number of cleaning Solutions 1 af gf tag cleay Ee GF Inlets to clean 51 Installation Test ODA m 52 Result A method plan for cleaning system is now ready to be run on a single or on several AKTAxpress separation systems To run the method plan for cleaning the system a Wash the outside of the inlet tubing with water and or ethanol Immerse the tubing ends to be used in the container with cleaning solution 1 see also the Summary page If the system will not be used for a longer time use a bacteriostatic solution e g 20 ethanol as the final wash solution 3 If the column valve is to be cleaned remove the columns and recon nect the tubing to the column block If the outlets are to be cleande insert a microplate Run the cleaning method plan as any other method plan The tubing can also be flushed using the following manual instructions not column or outlet tubing e PumpWash e SystemWash e LoopWash These instructions can be executed from the Pump Instructions dialog box by selecting Manual Pump in SystemControl They also appear in the method plans where they are automatically executed Note The system is always in Pause mode during Pump Wash Hence it is only possible to click END or CONTINUE
13. HiLoad_16 60_Superdex_75_prep_grade Global Folder select a folder in Method plan tree Method plan name Cancel I Create Folder Delete Help 18 1178 00 AA ep 106 9 3 Introduction Editing a method plan Method Wizard 5 Editing a method plan This section describes how to edit an existing method plan To edit a method plan In the Method plan list in the Method Wizard select the method plan to be edited Method Wizard Create or Change Method Plan Method plan Note 2004 01 07 Installation Test Punfication Protocol n a A Affinity Step Gel Filtration a AC DS IE GF Normal Affinity Column Sample Loading Level 1 GF T sC GF Running Condition Room Temperature repare ng HisTrap_HF_1_rml Global HiLoad_167 60_S5uperdes_r5_prep_grade Global 2 Click Next Result The Main Selections page appears The parameters values cannot be edited in this part of the Method Wizard Hain Selections Hain Selections Purity Man Selection Last Page Purity Click Next repeatedly to enter Advanced Zone Make the appropriate selections and edit the desired parameter values as described in 5 5 1 Advanced Zone introduction on page 110 and the subsequent sections Save the method plan as described in 5 2 5 Saving the method plan on page 106 Note The changes can be written in the Notes field in the Save As dialog ep 107 5 Method Wizard 5 4 Deleting a method plan 0 4 Delet
14. s instructions Wait for the computer to start up lt bers SI AES Ss SEE LISP I A ER Verify that the power indicator on the USB CAN converter is on when the computer has been turned on When the Windows desktop appears start UNICORN by double clicking on the UNICORN shortcut icon ak UNICORN logon User name defaut Result UNICORN starts with the UNICORN Manager on top Note See the UNICORN user documentation for instructions about how to create new users ep 145 6 Operation 6 3 Connecting a system 6 3 Connecting a system Introduction This section describes how to connect to a separation system from UNICORN Up to twelve separation systems can be connected in the System Control module Connecting a sep The separation systems that are available in UNICORN are shown in the vertical Brouronsyotem bar at the left hand side of System Control Disconnected systems are identified by a blue connector symbol SYS 1 v4 Note To add more systems in UNICORN see AKTAxpress Installation Guide e Left click the symbol of the system to be connected Result The system is connected and the symbol changes to a white status indicator The run data curves and logbook for the system is shown ds System Control SY 1 Method Result File iew Manual System Help Hold Continue End ES Instant Run E gt gt F gt gt Conc gt gt Cond gt ii A Li Conds Pressure
15. tion Protocols for AKT Axpress Methodology 4 Protocol descriptions and choice This section describes the different protocols that can be used with AKTAxpress and gives guidance on how to choose protocol for a purification The name of a protocol is an abbreviation of the combination of chromatography techniques used in the purification Example The protocol AC DS IEX includes purification with affinity chromatography followed by desalting and finally ion exchange chromatography Abbreviations found in the protocol names Abbrevi Chromatography technique ation Affinity chromatography For AKTAxpress there are seven protocols that can be used All of the automated protocols can be combined with automatic tag removal See sections 4 1 2 Description of affinity tags on page 59 to 4 1 5 Conditions for cleavage on page 64 for more information on affinity tags and removal of affinity tags Ce eee DS Note In GF and DS 1 step protocols the sample is loaded manually with a syringe before starting the run In automated multi step protocols the sample is automatically loaded via the system pump e p 55 4 Methodology 4 1 Protocols and affinity tag removal 4 1 1 Protocol descriptions and choice Consider when choosing protocol Guidance for choice of protocol 18 1178 00 AA ep 56 Questions to be considered before choosing a purification protocol What should the protein be used for after
16. 5 2 2 SING the Prepare ODT OM aister ene aaia 92 D225 USINE Me FUY ODON e aca tmnienabeusuchsccumesantenscenaaeanenuasemunen 96 OZ 4 USME Tne Mantaih ODUON asas TE 102 i249 SAVING he metod Dlane e T O a A 106 Doe POIUAS a Metod Plaissan e a A nE nat 107 D4 Deleting a method Plaisirs E EA 108 Jide AVANEN ZONE iioi aa a duaieeneeuneeunaenaredeneat 109 Dole Advanced Zone IMtroductONceneerinisreninii nn a a a OTELE 110 5 5 2 Adva nced Zone for PIED Ale iaicsicuanvemievessvidaadeeanehi tadaiagsabee a ania 111 Doo AGVANC OG Zone Tor PULT ariana a a EE E 115 5 5 4 Advanced Zone for Purify ATtinity cccccecseeeseeeeeteeteeeeeeeeseeateeeseeateatanes 120 5 5 5 Advanced Zone for Purify DESAI TING ics cautitate tahini oapninsiraeotitateaaaianteaduneiaeyas 125 5 5 6 Advanced Zone for Purify lon exXChange ccccceceeeeeeeeeeeeeeeeetseeseeteaeanes 128 5 5 7 Advanced Zone for Purify Gel filtration ccccceceeeeeeeeeeeeeeeeeeteetseeaneataees 132 5 5 8 Advanced Zone for Purify Prepare system and COIUMMNS cecceeeeeeeeaeeees 134 5 5 9 Advanced Zone for Purify General advanced SettingS ccccceeeeeeeeeeeeeeees 136 979 10 AGVanCed ZONe for WiGINTal NM sevinarcotewvessinioradvenat ei tacanvababoiusinerbetsduniueriexthin 138 5 5 11 Hints on optimizing a method plan cccecceseeeeceeeeeeeeeeteeeseegeeeeaeeaneatanes 140 6 OGRA OM estesct sie tecrtacaceteaulaieutauscemadiadetue van cena cid aun
17. B2 e p37 3 AKTAxpress overview 3 2 Separation system overview 3 2 4 Functional descript Pump and related items 18 1178 00 AA ep 38 ion Switch valves The switch valves are mainly used for gradient formation They have four buffer inlets two on each valve labelled A1 B1 A2 and B2 Inlet valve The flow from the switch valves continues to the inlet valve which provides six additional buffer inlets labelled A3 A8 The inlet valve also provides four sample inlet tubings labelled 1 S4 Injection valve A sample volume can also be injected manually into the flow path through the injection valve using a syringe The injection valve is located above the inlet valve Note Buffer containers sample tubes and other external hardware are not included at delivery Air sensor An air sensor is connected between the inlet valve and the pump In method plans the air sensor is activated by default and used for e Detecting when the sample supply is empty during sample loading The inlet valve then switches to a buffer inlet e Detecting when the buffer supply is empty during a run The system is then set to PAUSE mode The pump related items are located at the lower left hand side of the system Pressure sensor Mixer Pump heads Pump The pump is located at the lower left hand side of the separation system It is a low pulsation pump equipped with two pump heads which can deliver up to 65 ml min
18. Install Hardware Device Drivers A device driver is a software program that enables a hardware device to work with i an operating system This wizard upgrades drivers for the following hardware device Be Amersham URI45 Upgrading to a newer version of a device driver may add functionality toor improve the performance of this device What do you want the wizard to do Display alist of the known drivers for this device so that can choose a specific driver lt Back Cancel e Click Next e Select the option Specify a location Upgrade Device Driver Wizard Locate Driver Files Where do you want Windows to search for driver files Search for driver files for the following hardware device Be Amersham URJ45 The wizard searches for suitable drivers in its driver database on your computer and in any of the following optional search locations that you specify To start the search click Next Ifyou are searching on a floppy disk or CD ROM drive insert the floppy disk or CD before clicking Next Optional search locations CD ROM drives M Specify a location Microsoft Windows Update lt Back e Click Next e p 305 9 Troubleshooting and corrective actions 9 7 Checking USB CAN driver e Browse to the folder C Program Files KVASER Drivers and click OK Upgrade Device Driver W Locate Driver Files Where do you want Windows to search for driver files Insert the manufacturer s installation disk
19. Last Page Description related action Equilibrates the column s During the run the column s will be equilibrated with 5 column volumes CV of the appropriate buf fer except for the gel filtration column which will be equilibrated with 2 CV Note A more customized equilibration can be made using the Maintain option See 5 2 4 Using the Maintain option on page 102 Method Wizard 5 Description related action lon Exchange Blank Run Provides the ion exchanger with exchangeable counter ion exchange columns ions A blank run should be performed before first only time usage and after long term storage The column will be washed with e 5 CV of buffer A e 5 CV of buffer B e 10 CV of buffer A Metal lon Charge affin Metal ion charging of HiTrap Chelating HP and ity columns only HisTrap HP columns The inlet tubing has to be filled with metal ion solu tion before starting the run manually or within a method plan The column will be washed with e 5 CV deionized water e 1CV0 1M metal ion solution e 5 CV deionized water The metal waste will be collected through F11 Affinity Blank Run affin Avoids leakage of loosely bound metal ions during ity columns only the purification run A blank run should always be performed after metal ion charging or stripping of a column The column will be washed with e 5 CV of buffer A e 5 CV of buffer B e 10 CV of buffer A Finishing creating Con
20. ON Buffer inlet switch valve 2 port NC Buffer inlet 1500 Teflon inlet valve port 3 Buffer inlet 1500 Teflon inlet valve port 4 1500 Teflon ON Buffer inlet inlet valve port 5 1500 Teflon ON Buffer inlet inlet valve port 6 1500 Teflon ON Buffer inlet inlet valve port 7 1500 Teflon ON Buffer inlet inlet valve port Teflon ON Sample inlet 500 inlet valve port 12 e p25 8 5 8 Maintenance 8 6 Replacement procedures 8 6 2 Replacing tubing and connectors Material Inner diamet Label bold Description Length mm or designa tion 2 yellow Sample inlet 500 inlet valve port 11 S3 blue Sample inlet 500 inlet valve port 10 S4 green Sample inlet 500 inlet valve port 9 Switch valve 350 1 port IN to inlet valve port 1 Switch valve 350 2 port IN to inlet valve port 2 er mm Teflon ON Teflon ON Teflon ON Teflon ON Teflon Inlet valve 190 Teflon port IN to air sensor Pump Air sensor to pump mani fold Mix1 Pump head 2 to mixer Mix2 Pump head 1 to mixer Press Mixer to pressure sensor MixInj Pressure 260 sensor to in jection valve port 5 18 1178 00 AA ep 256 230 Teflon ON ON Tefzel Tefzel 120 120 160 Tefzel Tefzel ON Maintenance 8 Material Inner diamet Label bold Description L
21. Pump FlowR ate 0 00 65 00 inset C Flowpath 15 mimin Dette Frac Mode Alarms amp Mon Buffer C Sample Cie Other MinFl 0 01 65 00 INF iow H 4 Help 0 10 mimin men FlowContr No Yes Auto update If this is checked the parameter fields will be updated during method run e Select an option in the Instructions field and an instruction e Select parameter or set a suitable value in the Parameters field and click Execute to start the instruction For information about the instructions in a dialog box click Help Each system has a set of default system settings which can be changed e To change a system setting select System Settings in System Control The system must be in End mode Make the appropriate changes on parameter values and click OK S S_3 Monitors Instructions xi m Instructions AirSensor Parameters Averaginal imeLY Mode L ania AyvTimeU Normal aE SetCondScale0 Cond alue 0 00 mS cm SetCondScale1 00 Cond alue 100 00 mS cm Specials Monitors Curves Set Selected Parameter To Strategy Default Value Cancel Help 18 1178 00 AA ep 192 6 7 Overview Viewing the run Operation 6 During a run This section describes how to view the progress of the run and change parameters during a run The progress of the run can be viewed in detail in System Control By clicking the s
22. Sample 21D System 2 Sample 3 Sample 31D System 2 Sample 4 Sample 410 System 2 18 1178 00 AA ep 188 Operation 6 6 For each sample e Enter the isoelectric point in pl the extinction coefficient for the protein in Ext Coeff and the molecular weight of the protein in MW The extinction coefficient is the absorbance of 1 mg ml solution of the purified protein at 280 nm in a 1 cm cuvette Note If an import file exists containing these data for the proteins to be used the fields might automatically be filled The path to the import file folder must be spe cified in General Advanced Settings in Advanced Zone in the Method Wizard in Method Editor For more in formation on the import file see 10 3 Import file format on page 331 The import of data is done only once each time this wizard is used To perform a new import of data click Set Default on the first page or click cancel and then Instant Run See also 5 5 9 Advanced Zone for Purify General advanced set tings on page 136 Enter optional text for example running data in the text box It will appear in the pool table and in the result file in Documenta tion Notes Method Notes in Evaluation Method Wizard S xj Settings SYS 1 Systems Samples SamplelD System 1 4 Settings SYS 1 pl Settings SYS 2 Ext Coeff corr to Settings SYS 3 ht Settings SYS 4 Result Hame Summary pl Ext Coet corr to hat Free Text Sample 1 Free Text Sample 2 Free Test Sample
23. Summary Column position 1 Loop position S75 1 5 8 hour max Yes Volume needed max 364 ml O ml 104 ral tif ml O mil O ml 69 ml O mil O ml O mil O ml O mil O ral O ml Solution Buffer Affinity 4 Buffer Affinity B Buffer Affinity Regene Desalting Buffer Protease Cleavage Buffer Column HisTrap_HP_1_ ml Global His Trap HP_1_ rm Global HisTrap_HF_1_ ml Global Type of column Affinity Affinity Affinity Desalting HiPrep_26 10_Desalting Type of loop Capillary Loop Cerise Daa Click Run to start the run on the selected systems Result The systems will enter Pause mode Make a final check of the system setup using the list from the Sum mary page Click Continue or Continue all in System Control or press the Continue button on the system to continue the method ep 191 6 Operation 6 6 Starting a run 6 6 2 Starting a manual run 6 6 2 Introduction Manual control System settings Starting a manual run This section describes how to run a system manually For more information see the UNICORN user documentation Besides running method plans on the systems it is also possible to control the system manually for example starting and stopping the pump switching valve positions etc Follow the steps below to control the system manually 1 Select Manual Pump in System Control A dialog box containing manual instructions appear Instructions Parameters
24. gassed buffers for best performance Dirt or residues in the flow path from previous run Clean the system using a method plan for System Cleaning see 6 8 2 Cleaning system on page 198 Residues in the columns from previous run Replace or clean the columns using a method plan for Column cleaning see 6 8 3 Cleaning columns on page 202 Troubleshooting and corrective actions 9 Possible cause and action Noisy signal Locking nut in optical unit not properly tightened Turn the locking nut to the stop position See also 8 6 4 Replacing UV lamp on page 264 Air in buffers Check that there is no air in the buffers Degas the buffers if necessary The buffers might be impure Make sure the buffers have been filtered Air in the UV flow cell There may be air in the flow cell Check that the flow restrictor gives a back pressure of 0 2 MPa see 8 2 1 Checking flow restrictor on page 223 Dirty UV cell Clean the UV cell see 8 3 2 Cleaning UV cell on page 228 Air might be trapped in the pump Purge the pump according to the procedure in 6 5 3 Purging the pump and inlet tubing on page 156 Signal drift See Noisy signal above Instability See Noisy signal above Low sensitivity e Aging UV lamp Check the lamp run time see 8 3 1 Checking UV lamp run time on page 227 and replace it if neces sary see 8 6 4 Replacing UV lamp on page 264 UV lamp in wrong position Check that the lamp position and the filter
25. to seal and hold the pressure To solve the problem perform e Inplace cleaning If the problem remains continue with e Offline cleaning Performing in To perform inplace cleaning of the check valves place cleaning Note It is recommended to use degassed buffers and solutions Pump deionized water at a flow rate of 40 ml min for 2 minutes Change solvent to deionized water and flush the system If this does not correct the problem follow the instructions for per forming offline cleaning 2 Change solvent to 100 methanol and pump at a flow rate of 40 ml min for 4 minutes Performing offline To perform offline cleaning of the check valves cleaning CAUTION Check valves have precision matched components and should only be disas sembled by a trained person If the problem cannot be corrected the check valve should be replaced completely Required tools and material e 13 mm wrench e 18 mm wrench e Ultrasonic bath e 100 methanol 18 1178 00 AA ep 234 Maintenance 8 Change solvent to deionized water and flush out all used buffers solu tions Move all input buffers bottles below the level of the pump heads to prevent siphoning Disconnect electrical power from the system Remove the tubing from the pump heads Loosen the upper valve from the pump head using the 18 mm wrench Gently remove the check valve CAUTION Handle the check valves with care when they have been re moved f
26. 0 00 65 00 Insert Flowpath Frac C Alarms amp Mon Other 20 aa ml min Mode Buffer C Sample MinFlow 0 10 FlowContr 0 01 65 00 al aa ml min e No C Yes I Auto update If this is checked the parameter fields will be updated during method run e Click Execute to start the flow Select a flow path matching the tubing to be checked for example e InletValve and A1 e OQutletValve and F12 e LoopSelection and LP1 Instructions a m Parameters i C Pump Injection alve Position Insert ColumnPosition E tee Flowpath OutletValve Inlet alve Frac C Other C Alarms amp Mon ca te 7 Auto update If this is checked the parameter fields will be updated during method run e Click Execute to set the valves into position Inspect the UV curve for abnormal peaks see below 18 1178 00 AA ep 238 Maintenance 8 Abnormal pres Below is an examples of abnormal UV curve due to leaking connectors causing air sure curves bubbles in the tubing mAL 24 0 220 20 0 13 0 ep 239 8 Maintenance 8 4 Maintenance when required 8 4 5 Running installation test 8 4 5 Running installation test Introduction To check the function of the separation system an installation test can be performed on all separation systems e g after a prolonged stop The test can be started to run simultaneously on all systems if
27. 199 6 Operation 6 8 Procedures after a run 6 8 2 Cleaning system Method Wizard Ea Clean System Main Selections Maintain Sample Inlets Clean System Iv S1 Last Page Buffer Inlets Iv Al IV A2 AS IV AG Iv B1 Iv B2 Inlet 41 will be used for the options below V Clean Outlets Insert a Microplate MV Clean Loop 1 4 Clean Loop 5 Must be a capillary loop Clean Column Valve All Column Valve positions need bypass tubing C Select All C Deselect All Number of Cleaning Solutions 1 z Note The system will Pause during cleaning 4 message will appear when a new cleaning solution is needed Sample Inlets The sample inlets to be cleaned Buffer Inlets The buffer inlets to be cleaned Clean Outlets The outlets will be cleaned from inlet A1 a microplate must be inserted Clean Loop 1 4 The capillary loops to be cleaned from inlet A1 If this option is selected the option to Clean Loop 5 will be displayed Clean Column valve The column inlet tubing will be cleaned from inlet A1 Note Check that all column valve positions have bypass tubing Number of Cleaning Solutions Up to five different cleaning solutions can be used The tubing chosen in the method plan will be cleaned with one cleaning solution at the time The system will pause and a message appear when the inlets shall be inserted into a new cleaning solution Click Next and Finish 18 1178 00 AA ep 200 Cleaning the sys tem
28. 288 Troubleshooting and corrective actions 9 No satisfactory elution Possible cause and action from Gel filtration column e Check that the eluted peak from the previous step was collected in a capillary loop by inspecting the peak start and peak end markers in the chromato gram If not collected Open the used method plan and enter the Ad vanced Zone Change the watch parameters used in peak collection and run the sample again If the separation is not good enough change to a more suitable gel filtration column or divide the start sample into aliquots Perform repetitive or parallel purifications where less protein is applied in each run For manual gel filtration protocols it is only recom mended to load up to 5 ml sample per samle loop ep 289 9 Troubleshooting and corrective actions 9 3 Monitoring problems 9 3 Monitoring problems Introduction This section specifies troubleshooting for monitoring problems during a run The monitoring problems can be related to e UV curve e Conductivity curve e Pressure curve e Air sensor Faulty UV curve Find the possible cause and action for a specific UV curve fault in the table below If the problem remains contact the local Amersham Biosciences representative Ghost peaks 18 1178 00 AA ep 290 Possible cause and action e Air in buffers Check that there is no air in the buffers Degas the buffers if necessary It is recommened to use de
29. 3 SIX monthly MAINTENANCE ccceeeceeeeeeeeeeeeeeeeeeeeeeeeeeeneeuseeuseeeaeeenseeseesesuaeeseeeaeenaees 226 Oro le CNECKING UV lamp run TING nssins enatadcomaiasonianwneainntedasainamaibie bediabontaumeenhoutedahaen 22 Zo Gleanna UN TENera nites Seemed add N 228 6 5 5 Clednine CONGUCTIVITY CClliccaitatccewsainaulancmaddewaiamateabnidaiaamiamndangma darian raanece 231 8 4 Maintenance when required cccsccesecceeeccseeceeeeeeeceseeseeeeeeenseceeeneseeaeenaeenaesnsesaes 232 8 4 1 Externally cleaning the SYSt M ccccceceeseseeeeeteeeeeeteeesteateaeateatsaeaneateaeass 233 o4 Cleaning CHECK VaVe Sadice aia tenn 234 oA Checkine PUMO LESS Uessa na a EE arb hete latnanionts tes aaetaniteradaatornt 237 8 4 4 CHECKING tubing leakage sirsiran tiie E eei 238 SA Da RUNDINE MStalatiom TES sern EE 240 8 9 Calibration DrocedUre S cicsisninis ia a 241 oo yao mae Calibration Or Zero Presse sirara a E 242 epd Table of Contents 0 0 22 CallOraulon On CONGUCTIVITY Celh ecwctcsctusnattacadmenisee aaa lumenal el 243 8 6 Replacement DIOCCOUICS jiceictacientivedniudincheesiawntenaseeesdvesievatenteunddadiensd teedesanssieasaseieeeds 246 SOx lhe INMEDIACING Capillary OOD S snee a 247 8 6 2 Replacing tubing and connectorS ssesssssssssrrrsrrrrrnrrrrrnrrrrrnrrrrenrrrrerrrrrere 249 8 0 3 REDIACING TOW TeStriCTO sesten er ernea a eeina 262 Oi OAs Repa mne UV IAIN D e O 264 3 62 REDI ME CNECK Valve Saser AEE 269 8 60 6 Replaci
30. EMC EN 61326 and EN 55011 CISPR 11 Group I Class A LVD EN IEC 61010 1 UL 61010A 1 2003 and CAN CSA C22 2 No 10101 1 92 Column holders Column holder data of AKTAxpress Column block capacity Max 5 small columns Column holders 2 left hand side and 2 right hand side designed for 30 mm waist columns Microplate spe The microplates used in AKTAxpress must comply with the data cifications Parameter Data Type Deep well plate 96 wells height 45 mm Well shape Square wells 18 1178 00 AA ep 324 Reference information 10 10 2 2 Components specifications Introduction This section specifies the operating data of the components in AKTAxpress For general data of the system see 10 2 1 System specifications on page 323 Pump data Pump data Flow rate accuracy 2 or 0 02 ml min whichever is greater Flow reproducibility RSD lt 0 5 Mixer data Mixer data Parameter Internal volume Flow restrictor Flow restrictor data data UV monitor data UV monitor data Wavelengths 280 and 254 nm set by lamp position and filter Optical path length Optical cell volume Detector cell volume Absorbance range 0 01 to 5 AU p 325 10 Reference information 10 2 Technical specifications 10 2 2 Components specifications Linearity deviation Static noise short and long term Hg lamp lifetime at 254 nm Lifetime in room temperature 7000 hours in cold room 2000 hours
31. Maintain Prepare Last Page Purify Select Maintain and click Next 18 1178 00 AA ep 202 Operation 6 Select one of the two cleaning in place methods e Regular The standard CIP procedure for the chosen columns two solutions per column The solutions and inlets are specified on the Summary page in the wizard in System Control when starting the run They are also described in the dialog Help Rigorous Customized Equilibration A method plan that gives a range of options to design a protocol for cleaning or equilibration with up to nine cleaning solutions Equilibration is recommended for removing ethanol from new columns Click Next Maintain Main Selections Hd aintain Installation Test CIP Columns Regular Strip Affinity Columns Last Page f CIP Columns Regular CIF Columns Rigorous 4 Customized E quilibration Clean System ep 203 6 Operation 6 8 Procedures after a run 6 8 3 Cleaning columns Select suitable cleaning options and click Next Also refer to the User Instruction supplied with each column for column cleaning proced ures and column storage instructions Regular CIP Columns Regular Main Selections Note Max 5 columns allowed Maintain CIP Columns Regulz Running Condition Last Page Room Temperature Cold Room IV Wash Columns with Water Before CIP Inlet 45 I Affinity HisTrap_HP_1_ml Global v Number of Affinity Columns 1 z IV Desa
32. Performing auto matic affinity tag removal with AKT Axpress Methodology 4 The image below shows the different stages on the affinity column The numbers indicate the steps described in the table above Tag cleavage using AKTAxpress 2 4 5 6 7 8 9 Load sample Inject protease Incubate Elution Affinity and wash from Superloop cleaved protein regeneration Cleaved Affinity tag protein tagged protease amp non cleaved protein Loop Outlet F7 F10 See 6 5 8 Performing automatical affinity tag removal on page 175 for instructions on how to perform automatic affinity tag removal on KTAxpress e p63 4 Methodology 4 1 Protocols and affinity tag removal 4 1 5 Conditions for affinity tag cleavage 4 1 9 Introduction Conditions affect ing cleavage Cleavage time Raising cleavage temperature Buffer suggestions Further informa tion 18 1178 00 AA ep 64 Conditions for affinity tag cleavage This section describes conditions important for affinity tag cleavage Protease protein ratio buffer composition time and temperature are all important factors for efficient cleavage All these conditions can be optimized to fit the specific protein Different proteases need different amounts of time for cleavage If cleavage is not performed at the protease s optimal temperature the cleavage time will be longer Note Try to keep the glycerol concentration low in the protease solution since high glycerol c
33. The volume used for equilibrating the affinity column wo OO Om AA Fe w MM lt k Enable NaOH Wash Between Samples To wash the pump mixer and injection valve with NaOH inlet A6 followed by affinity buffer A between the sample loadings 1 st Wash Volume The volume of affinity buffer A used for washing out unbound sample Enable Watch Stable Baseline To check the stability of the UV signal during a specified time interval The Stable Baseline condition is met when the signal fluctuates less than the set Delta Base value during the set Stable Time interval If Watch Stable Baseline is enabled the first wash step will be completed when the baseline is stable Elution Volume The volume used for elution of the affinity column The second wash is a harsher wash used to remove contaminants loosely bound protein residues etc from the affinity column The volume options and parameters are described below Method Wizard Affinity 2 nd Wash Advanced one Page M Define Wash Concentration B 1 Concentration 0 D 100 28 2 2nd Wash Volume fi U D 999999 Cy Define Wash Concentration B To wash out contaminants from the column s Choose Concentration of B to use in the wash step and the 2 nd Wash volume Note The default setting for the second wash is 0 B which means that affinity buffer A is used e p 121 5 Method Wizard 5 5 Advanced Zone 5 5 4 Advanced Zone for Purify Affinity Peak collecti
34. Use in DS step HiPrep Desalting Note Sample loading volume is limited to the loop volume i e 10 ml Choosing columns Recommended column choice when running a GF one step protocol with manual for GF sample loading Which My has the target protein gt 7O kDa Use in GF step HiLoad 16 60 Superdex 200 pg Note Maximum sample loading volume is 5 ml 18 1178 00 AA ep 76 Methodology 4 4 3 Buffer alternatives About this section This section contains general descriptions of different buffers and gives guidance on how to choose buffers for a protocol See also buffer suggestions in the column instructions In this section This section contains the following sub sections Topic Description of buffers Buffer suggestions ep 4 Methodology 4 3 Buffer alternatives 4 3 1 Description of buffers 4 3 1 Description of buffers Introduction This section gives general guidance on how to choose buffers for different chromatography steps See also buffer suggestions in the column instructions Affinity chromato There are 3 steps in the affinity chromatography binding wash and elution In graphy buffers addition to these steps affinity tag removal can also be performed on the AC column e Binding buffer The buffer for binding has to match the binding properties of the used affinity column e Extra wash buffer second wash The extra wash is used to wash out non specifically bound impurities that d
35. V Peak Collection Desalting Intermediate l Flow Rate Pressure 7 Volumes J Peak Injection Peak Collection lon Exchange Intermediate IV Flow Rate Pressure I Volumes Wash Conc B l Gradient Peak Injection Peak Collection Gel Filtration IV Flow Rate Pressure Volumes Peak Injection Peak Fractionation MV Prepare System Prepare Column V General Advanced Settings C Select All C Deselect All Cancel Help Click Next to go through the subsequent pages On Last Page click Next to save the method plan See 5 2 5 Saving the method plan on page 106 e p115 5 Method Wizard 5 5 Advanced Zone 5 5 3 Advanced Zone for Purify Changing Purify The pages appearing in the Advanced Zone corresponds to the selected options on Lerares Page 1 The following sections describe all options and parameters available for each of the chromatography techniques respectively The options related to preparing the system and columns and General Advanced Settings are described in separate sections See the table below For parameters regarding See affinity 5 5 4 Advanced Zone Purify AC on page 120 5 5 5 Advanced Zone Purify DS on page 125 desalting 5 5 6 Advanced Zone Purify IEX on page 128 ion exchange System on page 134 5 5 8 Advanced Zone Purify Prepare Colums on page 134 preparing the columns 5 5 9 Advanced Zone Purify General Adv Settings on page 136 general adva
36. Watch Stable Baseline is enabled the first wash step will be completed when the baseline is stable Elution Volume Affinity regeneration The volume of affinity elution buffer used to regenerate the affinity column i e elution of tags tagged protease and uncleaved protein 2 nd Wash parameters Affinity 2 nd Wash 7 Elution Cleaved Protein Advanced Zone V Define Wash Concentration B Before Protease Injection Page Wash Concentration fo 0 100 B 1 Wash Volume fio 0 999999 CV 2 3 V Define Elution Concentration B 4 Concentration fo 0 100 B 5 Elution Volume Cleaved Protein fio 0 999999 CV Define Wash Concentration B Before Protease Injection To wash out the unbound sample from the column s before the protease is injected onto the column s Choose Wash Concentration B in the wash solution and the Wash volume Define Elution Concentration B Choose Concentration B used during the elution of the cleaved protein Elution Volume Cleaved Protein is the buffer volume used For more information on tag cleavage see 4 1 Protocols and affinity tag removal on page 54 18 1178 00 AA p 124 9 9 9 Introduction Flow rate Pres sure Volumes parameters Peak injection parameters Method Wizard 5 Advanced Zone for Purify Desalting This section describes the parameters used in the desalting step for the protocols B F Some of the default parameter values depend on the chosen column
37. a 0 010 50000 mL min Injection Flush Volume The extra volume besides the peak volume used to flush the loops when loading the sample onto the ion exchange column e p 129 5 Method Wizard 5 5 Advanced Zone 5 5 6 Advanced Zone for Purify lon exchange Peak fractionation The peak fractionation options and parameters for protocol C are described below The parameters define the final fractionation Peak Fraction Size The maximum volume of each peak fraction Peak Fractionation Algorithm Choose to start and stop the peak fractionation based on the signal curve e Level e Slope e Level _AND Slope e Level_OR_ Slope AND means that both conditions have to be met OR means that only one condition has to be met The fractionation start values are set by Start Level and Start Slope and the stop values by Stop Level and Stop Slope Different start and stop values for level and slope can be set The Stop Slope condition cannot be met unless the Peak_Max condition is met Stop Level can be met before Peak_Max The Peak_Max condition occurs when the UV signal has fallen to a fraction specified by Peak Max factor of the most recent peak maximum Note If the flow rate is changed the shape of the peak will change as well Hence the slope values might need to be changed accordingly Minimum Peak Width sets the minimum fractionation time of a single peak The fractionation of the current peak continues at least until the Minim
38. a run using a method plan for metal ion charging The table below describes how to start a run using the method plan See also the standard procedure described in 6 6 1 Starting a run using a method plan on page 186 Click Instant Run in System Control Result The Method Wizard dialog box appears 1 Select the required method plan in the Method plan field and click Next e p 167 6 Operation 6 5 Preparing the system for a run 6 5 5 Conditioning columns On each new page select the appropriate system and type the reques ted information Click Next to continue not be saved Click Run on the last page Click Continue to start the run On the Summary page check that the tubing and liquid supply fulfills the requirements listed on the page Note It is recommended to print the Summary page since it will Blank run on affin Before the first use of an affinity column GSTrap FF or newly charged HiTrap ity columns Chelating or HisTrap a blank run should be performed This results in a well conditioned and equilibrated column During the procedure each column is flushed with 5 CV of affinity buffer A 5 CV of buffer B and finally with 10 CV of buffer A Creating a method plan for an affinity blank run The procedure for creating a method plan for an affinity blank run is the same as the metal ion charging procedure except for the following changes on the Prepare Columns page e Click Affinity Blank Run Iv Affi
39. accordingly Minimum Peak Width sets the minimum fractionation time of a single peak The fractionation of the current peak continues at least until the Minimum Peak Width is reached even if the signal is fluctuating For more information on peak handling and watch conditions see 5 5 3 Peak handling and watch conditions on page 116 ep 133 5 Method Wizard 5 5 Advanced Zone 5 5 8 Advanced Zone for Purify Prepare system and columns 9 9 8 Advanced Zone for Purify Prepare system and columns Introduction This section describes the parameters used in the system and column preparation steps before a purification run Prepare system The Prepare System page contains options and parameters related to system eae preparation before and after the sample loading Method Wizard Prepare System Page Sample Inlet Fill Yolume 0 999999 ml 1 Buffer Inlet Fill Volume fi 5 0 999999 ml 2 3 Volume Needed to Remove Air 4 20 0 999999 mi Sample Inlet Fill Volume Volume used for filling the sample inlet tubing with buffer before immersing the tubing in the samples Note The system will Pause during the run and a message appear requesting the inlet tubing to be moved from the buffers to the samples Sample Inlet Clean Volume Volume used for cleaning the sample inlet tubing after loading the sample onto the affinity columns The system will Pause during the run when all samples are loaded onto the columns and a m
40. affected by long term use Avoid Avoid ETFE PP PE and PVDF are af fected by long term use wo OK Avoid Silicone not resist ant Pressure limit for PEEK decreases OK OK Silicone not resist ant OK Avoid Silicone not resist ant Pressure limit for PEEK decreases OK OK Silicone not resist ant ep 333 10 Reference information 10 4 Chemical resistance guide Exposure lt 1 day Exposure up to 2 Remarks months Chemical Hydrochloric acid gt 0 1 M Silicone not resist ant Avoid Titanium is af fected by long term use Isopropanol OK Methanol OK Avoid Silicone not resist ant Nitric acid di luted Nitric acid 30 Avoid Avoid Elgiloy is affected by long term use Phosphoric acid Avoid 10 Titanium alu minium oxide and glass are af fected by long term use Potassium carbon OK ate Potassium chlor OK ETFE PP and PE not resistant Sodium acetate Sodium bicarbon ate O O O Sodium borate O Sodium carbon O ate Sodium bisul phate OK OK OK OK OK OK OK OK OK OK OK OK K K K K K K K Sodium chloride O 18 1178 00 AA ep 334 Reference information 10 Chemical Exposure lt 1 day Exposure up to 2 Remarks months Avoid PVDF and boro silicate glass are affected by long term use OK OK OK K K K K K Sodium hydrox ide 2 M Avoid PEEK and titani um are affected by long term us
41. and PAUSE flashing The ee display alternates between the error code and the system unit number Run Lamp test All indicators are lit for 3 PAUSE seconds during the starting up se quence RUN Program update A dialog is also shown PAUSE on the UNICORN computer during the program update ree control from The separation system is mainly controlled and monitored by UNICORN A a communication converter box is connected between the USB port in the computer and the UniNet port in the separation system as communication interface UNICORN can control up to twelve separation systems simultaneously More information about controlling the separation system from UNICORN is found in 6 Operation on page 142 e p 45 3 AKTAxpress overview 3 2 Separation system overview 3 2 6 Labels 3 2 6 Labels Introduction This section describes the labels on the separation systems For information about the computer equipment refer to the manufacturer s instructions Labels Labels for safety product no CE C tick and rating are located on the rear panel of the separation systems A system no label is located on the left front rail The safety label warns the user of risk for personal injury Do not proceed until the instructions are clearly understood and all stated conditions are met CE Code No A 18 6645 01 18 1178 00 AA ep 46 AKTAxpress overview 3 3 3 Purification process overview About this s
42. and inlet tubing 6 5 3 Purging the pump and inlet tubing Overview Before starting a run all inlet tubing that will be used must be filled to remove air bubbles In addition both pump heads must be purged with methanol if the system has been left unused for a week or longer or the pump has been run dry CAUTION To protect the piston seals in the pump the pump must never be run with air in the inlet tubing Filling inlet tubing To fill the inlet tubing A1 A8 and 1 S4 A1 A8 and 1 S4 Check that the inlet tubing to be filled is properly immersed in the correct containers flasks tubes 2 Connect a male Luer syringe of at least 20 ml to the left purge valve Two syringes are included in the accessory kit supplied with the system 3 S JANN O ta 18 1178 00 AA ep 156 Filling the inlet tubing B1 and B2 Operation 6 Slowly draw buffer A1 with the syringe When fluid starts entering the syringe close the purge valve Check that there is no visible air left in the Al tubing Note If air in the other pump head is suspected draw buffer A1 through that pump head as well If required remove the syringe empty it and insert it in the purge valve again before continuing To fill the A2 inlet tubing or any other inlet tubing to be used first switch the valves e Start UNICORN and select System Control Manual Flowpath e Select InletValve and A2 or whichever inlet tubing to be filled e Click Execu
43. be shortened if required For a specification of the tubing see 8 6 2 Specification of system tubing on page 260 The cutting tool is included in the accessory kit Put the old labels on the new tubing 18 1178 00 AA ep 250 Maintenance 8 6 Mount the connectors on the tubing For fingertight connectors 1 Slide the nipple onto the tubing For M6 and UNF 5 16 connectors 1 Slide the nipple onto the tubing 2 Slide the ferrule onto the tubing with the thick end towards the end of the tubing e p 251 8 Maintenance 8 6 Replacement procedures 8 6 2 Replacing tubing and connectors 7 Attach the new tubing to the system e Insert the tubing with nipple into the port Make sure to insert the tubing all the way into the bottom of the port before tighten the connector NOTE Insert the tubing fully to the bottom of the port Ferrule NOTE Insert the tubing fully to the bottom of the port e Tighten the connector properly For areas difficult to access use the fingertight key available in the accessory kit Fingertight connectors should be tighten by fingers only but for areas difficult to access use the special key available in the accessory kit M6 and 5 16 should be tighten using the special key for M6 8 Press the On button to reconnect the system to UNICORN 18 1178 00 AA ep 252 Maintenance 8 9 Verify the new tubing by e Running a flow through the replaced capillary tubing and che
44. buffer cri using HisTrap 50 mM Tris HCl pH 7 5 0 5 M NaCl 40 mM im idazole binding using HiTrap 50 mM Tris HCl pH 7 5 0 5 M NaCl 20 mM im Chelating idazole extra wash second 50 mM Tris HCl pH 7 5 0 5 M NaCl 100 mM im wash idazole Note x of the extra wash buffer is added to the binding buffer This value can be changed in the Advanced Zone See 5 5 4 Advanced Zone for Purify Affinity on page 120 for details Example If using a binding buffer with 20 mM im idazole and 20 of extra wash buffer 100 mM im idazole the total concentration of imidazole during the extra wash will be 40 mM cleavage using TEV 50 mM Tris HCl pH 7 5 0 5 M NaCl 50 mM im protease on HisTrap idazole HP or HiTrap Chelat Note ing HP The imidazole concentration might need to be optimized especially if a different His tagged protease than TEV is used step elution 50 mM Tris HCl pH 7 5 0 5 M NaCl 500 mM im idazole In the affinity tag removal protocols the affinity columns can be equilibrated with either AC binding wash buffer extra wash buffer or an alternative cleavage buffer before protease injection AC buffer sugges tions for GST tagged proteins DS buffer sugges tions IEX buffer sugges tions Methodology 4 Suggested buffers for AC runs with GST tagged proteins When performing suggested buffer binding using GSTrap 50mM Tris HCl pH 7 5 150 mM NaCl 1 mM DTT HP or FF cleavage using 5
45. collector Capillary loops Column block pom Sea Control panel Column valve Loop valve UV optical unit al UV cell Column holder lt mh lt L apy _ gt lt if Conductivity cell ean Pressure sensor Mixer Inlet valve Pump Air sensor Electrical connect Connectors for power and communication are located on the rear of the system ors UniNet network Conductivity flowcell UV monitor optical unit Air sensor AC power inlet UV monitor lamp 18 1178 00 AA ep 28 Location of columns AKTAxpress overview 3 The columns can be attached to AKTAxpress in different places depending on the size of the column Small columns are connected to the column block through different unions See 6 5 4 Connecting columns and tubing on page 163for information about the unions The block has five ports In some applic ations it is possible to use two columns connected in series Column type EEI ll mL amp sf a lt K f adel a Z adel ae zdal no a a ret Q eo KTA x A Amersham Biosciences e p29 3 AKTAxpress overview 3 2 Separation system overview 3 2 1 Location of the components Large columns 30 mm Large columns are attached by using the left hand waist diameter column holder with snap locks The holder can hold two columns j gt i A A 18 1178 00 AA ep 30 AKTAxpress overview 3 Superloo
46. d 1 6 mm o d 1 8 3 18 1121 16 Desalt tubing Tefzel tubing i d 1 6 11 0004 04 mm o d 1 16 0 35 m Columns The following columns are available Affinity chromatography columns HisTrap HP 5 x 1 ml 17 5247 01 HisTrap HP 100 x 1 ml 17 5247 05 HisTrap HP 5 x 5 ml HisTrap HP 100 x 5 ml HiTrap Chelating HP 5 x 1 ml HiTrap Chelating HP 1 x 5 ml ep 339 10O Reference information 10 5 Ordering information GSTrap HP 100 x 5 ml 17 5281 05 GSTrap FF 5 x 1 ml 17 5130 01 GSTrap FF 2 x 1 ml 17 5130 02 GSTrap FF 1 x 5 ml 17 5131 01 Desalting columns Ion exchange columns anion IEX Ion exchange columns cation IEX RESOURCE S 1 x 1 ml 17 1178 01 RESOURCE S 1 x 6 ml 17 1180 01 HiTrap SP HP 5 x 1 ml 17 1151 01 Mono S 5 50 GL 1 x 1 ml 17 5168 01 18 1178 00 AA ep 340 Reference information 10 Gel filtration HiLoad 16 60 Superdex 75 prep 17 1068 01 grade 1 x 120 ml HiLoad 16 60 Superdex 200 prep 17 1069 01 grade 1 x 120 ml ep 341 18 1178 00 AA ep 342 A Accessories 336 Adjust pools 214 Advanced zone introduction 110 prepare system 112 prepare columns 113 define a folder for storing the results 114 introduction 115 changing parameters 116 peak handling 116 watch conditions 116 peak collection 117 changing parameters affinity 120 changing parameters tag cleavage 123 changing parameters desalting 125 changing parameters ion excha
47. deionized water Make a calibration of the conductivity monitor see 8 5 2 Calibration of conductivity cell on page 243 e p 231 8 Maintenance 8 4 Maintenance when required 8 4 Maintenance when required About this section This section describes the maintenance activities to be made when required In this section This section contains the following sub sections Topic Externally cleaning the system Cleaning check valves Checking pump pressure Checking tubing leakage Running installation test 18 1178 00 AA ep 232 Maintenance 8 8 4 1 Externally cleaning the system Maintenance inter When required val Reason for main The system should be kept dry and clean to ensure proper functioning of the system tenance Chemical stains and dust should be removed Actions To clean the system follow the procedure below 1 Check that no active run is in progress on the system The run indic ator should be off a Disconnect the mains power cable To remove stains spray the system with 20 ethanol and wipe off the excess Note Be careful not to bend any tubing ep 233 8 Maintenance 8 4 Maintenance when required 8 4 2 Cleaning check valves 8 4 2 Cleaning check valves Introduction Faulty operation of the check valves is usually indicated by e irregular flow e very low flow e unstable pressure traces Probable causes of this are air or dirt in a check valve preventing it from closing
48. fields will be updated during method run Connect a male Luer syringe of at least 20 ml to the left purge Turn the left purge valve counter clockwise half a turn to open it Slowly draw eluent B1 with the syringe When the B1 tubing is full up to the pump head and eluent starts entering the syringe close the purge valve Check that there is no visible air left in the B1 tubing If required remove the syringe empty it and insert it in the purge valve again before continuing 18 1178 00 AA ep 158 Operation 6 To fill the B2 inlet tubing first switch the inlet valve Select System Control Manual Pump Select Gradient and Target O B and Mode A1 B1 Click Execute Select Gradient and Target 100 B and Mode A2 B2 Click Execute to set the valve to position B2 1 Pump Instructions m Instructions Pump C Flowpath C Frac C Alarms amp Mon C Other Parameters Target 100 Length 0 00 Mode 0 0 100 0 E zB 0 00 9999 99 E min I Auto update If this is checked the parameter fields will be updated during method run A1 B1 A2 B2 a Repeat step 5 7 to fill inlet tubing B2 Stop the pump by clicking END in System Control Purging the pump Air remaining in the system may be removed by purging the pump and system by and system manually running Pump Wash or System Wash Note When performing a run using a Prepare Purification or Maintain method plan an
49. finally with 5 CV of deionized water The waste is collected through outlet F11 Creating a method plan for metal ion charging To create a method plan for metal ion charging 1 Click the Method Wizard icon in the Method Editor Result The Method Wizard dialog box appears Select New method plan in the Method plan field and click Next Click Prepare and then Columns on the Main Selections page Click Next 18 1178 00 AA ep 166 Operation 6 4 On the Prepare Columns page make the following selections Click Affinity Select column type Click Metal lon Charge Select the number of affinity columns Click Next Prepare Columns Main Selections Mote Max 5 columns allowed Prepare System Prepare Columns Running Condition Last Page f Room Temperature Cold Room I Affinity Hie Trap HF_1_ml Global Column Equilibration f Metal lon Charge Affinity Blank Run Metal lon Charge Affinity Blank Aun Number of Affinity Columns to Aun 1 F Note Make sure the metal ion charge inlet tubing 47 haz been filed with the corect solution prior start On Last page click Finish and save the metod plan Note The Metal Ion Charge inlet tubing A7 has to be filled manually before starting the run See 6 5 3 Purging the pump and inlet tubing on page 156 Note The Method Wizard also provides a combined method plan that includes metal ion charging automatically followed by a blank run Starting
50. flow rate used when eluting the cleaved protein after protease incubation Equilibration Cleavage Buffer Flow rate when loading the cleavage buffer onto the column before loading the protease solution Elution Affinity Regeneration The flow rate used to regenerate the affinity column i e elution of tags tagged protease and uncleaved protein using affinity elution buffer Pressure Limit Upper pressure limit during the affinity step Volume parameters Method Wizard ep 123 5 Method Wizard 5 5 Advanced Zone 5 5 4 Advanced Zone for Purify Affinity Cleavage Buffer Volume Cleavage buffer volume applied before loading the protease solution Load same volume of protease on all columns To load the same protease volume on all columns as entered in Default Protease Volume To use separate volumes for different columns deselect this option The separate volumes have to be entered in the System Control wizard before starting the run The Protease Injection Delay Volume is the volume between the loop valve and the column valve Enable NaOH Wash Between Samples To wash the pump mixer and injection valve with NaOH inlet A6 followed by affinity buffer A between the sample loadings Enable Watch Stable Baseline To check the stability of the UV signal during a specified time interval The Stable Baseline condition is met when the signal fluctuates less than the set Delta Base value during the set Stable Time interval If
51. initial system wash will be included in the system To purge the pump and or system 1 Carefully immerse the inlet tubing A1 in a flask containing the buffer to be used ep 159 6 Operation 6 5 Preparing the system for a run 6 5 3 Purging the pump and inlet tubing 2 Run a Pump Wash or a System Wash e Start UNICORN and select System Control Manual Pump e Select PumpWash and inlet A1 or SystemWash SYS_1 Pump Instructions m Instucton _ Parameters Insert O Pump Inlet C Fowpoh TRE bette ystemW as C Frac LoopWash Execute Alarms amp Mon Close C Other __ Chose gt Help 7 Auto update If this is checked the parameter fields will be updated during method run e Click Execute Purging the pump Both pump heads must be purged with methanol if with methano e The system has been left unused for a week or longer or e The pump has been run dry The purging will maintain the pumping capacity and protect the pump piston seals Note All inlet tubing that will be used in the next purification run should be filled manually before purging the pump The purging procedure in short 1 Flush out buffer using deionized water 2 Purge the pump using methanol 3 Flush out methanol using deionized water Preparation 1 Prepare two flasks containing 200 ml of methanol and 300 ml of deionized water respectively Put both Waste tubings from the injection valve into a
52. loop Uncleaved protein and tags are flushed from the AC column with buffer A3 and collected in collection tubes F7 F10 Methodology 4 4 Methodology About this chapter This chapter gives a brief introduction to the methodology for AKTAxpress It contains information and guidelines on how to choose protocols perform affinity tag removal choose columns and finally suggests buffers that can be used Developing the perfect scheme for purifying a protein remains though an empirical process and therefore has to be optimized In this chapter This chapter contains the following sections Protocols and affinity tag removal Buffer alternatives 4 3 Documents for further information ep 53 4 Methodology 4 1 Protocols and affinity tag removal 4 1 Protocols and affinity tag removal About this section This section contains general descriptions of the current protocols that can be used with AKTAxpress and guidance for how to choose a protocol It also describes the affinity tag removal process that can be automatically performed in AKTAxpress in combination with all supported multi step protocols In this section This section contains the following sub sections Topic Protocol descriptions and choice Description of affinity tags Description of proteases Description of the affinity tag removal process Conditions for affinity tag cleavage 18 1178 00 AA ep 54 4 1 1 Introduction Protocol defini
53. manifold J 7 Using the hex key unscrew and completely remove one of the two hex screws locking the pump head in position When unscrewing the second locking screw push firmly on the front face of the pump head to compensate for the pressure of the piston return spring Hold the pump head firmly to prevent it from twisting e Remove the second screw and without allowing the pump head to twist sideways carefully pull it out ep 2 3 8 Maintenance 8 6 Replacement procedures 8 6 6 Replacing pump piston seal Place the pump head face down on the bench Pull out the piston together with the return spring Wipe the piston with a clean cloth If salt solutions have been used the piston may be slightly corroded This corrosion can be removed with a rubber eraser If it cannot be wiped or rubbed clean scrape off any deposits with a scalpel or razor blade Inspect the piston with a magnifying glass for scratches Replace with a new piston if any scratches or cracks are found Remove the two screws securing the drain plate and the rinse chamber Remove and discard the rinsing membrane Remove the rinse chamber Drain plate Rinsing membrane Rinse chamber 18 1178 00 AA ep 274 Maintenance 8 Gently withdraw the piston seal Discard the used seal Piston seal T The pump head rinse chamber and drain plate should be carefully rinsed or cleaned in an ultrasonic bath if available If dirt can b
54. method plan for an ion exchange blank run Follow the description for the metal ion charging procedure or see the standard procedure described in 6 6 1 Starting a run using a method plan on page 186 The purpose of equilibrating a column is to remove ethanol and to equilibrate the column with buffer Note When using a HiLoad gel filtration or HiPrep desalting column for the first time or when changing buffer the column must be equilibrated During the equilibration the column is flushed with 5 CV of the appropriate buffer except for the gel filtration column which is flushed with 1 5 CV Creating a method plan for equilibration The procedure for creating a method plan for equilibration is the same as the metal ion charging procedure except for changes on the Prepare Columns page according to the following illustration e Click the check buttons to select the column types you want to equilibrate In the illustration all four column types are selected e Besides columns the required Column Position and Buffer Inlet should be selected for the desalting column Note If the gel filtration column is filled with ethanol it must be washed with water before equilibration with gel filtration buffer To do it automatically create a Customized Equilibration method plan for two solutions in Maintain in the Method Wizard ep 169 6 Operation 6 5 Preparing the system for a run 6 5 5 Conditioning columns M Affinity f Column Equi
55. oo Di oz 03 O4 D05 D06 oF O08 of 410 Check that the pressure difference is within the range 0 1 to 0 3 MPa If this is not the case the flow restrictor should be replaced see 8 6 3 Replacing flow restrictor on page 262 18 1178 00 AA ep 224 8 2 2 Maintenance inter val Reason for main tenance Changing solution Maintenance 8 Changing pump rinsing solution Every month The pump rinsing solution should be changed to avoid bacteriological growth The rinsing solution can be placed in a 50 ml test tube mounted in the right hand column holder The pump piston seal rinsing system Check valve Optional path TE without i 4 recirculation 3 Rinsing solution 20 ethanol To change the pump rinsing solution Empty the liquid container and fill it with 20 ethanol Place the test tube for example in the right hand column holder Repeat the procedure for all systems If required several systems can use the same liquid container Note Do not connect rinsing system tubing from several systems in serial The tubing from each system must be routed directly to the liquid container p 225 8 Maintenance 8 3 Six monthly maintenance 8 3 Six monthly maintenance About this section This section describes the six monthly maintenance activities In this section This section contains the following sub sections Topic Checking UV lamp run time Cleaning UV cell Cleaning conductivit
56. para meters Air sensor con trolled sample loading paramet ers Advanced Zone for Purify General advanced settings This section describes the options and parameters available on the General advanced settings page in all protocols The content of the page differs depending on the protocol used The General Advanced Settings page contains a number of parameters and miscellanous options Method Wizard General Advanced Settings Advanced Zone Flush Yolume Empty Loops id 0 100 ml Fage Loop wash Volume 20 10 999 mi 1 3 W Enable AirSensor Controlled Sample Loading Last Page Max Sample Volume S393 fp 9999 ml Purp Air Removal Volume 20 D 999999 ml Between Samples Extinction Coefficient comesponds to i mg ml cM Unit Molecular Weight Preferred Target Concentration Import File Location CAUNICORN S enverMethody izard mport W Store Results in a Main Folder Main Folder Name projectname user O KTAxpress Back Hest Finish Cancel Help Set Wetault The options and parameters for emptying loops Flush Volume Empty Loops The volume used to flush the excess and discarded sample from the loops to the outlets F7 F10 Loop Wash Volume The volume used to wash the loops The options and parameters defining air sensor controlled sample loading Enable AirSensor Controlled Sample Loading Used to provide loading of the entire sample volume The sample loading will
57. the chosen columns with two different cleaning solutions The type of solutions and the amount for each column depend on the column choices See the Summary page in the wizard when starting a run or the instructions supplied with the column Select running conditions and whether to wash the columns with water before the CIP or not Select columns and column positions only the DS column Proceed to 5 2 4 Finishing the Maintain method plan on page 105 ep 103 5 Method Wizard 5 2 Creating a method plan 5 2 4 Using the Maintain option CIP Columns rig The table below contains a description and instructions for the CIP Columns orous Custom Rigorous and Customized equilibration option ized equilibration options Maintain operation Description Instruction CIP Columns Rigor Cleans the chosen column with up to nine cleaning ous and Customized solutions or equilibration e Equilibrates the column with an optional number of solutions Recommended for removing ethanol from new columns and for adding ethanol to columns for storage For the required amounts of solutions see the Sum mary page in the wizard when starting a run Note Only one column type can be included 1 Select column type and the number of columns 2 Select CIP or Equilibration 3 Select the number of steps For each step make the following selections if there is more than one cleaning step additional pages appear by cl
58. the piston and to prolong the life of the seals the low pressure chamber behind the pump head can be flushed continuously with a low flow of rinsing solution The flexible rinsing membrane sweeps the piston and seals the rinsing system Check valve uy eee eee ew Optional path without Rinsing solution recirculation 20 ethanol e p311 10 Reference information 10 1 System description 10 1 1 Pump and related components Mixer Flow restrictor 18 1178 00 AA ep 312 The mixer is a static mixer with a volume of 0 25 ml Note Do not use organic solvents in buffers The mixer is designed for use with water based solutions only If organic solvents are used incorrect gradients or incomplete mixing may be the result The flow restrictor is positioned directly after the UV and conductivity monitors The restrictor generates a steady backpressure of 0 2 MPa to prevent air bubbles being formed in the monitors The flow restrictor is closed for pressures below 0 2 MPa Reference information 10 10 1 2 Valves Introduction This section describes the valves in AKTAxpress Outlet valve Se Column valve a Loop valve Injection valve Switch rewa N SW1 and S w2 DN HO valve Switch valves The switch valves SW1 and SW2 are 2 way 3 port valves used for buffer switching and gradient formation The valves have one port marked IN and two ports marked NC normally closed and NO normally
59. the system name sys1 Q In the menu select System Calibrate and choose Cond_Calib under Monitor r Calibration procedure Monitor Determination of cell constant for cond cell See Help Cond _Calib mS cm 1 Reference value 1 86 0000 1 0 300 0 9 Read value 1 Measured value fi 5 0 mS cm Cell constant 1 cm Close Help ep 243 8 Maintenance 8 5 Calibration procedures 8 5 2 Calibration of conductivity cell Start a manual run at 10 ml min to fill the conductivity cell with the calibration solution Pump calibration solution through the cell until the conductivity signal is stable and then set the system in Pause Read the conductivity value displayed under Measures value and compare it with the theoretical value from the graph below mS cm 97 aa En ECT eee ELEC PPE HSH eu PEAREN tar ERNI tat PEE ETE CSE eee egy tttpffpe en rina A i EE Sst E ANENE R SECT EEE REEN Var Gast aE ESTEE eS eC ACES eee EEC ESET EE If the displayed value corresponds with the theoretical value No further action is required Proceed to step 8 If the displayed value differs from the theoretical value E ce a a oon L EN PEER EE EEE AEEA a EEE EE E E E ete ee Proceed to step 6 Enter the theoretical conductivity value according to the graph in the field Reference value 1 Click Read value 1 Read value 1 The new cell constant is saved in the system 18 1178 00 AA ep 244
60. to the lower port of the superloop It is also possible to connect the superloop to the loop valve as usual and manually setting the injection valve and loop valve to the correct positions To set the valve positions manually Select Manual Flowpath in System Control Set LoopSelection to LP5 and click Execute and click Execute To inject liquid into the superloop set InjectionValve to Load and click Execute Before fitting or removing the syringe set InjectionValve to Inject If a system is used in a cold room it is possible to raise the temperature in the affinity column by using a column heater The efficiency of some proteases increases with increased temperature thus decreasing the incubation time Note Carefully raise the temperature if the protein is temperature sensitive The column heater is not controlled from AKTAxpress but can be physically placed on top of the system Extra long capillaries might be needed between the column valve and the affinity column used The protease volume then must be changed in the Advanced Zone in the Method Wizard considering the new delay volume e p 179 6 Operation 6 5 Preparing the system for a run 6 5 9 Checking the tubing 6 9 9 Checking the tubing Introduction This section describes how to check the tubing before a run Checking the The tubing must be checked for tubing e Air bubbles e Leakage If there are air bubbles or leakage the run might be adversely
61. trapped in the UV cell The flow restrictor creates a backpressure of 0 2 MPa AKTAxpress overview 3 Intermediate frac The intermediate fraction collection is handled by a loop valve and five capillary tion collection loops Capillary loops Loop valve Loop valve The loop valve is located at the upper right hand side It is used to direct an intermediate peak volume from the UV cell and the outlet valve into a suitable loop where it is stored for the next purification step Capillary loops Five loops each one with a volume of 10 ml are connected to the loop valve The loops have i d 1 0 mm and are made of Tefzel tubing ae fraction col The final fractionation parts are the outlet valve and fraction collector located at ection the top of the system Outlet valve Fraction collector Waste Fraction outlets F3 F11 ep4l 3 AKTAxpress overview 3 2 Separation system overview 3 2 4 Functional descript ion Outlet valve During the last purification step the outlet valve directs the peak volume to the fraction collector The outlet valve also provides up to nine outlets for collecting flowthrough volumes and nonselected peaks and a waste outlet The outlet valve is also a part of the intermediate fraction collection where it is used to direct the peak volume to the loop valve The flow through and nonselected peaks containers should be placed on a shelf under the bench or in a flask
62. under Microsoft Windows The Method Wizard in UNICORN provides easy creation of method plans for purification and preparation and maintenance of the separation system and columns Before a run the operator chooses purification method plan and the number of samples for each separation system The systems can then be started separately or simultaneously from UNICORN or from the control panel on each system The illustration below shows the System Control module in UNICORN when using four separation systems oe System Control SYS_1 Result o 4 O0per_1 Wizard verification 20031030 ID56 SYS_1001 res File View Manual T Hold End W Instant Run Ci ei Instruments eis Connection Pei Run Status ar Volume CEFE Yolume Acc Time Crime Gites Block Time ponu Inject gt gt Bed Flow Pressure gt gt uv gt gt UVLamp gt gt Conc gt gt Cond gt gt Cond gt gt p gt gt InletValve gt gt LoopSelect ColPos gt gt Outlet gt gt RemainTime AirSensor gt gt ontinue intinue all End all Cond Conc Pressure Flow maAU ss3 Q 700 600 500 400 300 200 100 263 12 ml Watch_Off UY 263 12 ml End Block 263 12 ml Block Fractionation_IEX 263 12 ml Base SameAsMain 263 12 ml Block Peak_Frac_Parameters 263 12 ml Base SameAsMain 263 12 ml Peak_FracParametersUY Level AND_Slope 0 50 min 30 000 mAU 30 000
63. use the unions supplied with the system to connect tubing with the columns Note The gel filtration and HiPrep Desalting columns might require longer 18 1178 00 AA ep 164 tubing A tubing especially made for connecting these columns is supplied with the system Operation 6 1 16 Male 1 16 Female M6 Female M6 Female 1 16 Female On line filter M6 Male Z a LO lon E and E GSTrap HisTrap e 1 16 Male M6 Female Column block 1 16 Female M6 Male 9 x lt eD pS e9 O op O O x tO mi ge O _ I 6 mI o0 e 5 a D Qa oO i O N o D _ Q Lu Li O O a es oc D O O aN 79 Lu Li a oc 1 16 Male 1 16 Female 1 16 Male einer M6 Female 1 16 Female M6 Male Column block e p 165 6 Operation 6 5 Preparing the system for a run 6 5 5 Conditioning columns 6 5 9 Conditioning columns Introduction This section describes how to perform metal ion charging blank runs and equilibration of the columns used in AKTAxpress Metal ion char New or stripped HiTrap Chelating columns and stripped HisTrap affinity columns Bae ora must be charged with metal ions The software allows up to five columns to be charged automatically During the procedure each column is flushed with 5 column volumes CV of deionized water 1 CV of metal salt solution and
64. when the signal level and or the slope of the signal curve falls below a defined value The Stop Slope condition cannot be met unless Peak Max has been detected The illustration below describes this UV max 1 2 Start Slope OR Start Level 3 UV max Peak Max Factor 0 5 DG Stop Level OR Stop Slope Note Ifthe flow rate is changed the shape of the peak will change as well Hence the slope values might need to be changed accordingly Note When the fractionation of a peak is finished it will in the chromatogram appear as the subsequent fraction volume is very large This is because the position of the fraction collector is shown and only the delay volume for the next peak if any actually is collected in this well e p 119 5 Method Wizard 5 5 Advanced Zone 5 5 4 Advanced Zone for Purify Affinity 9 0 4 Advanced Zone for Purify Affinity Introduction This section describes the parameters used in a standard affinity step during a purification run Some of the default parameter values depend on the chosen column Note Make sure that the parameter values do not exceed the specification of the chosen column Tag cleavage parameters are described separately since they appear only when tag cleavage is selected Flow rate Pressure The first affinity page that appears contains options and parameters related to flow PAES rate and pressure as described below Method Wizard Affinity Flow Ra
65. 0 AA ep 128 Method Wizard 5 Gradient paramet The gradient options and parameters are described below ers Peak injection parameters Method Wizard lon Exchange Intermediate Gradient Advanced one fe Yes i Mo gn m1 ID fat Gradient Segment 1 Target Concentration Gradient Length Gradient Segment 2 Target Concentration Gradient Length oo MM AA eee Clean after Elution at 100 6 Collect Flowthrough in Outlet F F10 S0 0 100 8 20 0 999999 Civ 100 0 100 B D 999999 CY TEET 0 999999 CV Collect Flowthrough in Outlet F7 F10 To collect the wash volume in outlets F7 F10 Sample 1 in F7 sample 2 in F8 etc Gradient Segment 1 and 2 The Target Concentration and the Gradient Length are used to define two different gradient segments Clean after elution at 100 B Volume of buffer B2 used to wash the column after the gradient segments The peak injection options and parameters are described below Method Wizard lon Exchange Final Peak Injection Peak Fractionation Advanced one Peak Injection Page Injection Flush Volume E 0 100 ml 1 2 3 Peak Fractionation 4 Peak Fraction Size fi 0 2 2 ml 5 Peak Fractionation Algorithm JLevel_AN D_Slope 5 Start Level fao 6000 6000 mau 7 Start Slope a 0 070 50000 mL min z Peak Max Factor 0 5 0 1 9 Minimum Peak width D5 0 15 1500 min 10 Stop Level fao 6000 6000 mau 11 Stop Slope
66. 0 mM Tris HCl pH 7 5 150 mM NaCl 1 mM PreScission on GSTrap EDTA 1 mM DTT HP or FF 50 mM Tris HCl 10 mM reduced gluthathione pH 8 0 In the affinity tag removal protocols the affinity columns can be equilibrated with either AC binding wash buffer or an alternative cleavage buffer before protease injection Suggested buffers for DS runs If for example suggested buffer preparing for AIEX 50 mM Tris HCl pH 8 0 preparing for CIEX 20 mM MES pH 6 0 preparing for storage include e g 10 glycerol in a suitable buffer e g 50 mM Tris HCl pH 7 5 150 mM NaCl Suggested buffers for IEX runs If for example suggested buffer binding to AIEX 50 mM Tris HCI pH 8 0 binding to CIEX 20 mM MES pH 6 0 elution from AIEX 50 mM Tris HCl pH 8 0 1 M NaCl elution from CIEX 20 mM MES pH 6 0 1 M NaCl Note The chosen buffer and its pH will depend on the pI of the target protein e p81 4 Methodology 4 3 Buffer alternatives 4 3 2 Buffer suggestions GF buffer sugges tions Further informa tion 18 1178 00 AA ep 82 Suggested buffers for GF runs If for example suggested buffer preparing for further 50 mM Tris HCl pH 7 5 150 mM NaCl studies preparing for storage include e g 10 glycerol in a suitable buffer e g 50 mM Tris HCl pH 7 5 150 mM NaCl For further information see method handbooks in 4 4 Documents For Further Information on page 83 and product specifications 4 4 I
67. 000 ml Stop Slope p00 0 010 10000 mU min Start Slope om cm E Di Mi Peak Fraction Size The maximum volume of each peak fraction Peak Fractionation Algorithm Choose to start and stop the peak fractionation based on the signal curve e Level e Slope e Level_AND Slope e Level_OR_ Slope AND means that both conditions have to be met OR means that only one condition has to be met The fractionation start values are set by Start Level and Start Slope and the stop values by Stop Level and Stop Slope Different start and stop values for level and slope can be set The Stop Slope condition cannot be met unless the Peak_Max condition is met Stop Level can be met before Peak_Max The Peak_Max condition occurs when the UV signal has fallen to a fraction specified by Peak Max factor of the most recent peak maximum Note If the flow rate is changed the shape of the peak will change as well Hence the slope values might need to be changed accordingly Minimum Peak Width sets the minimum fractionation time of a single peak The fractionation of the current peak continues at least until the Minimum Peak Width is reached even if the signal is fluctuating epl2 5 Method Wizard 5 5 Advanced Zone 5 5 6 Advanced Zone for Purify lon exchange 9 9 6 Advanced Zone for Purify lon exchange Introduction This section describes the parameters used in the ion exchange steps for the protocols C E Some of the def
68. 216 exporting 216 Pools adjusting 214 Power on 144 Preparing for automatic affinity tag removal 175 Pressure checking 237 calibration 242 Index ep 349 Index curve 296 Print report 218 Printing pooling protocol 216 Process description 49 Process flow chart 48 Product numbers 336 Protease descriptions 61 types 61 calculation 175 Protocols 55 descriptions 55 for AKTAxpress 55 number of samples 57 Pump synchronization 43 description 309 Pump piston replace 280 Pump piston seal replace 2 2 Pump pressure checking 237 Purging the pump 156 Purging the pump with methanol 160 R Report printing 218 Reset the system 43 Results viewing 212 S Safety 11 Samples 18 1178 00 AA ep 350 preparing 171 tubing 171 manual loading 172 flow control 184 Schematic drawing of the flow path 15 Software 303 Spare parts 336 Superloop filling 177 Syringe 156 Syringe 160 Syringe 2 8 system Control expand 193 collapse 193 T Tag cleavage parameters 123 Tag cleavage see affinity tag removal 175 Technical specifications 323 Troubleshooting 282 Tubing dimensions 35 fittings types 36 checking 180 leakage 238 replace 247 replace 249 specification 254 piston rinsing 260 Typographical conventions 22 U UNICORN introduction 16 software modules 24 operation overview 25 Index ep 351 Index accessing help functions 26 log on 1
69. 3 e Click Next Proceed to 6 6 1 Final setup and starting the run on page 191 ep 189 6 Operation 6 6 Starting a run 6 6 1 Starting a run using a method plan Final setup and For final setup and starting the run starting the run Step If required edit the folder path and names of the result files Click Next By default the result files will be saved in Home folder Date System name 001 If there are several result files created on the same date the files will be named 001 002 and so on after the identical name If Main Folder is chosen in General Advanced Settings in the Method Wizard the result file will be saved in Home folder Main folder Date System name 001 x Folder Name and Result Name System Free Test Folder Fath Home Result Hame Folder Mame Summary 20031126 Systems Result File Hame GYG os 1 oye 2 evs 2 18 1178 00 AA ep 190 Operation 6 The Summary page specifies the method requirements The tabs for each system specifies the required columns loops buffers solutions etc and where they should be connected The total requirement of buffers and solutions is listed on the Summary tab If required make a printout of the list by clicking Print Note that the list will not be saved Method Wizard Summary Systeme Samples SamplelD System 1 4 System Settings S5 1 Settings S5 2 Settings S5 3 Settings SYS 4 Total run Alr sensor Inlet position Result Mame
70. 45 indicator colors 146 indicator colors 194 User documentation package 20 UV curve 290 UV cell cleaning 228 UV lamp checking run time 227 replacing 264 checking run time 264 V Valves description 313 View results 212 Viewing a run 193 W Warnings 11 Waste tubing 155 Watch conditions 117 18 1178 00 AA ep 352 IS bbe Mle Np gy ejesddn X L Aq uepems ul payUld ejesddn qe uolewsojul 9L losciences Amersham gt B
71. 867 0 5000 0 0000 B64 0 7500 0 0000 Show all fractions Show all columns Delete Delete all Export Help 18 1178 00 AA ep 216 Evaluation 7 4 e Click Show all fractions to display the individual fractions instead of fraction ranges for the pools e Click Show all columns to display all the information columns from the Pool table Possible actions in the Pooling Protocol Note The information in the Pooling Protocol is saved for the individual UNICORN user The Pooling Protocol can only be cleared by clicking the Delete or Delete all button To delete a single pool select a pool and click the Delete button To clear the whole protocol click the Delete all button To print the protocol click the Print button to print the protocol on the default Windows printer To save the protocol as a file click the Export button to save the protocol in one of the following formats text txt Excel xls HTML htm XML xml e Click the Close button to close the Pooling Protocol dialog box Result If the protocol was exported or only edited the dialog box will close If the protocol was printed a dialog box will open asking if the list is to be deleted and a new one started e p217 7 Evaluation 7 6 Printing report 7 6 Printing report Introduction Reports can be printed after a completed run To find information on how to create reports see the UNICORN Online Manual This s
72. Advanced Zone for Purify chromatographic technique e Advanced Zone for Maintain 18 1178 00 AA ep 110 Method Wizard 5 9 9 2 Advanced Zone for Prepare Introduction This section describes the Advanced Zone for a Prepare method plan Note Make sure that the parameter values do not exceed the specification of the chosen column Selecting paramet To select parameters ers 1 Select the required options on Page 1 Select General Advanced Settings to define a main folder for storing the results Method Wizard Advanced one Select type of variables to change Advanced one Page V Prepare System IM Prepare Column I General Advanced Settings Click Next Result The next selected page appears e p111 5 Method Wizard 5 5 Advanced Zone 5 5 2 Advanced Zone for Prepare Changing Prepare To change Prepare System parameters if the options have been selected in the first System parameters part of the Method Wizard 1 Change the desired values on Page 2 Method Wizard Prepare System Advanced one Page Sample Inlet Fill Volume fis 0 999999 ml 1 Buffer Inlet Fill Volume fis D 999999 mi 3 Volume Needed ta Remove Air 4 20 10 999999 ml Sample Inlet Fill Volume Volume used for filling the sample inlet tubing Buffer Inlet Fill Volume Volume used for filling the buffer inlet tubing with buffer before immersing the tubing in the samples Volume Needed to Remove Air
73. DS His or G5 T tagged target protin amount high Which target protein tag Use in ACstep HisTrap HP GSTrap FF HisTrap HP GS Trap FF 1 ml 1 ral 5 ml D mil HiPrep 26 10 Desaltng Which pl has the Which pl has the target protein target protein Wes per RESQURGE 5 6 mil i RESOURCE 5 RESOURCE 1 ral 6 ml or any one of or any one of or any one of or any one of Hilrap GQ HP 1 ml HiTrap sP HP 1 mi 2xHiTrap Q HP 1 ml 2xHiTrap SP HP 1 ml 2xHiTrap Q HP 1 mi 2xHiTrap SP HP 1 ml Mono Q 5 50 GL 1 mif Mono 5 5 50 GL 1 ml Mono O 5 50 GL 1 m Mono s 5 30 GL 1 ml HiPrep 26710 Desalting Comments on alternative column choices Comments on alternative column choices AC Overloading of an affinity column can be performed to minimize non specific binding often results in a purer end product Which target protein tag Use in OSstep Use in IEX step RESOURCE G 1 mal r Use in O S step If there is a low expressed target protein in a large sample volume consider using a 5 ml HiTrap column to reduce the risk of column clogging or protein degradation ep 3 4 Methodology 4 2 Column alternatives 4 2 2 Choosing columns Comments on alternative column choices 1 ml HiTrap affinity columns can be combined with 2 x HiTrap desalting columns Band broadening effects in the system combined with a limited sample loading volume on the 2 x
74. Hg lamp lifetime at 280 nm Lifetime in room temperature 3500 hours in cold room 2000 hours Conductivity Conductivity monitor data monitor data Conductivity range 0 01 300 00 mS cm Accuracy Max 0 4 mS cm typically lt 0 1 mS cm Reproducibility short and long term Max 3 or 15 pS cm whichever is greater Noise Max 0 5 of full scale calibrated range typically 0 1 Response time lt 3 s 0 95 of step Internal volume 24 pl Pressure sensor Pressure sensor data data Parameter Pressure range 18 1178 00 AA ep 326 Reference information 10 Valve data Valve data Inlet valve Internal volume in out lt 33 pl Maintenance interval 10 000 positions 1 year Injection valve Internal volume in out lt 17 ul Maintenance interval 10 000 positions 1 year Loop valve and column valve Internal volume in out lt 16 nl Maintenance interval 10 000 positions 1 year Outlet valve Internal volume in out lt 15 nl Maintenance interval 10 000 positions 1 year Capillary loops Capillary loop data Fraction collector Fraction collector data Collector type X Y collector for 96 deep well micro plates p 327 10 Reference information 10 2 Technical specifications 10 2 3 Wetted materials 10 2 3 Wetted materials Introduction This section specifies the wetted materials of AKTAxpress 18 1178 00 AA ep 328 Reference information 10
75. HiTrap desalting column might however reduce protein recovery A longer IEX column or a column with smaller bead size can be used to increase resolution Make sure to use an IEX column with sufficient capacity for the protein amount Only the same desalting column can be used for all DS steps in this protocol DS 5 ml HiTrap affinity columns should not be combined with 2 x HiTrap desalting columns since the eluted affinity peak might be larger than the volume possible to load on the 2 x HiTrap desalting column 18 1178 00 AA ep74 Methodology 4 Choosing columns Recommended column combinations when running an AC DS IEX GF protocol for AC DS IEX GF His or G5 T tagged target protein amount high Which target Which target protein tag protein tag Use in ACstep HisTrap HF GSTrap FF HisTrap HP GSTrap FF 1 ral 1 ral 5 ral 5 ml HiPrep 26 10 Desaltng Which pl has the Which pl has the target protein target protein Tors e75 RESOURCE 5 6 ral RESOURCE 5 RESOURCE Q 1 ral a 6 ml or any one of or any ane oF or any ane af or any ane of HiTrap Q HF 1 ml HiTrap SP HF 1 ml 2xHiTrap O HF 1 ml H 2xHilrap SP HP 1 ml zxHiTrap Q HF 1 mi 2xHiTrap SP HF 1 mil Mono O SI50 GL 1 mli Mono s BBO GL 1 mi Mono O 5 50 GL 1 m Mono 5 8 60 GL 1 ml f Which Ml has the target protein 70 kDal gt 70 kDa Use in GF step Hiload 1686 650 Hiload 16 60 Superdex 75 pg Superdex 200 pg Co
76. Italian and Swedish UNICORN AKTAxpress User Refer Detailed instructions on the use of ence Manual UNICORN This document is also available as an on line manual 18 1178 00 AA ep 20 Document struc ture UNICORN Administration and Technical Manual Introduction Information for the system administrat or about for example user administra tion network installation and troubleshooting This document is also available as an on line manual This manual is divided into chapters Each chapter starts with a brief overview that presents the contents and the headings for the sections that the chapter contains The section begins with an introduction that summarizes the content 2 Each section is divided into blocks of information with separating lines The blocks are identified by labels in the margins This makes it easier for you to quickly scan a page to find the exact topic you are looking for AKTAxpress User The table below describes the content in each chapter in this manual Manual 2 Introduction 3 AKTAxpress overview 4 Methodology 5 Method Wizard Regulatory contact and safety inform ation General system overview of AKTAxpress and basic operating principles background to purification of recombinant proteins and informa tion about the user documentation Description of AKTAxpress and a purification process overview A guide to selecting purification pro tocol buffers and colum
77. Note Make sure that the parameter values do not exceed the specification of the chosen column The first desalting page that appears contains options and parameters related to flow rate pressure and volumes Method Wizard Ed Desalting Intermediate Flow Rate Pressure Yolumes Advanced Zone Flow Fate 10 D 65 ml min Fage Pressure Limit 0 35 0 3 MPa 3 3 Equilibration volume 0 1 0 999999 Cy 4 Elution volume Le D 999999 Cv Flow Rate Flow rate during the desalting step Pressure Limit Upper pressure limit during the desalting step Equilibration volume Volume used when equilibrating the desalting column Elution Volume Volume used during the elution of the desalting column The peak injection options and parameters Method Wizard Desalting Intermediate Peak Injection 7 Peak Collection Advanced one Peak Injection Page Injection Flush olume 0 100 ml 1 Max Injection Volume fio 0 100 ral Injection Flush Volume The extra volume besides the peak volume used to flush the loops when loading the sample onto the desalting column Max Injection Volume The maximum volume to be injected onto the desalting column Note The maximum injection volume is set up to make sure that the peak volume flush volume do not exceed the maximum sample loading volume of the column 10 ml for the HiPrep Desalting column e p 125 5 Method Wizard 5 5 Advanced Zone 5 5 5 Advanced Zone for Pur
78. S gulde serisinin us aieuneiedeuebi Gatos 332 EOS Ordern INTOA ON een acteun e detchatden siento eetesusmnndesatiiddetuntnden ETEO 336 18 1178 00 AA ep6 1 Preface Preface 1 About this chapter This chapter contains regulatory contact and safety information In this chapter This chapter contains the following sections Topic Important user information Product information Safety ep 1 Preface 1 1 Important user information 1 1 Important user in formation Warning symbols Caution notices Note signs Comments on manual Copyright 18 1178 00 AA ep8 Important user information All users must read this entire manual to fully understand the safe use of AKTAxpress The following warning symbol is used in the documentation WARNING N The Warning symbol highlights instructions that must be followed to avoid personal injury Do not proceed until all stated conditions are clearly understood and met The following caution notice is used in the documentation CAUTION The Caution sign highlights instructions that must be followed to avoid damage to the product or other equipment Do not proceed until all stated conditions are clearly understood and met The following note sign is used in the documentation Note The Note sign indicates information important for trouble free and optimal use of the product Should you have any comments on this manual we will be pleased t
79. The drip plate located below the microplate in the fraction collector should be emptied when necessary The purpose of the drip plate is to collect any overflow from the microplate Overflow might indicate an error see 9 1 Troubleshooting on page 282 If the drip plate becomes full there is a risk of damage to the system oe drip To empty the drip plate in the fraction collector follow the procedure below plate WARNING f Do not put any body parts in front of or close to the fraction collector The microplate can be ejected and retracted both manually and automatically by the system CAUTION Ensure that no parts like tubing or columns are positioned in front of the fraction collector hindering the ejection of the microplate The movement of the plate might destroy columns or tubing positioned in front of the fraction collector Check that the system is in Idle mode with no active run Press the Eject button to eject the collector plate out of the system if necessary Each time the button is pressed the sled switches between inside and outside the system 18 1178 00 AA ep 196 Operation 6 Carefully remove the drip plate by sliding it outwards gt o Empty any fluid into waste 3 Slide the drip plate back into position Check the function by pressing the Eject button to load the collector plate e p 197 6 Operation 6 8 Procedures after a ru
80. Trap column prepacked with lands mi Glutathione Sepharose High Perform ance For high resolution purification of GST tagged proteins GSTrap FF HiTrap column prepacked with Panini Glutathione Sepharose Fast Flow For fast purification of GST tagged pro teins Methodology 4 Desalting columns Desalting DS columns separate molecules with large size differences Desalting is a quick method for size separation and is often used to remove salts from proteins or to change buffers The following desalting columns are supported by AKTAxpress HiPrep 26 10 Desalting HiPrep column prepacked with Sepha een dex G 25 Fine For fast buffer ex change and desalting of samples with a volume of lt 15 ml 2 x HiTrap Desalting HiTrap columns prepacked with 2 x 5 ml see also 4 2 1 Coupled Sephadex G 25 Superfine For fast columns on page 69 buffer exchange and desalting of samples with a volume of lt 3 ml a exchange Ion exchange IEX columns separate proteins based on differences in charge The ye separation is mediated by a reversible interaction between a charged protein and an oppositely charged chromatographic media Anion exchange columns Anion exchange columns are used to separate negatively charged proteins The following anion exchange columns are supported by AKTAxpress Supported anion IEX columns and Characteristics volumes RESOURCE Q Columns prepacked with SOURCE 1 and 6 ml 15Q Fast purific
81. Volume used to remove air from the pump after the inlets have been filled Click Next Result The next selected page appears 18 1178 00 AA ep112 Method Wizard 5 Changing Prepare To change Prepare Column parameters if the options have been selected in the first cama paramet part of the Method Wizard Note The parameter available depend on the choices made earlier in the Method Wizard Method Wizard Prepare Column Advanced one Affinity Page Flow Rate Equil Blank Aun 0 65 mlmin Pressure Limit 0 3 MPa Equilibration Volume 0 999999 Cy Desalting Last Page Pressure Limit 0 3 MPa Flow Rate 0 65 ml min Equilibration Wolume 0 999999 Cy co lon Exchange Pressure Limit 0 3 MPa Flow Rate 0 65 ml min Equilibration Wolume 0 999999 Cy Gel Filtration Pressure Limit 0 3 MPa Flow Rate 0 65 ml min Equilibration Wolume 0 999999 C bs EB se i EB I e is Pressure limit Upper pressure limit when equilibrating a column Flow Rate Flow rate when equilibrating a column Equilibration Volume Volume used for equilibrating a column Click Next Result The next selected page appears ep 113 5 Method Wizard 5 5 Advanced Zone 5 5 2 Advanced Zone for Prepare Defining a folder To define a sub folder under the Home folder for storing the results for storing the res ults Enter the desired folder name Method Wizard Ea Advanced o
82. Wash after air detection the system will automatically go to the same mode as before the air detection 18 1178 00 AA ep 182 Air detection at sample loading Operation 6 Air detection during sample loading makes it possible to apply the entire sample volume and still prevent air from reaching the column By default air detection is always enabled during sample application in a method plan made in the Method Wizard When air is detected during sample loading the inlet valve switches to port Al The pump then uses a few ml of buffer Al to apply sample that is left in the tubing onto the column The pump then flushes the air out with affinity buffer A1 at 20 ml min The flush volume can be changed on the General Advanced Settings page in Advanced Zone in the Method Wizard The sample loss when using air detection is approximately 0 5 ml Setting a fixed sample volume It is also possible to set a fixed sample loading volume e To set a fixed volume the air sensor option must first be cleared in the method plan This is done on the General Advanced Settings page in Advanced Zone in the Method Wizard Method Wizard General Advanced Settings Advanced one Empty Loops Page Flush Volume B 0 100 ml 1 Wash Volume 20 10 999 ml 2 4 T Enable AirSensor Controlled Sample Loading 4 Note Sample volume must be entered in System Control e When the method plan is started in System Control the sample volume has to be
83. YS 3 Method Result File View Manual System Help arin H mm Expand gt gt bh fes a r Calibration procedure Monitor Sets the pressure reading to zero See Help PumpPress 1 Start calibrate Close Help Click Start calibrate and wait until it is enabled again 10 20 s Start calibrate Result The injection valve will automatically be switched to Waste and the pressure reading set to zero 2 In the menu select System Calibrate and choose PumpPress under Monitor Click Close to finish the operation 18 1178 00 AA ep 242 Maintenance 8 8 5 2 Calibration of conductivity cell Introduction Problems in the conductivity cell can cause abnormal conductivity readings in chromatograms There are two different procedures e Calibration of current conductivity cell e Setting the conductivity cell constant after replacement Calibrate current To calibrate the current conductivity cell conductivity cell l _ Note Calibration of the conductivity cell is not normally required The cell constant is calibrated and set at the factory Note Perform the test at room temperature 20 30 C Prepare 20 ml of calibration solution 1 00 M NaCl 58 44 g l and immerse the A1 tubing into the solution Note Make sure that the temperature of the calibration solution is the same as the room temperature 2 In the UNICORN System Control module select the system to be calibrated by clicking
84. acing a piston To replace the piston and the seal follow the instructions in 8 6 6 Replacing pump piston seal on page 272 18 1178 00 AA ep 280 Troubleshooting and corrective actions 9 9 Troubleshooting and corrective actions About this chapter This chapter describes the troubleshooting and corrective actions of AKTAxpress In this chapter This chapter contains the following sections Topic Introduction to troubleshooting Problems during a run Monitoring problems Leakage problems Alarms and connection problems Error code list Checking USB CAN driver ep 281 9 Troubleshooting and corrective actions 9 1 Introduction to troubleshooting 9 1 Introduction to troubleshooting Introduction This section describes the troubleshooting procedure and includes a general check list of common errors to be checked Troubleshooting To troubleshoot AKTAxpress Always start checking the General check list below 2 Find the fault and corrective actions in one of the different sections depending on the type of problem e Problems during a run Errors related to failure to load samples high back pressure and unsatisfactory elution from the columns see 9 2 Problems during a run on page 284 Monitoring problems These are errors detected during a run or when evaluating the resulting chromatograms For example ghost peaks noisy signal signal drift non linear gradients and unstable readings see 9 3 Monitoring problems
85. affected To check the tubing follow the procedure below CAUTION To protect the piston seals in the pump the pump should not run with air in the inlet tubing Follow the instructions for purging the pump CAUTION Before the start of each run ensure that there is an adequate supply of eluent in the reservoirs Never allow the pump to run dry because this will affect the lifetime of the piston seals 1 Leakage inspection Inspect the tubing for leakage Pay special attention to the tubing connectors that are related to columns If leakage is found tighten the connectors or replace the tubing if necessary see 8 6 2 Replacing tubing on page 249 18 1178 00 AA ep 180 Operation 6 Buffer tubing Check that the tubing is fully immersed in the buffer containers Fix the tubing in the cap for example by e Fitting the inlet filter weights on the tubing e Running the tubing through the rubber cap included in the access ory kit 2 Air bubbles inspection Inspect the inlet tubing for air bubbles All tubing up to the pump heads should be inspected Samples Buffers Buffers S1 S4 A1 A2 B1 A3 A8 B2 If air bubbles are found perform manual purging of the system see 6 5 3 Purging the pump and inlet tubing on page 156 e p 181 6 Operation 6 5 Preparing the system for a run 6 5 10 Using air detection 6 5 10 Using air detection Introduction This section describes the automatic air detection in AKTA
86. alve is identical to the column valve The valve has fingertight connectors The valve has no user replaceable parts L2 Bypass Injection valve ED L4 L3 Injection valve Bypass i2 Default position at startup For flow path see 10 1 2 Column valve on page 316 18 1178 00 AA e p316 Outlet valve Reference information 10 The outlet valve is a 12 way 13 port valve For each 12 positions an outlet port is connected to the central inlet port The valve has fingertight connectors The valve has no user replaceable parts Fraction collector Waste FS Injection valve SN ON F4 F11 O OJ F5 Flow restrictor F7 Default position F8 at startup The valve is used for redirecting samples from a column to a capillary loop passing samples to the fraction collector and passing liquids to flowthrough containers and waste For flow path details see 3 2 2 Liquid flow path on page 34 e p 317 10 Reference information 10 1 System description 10 1 3 Monitors 10 1 3 Monitors Introduction Pressure sensor UV monitor This section describes the monitors and flow restrictor in AKTAxpress Flow restrictor UV monitor Conductivity monitor Air sensor The UV optical unit houses the Hg lamp the wavelength filter and the UV flow cell The light beam is directed through a double conical or straight flowthrough cuvette of 2 pl illuminated volume to a photodetector The photo
87. ame of the result files etc This information will be requested in a wizard in the System Control module where the final preparation before starting the run is made When the wizard is finished a unique method for each of the systems is created and started See 6 6 1 Starting a run using a method plan on page 186 ep 85 5 Method Wizard 5 1 Method Wizard introduction Opening the Method Wizard The first page in the Method Wiz ard To open the Method Wizard 1 2 Click the Method Editor button located in UNICORN task bar Result The Method Editor module appears Click the Method Wizard icon located in the tool bar Alternatively choose File Method Wizard Result The Method Wizard appears The first page shown below contains a list of previously created and stored method plans and a New option for creating a new method plan The first time the Method Wizard is used the list only contains the Installation test The Note pane to the right gives a short summary of the chosen method plan Method Wizard E x Create or Change Method Plan Method plan Note Use thiz Method plan if you want to create a new plan 9 ACDS IEXDS H AC DS IEX GF fig AC GF ay GF FL Maintain J Prepare Method plan selected Delete Back Fintsh Cancel Help Set Default 18 1178 00 AA e p86 Method Wizard 5 The Method Wiz The table below describes the Method Wizard buttons ard bu
88. and action e Air bubbles passing through or trapped in pump Check that there is sufficient buffer in each liquid container Check all connections for leakage Use degassed solutions Purge the pump according to the procedure in 6 5 3 Purging the pump and inlet tubing on page 156 Check valves not functioning There might be dirt in the check valves Clean the valves see 8 4 2 Cleaning check valves on page 234 Blockage or partial blockage of flow path Flush through to clear any blockage by running e g System Wash If required replace the tubing and connectors see 8 6 2 Replacing tubing and connectors on page 249 Pump piston assembly leaking Replace the piston assembly in the pump head see 8 6 7 Replacing a damaged pump piston on page 280 See Irregular flow above See Irregular flow above e p 295 9 Troubleshooting and corrective actions 9 3 Monitoring problems Faulty air sensor Find the possible cause and action for a specific air sensor fault in the table below If the problem remains contact the local Amersham Biosciences representative Too high sensitivity No response Possible cause and action Wrong sensitivity setting Change to low sensitivity in System Settings in System Control Air might be trapped in the pump Purge the pump according to the procedure in 6 5 3 Purging the pump and inlet tubing on page 156 The air sensor is not connected Check the air sensor cab
89. anged the shape of the peak will change as well Hence the slope values might need to be changed accordingly Stop Collection The Peak_Max condition occurs when the UV signal has fallen to a fraction specified by Peak Max factor of the most recent peak maximum The Peak end is detected when the Peak_ Max condition is met AND e the signal falls below the Watch Level Less than value OR e a Valley is detected OR e a Watch StablePlateau condition is met The condition is met when the signal is stable within the limits of the Delta Plateau value during the Stable Time interval Peak SelectionSelect Largest Peak Based on either peak height or peak area to be transferred to the next column Area is the default value 18 1178 00 AA ep 126 Peak fractionation Method Wizard 5 For more information on peak handling and watch conditions see 5 5 3 Peak handling and watch conditions on page 116 The peak fractionation options and parameters for the protocols B D and F are described below The parameters define the final fractionation and how the final peaks are collected in the fraction collector Method Wizard E Desalting Final Peak Fractionation Advanced one Peak Fractionation Page Peak Fraction Size en 0 2 2 ml Peak Fractionation Algorithm Leve AN D_Slope x Start Level o 6000 6000 m U 100 0 010 10000 mU min Peak Mas Factor 0 5 0 1 Minimum Peak Width jas 0 15 1500 min Stop Level o 6000 6
90. any values in the Advanced Zone unless the consequences are fully understood 6 ep 149 6 Operation 6 4 Creating a method plan e On the Save As page select destination folder in the Method plan tree or create a new one if needed e Type any additional information in the Notes field Method plan CIP Regular Installation Test sea Purification Protocol E Affinity Step Desalting lon Exchange Gel Filtration Normal Affinity Column Sample Loading Level Running Condition Room Temperature Columns HisTrap_HP_1_ml Global HiPrep_26 10_Desalting Global RESOURCE_Q_1_ml Global HiLoad_16 60_Superdex_75_prep_grade Global Folder select a folder in Method plan tree Method plan name Cancel Create Folder Delete Help Note Itis not possible to move method plans between folders Type the name of the method plan and click OK Result The method plan is saved and can now be started in the System Control module 18 1178 00 AA ep 150 Operation 6 6 5 Preparing the system for a run About this section This section describes how to prepare the separation system for a run In this section This section contains the following sub sections Topic Preparing buffers and solutions Preparing outlet and waste tubing Purging the pump and inlet tubing Connecting columns and tubing Conditioning columns Preparing samples Preparing the fraction collector Preparing the system for automat
91. appears Continue with the instruc tions in 5 2 3 Prepare system options on page 100 Column Equilibration click Next The Column Equilibration page appears Continue with the in structions in 5 2 3 Column equilibra tion options on page 101 ep 99 5 Method Wizard 5 2 Creating a method plan 5 2 3 Using the Purify option Prepare system The options on the Prepare System page depend on the choice of purification options protocol In the table below identify the protocol used and follow the given instruction Protocol A E Prepare System Main Selections Purify Fill Sample Inlets with Buffer Prepare System Bice we Column Equilibration Last Page Clean Sample Inlets after Sample Loading i Yes C No Motel Enabling thie option will make the system pause after sample loading e Fill Sample Inlets with Buffer To fill the sample inlets with affinity buffer A before immersing them in the samples e Clean Sample Inlets after Sample Loading To clean all used sample inlets with a cleaning solution after sample loading Note During and after these operations the run will Pause and messages appear to guide through the operations The operator MUST click Continue to proceed the run Protocol F and G e Assisted Sample Loading To assist when filling the loops with sample Note When performing the filling the system will pause and messages appear to guide through the operation Glick Next 18 1178 00 AA ep 100
92. ase to each other by individual stepper motors controlled by a microprocessor Each outlet check valve houses a purge valve and a fingertight connector It is used for draining any unwanted liquid or to remove air from the system The purge valve is opened by turning it counterclockwise half a turn _ Purge valve pe Outlet check valve Rinse chamber outlet cae 18 1178 00 AA ep 310 Reference information 10 The inlet to each pump head is fed from a common manifold Liquid is drawn up into the pump head through a non return check valve by the action of the piston being withdrawn from the pump chamber On the delivery stroke of the piston the inlet valve is sealed by the pressure developed and eluent is forced out through a similar check valve at the outlet Outlet check valve Rinse chamber outlet Rinse chamber Return spring Inlet check valve Piston Piston seal Rinse membrane The pistons are actuated by cams eccentrics driven by the motors Force for the retraction of the pistons is provided by coil springs The length of stroke of the pistons is fixed and changes in the flow rate are made by varying the speed of the drive motor Piston seal rinsing system Leakage between the pump chamber and the drive mechanism is prevented by a piston seal The pistons and seals are continuously lubricated by the presence of liquid To prevent any deposition of salts from aqueous eluents on
93. at pressures up to 3 MPa The pump handles all liquid transport in the separation system Mixer The buffers used for creating gradients are mixed in a static mixer which is located directly after the pump The volume of the mixer chamber is 0 25 ml Pressure sensor Column handling AKTAxpress overview 3 A pressure sensor is connected directly after the mixer for continuous detection of the system pressure The column handling items are the column valve and two types of column supports a column block for small columns and column holder for larger columns Column block Column valve Column holder Holder for Superloop and rinsing solution tube Column valve The column valve is located at the upper left hand side of the front panel The valve makes it possible to select between up to five columns and a by pass tubing Column block The column block located directly above the column valve can take up to five small columns with fingertight fittings Column holder The rail on the left hand side is equipped with snap locks for fastening up to 2 large columns while the right hand side snap locks are used for a superloop and a tube for piston seal rinsing solution The online filter supplied can be connected to the top of the gel filtration columns using a 1 16 male M6 female connector UV and conductiv The UV cell and the conductivity cell are located at the middle part of the system ity detection Flow restrict
94. ation with high resol ution HiTrap Q HP Columns prepacked with Q Sepharose 1 and 2 x 1 ml see also 4 2 1 Coupled High Performance for high resolution columns on page 69 purification Mono Q 5 50 GL Column prepacked with MonoBeads 1 ml for purification with the highest resol ution e p 67 4 Methodology 4 2 Column alternatives 4 2 1 Descriptions of columns Gel filtration columns 18 1178 00 AA ep 68 Cation exchange columns Cation exchange columns are used to separate positively charged proteins The following cation exchange columns are supported in AKTAxpress Supported cation IEX columns and volumes RESOURCE S 1 and 6 ml HiTrap SP HP 1 and 2 x 1 ml see also 4 2 1 Coupled columns on page 69 Mono S 5 50 GL 1 ml Characteristics Columns prepacked with SOURCE 15S Fast purification with high resol ution Columns prepacked with SP Sepharose High Performance for high resolution purification Column prepacked with MonoBeads for purification with the highest resol ution Gel filtration GF columns separate proteins with differences in molecular size They are often used in a final polishing step Gel filtration separation results in high quality samples of homogenous size The method could also be suitable e g for the separation of protein monomers from protein dimers and trimers The following gel filtration columns are supported by AKTAxpress Supported GF columns and volume
95. ault parameter values depend on the chosen column Note Make sure that the parameter values do not exceed the specification of the chosen column Flow rate Pressure The first ion exchange page that appears contains options and parameters related paretets to flow rate and pressure as described below i lon Exchange Intermediate Flow Rate 7 Pressure Advanced one Flow Aate Fage E guilibration E 0 65 ml min 1 Wash J D 65 ml min 4 D 65 ml min Pressure Limit fi s 0 3 MPa Elution 1 Fa Di Md Equilibration Flow rate when equilibrating the ion exchange column Wash Flow rate when washing out unbound sample from the ion exchange column after sample injection Elution Flow rate during the elution Pressure Limit Upper pressure limit during the ion exchange step Volumes Wash The volume and wash options and parameters are described below Conc B paramet ers Method Wizard lon Exchange Intermediate Volumes 7 Wash Conc B Advanced Zone E quilibration volume f 0 399995 CH Fage 1 Wash Volume 2 0 999999 Cy W Define Wash Concentration B Wash Concentration fo D 100 8 N 2i M Equilibration Volume Volume used for equilibrating the ion exchange column Wash Volume Volume used for washing out unbound sample from the ion exchange column after sample injection Define Wash Concentration B Concentration B of ion exchange buffer B2 for washing out the unbound sample 18 1178 0
96. c Amount Teni Target conc Target Mw 120 6000 kDa 01 systemt 001 Sample1_ Coeff mg ml mg mg ml vol Baseli 1 A2 A4 5 9985 3656 6970 0 9520 3 3770 20 2568 10 0000 2 0257 aseline 2 A5 AB 3 4342 270 5575 0 9520 0 4138 1 4210 10 0000 0 1421 Zero Baseline x Fraction curve Add to Pooling protocol 01 systemt 001 Sample1_ x z z View Pooling protocol Note Only adjacent fractions will be pooled The fraction numbers for each pool are listed in the table as a range in retention order e g A6 A7 etc If the extinction coefficient has been entered in the wizard in System Control concentration and amount are automat ically calculated Otherwise enter the extinction coefficient manually by marking a pool and then typing the value in the extinction coefficient field The concentration is calcu lated in mg ml or M and Amount ts calculated in mg or mole Evaluation 7 2 The pooled fractions can be adjusted manually e To include or exclude adjacent fractions in a pool Click the numbered marker under the pool and drag the sideline To add more pools Click between the droplines under a fraction to create a new pool and drag the sidelines to include more adjacent fractions e To delete pools Click the numbered marker to select the pool and click the Delete button Click the Delete All button to clear all pools e To restore the pools created by UNICORN Click the Default Pool button 3 To select anot
97. c flow control has not been disabled in the Advanced Zone A 5 ml affinity column instead of a 1 ml column is recommended for large sample volumes more than 20 30 ml Clean or exchange columns Check the online filter if used It can become clogged if unfiltered buffers are used There might be dirt or residues in the flow path Clean the system using a method plan for system cleaning see 6 8 2 Cleaning system on page 198 If using a highly viscous sample dilute it to ease sample loading Check the calibration of zero pressure see 8 5 1 Calibration of zero pressure on page 242 18 1178 00 AA ep 284 Troubleshooting and corrective actions 9 Sample loading Find the possible cause and action for a specific problem in the table below problems Sample loading failure Possible cause and action Make sure that no air has entered the inlet tubing when moving the tubing from buffer solution to the sample Do not place the sample inlet tubing too close to the bottom of the sample tube Air bubbles might otherwise be created causing the sample loading to stop due to the air sensor Purge the pump to remove trapped air bubbles see 6 5 3 Purging the pump and filling the inlet tubing on page 156 Check the sample inlet tubing connectors A ferrule could be distorted or a connector tightened too hard Cut the tubing end using a tubing cutter and replace the ferrule If air bubbles keep appearing in the samp
98. ce 8 2 Monthly maintenance 8 2 Monthly maintenance About this section This section describes the monthly maintenance In this section This section contains the following sub sections Topic Checking flow restrictor Changing pump rinsing solution 18 1178 00 AA ep 222 Maintenance 8 8 2 1 Checking flow restrictor Maintenance inter Every month val Reason for main A flow restrictor opening at too high pressure will cause a high pressure alarm tenance from the system In exceptional cases this can result in column rupture A flow restrictor opening at too low pressure might cause air bubbles in the buffer solution in the flow path The position of the flow restrictor In addition a flow restrictor is integrated in the mixer Checking flow re To check the flow restrictor strictor Immerse inlet tubing A1 into deionized water Start a flow from A1 to outlet valve waste by e Select System Control Manual Flowpath e Select Injection Valve and Inject Click Execute 2 Start a flow from A1 to injection valve waste by e Start UNICORN and select System Control Manual Flowpath e Select Injection Valve and Waste Click Execute e Select System Control Manual Pump e Select a flow rate of 5 ml min Click Execute p 223 8 Maintenance 8 2 Monthly maintenance 8 2 1 Checking flow restrictor 4 Inspect the pressure curve in the UNICORN System Control window MPa Pressure 0 5 0 0
99. ck the connectors for leakage Running the tubing leakage test see 8 4 4 Checking tubing leakage on page 238 Adjusting the frac The fraction collector tubing should be adjusted in height when replaced tion collector tubing 1 Adjust the capillary tubing end to match the height of the microplate The capillary end should be positioned about 1 mm above the micro e p 253 8 Maintenance 8 6 Replacement procedures 8 6 2 Replacing tubing and connectors Specification of system tubing AKT Axpress has the following tubing connections for the system flow path For information about the piston rinsing tubing see 8 6 2 Specification of piston rinsing tubing on page 261 7 Waste cay Outlet Valve Fraction Collector Loop O Q ov po Bypass O ee Lease Loop Valve Manual LW LoopV2 inject ColV Column Valve MixInj Inlet Valve Press s3 Mixer Samples c A EFA a 2 AOA CSCA dela AirS Buffers Air sensor 18 1178 00 AA ep 254 Label bold or designa Maintenance Material Inner diamet Description Length mm er mm 1500 Teflon ON ON ON Buffer inlet switch valve 1 port NO 1500 Teflon ON Buffer inlet switch valve 1 port NC 1500 Teflon ON Buffer inlet switch valve 2 port NO 1500 Teflon
100. cols it is only possible to load up to 3ml per sample loop if using two HiTrap De salting columns in series 10 ml per sample loop if using a HiPrep 26 10 Desalting column ep 287 9 Troubleshooting and corrective actions 9 2 Problems during a run No satisfactory elution Possible cause and action from IEX column e Check that the eluted peak from the previous step was collected in a capillary loop by inspecting the peak start and peak end markers in the chromato gram If not collected Open the used method plan and enter the Ad vanced Zone Change the watch parameters used in peak collection and run the sample again Check that the appropriate size of desalting column was used in the previous step If the separation is not good enough Open the used method plan and enter the Ad vanced Zone Change the length and or slope of the salt gradient or Decrease the flow rate If the problem persists create a new method plan using a column that will give a higher res olution result see 4 Methodology on page 53 If the protein did not bind to the column it was directed to one of the fraction outlets F7 F10 To protein binding change the binding buffer or adjust buffer pH Check that the appropriate anion cation ex change column is used To collect all IEX peaks run the sample again us ing the AC DS IEX protocol All peaks will then be collected in the microplate 18 1178 00 AA ep
101. com 18 1178 00 AA ep 10 Preface 1 1 3 Safety Read the Safety Read the safety warnings and cautions before starting the installation Handbook l l l l i l l The warnings are available in English German Spanish French Italian and Swedish in the AKTAxpress Safety Handbook epll 2 Introduction 2 Introduction About this chapter This chapter contains an introduction to AKTAxpress and the basic operating principles It also gives a background to multi step purification of recombinant proteins and general information about the user documentation In this chapter This chapter contains the following sections Topic Introducing AKTAxpress Purification of recombinant proteins Basic operating principles AKTAxpress user documentation 18 1178 00 AA ep 12 Introduction 2 2 1 Introducing AKTAxpress Introduction This section gives a general overview of AKTAxpress AKT Axpress AKTAxpress is a liquid chromatography system intended for automated multi step purification processes The system has been developed and optimized for purification of His tagged and Glutathione S Transferase GST tagged recombinant proteins from clarified cell lysates It supports a number of purification protocols containing up to four chromatography steps for example affinity chromatography followed by desalting ion exchange and gel filtration All supported multi step protocols include tag cleavage as an option
102. cteoviseniesadvenngeicaednvamabobwuseremiadacineiontais 186 OO oane a manua APU eae a E 192 O DUNNE a a AEE 193 6 8 Procedures after a run asnssnsnnnnnnnnnnnusnnnnnnnnnunnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnn nnne 195 Gole IE MDLV IAS AHE Diale tseisean a a E eina 196 26 2 CleaMNe SYSTE Nissa A E NEA 198 676 55 Ceann e COUMANS ea hang atntenl genenasteacentdateieaiveaonnin 202 6 8 4 Performing metal ion stripping of affinity COIUMNS ccccceseeeeeeeeeeeeeeaeeees 206 Ta Evalia tioN iinn asada aun cece racy sdaueein cede a 208 7 1 Evaluation procedure OVELVICW cccceceeeeceeeeeteeeeeeeteeeaeeateaeaeeatsataeeaneataeeantatanes 209 732 Finding ANG ODEHING FOSUTSsiectei ai ccuads samtanidoenteawaade nes stadei aa meneanansavensadeaaaaneutuns 210 Jao VIC WINS FCS UNIS occas aiestaacen a nnd cannaaka tet aiede T R O 212 7 4 Pooling fractions and adjusting the POols ccccceceseseeeeeeeeeeeeeeeeeeeteaeaeeneataees 213 Tce COCAINE DOON DFOLOC ONS sinc sacsuayiaae seteneenseweayanisdiantiieddeainedenaparsouabercaresaenninn 216 KOs TINTING VE OOM sak tunecaareiaatnuddinatiea a a 218 8 Mante ANN G smsna a aAA 220 8 L Maintenance Progr aMrieoseresig anma a eea 221 8 2 MONTY MAINTE NANG ssnin sateaneu sade hccnduet teen vei netandeumeaeesstaneet 222 o2 va CMECKING TIOW TESIC Lawes 42cis anutein anton E E 223 8 2 2 Changing pump rinsing SOM TOM sxavwairsacathnacinanarudedeauindaaleutindeandcinad aaancsiaaents 225 8
103. ction of the microplate The movement of the plate might destroy columns or tubing positioned in front of the fraction collector 18 1178 00 AA ep1 6 Filling the superloop Operation 6 Attach the superloop to the column holder on the right hand side 2 Remove the loop connected between port OUT 5 and IN 5 in the loop valve Use the fingertight key if necessary 2 Connect the tubing from the top of the superloop to the loop valve port OUT 5 3 Connect the tubing from the bottom of the superloop to the loop valve port IN 5 The superloop should be manually filled with protease solution The Method Wizard contains an option for assisted manual loading of the superloop in preparation and purification runs Dialog boxes will then automatically appear when starting the run requesting the superloop to be filled To fill the superloop Connect a Luer fill port to the injection valve port MANUAL INJEC TION 2 Fill a syringe with at least 5 ml of affinity buffer A or the buffer used in the protease solution e p 177 6 Operation 6 5 Preparing the system for a run 6 5 8 Preparing the system for automatic affinity tag removal When starting a run the system enters Pause mode When pressing Continue a dialog box appears requesting the syringe to be fitted e Fit the syringe in the fill port and click Continue Do not inject the buffer before clicking Continue System Control SYS_1 Message fel Ez Inse
104. d with a step gradient using buffer inlet A3 and the peaks collected in loops The largest peak is selected 4 The nonselected peaks are emptied from the loops to the collection tubes in outlet F7 F10 one for each sample The loops are then flushed with buffer A2 to waste The table below describes the elution procedure of the AC column s The table below describes the desalting procedure of the collected peak volume A System Wash is performed to fill the system with buffer A2 2 The AC peak collected in the loop is loaded onto the DS column peak volume flush volume 3 The DS column is eluted with buffer A2 and the peak volume collec ted in a loop loop is then flushed with buffer A2 to waste 4 The excess of the selected peak from the AC column is emptied from the loop to the collection tubes F7 F10 one for each sample The Ion exchange Gel filtration AKTAxpress overview 3 The table below describes the ion exchange procedure of the collected peak volume A PumpWash is performed to fill the pump with buffer A2 2 The DS peak collected in the loop is loaded onto the IEX column peak volume flush volume The loop with the loaded peak is flushed with buffer A2 to waste 4 Unbound sample is flushed from the IEX column to waste with buffer A2 Peaks are collected in outlet F7 F10 The IEX column is eluted with a buffer A2 B2 gradient and the peak volumes are collected in loops The nonselected peak
105. detector current is fed to the signal processing circuitry in the system Optical unit Outlet Flow Photo Filter Lens Beam splitter cell detector Photo detector LC 18 1178 00 AA ep 318 Lamp voltage Microprocessor Conductivity monitor Pressure sensor Reference information 10 The reference signal comes from the same point in the lamp as the signal measuring the sample thus assuring a stable baseline by eliminating the effects of variations in lamp intensity The Hg lamp emits light only at certain wavelengths It does not emit light at 280 nm so for this wavelength the light is converted at a fluorescent surface before it passes the filter On the lamp housing there is a special exit for 280 nm light which means that the lamp position needs to be changed when working with this wavelength The conductivity monitor flow cell is positioned below the UV housing The flow cell has two cylindrical titanium electrodes positioned in the flow path of the cell An alternating voltage is applied between the electrodes and the resulting current is measured and used to calculate the conductivity of the eluent The monitor controls the AC frequency and increases it with increasing conductivity between 50 Hz and 50 kHz giving maximum linearity and true conductivity values The conductivity is automatically calculated by multiplying the measured conductance by the flow cell s cell constant The cell constant is
106. during Pump Wash For more information about the instructions click Help in the dialog box ep 201 6 Operation 6 8 Procedures after a run 6 8 3 Cleaning columns 6 8 3 Cleaning columns Introduction When running different types of purification methods and different samples after each other the columns should be cleaned between the runs or according to the column instructions This will remove unspecific bound proteins and prevent column clogging CIP Column is a Method Wizard option specifically made for cleaning columns The method is adapted to the columns used when setting up the method plan in the Method Wizard e g flow rate and pressure settings column volume etc Prerequisites The following are required for cleaning the columns e Cleaning solutions for example 0 5 M NaOH and deionized water Up to nine cleaning solutions can be used in one cleaning run See the column instructions e A method plan for cleaning the columns Creating a method To create a method plan for column cleaning in place plan for column cleaning in place Step Action In the Method Editor module in UNICORN start the Method Wizard by for example selecting File Method Wizard Click Next to create a new method plan Create or Change Method Plan Method plan Note MEN Use this Method plan if you af dz iex prep col plan af d prep ds at ds af gf tag cleay 2 GF gt DA a 1 Wa T 1 Hain Selections Hain Selections
107. e The requested information in the wizard is filled in See also 6 6 1 Starting a run using a method plan on page 186 The last page in the wizard Summary specifies the method require ments totally and for each of the systems It is recommended to print this list since it will not be saved When clicking Run on the Summary page all systems enter Pause mode A final check of the setup can be made 4 The run is started by clicking Continue or Continue all or by pressing the Continue button on the front panel ep 49 3 AKTAxpress overview 3 3 Purification process overview 3 3 2 Description of the process steps Loading the sample onto the AC column Elution of the AC column Desalting 18 1178 00 AA ep 50 The table below describes how the sample is loaded onto the AC column The sample S1 is loaded onto the first AC column The inlet valve switches to Al when the sample tube is empty air sensor detection Air is removed through a Waste port in the injection valve Unbound sample is flushed from the column with buffer A1 and collected in a flowthrough collection flask 4 When using several samples during the run step 1 3 are repeated serially for all samples before proceeding to the next step A LoopWash of all loops is performed with buffer A2 A second wash is performed with buffer A1 B1 to wash out unbound sample If peaks are detected they will collected in F7 F10 The AC column is elute
108. e Sulphuric acid diluted Sulphuric acid Avoid Avoid medium concen tration Tetrachloroethyl Avoid Avoid Silicone PP and ene PE are not resist ant Tetrahydrofuran Avoid Avoid ETFE CTFE PP and PE are not resistant Avoid Pressure limit for PEEK decreases ee ee OKT O OK PP and PE are af fected by long term use Toluene Trifluoroacetic acid 1 Urea Trichloroacetic O acid 1 ep 335 10O Reference information 10 5 Ordering information 10 5 Ordering information Introduction This section lists the systems accessories and user replaceable spare parts available for AKTAxpress If not stated otherwise the code number contains a pack of one 1 unit Systems The following systems are available AKTAxpress system for system exten 18 6645 01 sion AKTAxpress set of 4 systems 18 6645 04 AKTAxpress set of 4 systems with 18 6645 05 computer Holders The following holders are available for the system Pump parts The following spare parts are available for the pump Pump piston seal kit including 2 pis 18 1112 04 ton seals and 2 rinse membranes Check valve kit including 1 inlet and 18 1128 66 1 outlet check valve Piston kit including piston spring 18 1112 13 seal and rinse membrane 18 1178 00 AA ep 336 Reference information 10 Monitor parts The following spare parts are available for the monitors UV monitor Item Hg optics with 254 280 nm filte
109. e absorbance curve does not return com pletely to the base line after peak elution the quality of the imidazole might be poor Change to imidazole with higher quality or increase the Peak_Max factor to be able to detect peak end GSTrap HP and FF Many proteins bind better to GSTrap HP and FF at room temperature than in cold room whereas other protein shows the opposite Adding 1 mM DTT or other reducing agent to the buffer might increase the binding capacity Tag cleavage High glycerol concentrations in the buffer might decrease the activity of some proteases For operation and optimization hints see 4 Methodology overview on page 53 and 6 5 8 Performing automatic affinity tag removal on page 175 Troubleshooting and corrective actions 9 No satisfactory elution Possible cause and action from Desalting column e Check that the eluted peak from the previous step was collected in a capillary loop by inspecting the peak start and peak end markers in the chromato gram If not collected Open the used method plan and enter the Ad vanced Zone Change the watch parameters used in peak collection and run the sample again If the protein co elutes with the salt peak the re commendation is to add a small amount of salt for example 20 mM to the desalting buffer However too high salt concentration might affect the binding capacity when using ion exchange in the following step For manual desalting proto
110. e choice of buffer and its pH for IEX chromatography depends on the pI of the target protein and if an anion or a cation exchange column should be used Normally the pH of the buffer is selected to be at least 1 pH unit below or above the pI of the target protein e If using an anion exchange column for example Resource Q use a buffer with a pH gt pl of target protein e If using a cation exchange column for example Resource S use a buffer with a pH lt pl of target protein Gel filtration is used to perform a high resolution purification of the protein The buffer should be chosen to match the activity or application that will follow e g crystallization NMR studies functional studies or storage ep 9 4 Methodology 4 3 Buffer alternatives 4 3 2 Buffer suggestions 4 3 2 Introduction Guidelines AC buffer sugges tions for His tagged proteins 18 1178 00 AA ep 80 Buffer suggestions This section contains buffer suggestions that can be used as a starting point for purification For optimal behavior in a specific protein purification optimization might be needed See also buffer suggestions in the column instructions Several aspects need to be considered when choosing buffers Guidance is provided in the instructions for each column and in the purification handbooks see 4 2 1 Descriptions of columns on page 66 Suggested buffers for AC runs with His tagged proteins When performing suggested
111. e seen on any surfaces the inlet and outlet check valves should be removed and cleaned separately see 8 4 2 Cleaning check valves on page 234 Slightly wet the new seal with water Place the seal in the hole on the pump head and press it down into position with a hard flat object e p 275 8 Maintenance 8 6 Replacement procedures 8 6 6 Replacing pump piston seal 18 1178 00 AA ep 2 6 With the pump head still facing downwards on the bench place the rinse chamber onto the head with the rinse ports in line with the inlet and outlet check valves The conical depression in the rinse chamber should be facing upwards ready to accept the new rinsing membrane Fit the rinsing membrane with the conical face down wards Place the drain plate on top of the assembly Use the two screws to lock the complete assembly together Note Align the drainage hole in the drainage plate with the inlet check valve the opposite side of the pump head marked OUT UP Maintenance 8 16 Wipe clean the piston and remove all finger prints Wet the piston with water and then insert it into the return spring With the pump head facing downwards on the bench insert the piston into the pump head by pushing it gently but firmly vertically downwards into the seal Note Do not push the piston at an angle to the head and DO NOT twist the piston 17 Turn the head so that the inlet valve and drainage hole are facing downwards and the text UP OUT o
112. e table below A detailed specification can be found on the Summary page in the Method Wizard in System Control See also the connection guide in this section for how to use unions with the columns Gel filtration or desalting column Left hand side Gel filtration column and or HiPrep desalting column Right hand side Superloop see also 6 5 8 Performing automatic tag removal on page 175 and tube for pump rinsing solution Note The number of affinity columns equals the number of samples ep 163 6 Operation 6 5 Preparing the system for a run 6 5 4 Connecting columns and tubing Note Insert stop plugs or non used connectors into empty ports in the column block to prevent dirt from entering the flow path Note Some applications require two columns of the same type connected in Connecting the column tubing series This is marked x2 in the column list in the Method Wizard Connect the tubing from the column valve to the columns according to the table below To avoid introducing air into the column run the pump at a low flow rate e g 0 3 ml min when connecting the fitting also when connecting an online filter to the gel filtration inlet tubing See also the connection guide below for how to use unions to connect the tubing to the columns OUT 5 Column block lower port 5 or in column holder Note No column should be connected to the bypass ports Connection guide The connection guide below describes how to
113. ection This section gives an overiew of the steps during a typical purification process In this section This section contains the following sub sections Topic Process flow chart Description of the process steps ep4 3 AKTAxpress overview 3 3 Purification process overview 3 3 1 Process flow chart 3 3 1 Process flow chart Introduction This section contains a flow chart of the process steps during a typical run with AKTAxpress Process flow chart The example below shows the main steps during a four step purification of a single sample of a protein Affinity AC Desalting DS Ion exchange IEX Gel filtration GF which is equal to protocol E in the Method Wizard Loading sample on AC lon exchange column e Pump wash e Sample loaded onto e Peak in loop loaded onto IEX column AC column e Loop for the loaded peak emptied and e Unbound sample flushed flushed from column e Unbound sample flush e Elution of IEX A2 B2 gradient e Peaks collected in loops and largest peak selected e Nonselected peaks emptied and loops flushed AC as DS IEX GF A PAS f wash elution protein salt elution fractionation E ffs H Sample Non selected peaks microplate loading f Flow through Elution of AC E Desalting Gel filtration e Loop wash e System wash e System wash e 2 nd wash to wash out e Peak in loop loaded onto DS e Peak in loop loaded onto unbound sample column GF colu
114. ection describes how to print reports from runs with AKTAxpress How he Soy a The table below describes how to print a Standard report format in the Evaluation module Open a result file 2 e Select File Report or e Click the Report icon Format lobal Chromatograrn Global Chromatogram_Peaks Global Chromatograrn_ Quant Global Chromatogram_FReport Global Full Report Global Installation_T est Global Apress sample Global Mpress2samples Global pressssamples ki l standard report e Select Xpress1sample Xpress2samples Xpress3samples or Xpress4samples report format depending on how many samples are included in the result file The report format XpressActive Chrom can also be selected if only the active zoomed window should be included in the report e Click the Edit button to modify the report format if needed e Click the Print button Result The Print dialog box opens e Choose what pages and how many copies to print e Click OK Note Printers are set up in the File menu of the UNICORN Manager 18 1178 00 AA ep 218 Evaluation 7 Customized re For instructions on how to print customized reports see UNICORN Online Manual ports e p 219 8 Maintenance 8 Maintenance About this chapter This chapter describes the maintenance activities for KTAxpress In this chapter This chapter contains the following sections Topic Maintenance program Monthly maintenance
115. ed in front of the fraction collector hindering the ejection of the microplate The movement of the plate might destroy columns or tubing positioned in front of the fraction collect or e p 173 6 Operation 6 5 Preparing the system for a run 6 5 7 Preparing the fraction collector Follow the instruction in the table below to install a microplate Press the Eject button to eject the fraction collector plate if necessary 2 Place the microplate on the sled and check that the labelling H and A match the labelling on the system hni a ee Check that the gap between the end of the tubing and the microplate is 1 2 mm Press the Eject button to load the collector plate if necessary 18 1178 00 AA ep174 6 5 8 Introduction Operation 6 Preparing the system for automatic affinity tag removal This section describes how to prepare the system for automatic affinity tag removal in AK TAxpress system Protocols for affin Automatic affinity tag removal can be combined with all AKTAxpress multi step ity tag removal Adding affinity tag removal to a method plan Example on pro tease calculation protocols To add automatic tag removal to a method plan On the Purify page in the Method Wizard select Tag Cleavage to add an affinity tag removal step to the method plan Choose Incubation Time and select Fill Columns with Cleavage buffer if a specific cleavage buffer other than the ordinary affi
116. ee Adding systems in UNICORN in AKTAxpress Installation Guide If trying to connect in view mode from a remote computer check that the local UNICORN work station is running 9 ep 299 9 Troubleshooting and corrective actions 9 6 Error code list 9 6 Error code list Introduction This section contains a list of error codes and their meaning The error codes are presented on the display of the separation system as a flashing code EXxx RUN PAUSE To enable identification of the system unit number the display will alternate between flashing the error code and the system unit number Error codes The following error codes can occur 51 57 60 61 100 105 18 1178 00 AA ep 300 System error Internal error Restart system If problem remains contact service System error Internal error Restart system If problem remains contact service Illegal mode exchange It could be caused by a WATCH instruction with action CONTINUE that occurred when the system was in RUN state Check the method Otherwise there could be an internal error Restart system If the problem remains call service System error Internal error Restart system If problem remains contact service Pressure calibration error Check that the system is at zero pressure Pump synchronization error By pressing END the pump will be synchronized If this doesn t help restart the system If problem remains contact service
117. efault Hi aintain E Installation Test C Strip Affinity Columns CIP Columns Regular C CIP Columns Rigorous 4 Customized E quilibration Clean System See the description and follow the instructions for the chosen opera tion in the tables below 2 Select the requested type of maintenance procedure on the Maintain page Click Next Method Wizard Main Selections Maintain CIP Columns Regular Last Page 18 1178 00 AA ep 102 Installation test options Strip affinity columns options Method Wizard 5 The table below contains a description and instructions for the Installation test options Maintain operation Description Instruction Installation Test Tests the function of the pump and the switch valves e Proceed to 5 2 4 Finishing the Maintain method plan on page 105 The table below contains a description and instruction for the Strip affinity columns options Maintain operation Description Instruction Strip Affinity Columns Strips metal ions off the affinity column s using 5 CV of strip buffer followed by 10 CV of deionized water 1 Select type of column number of columns and running conditions 2 Proceed to 5 2 4 Finishing the Maintain method plan on page 105 CIP Columns regu The table below contains a description and setting instructions for the CIP Columns lar options Regular option Maintain operation Description Instruction CIP Columns Regular Cleans
118. efzel Tefzel Tefzel Tefzel Maintenance 8 Label bold Description Length mm Inner diamet Material or designa er mm tion L1 capillary Loop valve 12800 Tefzel loop port 2 to 8 10 ml L2 capillary Loop valve 12800 Tefzel loop port 3 to 9 10 ml L3 capillary Loop valve 12800 Tefzel loop port 4 to 10 10 ml L4 capillary Loop valve 12800 Tefzel loop port 5 to 11 10 ml L5 capillary Loop valve 12800 Tefzel loop port 6 to 12 10 ml Outlet valve 260 PEEK port 2 to fraction col lector 1500 Tefzel Fraction out let outlet valve port 3 Fraction out 1500 Tefzel let outlet valve port 4 Fraction out 1500 Tefzel let outlet valve port 5 Fraction out 1500 Tefzel let outlet valve port 6 Fraction out 1500 Tefzel let outlet valve port 7 Fraction out 1500 Tefzel let outlet valve port 8 e p 259 8 Maintenance 8 6 Replacement procedures 8 6 2 Replacing tubing and connectors Label bold Description Length mm Inner diamet Material or designa er mm tion F9 blue Fraction out 1 Tefzel let outlet valve port 9 F10 green Fraction out 1 Tefzel let outlet valve port 10 Fraction out 1 Tefzel let outlet valve port 11 Injection 1500 1 Tefzel valve port 2 Injection 1500 1 Tefzel valve port 3 Outlet valve 1500 1 Tefzel port 1 Specification of KTAxpress has the following tubing connections for the piston rinsing tubi
119. einject and the loop valve to the position of the new capillary loop 18 1178 00 AA ep 248 Maintenance 8 8 6 2 Replacing tubing and connectors anne inter When required for example when a tubing has clogged va Reason for main Clogged tubing may contaminate or cause high back pressure and hence preventing tenance a successful purification run Required material Extra tubing is included in the accessory kit CAUTION Only spare parts approved or supplied by Amersham Biosciences may be used for maintaining and servicing the system Replacing tubing To replace the tubing 1 Set the system in Standby mode by pressing the On button This will disconnect the system from UNICORN RUN a p 249 8 Maintenance 8 6 Replacement procedures 8 6 2 Replacing tubing and connectors Loosen the connectors to remove the tubing The system contains two types of tubing e 1mm capillary tubing with fingertight connectors e 1 6 mm capillary tubing with M6 or UNF 5 16 air sensor pump inlet connectors including ferrules If the tubing has labels remove the labels to be used with the new tubing later Discard the tubing and connectors Cut the new tubing to the same length as the old tubing Use the cutting tool to get a straight angle cut Note When replacing system tubing use the original length to ensure that the correct delay volumes are maintained Inlet and outlet tubing may
120. ell a seuann y s thy I P Og i A l alternative co ia superloop pressure injection sensor valve waste air sensor Wa buffers buffers buffers Samples Ai B1 A2 B2 A3 8 1 4 The main components are as follows Combined sample and buffer pump e Switch valves for buffer selection and gradient formation e Separate rotary valves for sample and buffer selection manual injection column selection loop selection and outlet flow direction e UV and conductivity cells e p 15 2 2 1 Introduction The UNICORN control system 18 1178 00 AA ep 16 Introducing KTAxpress e Loops for intermediate fraction collection e Fraction collector with deep well microplate The columns required for the chosen purification can be attached to the system either by threading them into the column block small columns or by securing them with snap locks in the column holder large columns The separation system is equipped with a control panel with push buttons and display The push buttons allow the operator to start and pause a run The display indicates running status identity of the system error codes etc UNICORN is a complete package for control and supervision of AKTAxpress It consists of software for interfacing the controlling computer to the separation system via the USB CAN converter box supplied The software runs
121. ength mm or designa tion er mm Tefzel ColV Injection 350 valve port 6 to column valve IN Clin Column valve 350 port 6 to column 1 Clout Column 190 block port 1 to column valve port 12 C2in Column valve 350 port 5 to column 2 C2out Column 190 block port 2 to column valve port 11 C3in Column valve 350 port 4 to column 3 C3out Column 190 block port 3 to column valve port 10 C4in Column valve 350 port 3 to column 4 C4out Column 190 block port 4 to column valve port 9 CSin Column valve 350 port 2 to column 5 Tefzel Tefzel Tefzel Tefzel Tefzel Tefzel Tefzel Tefzel Tefzel e p 257 8 Maintenance 8 6 Replacement procedures 8 6 2 Replacing tubing and connectors Label bold or designa tion LoopV1 LoopV2 18 1178 00 AA ep 258 Description Column block port 5 to column valve port 8 Column valve port 1 to port 7 Column valve port OUT to UV cell UV cell to conductivity cell Conductivity cell to restrict or Restrictor to outlet valve port IN Outlet valve port 12 to in jection valve port 7 Injection valve port center to loop valve port IN Loop valve port 1 to port 7 Loop valve port OUT to injection valve port 4 Length mm 190 160 200 200 350 160 500 260 160 260 Inner diamet er mm Material Tefzel Tefzel Tefzel Tefzel Tefzel Tefzel T
122. ents are available before attempting to replace a spare part Note It is not possible to reinstall a used piston seal after removal Note Always replace the piston seals on both pump heads at the same time Note It is recommended to use degassed liquids Spare part and The following spare parts and tools are required tools required E e Seal kit 18 1112 04 containing 2 piston seals and 2 rinse membranes e 1 4 inch wrench e 3 mm hex key e Screwdriver e 100 methanol for running in the new piston seal Replacing piston To replace the piston seal seal and mem brane Step Action 1 Change solvent to deionized water and flush out all salt Move all input buffers bottles below the level of the pump heads to prevent siphoning CAUTION REPLACING SPARE PARTS Read the instructions carefully Check the orientation of each part before continuing with the next instruction Beware that for example some individu al parts of the pump head can be assembled incorrectly 18 1178 00 AA ep 272 Step 2 Maintenance 8 Disconnect the electrical power to the system to be maintained Remove the tubing e Disconnect and remove the tubing from the pump head outlets upper ports e Remove the tubing of the piston seal rinsing system plug in fit Unscrew the two white knurled screws under the pump to release the support bracket that locks the inlet manifold into the inlet valves Gently lower and remove the complete
123. eparation system symbol on the left the current status for the particular system can be displayed ee System Control SYS_1 Method Wizard Generated Result o 0per_1 Wizard verification 20031030 ID56 SYS_1001 res File Yiew Manual Hold Continue End amp a Instant Run Continue all End all pes ia ie Status ee Volume Erem Volume io Time ie Time ao gt gt eres Pressure gt gt UV gt gt UVLamp gt gt Conc gt gt Cond gt gt Cond gt gt XYFRAC gt gt InletValve gt gt LoopSelect ssi EENE CT a Ee Eo E E E ColPos gt gt Outlet gt gt RemainTime i ensor gt gt ss2 Q iy Cond Cont Pressure Flow 7 Tea Na o e mAU 600 500 400 300 200 100 ml 10 0 10 20 30 40 50 60 70 80 90 4100 110 120 130 140 150 160 170 180 190 200 210 220 230 240 250 260 270 280 263 12 ml Watch_Off UV 263 12 ml End Block 263 12 ml Block Fractionation_lEX 263 12 ml Base SameAsMain 263 12 ml Block Peak_Frac_Parameters 263 12 ml Base SameAsMain 263 12 ml Peak_FracParametersUY Level AND_Slope 0 50 min 30 000 mAU 30 000 mAU 50 000 mAU min 100 000 mAU min 0 50 263 12 ml End Block 263 12 ml Block Fractionation_Volume 263 12 ml Base SameAsMain 263 12 ml Peak_Fractionation 0 100 ml 263 12 ml End Block 263 12 ml End Block 263 12 ml Block Elution_lEX 263 12 ml Base SameAsMain 263 12 ml Flow 1 00 ml min Buffer 0 10 ml min Y
124. er to get the set target concentration Import File Location The path to the folder where import file is stored The import file might contain information about e g extinction coefficient and molecular weight of the proteins to be used For more information on the import file see 10 3 Import file format on page 331 Store Results in Main Folder By default the results are stored directly under the Home folder To store the results in a sub folder under the Home folder fill in the name of the folder in the field If the name of the folder does not exist the folder is created automatically ep 137 5 Method Wizard 5 5 Advanced Zone 5 5 10 Advanced Zone for Maintain 5 5 10 Advanced Zone for Maintain Introduction This section describes the Advanced Zone for a Maintain method plan The following method plans allow parameter values to be changed e Stripping Affinity Columns e Clean System All method plans allow defining a main folder for storing the results Note Make sure that the parameter values do not exceed the specification of the chosen column Stripping affinity To change parameter values in a Stripping Affinity columns method plan columns Select the required options on Page 1 and click Next Select General Advance Settings to define a main folder for storing the results Method Wizard Advanced one Select type of varnables to change Advanced one Page M Maintain 1 4 IW General Advanced Se
125. es 263 12 ml Block Gradient_Segment_1 263 12 ml Base CY 1 92 ml HiTrap_SP_HP_1_ml_x2 a a 263 12 ml Gradient 50 0 B 20 00 C A2 B2 su gt For Help press F1 Run Gradient_Seqment_1 No watch Controlled By Pia By clicking Expand in the left hand upper corner more information on the current status of each system is displayed By clicking Collapse the information disappears System Control SYS 3 Method Wizard Generated Result c Default 005 res File Yiew Manual System Help Heid Continue End Wa Jui Instant Run Continueall End all lt lt Collapse EZES Method Step Logbook E oe svs1 sys2 ep 193 6 Operation 6 7 During a run Up to three view panes Run Data Curves and Logbook can be displayed showing different aspects of the run e The Run Data view pane displays the current values for selected run parameters e The Curves view pane displays the monitor signal values graphically e The Logbook view pane shows the actions as the run proceeds All actions and unexpected conditons are logged with date time and current user name The log book provides a complete history of the run and is saved in the result file To customize the view panes right click in the respective view pane and select Properties For more information about customizing the view panes see the UNICORN user documentation Ending the run To stop the run on a system before it is finished Chec
126. es universal computer functions will not be explained e UNICORN must be installed and configured correctly on the computer e You need to understand the basic concepts of liquid chromatography Terminology and functionalities will be explained only when they differ from normal practice e Before you try to operate AKTAxpress you must study and understand AKTAxpress Safety Handbook AKTAxpress overview 3 3 AKTAxpress overview About this chapter This chapter contains an overview of the UNICORN control system and a brief physical and functional description of the components in AKTAxpress It also contains an overview of the purification process steps In this chapter This chapter contains the following sections Topic UNICORN overview Separation system overview Purification process overview ep 23 3 AKTAxpress overview 3 1 UNICORN overview 3 1 Introduction The UNICORN control system Software modules Method Wizard 18 1178 00 AA ep 24 UNICORN overview This section is an overview of the UNICORN control system UNICORN is a complete package for control and supervision of AKTAxpress It consists of software for interfacing the controlling computer to the separation systems via the USB CAN converter box supplied The UNICORN software runs under Microsoft Windows UNICORN consists of four integrated modules which are described in the table below UNICORN Manager File handling and admin
127. essage appear requesting the inlet tubing to be moved to the cleaning solution After the cleaning the system will Pause The run then has to be manually continued Note The system will Pause during the run when all samples are loaded onto the columns A message will appear requesting the inlet tubing to be moved to the cleaning solution After the cleaning the system will Pause The run then has to be manually continued Volume Needed to Remove Air The volume used to remove air from the pump after the inlets have been filled 18 1178 00 AA ep 134 Method Wizard 5 Prepare column The Prepare Column page contains options and parameters related to column ponamnenees preparation before the sample loading Method Wizard Prepare Column Advanced one Affinity Page Equilibration Volume 0 999999 Cy 1 2 Desaking 3 E quilibration Volume 0 999999 Cy 4 Last Page lon Exchange Flow Rate 0 65 ml min Equilibration Volume 0 999999 Cy Gel Filtration Flow Rate 0 65 ml min Equilibration Volume 0 999999 Cy Bi Wi Equilibration Volume Volume used for equilibrating a column Optional for all columns Flow Rate Flow rate when equilibrating a column Optional for ion exchange and gel filtration columns only e p 135 5 Method Wizard 5 5 Advanced Zone 5 5 9 Advanced Zone for Purify General advanced settings 0 9 9 Introduction The General Ad vanced Settings page Empty loops
128. ext breakpoint in the running method Pressing the button in other modes will issue an error to UNICORN ep 43 3 AKTAxpress overview 3 2 Separation system overview 3 2 5 Indicators and controls Eject Load microplate In END mode Eject or load microplate In PAUSE mode Eject or move the microplate to the position it had when the system was paused In RUN mode No function Indicators The separation systems are equipped with the following indicators Run Green indicator A run is active Pause Yellow indicator The system is in PAUSE mode Status display Normal operation System unit number 1 12 for example 12 e Error 3 digit error code for example E416 UV lamp broken Status indication The indicators can show the following status Standby The system is in standby All active parts are turned off The system can be turned on by pressing the On button RUN Idle No activity The display shows PAUSE the system unit number e g 12 Run Run A run is performed PAUSE Run Pause The system is paused either PAUSE manually or automatically by UNICORN 18 1178 00 AA ep 44 AKTAxpress overview 3 RUN Communication error There is no con PAUSE nection with the AKTAxpress PC driver Two segments flashing RUN Error The system is paused and an er PAUSE ror code is displayed For a description of the error codes refer to 9 6 Error de list 300 Display
129. fication Protocol A Affinity Step Gel Filtration Tag Cleavage Affinity Column Sample Loading Level i Normal High Running Condition f Room Temperature Cold Room Columns Affinity Hie Trap HP_1_ rl Global Gel Filtration Hiload 16 60 Superdex 75 prep_grade Global Fost Run Instructions Reequilibrate Affinity Columns Get Default The table below describes the Purify dialog options and the related actions during Description related ac tions Includes step for remov ing the affinity tag from the recombinant protein See 4 1 4 Affinity tag re moval on page 62 for more information e p 97 5 Method Wizard 5 2 Creating a method plan 5 2 3 Using the Purify option To perform tag cleavage not avail able for Protocol F G tag cleavage not avail able for Protocol F G all types of purification runs except Protocol F G all types of purification runs all types of purification runs 18 1178 00 AA ep 98 Select appropriate Incubation Time Information on factors affecting the cleavage time can be found on 4 1 5 Condi tions for cleavage on page 64 Fill Columns with Cleav age Buffer appropriate Affinity Column Sample Loading Level actual Running Condition the Column s to be used For information about appropriate columns see 4 2 2 Choosing Columns on page 70 Description related ac tions The time required for the added protease to c
130. for many applications a single step purification using affinity chromatography is sufficient to achieve the desired level of purity However if there is no suitable fusion system or when higher degree of purity is required a multi step purification process will be necessary AKTAxpress has been developed to meet the growing demand for quick and easy purification of recombinant proteins with high purity The system is based on purification strategies that involve combinations of protein capture on affinity columns buffer exchange in desalting columns intermediate purification in ion exchange columns and polishing in gel filtration or ion exchange columns Consequently AKTAxpress supports a number of purification protocols containing up to four steps The intermediate protein peaks between the steps are collected in loops The purification protocols in AKTAxpress are primarily designed for purification of the most common fusion proteins His tagged and Glutathione S Transferase GST tagged proteins Tag removal is optional in all protocols Up to twelve AKTAxpress systems can work in parallel where each system can handle up to four different samples through the whole purification procedure independently of the other systems This increases the protein production throughput which is a key issue for example in the field of structural and functional genomics 2 3 Introduction Sample applica tion and wash Elution Inter
131. he syringe When fluid starts entering the syringe continue to draw a few milliliters before closing the purge valve Check that there is no visible air left in the A1 tubing ta oh Repeat the steps 2 3 and 4 for the right pump head Check that the outlet tubing is not blocked Connect a thin capillary or a column that will give sufficient back pressure see below Reconnect the mains power cable to the system Run at a flow rate of 1 ml min at a back pressure lt 0 5 MPa for 15 minutes Run at 20 ml min at a back pressure of 2 3 MPa for 15 minutes Finally perform system cleaning see 6 8 2 Cleaning system on page 198 ep2 9 8 Maintenance 8 6 Replacement procedures 8 6 7 Replacing a damaged pump piston 8 6 7 Replacing a damaged pump piston Introduction Typical symptoms of a damaged piston are observed as e excessive piston seal wear e unstable pressure e a reduction in the flow e noise as the piston moves The piston should be removed examined for damage or salt precipitation and then replaced with a new piston if necessary If a damaged piston has been in operation the piston seal will be destroyed and should also be replaced Spare parts and The following spare parts and tools are required tools required l l E l l l Piston kit 18 1112 13 containing piston spring seal and rinse membrane e 1 4 inch wrench e 3 mm hex key e Screwdriver e 100 methanol for running in the new piston seal Repl
132. her baseline curve click the Default Pool button in the Baseline droplist Note To calculate concentration and amount for peaks that have not been fractionated perform a simulated peak fractionation See UNICORN Online Manual for more information e p215 7 Evaluation 7 5 Creating pooling protocols 7 5 Creating pooling protocols Introduction Information about pooled fractions from different result files can be assembled in the pooling protocol The pooling protocol can be printed for use when pooling the samples physically This section describes how to add pools to the pooling protocol and how to print or export the pooling protocol Creating pooling Once the pools have been satisfactorily adjusted they can be added to the pooling protocol protocol The table below describes how to add pools to the Pooling Protocol and send the list to a printer or export the list to a file Open a result file in the Evaluation module Make sure the pools are satisfactorily adjusted See 7 4 Pooling and adjusting the pooling on page 213 Click the Add to Pooling Protocol button Result The pooled fractions from the active result file is added to the Pooling Protocol e Click the View Pooling Protocol button Result The Pooling Protocol dialog box opens Fractions to be pooled System Result Sample Id Pool Conc Text End vol RN LAB 21 Example Result002 A11 B12 0 7500 0 0000 B11 B10 0 5000 0 0000 B8
133. hly specific and a one step purification therefore often results in a reasonably pure material Purification of His or GST fusion proteins is simple and uses mild elution conditions that minimize the risk of damage to the functionality of the target protein Methodology 4 4 1 3 Description of proteases Introduction This section describes different types of proteases that can be used for tag cleavage About proteases Proteases can be tagged or non tagged When using a His or GST tagged protease the cut off target protein can be eluted while the protease is still bound to an immobilized metal affinity chromatography column IMAC or a Glutathione affinity chromatography column When using a non tagged protease further purification is necessary to separate the target protein from the protease Protease types The following list describes a few proteases commonly used for tag cleavage Optimal in Codenumber Comment cubation tem perature Common re cognition se quence Protease marks the cleavage site Thrombin non tagged Factor Xa non tagged PreScission LVPR GS 27 0846 01 Cuts with clean ends if an N termin al affinity tag is used 27 0849 01 27 0843 01 LEVLFQ GP GST tagged e TEV protease His tagged ENLYFQ G Invitrogen Highly specif ic Low op timal temper ature Highly specif Ic Note Low incubation temperature reduces risk of p
134. holder optional WARNING N Never place flasks on the top of the separation system If they become full and overflow liquid might penetrate the system causing a short circuit 18 1178 00 AA ep 42 Fraction collector The fraction collector is a X Y collector made for 96 square well microplates 8x12 well positions The volume of a well is typically 2 2 ml Nae Lip 5 K O 3 2 9 Introduction Overview Push buttons AKTAxpress overview 3 Indicators and controls This section describes the indicators and controls on AKTAxpress For information about the computer equipment refer to the manufacturer s instructions The separation systems are equipped with the following indicators and controls Run indicator Pause Continue x gael Pause indicator jer cag Status BN O RUN m E j i W On Next T Standby breakpoint Reset Eject Load fraction collector The separation systems are equipped with the following push buttons On Standby Reset In STANDBY mode Power on Reset of separation system incl pump synchronization In END mode Standby No power to active com ponents Pressing the button for 5 seconds in END RUN PAUSE mode Reset of separation sys tem incl pump synchronization Pause Continue In RUN mode Pause In PAUSE mode Continue Next breakpoint In RUN mode Go to n
135. housing and discard it WARNING N The UV monitor has a mercury Hg lamp that contains small amounts of mercury The lamp 18 1178 00 AA ep 266 Maintenance 8 Slide the lamp housing firmly into place and check that the detector housing is in its right hand position oh gawk oy LF NO YO diy Y A click will indicate that the housing is in position OURO 4 10 Open the cap covering the filter wheel and check that the filter wheel is in the 280 nm position If this is not the case turn the wheel into the correct position A click will indicate that the wheel is in position Fix the new lamp cable in the cable duct and connect it at the back of the system A rs II JA T C D gt H i j uy A QZ J f T B J We YZ LANN PA TA D E IV 7 f HEN a gt g D S A AD y a A y N Hy Ji jj LL A A i jj UN M AA JN l N L lt n 2 ae Si x eo S TN NAI i y y i j ee T M A S i N A Y di J ep 267 8 Maintenance 8 6 Replacement procedures 8 6 4 Replacing UV lamp 12 Connect power to the system and set the unit in Idle mode Note If alow intensity warning is given even after a change of UV lamp the filter may need to be changed Contact a local Amersham B
136. ic affinity tag removal Checking the tubing Using air detection Using flow control during sample loading e p 151 6 Operation 6 5 Preparing the system for a run 6 5 1 Preparing buffers and solutions 6 5 1 Preparing buffers and solutions Introduction This section describes where the inlet tubing from buffers and solutions should be connected and where the containers should be placed Liquid quality re For best purification results use deionized water and high purity chemicals Filtering commendation of liquids through a 0 45 pm filter and degassing the liquids is recommended Note Do not use organic solvents in buffers The mixer is designed for use with water based solutions only If organic solvents are used incorrect gradients or incomplete mixing might occur Preparing the con e Prepare required buffers according to the chosen purification protocol and fill aus the containers e Place the containers on a suitable shelf under the bench Inlet supply fora Equilibration binding elution and washing buffers as well as cleaning solutions purification run 6 introduced into the system through the inlet tubing A1 A8 B1 and B2 Running a purification method The table below shows the standard buffer and solution supply for a normal purification run See also 4 Methodology on page 53 e Immerse the inlet tubing in the containers according to the table also shown on the Summary page in the Method Wizard i
137. icking Next A To perform a pump wash before the cleaning step select PumpWash Enter an appropriate Flow Rate according to the instructions supplied with the column Select the buffer Inlet to be used Enter an appropriate Volume in column volumes according to the instructions supplied with the column Enter a pause time in Pause before Next Step if in cubation of the column in the chosen solution is required Proceed to 5 2 4 Finishing the Maintain method plan on page 105 18 1178 00 AA ep 104 Method Wizard 5 Clean system op The table below contains a description and instructions for the Clean System option tions Maintain operation Description Instruction Clean System Cleans the chosen tubing loops and valves with one cleaning solution at a time When running the meth od the method will pause and a message appear when the inlets should be inserted into a new cleaning solution The loops column valve and outlets will be cleaned using inlet A1 Select the Sample InletsS 1 S4 and the Buffer Inlets A1 A8 B1 and B2 to clean Select Clean Outlets to clean all outlets A micro plate must be placed in the fraction collector Select Clean loop 1 4 to clean all four loops If Loop 5 is selected it must be a capillary loop Select Clean Column Valve to clean all column pos itions All positions require bypass tubing 5 Select Number of Cleaning Solutions to use 6 Proceed t
138. ification of recombinant proteins This section introduces the reader to purification of recombinant proteins and thus gives a background to the development of AKTAxpress Success in isolation and purification of a target protein is highly based on the knowledge of the protein characteristics Protein purification serves to deliver a suitable amount of pure target protein in its native biological state for use in further studies The requirements on the end product may vary considerably For instance a certain enzyme may be considered pure enough for activity studies at a relatively moderate mass purity provided that the end product is free of substances interfering with the activity of the enzyme Pure enough for structure analysis of a protein on the other hand normally means a rather high mass purity since interfering substances may affect the techniques used in structure determinations The purification itself is nearly never a final goal but rather a necessary step on the way to perform protein research in many different scientific areas The need to produce pure target proteins quickly and easily has increased the usage of recombinant proteins greatly in recent years So has also the wealth of techniques and products used for their amplification and purification The advantage of using a fusion protein to facilitate purification of a recombinant protein is widely recognized Fusion proteins are simple and convenient to work with and
139. ify Desalting Peak collection The peak collection options and parameters for the protocols C E are described parameters below The parameters define how the peaks are collected in the loops Peak Collection Max Volume in Each Loop 75 0 5 10 mi Mas Number of Loops a x Allow Collection of Single Peaks in Several Loops E Yes C No co sm oO mm A ty Max Volume in Each Loop The maximum volume allowed in each loop Using volumes above 7 5 ml might cause sample loss due to band broadening Max Number of Loops The maximum number of loops used to collect peaks in the desalting step Allow Collection of Single Peaks in Several Loops To collect single peaks with a volume larger than Max Volume in Each Loop in several loops Method Wizard Desalting Intermediate Peak Collection Advanced one Start Collection Page Watch Level Greater than m 6000 6000 m l 1 Watch Slope Greater than fioo 50000 50000 mAU min 2 3 Stop Collection 4 Peak Max Factor oe 0 1 5 watch Level Less than o 6000 6000 mL E Watch Stable Plateau T Stable Time 1 0 9999 min 8 Delta Plateau B 00 i0 6000 mAn J 10 11 Peak Selection Desalting gt lon Exchange 1 Select Largest Peak Based on 13 i Area Height Start Collection The peak collection starts when both the UV level exceeds the Watch Level Greater than value AND the UV slope exceeds the Watch Slope Greater than value Note If the flow rate is ch
140. in the wizard The import file may contain the following parameters for each sample ID Sample volume Isoelectric point Extinction coefficient Molecular weight Free text This illustration is an example of an import file containing information on three different samples FP GFP aFP GFP GFF APB APB APB APB APE APE APE APE APE APE His Volume 10 His pI 6 19 Hi5 Extcoett Oo 746 His Mw JB His FreeText Fi Yolume 10 7 pI 5 95 Extcoett o 4 Fi Mw 2a Green Fluorescent Protein 7 FreeText Unstable at room temperature 13 volume 10 13 pI 5 56 l3 Extcoeff 1 175 1s Mw r 13 FreeText Avoid pH below 5 5 The import file can be created in a text editor for example Notepad Follow the for mat description below The file name is optional but the extension must be txt Column 1 Sample ID Column 2 Parameters Column 3 Parameter values The parameter names must be written according to the example below Separate the columns by pressing Tab once Press Enter to create a new line The storage location of the import file is by default C UNICORN Server MethodWizardImport The path can be changed in each method plan see 5 5 9 Advanced Zone for Purify General advanced settings on page 136 ep 331 10O Reference information 10 4 Chemical resistance guide 10 4 Chemical resistance guide Introduction This section specifies the chemical resistance of AKTAxpress to some of the mo
141. ing Run the pump at 20 ml min for 1 minute e Set the flow rate to 20 ml min and click Execute e Click Continue to start the flow Run the pump for 1 minute e Stop the pump by clicking Pause Carefully move the inlet tubing A1 back to the flask containing water e Stop the pump by clicking End Carefully move the inlet tubing A1 to the flask containing the buffer that will be used during the next purification run Note Remember to fill inlet tubing A1 with the buffer to be used as described previuosly in this section 6 Run the pump at 10 ml min for 10 minutes e Set the flow rate to 10 ml min and click Execute e Click Continue to start the flow Run the pump for 10 minutes 18 1178 00 AA ep 162 6 5 4 Introduction Cautions Attaching the columns for a purification run Operation 6 Connecting columns and tubing This section describes how to connect columns and tubing that are used in a purification run CAUTION Tighten the columns properly in the column block to avoid leakage Over tightening might though rupture the column connectors CAUTION Make sure that no parts e g tubing or columns are positioned in front of the fraction collector hindering the ejection of the microplate The movement of the plate might destroy columns and tubing positioned in front of the fraction collector Attach the columns to the column block or the column holder for a purification run according to th
142. ing a method plan Introduction This section describes how to delete an existing method plan Deleting a method To delete a method plan plan In the Method plan list in the Method Wizard select the method plan to be deleted Method Wizard E Create or Change Method Plan Method plan Mote 2004 01 07 Installation Test Purification Protocol unification Protoco gt i m A Affinity Step Gel Filtration a AC DS IEX GF Normal Affinity Column Sample Loading Level EL GF i w Running Condition Room Temperature Columns HizTrap_HF_1_ml Global Hiload_16 60_Superndex_ 75 prep_grade Global Method plan selected JAC GF Delete i lt Back Next gt Finish Cancel Help Set Default Click Delete Result A confirmation dialog appears Click Yes in the dialog Result The method plan is deleted and disappears from the Method plan list 18 1178 00 AA ep 108 Method Wizard 5 5 5 Advanced Zone About this section This section describes how to change parameter values in the Advanced Zone in the Method Wizard In this section This section contains the following sub sections ep 109 5 Method Wizard 5 5 Advanced Zone 5 5 1 Advanced Zone introduction IIl Introduction The Advanced Zone Advanced Zone content Entering the Ad vanced Zone Advanced Zone introduction This section describes parameters in Advanced Zone and how to change them The Advanced Zo
143. ing report 18 1178 00 AA ep 208 Evaluation 7 7 1 Evaluation procedure overview Introduction This section is an overview of the normal procedure for evaluation of results after a run with AKTAxpress The normal evalu Normally 5 steps are performed after a run Find and open results see 7 2 on page 210 View results and adjust the viewing see 7 3 on page 212 ation procedure Adjust pooling see 7 4 on page 213 Create and print pooling protocol see 7 5 on page 216 Print report see 7 6 on page 218 Optional evalu Optional actions can be performed in the evaluation part of UNICORN For more ANOM ACHONS information see UNICORN Online Manual ep 209 7 Evaluation 7 2 Finding and opening results 7 2 Finding and opening results Introduction This section describes how the result files can be found and opened in the UNICORN folders Recommended In UNICORN it is recommended to find and open result files in the Evaluation finding and open ing of files module the following way to receive automatic pooling Use the Recent Runs tab or the Find tab in the File Navigator on the left hand side of the Evaluation window to locate the result file Recent Runs Files Find Result file name Fo Value of variable Sample_ID p450 Quick Find Find LL System1001 o Home ali System1002 o Home Note See How to use the File Navigator in the UNICORN Online Manual for detai
144. into the drive selected and then click OF Copy manufacturers files frorn g Program FilessE VASE AAD nvers Hert gt Cancel e Click the Next button Upgrade Device Driver Wizard Driver Files Search Results The wizard has finished searching for driver files for pour hardware device The wizard founda driver forthe following device Amersham URJ45 A suitable driver for this device is aleady installed To keep the currently installed diver click Cancel To search another location for a different driver click Back or to reinstall the current driver click Next c winntinfvoent2 int Cancel e Click Next and then Finish Close the Device Manager and the System Properties dialog The wizard should find the correct driver 18 1178 00 AA ep 306 Reference information 10 10 Reference information About this chapter This chapter includes reference information for AKTAxpress In this chapter This chapter contains the following sections Topic System description Technical specifications Import file format Chemical resistance guide Ordering information ep 307 10O Reference information 10 1 System description 10 1 System description About this section This section describes the components of AKTAxpress For a system overview see 3 2 Separation system overview on page 27 In this section This section contains the following sub sections Topic Pump and related comp
145. ion 10 2 Technical specifications 10 2 Technical specifications About this section This section contains specifications of AKTAxpress and its components In this section This section contains the following sub sections Topic System specifications Components specifications Wetted materials 18 1178 00 AA ep 322 10 2 1 Introduction Performance data Gradient forma tion Physical data Reference information 10 System specifications This section specifies the general operating data of AKTAxpress For components data see 10 2 2 Components specifications on page 325 General performance data of AKTAxpress Gradient formation data of AKTAxpress Linear gradients Conditions e Water based buffers with different concentrations of salts Linearity 2 B within 20 80 B Step gradients quasi isocratic mixing Conditions e Water based buffers with different concentrations of salts e Mix proportions 5 95 Physical data of KTAxpress Supply voltage 100 240 V AC 50 60 Hz Power consumption Max 120 VA Degree of protection EN 61010 1 Dimensions w250 x d490 x h660 mm ep 323 10 Reference information 10 2 Technical specifications 10 2 1 System specifications Ambient temperature Temperature operation 4 to 40 C storage 25 to 60 C Relative humidity 20 95 non condensing See 10 2 3 Wetted materials on page 328 Standards Compliance of AKTAxpress
146. ions The largest peak is then loaded directly from the loop onto the next column for the next purification step When the last purification step is finished and the eluted protein peak has been detected by the UV cell the outlet valve directs the peak volume to the deep well microplate in the fraction collector ep 19 2 Introduction 2 4 AKTAxpress user documentation 2 4 AKTAxpress user documentation Introduction This section gives an overview of the user documentation for AKTAxpress AKTAxpress user The table below describes all user manuals included in AKT Axpress user documentation documentation package package AKTAxpress User Manual How to use the system including concepts methodology operation evaluation troubleshooting and maintenance This document is also available as an on line manual AKTAxpress Cue Cards Short step by step instructions and tables for the daily work such as cre ating and running a method plan evaluating the results preparation and maintenance of the system and columns Video sequences are supplied showing the basics of operation AKTAxpress Installation Guide How to prepare for the initial installa tion to perform the installation to run the installation test and to expand an existing installation with additional systems AKTAxpress Safety Handbook Safety instructions that must be fol lowed for the use of AKTAxpress in 6 languages English German Span ish French
147. iosciences Limited Amersham and Amersham Biosciences are trademarks of Amersham plc Microsoft and Windows are either registered trademarks or trademarks of Microsoft Corporation in the United States and or other countries AcTEV is a trademark of Invitrogen Corporation Teflon and Tefzel are registered trademarks of E I du Pont de Nemours and Company UNICORN Any use of this software is subject to Amersham Biosciences Standard Software End User Agreement HiTrap Chelating HP Purification and preparation of fusion proteins and affinity peptides comprising at least two adjacent histidine residues may require a license under US pat 6 284 933 and US pat 5 310 663 including corresponding foreign patents assignee Hoffman La Roche Inc Sweden Amersham Biosciences AB Bj rkgatan 30 SE 751 84 Uppsala Sweden epg 1 Preface 1 2 Product information Great Britain Amersham Biosciences UK Limited Amersham Place Little Chalfont Buckinghamshire England HP7 9NA USA Amersham Biosciences Corp 800 Centennial Avenue PO Box 1327 Piscataway NJ 08855 USA Germany Amersham Biosciences Europe GmbH Munzinger Strasse 9 D 79111 Freiburg Germany Japan Amersham Biosciences K K Sanken Building 3 25 1 Hyakunincho Shinjuku ku Tokyo 169 0073 Japan Hong Kong Amersham Biosciences Limited 13 F Tower I Ever Gain Plaza 88 Container Port Road Kwai Chung New Territories Hong Kong Web address www amershambiosciences
148. iosciences representative Reset the UV lamp run time counter in UNICORN Note This step can only be made by an Amersham Biosciences service engineer 18 1178 00 AA ep 268 8 6 5 Introduction Spare part and tools required Replacing the check valves Maintenance 8 Replacing check valves A check valve should be replaced if dirt etc cannot be removed by cleaning the check valve The following spare parts and tools are required e Check valve kit 18 1128 66 containing one inlet check valve and one outlet check valve e 13 mm wrench e 18 mm wrench CAUTION The check valves should only be disassembled by a trained person To replace the check valves Change solvent to deionized water and flush out all salt Move all input buffers bottles below the level of the pump heads to prevent siphoning Disconnect electrical power from the system Remove the tubing from the pump heads 4 Loosen the upper valve from the pump head using the 18 mm wrench x 2 B ws E P 9 ep 269 8 Maintenance 8 6 Replacement procedures 8 6 5 Replacing check valves Gently remove the check valve and replace it with a new one CAUTION Handle the check valves with care when they have been re moved from the pump heads to prevent loss of any internal components Unscrew the two white knurled screws under the pump to release the support bracket that locks the inlet manifold into the inlet valve
149. istration Method Editor To create and edit methods and method plans System Control To start method plans and to control and monitor the separations processes on line Evaluation To evaluate and present stored results from the separ ation processes The Method Wizard included in UNICORN provides easy creation of method plans for purification preparation and maintenance of the separation systems and columns The Method Wizard also allows editing of stored method plans Before a run the operator chooses method plan and the number of samples for each separation system The systems can then be started separately or simultaneously from UNICORN For more information about the Method Wizard see 5 Method Wizard on page 84 Operation over view AKTAxpress overview 3 The step by step operation of the separation system is described below Run preparations Starting a run Separation systems Samples IOD una VOODOO Evaluating the results Run procedures Create A new method plan is created using the Method wizard in the Method Editor module If required a traditional method can also be created in the Method Editor Run Method plans are started on single or multiple systems using a wizard in the System Control module Methods can only be started on a single system A method cannot be started using the wizard Evaluate Result files are created continuously and when
150. k that the correct system is selected on the screen Click End above the Run data view pane Status indicator The table below shows how the indicator colors relate to the run status colors White a C Error indication When a warning or an alarm is issued from a system the background of the system symbol starts flashing and the background color turns yellow Changing paramet The separation system can be controlled with manual instructions issued from the SiR Manual menu in System Control These instructions can be used during the run to change system conditions in response to the results observed e When changing or loading a microplate during a run the system must first be set to Pause before pressing the Eject button on the system control panel Problems duringa In case of problems during the run see 9 1 Troubleshooting on page 282 run 18 1178 00 AA ep 194 Operation 6 6 8 Procedures after a run About this section This section describes how to clean the system tubing and columns after performed protein purification It also includes how to perform metal ion stripping of the affinity columns In this section This section contains the following sub sections Topic Emptying drip plate Cleaning system Cleaning columns Performing metal ion stripping of affinity columns e p 195 6 Operation 6 8 Procedures after a run 6 8 1 Emptying drip plate 6 8 1 Emptying drip plate Introduction
151. le Not functioning Check the function of the air sensor Replace if necessary The air sensor is disabled Check that the air sensor has not been disabled 18 1178 00 AA ep 296 Troubleshooting and corrective actions 9 4 Leakage problems Introduction This section specifies troubleshooting for when there are leakage problems The problems can be related to e Pump leakage e Other leakage in valves tubing connectors sensors mixer and column block Pump leakage Find the possible cause and action for a specific problem below Low buffer flow and dis trurbing noise Possible cause and action e Bad piston spring Disassemble the pump head and examine the pis ton spring If the spring is corroded check the piston seal and rinse membrane Make sure that the rinsing system is always used when working with aqueous buffers with salt concentration see 8 6 2 Specification of piston rinsing tubing on page 261 If the piston is damaged replace it see 8 6 6 Re placing pump piston seal on page 272 Flow path leakage Find the possible cause and action for a specific problem below To perform a tubing leakage test see 8 4 4 Checking tubing leakage on page 238 Leakage around a pump head Leaking connection or crystallized material around tubing connect ors Internal leakage Possible cause and action e Piston seal or rinsing membrane incorrectly fitted or worn Replace the pump piston seals in
152. le loading tubing replace the tubing see 8 6 2 Replacing tubing on page 249 If using a highly viscous sample dilute it to ease sample loading Check that the flow restrictor generates a backpres sure of 0 2 0 05 MPa as follows Set the injection valve to one of the Waste tubings Run the pump manually at 10 ml min with water Note the backpressure in the Run Data window Connect the flow restrictor to the open end of the Waste tubing Note the IN marking Run the pump manually at 10 ml min with water Note the backpressure Calculate the backpressure difference If it is not within limit replace the flow restrictor See 8 6 3 Replacing flow restrictor on page 262 e p 285 9 Troubleshooting and corrective actions 9 2 Problems during a run Elution problems Find the possible cause and action for a specific problem in the table below 18 1178 00 AA ep 286 No satisfactory elution from Affinity column Possible cause and action If the protein did not bind to the column it will be found in the flowthrough that was collected in one of F3 F6 Verify that the tagged target protein is present in the start sample Check that the sample has been applied onto the affinity column Check that appropriate wash and elution buffers were applied HisTrap HP and HiTrap Chelating HP If the target protein elutes during Wash 2 de crease the imidazole concentration of wash 2 solution If th
153. leaning Use this procedure for general cleaning in case of problems e Offline cleaning Use this procedure as preventive maintenance every 6 months Performing in To make an inplace cleaning of the UV flow cell place cleaning 1 Pump a cleaning or sanitizing agent through the flow cell WARNING N CORROSIVE CHEMICALS NaOH is corrosive and therefore dangerous to health Avoid spillage and wear protective glasses The standard recommendation is to pump 1 M NaOH at a flow rate of 1 ml min for 2 minutes Stop the flow and leave the cell filled with liquid for 30 minutes Rinse with buffer or deionized water If this does not correct the problem follow the instructions for making offline cleaning 18 1178 00 AA ep 228 Maintenance 8 Performing offline To perform offline cleaning of the UV flow cell cleaning WARNING N The UV monitor uses high intensity ultra violet light Do not remove the optical unit while the lamp is ON to prevent injury to eyes Required tools and materials e Syringe e Surface active detergent solution like Decon 90 Deconex 11 RBS 25 or equivalent e Deionized water Set the system in Standby mode by pressing the On Standby button This will disconnect the system from UNICORN RUN _ a Fill a syringe with deionized water 3 Remove the UV monitor capillary tubing from the conductivity cell and the column valve Connect a syringe to the upper capillary tubing using the co
154. led instructions on how to locate files and set up File Navigator preferences Click the button to expand the list for the result file LL System1 001 or Home FLL System1002 0 4 Home 30311244 Double click the sample file to open it E pu System1 001 o 4Home a sane F450 ia Sample2 APB 7 HM Systern1002 o Home Result The chromatogram of the sample is displayed and the frac tions are automatically pooled in the software By default the chromatograms in a run are shown as opened windows The chro matogram window on top is the active window There is also a minimized Temporary chromatogram window See Basic presentation of chromatograms in the UNICORN Online Manual for further in formation about chromatograms 18 1178 00 AA ep 210 Evaluation 7 Alternative finding See How to open a result file in the UNICORN Online Manual for alternative oe opening of ways to open result files Automatic pooling will not be performed if result files are opened in any of the alternative ways e p211 7 Evaluation 7 3 Viewing results 7 3 Viewing results Introduction This section describes how to view the results Zooming inthe If the window is zoomed to show the peaks only it is easier to evaluate the chromatogram chromatogram In the active chromatogram window zooming in on a designated area of the chromatogram is the easiest and quickest way to enlarge different parts of a curve
155. les for simplified column selections for the different protocols In this section This section contains the following sub sections Topic Descriptions of columns Choosing columns e p 65 4 Methodology 4 2 Column alternatives 4 2 1 Descriptions of columns 4 2 1 Introduction Column types Affinity chromato graphy columns 18 1178 00 AA ep 66 Descriptions of columns This section describes the different types of columns currently supported by AKTAxpress Four main column types are used for chromatography in AKTAxpress Column type abbreviation Separation property More information on the main column types is found below Affinity chromatography AC columns separate proteins on the basis of a reversible interaction between a protein or a group of proteins and a specific ligand attached to a chromatographic matrix Affinity chromatography can be used whenever a suitable ligand is available The AC column binds specific molecules from even large sample volumes very efficiently The following AC columns are supported by AKTAxpress HisTrap HP HiTrap column prepacked with Ni 1 and 5 ml Sepharose High Performance a Ni precharged medium optimized for purification of His tagged proteins HiTrap Chelating HP HiTrap column prepacked with Che iaid Smi lating Sepharose High Performance to be charged with the metal of choice for purification of His tagged proteins GSTrap HP Hi
156. libration Metal lon Charge Affinity Blank Run Metal lon Charge Affinity Blank Aun Number of Affinity Columns to Aun 2 F M Desalting Column Equilibration HiPrep_ 26 10 Desalting Global hi Column Position Fosition3 x Buffer Inlet AA is RESOURCE _ _1_ral Global g C lon Exchange Blank Aun M_ lon Exchange f Column Equilibration M Gel Filtration Column E quilibration Hiload_16 60_Superdes_ 75 prep_grade Glob Starting a run using a method plan for an equilibration run Follow the description for the metal ion charging procedure or see the standard procedure described in 6 6 1 Starting a run using a method plan on page 186 18 1178 00 AA e p 170 6 5 6 Introduction Preparing samples Filling the sample inlet tubing Setting the sample volume Operation 6 Preparing samples This section describes how to prepare the samples To prepare the samples Prepare the samples and clarify them using centrifugation and or filtration through a 0 45 pm filter The tube holder is used for storing tubes with small sample volumes The optional flask holder can be used for sample flasks with larger volumes Immerse the sample inlet tubing in affinity buffer A the pump and inlet tubing on page 156 Performed automatically during the run if selected in the method plan Gently move the sample inlet tubing to the sample flasks or tubes tubing 1 to sample 1 tubing 2 to sample 2 e
157. llary tubing from the old restrictor Lift off the restrictor from the holder 18 1178 00 AA ep 262 Maintenance 8 Refit the capillary tubing to the new restrictor Make sure that the tubing from the conducitivity cell is attached to the IN port on the flow restrictor 4 Press the On button to reconnect the system to UNICORN Check the function see 8 2 1 Checking flow restrictor on page 223 e p 263 8 Maintenance 8 6 Replacement procedures 8 6 4 Replacing UV lamp 8 6 4 Replacing UV lamp one inter Replace the UV lamp when va e The UV signal trace has excessive noise e The run time has exceeded the typical life time of the lamp The UV lamp has a life time of typically i Wavelength 254 nm Wavelength 280 nm The lamp run time can be checked from UNICORN see 8 3 1 Checking UV lamp run time on page 227 Required material e Hg lamp in housing including cable 18 1128 22 e Cross head screwdriver CAUTION Only spare parts approved or supplied by Amersham Biosciences may be used for maintaining and servicing the system Replacing the UV When replacing the lamp the complete lamp housing including UV lamp should lamp be replaced Lamp housing UV lamp Filter housing Detector housing Flow cell To change the UV lamp including housing Note It is recommended that an Amersham Biosciences service engineer changes the UV lamp The user can change the lamp but cannot reset the lamp run ti
158. llowing watch conditions are used 1 Greater_Than AND Slope_Greater_Than 2 Peak Max with Factor 3 Less_Than_or_Valley OR Stable_Plateau e p 117 5 Method Wizard 5 5 Advanced Zone 5 5 3 Advanced Zone for Purify The illustration below shows where the watch conditions are met mAU UV max Example of using The Peak_Max factor is important for dectecting a valley since Peak_End cannot ua hae be detected unless Peak_Max has been detected The illustration below shows how two different Peak Max factors affects the detection of a Valley and collection of double peaks Peak Max factor 0 5 Peak Max factor 0 8 Example of using The condition Stable_Plateau is met if the UV signal does not deviate by more than Stable_Plateau the Delta_Plat value during the time interval specified for the watch The illustration below shows an example of this Stable Time Stable Time condition met Va Delta Plateau Delta Plateau condition not met 18 1178 00 AA ep 118 Method Wizard 5 Peak fractionation The final peak fractionation does not involve any watch instructions The parameters fractionation simply starts when the signal level and or the slope of the signal curve exceeds a defined value During the fractionation a specified maximum volume is collected in each well in the fraction collector The fractionation continues at least until the Minimum Peak Width time has elapsed The fractionation stops
159. lting HiPrep_26 10_Desalting Global X Column Position Position3 x IV lon Exchange RESOURCE _Q_1_ml Global X IV Gel Filtration HiLoad_16 60_S5uperdex_75_prep_grade Glob Rigorous Customized Equilibration CIP Columns Rigorous Customized Equilibration Main Selections Maintain Column Type CIP 7 Equil RESOURCE_Q_1_ml Global F Last Page CIP Equilibration Number of Columns a E Number of Steps Flow Rate o o 0 65 ml min Inlet fa xj Volume o o 0 999999 CV Method plan af ds prep ds CIP Columns Regular j ial al ds Columns HisTrap_HP_1 ml Global ma Result A method plan for cleaning columns is now ready to be run on a single or on several separation systems 18 1178 00 AA ep 204 Operation 6 Cleaning the To run the method plan for column cleaning in place columns Run the cleaning in place method plan as any other method plan e p 205 6 Operation 6 8 Procedures after a run 6 8 4 Performing metal ion stripping of affinity columns 6 8 4 Performing metal ion stripping of affinity columns Introduction Before regenerating HiTrap Chelating and HisTrap affinity columns metal ions must be removed from the columns Note Always remove metal ions before or directly after storing the columns in ethanol During the procedure the column is flushed with 5 CV of EDTA solution and then washed with 10 CV of deionized water
160. lts Step Action Enter the desired folder name on the page Method Wizard x General Advanced Settings Advanced one Page I Store Results in a Main Folder 1 Main Folder Name proiectnameuser 2 3 4 Store Results in Main Folder To store the results in a sub folder under the Home folder fill in the name of the folder in the field If the name of the folder does not exist the folder is created automatically Click Next to save the method plan See 5 2 5 Saving the method plan on page 106 ep 139 5 Method Wizard 5 5 Advanced Zone 5 5 11 Hints on optimizing a method plan 9 5 11 Hints on optimizing a method plan Introduction This section contains hints and directions for how to optimize method plans for troublefree operation of KTAxpress Most of the adjustments are made in Advanced Zone in the Method Wizard Peak volume Peak volumes for peaks eluted from affinity and ion exchange columns should be as small as possible It will increase the possibility to e load the entire peak volume onto any desalting or gel filtration column in the next step since their maximum loading volumes are limited e optimize the recovery in the ion exchange step by making sure that the entire peak volume is collected when eluted in the ion exchange gradient Only one loop is available for each peak so peak volumes larger than 7 5 ml will cause sample loss To minimize the peak volume e Choose a suitable column t
161. mAU 50 000 mAU min 100 000 mAU min 0 50 263 12 ml End Block 263 12 ml Block Fractionation_Volume 263 12 ml Base SameAsMain 263 12 ml Peak_Fractionation 0 100 ml 263 12 ml End Block 263 12 ml End Block 263 12 ml Block Elution_IEX 263 12 ml Base SameAsMain 263 12 ml Flow 1 00 ml min Buffer 0 10 ml min Yes 263 12 ml Block Gradient_Seqgment_1 263 12 ml Base CY 1 92 ml HiTrap_SP_HP_1_ml_x2 263 12 ml Gradient 50 0 B 20 00 CV A2 B2 For Help press F1 Run Gradient_Segment_1 No watch Controlled By Pia Introduction 2 Several purifica A single separation system can handle one purification protocol at a time The ae lel tocolsin Standard AKTAxpress delivery package includes four systems thus allowing up to four different purification protocols to be run simultaneously Totally UNICORN provides individual and simultaneous control of up to twelve separation systems by one single computer The individual systems can also be separated from each other thus allowing operation in cold room and room temperature in parallel Required installa AKTAxpress separation system and the UNICORN computer should be installed tions and tested by Amersham Biosciences personnel epl 2 Introduction 2 2 Purification of recombinant proteins 2 2 Introduction Protein purifica tion Multi step purific ation of recombin ant proteins AKTAxpress for multi step purifica tion 18 1178 00 AA ep 18 Pur
162. matogram appear as the subsequent fraction volume is very large This is because the position of the fraction collector is shown and only the delay volume for the next peak if any actually is collected in this well Note The collection in the microplate is performed in a serpentine manner i e first in A1 A2 A11 A12 and then in B12 B11 B2 B1 and so on To display all fractions used select Show all fractions in the Pooling Protocol dialog e p213 7 Evaluation 7 4 Pooling fractions and adjusting the pools Pooling fractions The table below describes how to view the suggested pool fractions and adjust the and adjusting the pools 18 1178 00 AA ep 214 pools If the results were opened via Recent Runs or Find tab in the Evalu ation module UNICORN will automatically display a suggested pooling of the fractions The pooled fractions are listed in a table below the chromatogram and the pooled peaks are numbered sequen tially in the chromatogram Note If the results were opened any alternative way the pooling is not automatically performed Choose Operations Pool to pool the fractions before continuing below UttryckskollO01 Sample1 UY UttryckskollO01 Sample1 Fractions UttryctskollO01 Sample1 Logbook A2 A3 A4 A5 A6 10 0 15 0 20 0 25 0 30 0 35 0 40 0 46 0 mil pI Delete Delete All Default Pool Help 1 Pool table Source curve Theoretical pl 5 3000 i Ext Con
163. me in UNICORN 18 1178 00 AA ep 264 Maintenance 8 WARNING N The UV monitor uses high intensity ultra violet light To prevent injury to eyes do not remove the optical unit while the lamp is ON Disconnect the power to the system for example by unplugging the mains power cable from the system CAUTION To prevent equipment damage the mains power to the sep aration system must be disconnected before any cable is plugged or unplugged at the rear of the system Remove the UV monitor assembly from the system by moving it upwards until the unit is released from its holder A7 GY 2 Remove the connector UV monitor lamp at the rear of the unit and loosen the cable from the cable chute under the system AA AT e p 265 8 Maintenance 8 6 Replacement procedures 8 6 4 Replacing UV lamp Use a cross head screwdriver to detach the end plate by removing one and loosening the other of the two holding screws on the lamp housing to be removed Lamp housing end plate must be handled with care and disposed of ac cording to national and local environmental regulations On the new UV lamp housing detach the end plate Slide the new lamp housing onto the filter housing making sure that the cable is on the right side When sliding the lamp housing depress the two pressure pads on the filter housing in turn to facilitate the installation Refit the lamp housing end plate 5 Slide the old lamp housing off the filter
164. mediate frac tion collection Final fraction col lection Introduction 2 Basic operating principles This section gives a brief introduction to the function of AKTAxpress It describes the main steps during a purification run as shown in the table below Sample application and wash Intermediate fraction collection Final fraction collection A functional description of AKTAxpress and a detailed description of the steps during the purification process can be found in section 3 3 Purification process overview on page 47 sep The inlet valve selects the appropriate sample inlet The pump then loads the sample onto the affinity column which captures the protein If using several samples during the run the sample loading and the subsequent wash steps will be executed serially for all samples before elution of the first protein starts Each sample is therefore loaded onto its own affinity column and the captured proteins will be stored on the columns while waiting for the elution step The inlet valve selects the appropriate buffer inlet If a gradient is required two buffer inlets can be selected simultaneously using a switch valve The pump transports the buffer through the column to elute the captured protein The eluted protein peak is detected by the UV cell If the protein is to be further purifed in a subsequent step the peak volume will be directed to one of the loops The loops are used to store the intermediate fract
165. mments on alternative column choices Comments on alternative column choices Overloading of an affinity column can be performed to minimize non specific binding often results in a purer end product Use in DSstep Use in IEX tep RESOURCE Q 1 ml J 1 ml HiTrap affinity columns can be combined with 2 x HiTrap desaltingcolumns Band broadening effects in the system combined with a limited sample loading volume on the 2 x HiTrap desalting column might however reduce protein recovery AC If there is a low expressed target protein in a large sample volume consider using a 5 ml HiTrap column to reduce the risk of column clogging or protein degradation e p 75 4 Methodology 4 2 Column alternatives 4 2 2 Choosing columns Comments on alternative column choices 5 ml HiTrap affinity columns should not be combined with 2 x HiTrap desalting columns since the eluted affinity peak might be larger than the volume possible to load on the 2 x HiTrap desalting column Make sure to use an IEX column with sufficient capacity for the protein amount To get sufficient separation it is important to choose a gel filtration column with appropriate selectivity IEX A longer IEX column or a column with smaller bead size can be used to increase resolution Choosing columns Recommended column choice when running a DS one step protocol with manual for DS sample loading Which sample volume gt 3 mi
166. mn e Elution of AC e Elution of DS e Elution of GF e Peaks collected in loops and e Peaks collected in loops and e Peaks collected in largest peak selected largest peak selected fraction collector e Nonselected peaks emptied e Excess of selected peak from e Excess of selected peak and loops flushed AC column emptied and from injection emptied loops flushed and loops flushed A detailed description of the process steps can be found in the section 3 3 2 Description of the process steps on page 49 18 1178 00 AA ep 48 3 3 2 Introduction Manual system setup before start ing the run Starting the run AKTAxpress overview 3 Description of the process steps This section contains a detailed description of the process steps during the four step purification process described in the flow chart The description is generalized to cover up to four samples A separate description of the tag cleavage procedure is also included Note The description covers the most important process steps only The table below briefly describes how the operator prepares AKTAxpress for a run 4 The correct columns are fitted and equilibrated if not included in the method plan The inlet tubing is filled if not included in the method plan and the pump heads purged The table below briefly describes the procedure for starting a purification process A wizard dedicated for initializing the run is started in the System Control modul
167. n 6 8 2 Cleaning system 6 8 2 Cleaning system Introduction When running different types of samples or purification methods after each other the sample inlet tubing and the system flow path should be cleaned between the runs This will prevent sample contamination protein precipitation and buffer mixing When leaving the system for the weekend or for a longer time the cleaning procedure should be finished with 20 ethanol to prevent bacterial growth in the tubing Note Do not leave the system with salt buffer in the flow path It might damage the pump Prerequisites The following are required for automated system cleaning e Cleaning solutions for example NaOH and deionized water Up to five different cleaning solutions can be used in one cleaning run The operator moves the inlet tubing from solution to solution e 20 Ethanol when the system will be unused for a longer time e A method plan for cleaning the system Creating a method To create a method plan for cleaning the system plan for cleaning system l Step Action In the Method Editor module in UNICORN start the Method Wizard by for example selecting File Method Wizard af ds af gf tag cleav GF gt Tes n 2 Click Next to create a new method plan Create or Change Method Plan Method plan Hote New Use this Method plan if you af ds iex prep col plan af ds prep ds 18 1178 00 AA ep 198 Operation 6 Select Maintain and click Next ep
168. n System Control Inlet Buffer or solution tubing C e Ang wate a e reee 1 2 3 4 5 6 7 8 B1 B2 18 1178 00 AA e p152 Operation 6 Note The affinity elution buffer is fed from inlet A3 not B1 The step gradient made with A3 buffer will therefore not be shown in the chromatogram because it only shows the concentration of buffer B Inlet supply for The liquid supply differs from the purification methods when preparation and E l N maintenance e stripping metal ions from affinity columns e charging new or stripped affinity columns with new metal ions or e running Cleaning in place CIP method plans Stripping and charging affinity columns The table below shows the standard buffer and solution supply for stripping and charging affinity columns e Immerse the inlet tubing in the containers according to the table also shown on the Summary page in the Method Wizard in System Control Metal ion eee solution e g 0 1 M NiSO Metal stripping solution e g His Affinity A buffer with 50 mM Note Inlet tubing A7 metal ion charging solution must be manually filled before starting the charging run Note The waste from the metal ion charging and stripping runs is collected through outlet tubing F11 ep 153 6 Operation 6 5 Preparing the system for a run 6 5 1 Preparing buffers and solutions Cleaning in place The table below shows the standard buffer and solution supply for performing Cleaning i
169. n place CIP e Immerse the inlet tubing in the containers according to the table also shown on the Summary page in the Method Wizard in System Control Buffer or solution Purging the pump If there is air in the flow path the pump and the inlet tubing to be used must be ane inlet filled manually before starting a run See 6 5 3 Purging the pump and inlet tubing 5 on page 156 Vent opening on If the containers have caps each cap must have a vent opening to prevent a vacuum container caps from forming as buffer or solution is extracted 18 1178 00 AA ep 154 Operation 6 6 9 2 Preparing outlet and waste tubing Introduction This section describes where the outlet and waste tubing should be placed ae the e Immerse the tubing from the outlet valve in appropriate flasks or containers as tubing shown in the table below also shown on the Summary page in the Method Wizard in System Control ve a ee ometene F12 Connected to injection valve port 7 to redirect inter mediate peaks for storage in loops Note The optional flask holder can be used for storing samples and flasks holding F3 F11 e Immerse the waste tubing from the injection valve in appropriate flasks or containers as shown in the table below C Waste 2 used for manual sample injection Waste 3 used for e g system wash and intermediate loop collection e p 155 6 Operation 6 5 Preparing the system for a run 6 5 3 Purging the pump
170. n the pump head is facing upwards Mount the complete pump head over the locating pins on the front panel Locate the metal end of the piston and the spring towards the drive cam Hold the pump head firmly against the side panel of the housing with one hand Do not allow the assembly to twist under pressure from the return spring Using the hex key fit and tighten one of the hex screws e Fit and tighten the remaining screw ep2 8 Maintenance 8 6 Replacement procedures 8 6 6 Replacing pump piston seal Refit the tubing e Reconnect the outlet tubing to the outlet check valves e Reconnect the inlet manifold e Refit the tubing of the piston seal rinsing system 19 Purge the pump and run in the new piston seal carefully following the instructions below Running in the The piston seal should be run in using 100 methanol new piston seal CAUTION Before the start of each run ensure that there is an adequate supply of solution in the reservoirs Never allow the pump to run dry since this will affect the lifetime of the piston seals fE Fill a reservoir with 100 methanol and immerse the inlet tubing A1 Connect a male Luer syringe of at least 30 ml to the left purge valve Two syringes are included in the accessory kit supplied with the system 18 1178 00 AA e p278 Maintenance 8 oo a left purge valve counter clockwise half a turn to open it K ZA ANIS We AD Slowly draw buffer A1 with t
171. nced settings gel filtration 5 5 7 Advanced Zone Purify GF on page 132 preparing the system 5 5 8 Advanced Zone Purify Prepare Peak handling in All purification steps in the standard protocols in the Method Wizard include peak AKT Axpress handling In Advanced Zone some of the parameters that control peak handling can be changed The parameters are divided in two categories e Peak collection e Peak fractionation The peak collection parameters are used in watch conditions to determine how peaks are collected between the purification steps These peaks are collected in one or several of the five loops One peak can also be divided into several loops depending upon the volume The largest peak is then used in the next purification step Peaks that are detected when all loops are occupied are collected in a separate outlet tube The peak fractionation parameters determine how peaks are collected at the final fractionation during the last purification step These peaks containing the final product of the purification run are collected in the fraction collector The collection in the microplate is performed in a serpentine manner i e first in A1 A2 A11 A12 and then in B12 B11 B2 B1 and so on 18 1178 00 AA ep 116 Peak collection and watch condi tions Example of using watch combina tions Method Wizard 5 The peak collection is controlled by a series of UV signal watch conditions included in the meth
172. ne Page 1 2 3 4 General Advanced Settings W Store Results in a Main Folder Main Folder Hame projectname user Store Results in Main Folder To store the results in a sub folder under the Home folder fill in the name of the folder in the field If the name of the new folder do not exist the folder is created automatic ally Click Next to save the method plan See 5 2 5 Saving the method plan on page 106 18 1178 00 AA ep114 9 9 3 Introduction Entering the Ad vanced Zone Method Wizard 5 Advanced Zone for Purify This section gives an introduction to the Advanced Zone for a Purify method plan It also describes the principles of peak handling and how watch conditions are used for peak collection and fractionation Note Avoid changing default parameter values in a method plan unless the result is clearly understood Changing a parameter value might require adjustments of other parameter values as well The first page that appears in the Advanced Zone contains a list of parameters that can be changed in the current method plan The method plan is based on the selections made in the first part of the Method Wizard To select parameters 1 Select the required options on Page 1 Advanced Zone Page 1 2 3 4 5 6 7 Last Page ONS lt Back Next gt Advanced Zone Select type of variables to change Affinity I Flow Rate Pressure Volumes IV 2nd Wash
173. ne in the Method Wizard is used for viewing and changing parameter values in method plans In new method plans the default values are shown They are preoptimized but can be changed to meet specific demands Values in new method plans as well as in previously made method plans can be changed Note Avoid changing default parameter values in a method plan unless the result is clearly understood Changing a parameter value might require adjustments of other parameter values as well The content in Advanced Zone depends on the parameters selected on the first page in Advanced Zone Only pages that are related to these parameters will be shown If a selection or parameter value in Advanced Zone needs to be changed the Back and Next buttons can always be used to return to the Advanced Zone page in question Entering the Last Page in Advanced Zone will though lock the main selections made in the first part of the Method Wizard To enter the Advanced Zone 1 When all selections are made in the Method Wizard the Last Page appears e Click Next Method Wizard Last Page Main Selections Purify All required selections are made Prepare System Column Equilibratiors Press Finish to save the selections as a Method Plan Last Page Press Next to continue to the Advanced one On Page 1 select the parameters that should be changed The following sections describe the main procedures for changing parameter values e Advanced Zone for Prepare e
174. ng piston rinsing tubing Rinse Rinse In Rinse Out HU memmen mn re ol se ee mT A i ae l Designation Description Length mm Inner diamet Material er mm Rinse In Rinsing solu 1350 PTFE tion inlet to pump head 2 lower port 18 1178 00 AA ep 260 Designation Rinse Rinse Out Description Pump head 2 upper port to pump head 1 lower port Pump head 1 upper port to rinsing solu tion outlet Length mm Inner diamet er mm 200 including check valve Maintenance 8 Material Silicone e p 261 8 Maintenance 8 6 Replacement procedures 8 6 3 Replacing flow restrictor 8 6 3 Replacing flow restrictor one inter When required for example when the restrictor opens at too high or too low ae pressure Reason for main A flow restrictor opening at too low pressure might cause air bubbles in the UV tenance flow cell A flow restrictor opening at too high pressure will cause a high pressure alarm from the system In exceptional it might cause column rupture Required material Flow restrictor FR 902 18 1121 35 CAUTION Only spare parts approved or supplied by Amersham Biosciences may be used for maintaining and servicing the system Replacing flow re To replace the flow restrictor strictor 1 Set the system in Standby mode by pressing the On button This will prevent runs from being started from UNICORN RUN _ a Remove the two capi
175. ng DUMP PISTON Sedlare en a a a E A 212 8 6 7 Replacing a damaged pump PISLOM ss scdanserinetiernnned resdwnenneede nenwenelveasnmmaentebidd 280 9 Troubleshooting and corrective ACTIONS ccceccceecceecceeecneeeeeeceeeeeeeeuseeseeuaeeeeseneseeeseenes 281 9 Tee Introduction to troubleshooters e a a ea 282 DZ Problems durine a T nonisonanniieenn nna oaa aoa 284 9 3 Monitoring PropleMSesrnsusei aa a a ad 290 94A Leakage DODICM crcr aeaa a O E EE 297 9 5 Alarms and connection problemS ssesssssesssrrrssrrrrsrrrrrnrrrnrnrrrrenrrrrenrrrrenrrerenne 298 DOs ETOF COC IS Teco A E E AN eae 300 9 7 Checking USB CAN ATIVE sesser tiaue atten emed cee tuvenlaet a A tee Sah cnutel 2a Sued 303 TO RETERENCE UNIGEIN AUG icira a a 307 10 k SYSTEM GOSCHI TION sarcina a 308 10 1 1 Pump and related COMPONENTS ccceceececeeeeeteeeeeeeteeeateatseeaneanseeaneataees 309 Oe WIC Saesnes ate bla ate na hbase tea Aird eaS S13 TOS red MONO aa sce aactntiecitee maaan detad tsaiclen iad asia ana teeta sausanh heats sade becaane te mceee batons 318 EO SLAs VAC TION COMO Ol eaa EE 321 10 2 Technical specifications s sisien 322 tO SSYSLOM SDOCITIC ANIONS sinister scetvaesraceneratedreeiace eiucacdtsltaled Eaa 323 10 2 2 COMPONENTS SPCCITICATIONS ince sitaseeracente vxiuicn aa 325 FO R729 WV CILOC Malena sieacesrecctatestianasalectcatwcincc ceeds arian a a aaa 328 LO 232 IMAC LIS TOF AL aenn Guede iia te recaa ae T N 331 LO 4 CAemMiCal TCSISLANC
176. ng the run with instructions for filling the loop See also 6 5 8 Performing affinity tag removal on page 175 for a description of the procedure Note The sample volume must be lower than the maximum sample loading volume of the desalting or gel filtration column used In addition the sample volume must always be lower than the loop volume which is 10 ml Observe that the sample volume should be entered when starting the method plan in System Control as well Example The HiPrep Desalting column alone can take up to 15 ml but when used in AKTAxpress the maximum sample loading volume is 10 ml 18 1178 00 AA ep 172 6 5 7 Introduction Microplate require ments Installing a micro plate Operation 6 Preparing the fraction collector This section describes how to load a microplate in the fraction collector The microplates for the fraction collector in AKTAxpress must fulfill the following requirements Deep well plate with 96 wells height 45 mm e Square well design not cylindrical wells The following manufacturer s microplates are tested and approved by Amersham Biosciences to be used with AKT Axpress e Greiner 780201 e Eppendorf 7605662 e Nunc P7616 e Corning A9347 WARNING Do not put any body parts in front of the fraction collector The microplate can be ejected both manually and automat ically by the system WARNING N Ensure that no parts e g tubing or columns are position
177. nge 128 changing parameters gel filtration 132 changing parameters system adn column preparation for Purify 134 changing parameters general advanced setttings for Purify 136 maintain system 138 Affinity columns metal ion charging 166 Affinity tag description 59 types 59 His tag 59 GST tag 59 removal process 62 Affinity tag removal description of proteases 61 conditions 64 cleavage time 64 cleavage buffers 80 assisted manual loading of Superloop 93 tag cleavage parameters 123 automatic 175 protease calculation 175 Air bubbles 180 Air detection 182 Index ep 343 Index Air sensor detection 182 sensitivity 182 Article numbers 336 Automatic removal of affinity tags 175 Background to AKTAxpress 18 Bar code reader 186 Basic operating principles 19 Buffer alternatives 78 descriptions 78 for AC 78 for DS 78 for IEX 79 for GF 79 choosing 80 for AC 80 for DS 81 for IEX 81 for GF 82 C Calibration pressure 242 conductivity cell 243 Cautions 11 CE certification 9 Cell constant 245 Changing parameters 194 Check valve replace 269 Check valves cleaning 234 Checking USB CAN driver 303 18 1178 00 AA ep344 Chemical resistance 332 Choosing buffer 80 Choosing columns 70 Choosing protocol considerations 56 Chromatography abbreviations 55 Class A 9 Cleaning system 198 columns 202 system 233 Cleaning in place buffer and soluti
178. nity HiTrap_Chelating_HP_1_ml Global b C Column Equilibration Metal lon Charge Affinity Blank Run Metal lon Charge Affinity Blank Run Number of Affinity Columns to Run 3 Y Note The Method Wizard also provides a combined method plan that includes metal ion charging automatically followed by a blank run Starting a run using a method plan for an affinity blank run Follow the description for the metal ion charging procedure or see the standard procedure described in 6 6 1 Starting a run using a method plan on page 186 18 1178 00 AA ep 168 Blank run on ion exchange columns Equilibrating columns Operation 6 Before the first use of an ion exchange column e g HiTrap SP or RESOURCE Q or after long term storage a blank run should be peformaed The purpose is to provide the ion exchange column with exchangeable counter ions During the procedure each column is flushed with 5 CV of ion exchange buffer A 5 CV of buffer B and finally with 10 CV of buffer A Creating a method plan for an ion exchange blank run The procedure for creating a method plan for an ion exchange blank run is the same as the metal ion charging procedure except for the following changes on the Prepare Columns page e Click lon Exchange e Select column type e Click lon Exchange Blank Run V lon Exchange MonoQ_5 50_GL Global lon Exchange Blank Run Column Equilibration Starting a run using a
179. nity binding buffer should be used During the run the software will give guidance to filling the superloop The filling is performed manually with a syringe in the injection valve See 6 5 8 Filling the superloop on page 179 The rest of the tag cleavage procedure is performed automatically The protease volume and concentration can be automatically calculated by using the Protease Calculator sheet found on the UNICORN CD AKTAxpress Protease Calculator Your Data What is the column volume CY of your HiTrap affinity column HT Smi How many samples on this system will you be running How much protein will be applied to each HiTrap affinity column 20 mg How many CV of protease will be applied 0 7 is default 0 7 CV How much protease do you need to cleave 1 mg of protein 200 units or mg Calculated Calculated volume of protease required for filling the superloop ml Calculated required concentration of protease units or mg ml Calculated total amount of protease required units or mg e p 175 6 Operation 6 5 Preparing the system for a run 6 5 8 Preparing the system for automatic affinity tag removal The following example describes how to perform a manual calculation Conditions e Protein type His tagged with TEV protease cleavage site e Number of samples 4 e Estimated amount of protein 20 mg e Affinity column HisTrap HP 5 ml e Protease TEV protease 200 units mg target protein e Dela
180. nnecting pieces A and B A Luer female to 1 16 male 18 1112 51 B Union 1 16 to 1 16 18 3855 01 p 229 8 Maintenance 8 3 Six monthly maintenance 8 3 2 Cleaning UV cell 5 Inject water through the cell in small amounts while holding the A a i OS g J i Fill the syringe with a 10 surface active detergent solution like Decon 90 Deconex 11 RBS 25 or equivalent and inject through the cell 5 times For the last injection leave the detergent solution in the flow cell for at least 20 minutes Rinse the syringe and then flush the flow cell with water 10 ml Reconnect the capillary tubing 18 1178 00 AA ep 230 Maintenance 8 8 3 3 Cleaning conductivity cell Maintenance inter Every 6 months val Reason for main A clean flow cell is essential for ensuring correct measurements If the conductivity peer measurements are not comparable to previous results the electrodes in the flow cell may be contaminated and requires cleaning Performing in To make an inplace cleaning of the conductivity flow cell 1 Pump a cleaning or sanitizing agent through the flow cell WARNING N CORROSIVE CHEMICALS NaOH is corrosive and therefore dangerous to health Avoid spillage and wear protective glasses The standard recommendation is to pump 1 M NaOH at a flow rate of 1 ml min for 2 minutes place cleaning Stop the flow and leave the cell filled with liquid for 15 minutes Rinse with buffer or
181. ns Description of the Method Wizard Prepare Purify and Maintenance Instructions for preparing the system for a run and performing a run on one or several systems Viewing and evaluating the results printing chromatograms and generat ing reports ep2l 2 Introduction 2 4 AKTAxpress user documentation Typographical conventions Prerequisites 18 1178 00 AA ep 22 8 Maintenance Maintenance schedules and instruc tions for preventive maintenance and replacing spare parts 9 Troubleshooting and corrective ac Overview of error symptoms possible tions causes and corrective actions Error codes 10 Reference information Detailed hardware description technic al and chemical specifications order ing information Menu commands field names and other text items from the software or on control buttons are quoted exactly as they appear in a bold typeface e Example Run Setup Search paths are shown in a bold typeface with a separating colon between each level e Example View Windows Customise i e the menu command Customise in the sub menu Windows from the View menu Text entries that UNICORN generates or that the user must type are represented by a monotype typeface e Example Connection change The following prerequisites must be fulfilled before you can use this manual in the way it is intended e You need to have a general understanding of how the PC and Windows work In most cas
182. nse to B See Non linear gradient above changes Note A delay of 3 ml plus the column volume is normal Incorrect absolute con Bad calibration ductivity value Calibrate the conductivity cell see 8 5 2 Calibra tion of conductivity cell on page 243 e Incorrect calibration solution Calibration solution 1 00 M NaCl not correctly prepared Prepare a new calibration solution and recalibrate the conductivity cell see 8 5 2 Calibra tion of conductivity cell on page 243 ep 293 9 Troubleshooting and corrective actions 9 3 Monitoring problems Possible cause and action Incorrect or unstable e Cable not connected properly reading Check that the conductivity flow cell cable is con nected properly to the rear of the system see the AKTAxpress Installation Guide Air might be trapped in the pump Purge the pump according to the procedure in 6 5 3 Purging the pump and inlet tubing on page 156 Column not equilibrated Equilibrate the column If necessary clean the columns using a method plan for Column cleaning see 6 8 3 Cleaning columns on page 202 18 1178 00 AA ep 294 Faulty pressure curve Troubleshooting and corrective actions 9 Find the possible cause and action for a specific pressure curve fault in the table below If the problem remains contact the local Amersham Biosciences representative Irregular flow Noisy baseline signal Irregular pressure trace Possible cause
183. nt update failed Contact Service Two segments flashing Check that the UNICORN computer is turned on and system unit no Start UNICORN and check the connection status steady as below 18 1178 00 AA ep 298 UNICORN Connection status NO 1 UNICORN Connection status NO 2 UNICORN Connection status NO 3 Troubleshooting and corrective actions No contact with the systems e Check the cables see AKTAxpress Installation Guide Cables to the USB CAN converter UniNet cables between the systems and the USB CAN converter Check that the last system on the UniNet chain has the termination plug inserted see AKTAxpress Installation Guide Remove and insert the USB cable to the USB CAN converter If problem persists restart the computer and reset the separation system No contact with the AKTAxpress driver e Check the cables see AKTAxpress Installation Guide Cables to the USB CAN converter UniNet cables between the systems and the USB CAN converter Check that the last system on the UniNet chain has the termination plug inserted see AKTAxpress Installation Guide Remove and insert the USB cable to the USB CAN converter Check the USB CAN driver installation see 9 7 Checking USB CAN driver on page 303 If problem persists restart the computer and reset the separation system Internal software error OCI e Check the system installation by using the UNICORN CD supplied S
184. ntroduction Further informa tion Methodology 4 Documents for further information This section includes a list of the different documents that can be used to find more information on protein separation and related topics Further information on protein separation can be found in the following documents Affinity Chromatography Handbook 18 1022 29 Principles and methods Gel Filtration Principles and methods 18 1022 18 GST Gene Fusion System Handbook 18 1157 58 Ion Exchange Chromatography Prin 18 1114 21 ciples and methods Protein Purification Handbook 18 1132 29 Recombinant Protein Handbook 18 1142 75 e p 83 5 Method Wizard 5 Method Wizard About this chapter This chapter describes the Method Wizard in UNICORN In this chapter This chapter contains the following sections Topic Method Wizard introduction Creating a method plan Deleting a method plan Advanced Zone Editing a method plan 18 1178 00 AA ep 84 J 1 Introduction The Method Wiz ard The method plan Using a method plan Method Wizard 5 Method Wizard introduction This section gives a general description of the Method Wizard in the Method Editor module and instructions for how to start the wizard The Method Wizard in Method Editor is a step by step guide for creating method plans It consists of a number of pages with questions and instructions on how to create the method plan The option
185. o not come off with the normal binding buffer wash The extra wash buffer should include a small amount of elution buffer to release more of the impurities from the column but not enough to release the target protein Note The extra wash is recommended when running HisTrap or HiTrap Chelating columns The use of extra wash is not recommended when purifying GST tagged proteins e Cleavage buffer If affinity tag cleavage is performed the binding buffer can often be used If preferred an alternative cleavage buffer can be used to condition the affinity column before injection of the protease e Elution buffer The elution buffer should be chosen to elute the target protein as specifically as possible For AKTAxpress step elution of the bound protein from the AC column is used to achieve an elution volume suitable for the next purification step Desalting chroma Desalting can be performed in preparation for an ion exchange chromatography tography buffers step or as a last buffer exchange step e DS before IEX If IEX is to be performed after DS use the IEX binding buffer for the desalting step e DS as last step If DS is used as a final purification step the buffer should be chosen to match the activity or application that will follow e g crystallization NMR studies functional studies or storage 18 1178 00 AA ep 78 Ion exchange chromatography buffers Gel filtration chromatography buffers Methodology 4 Th
186. o 5 2 4 Finishing the Maintain method plan on page 105 Finishing the To finish the Maintain method plan Maintain metho plan Step Action When all selections are made click Next Result The Last Page appears Proceed to 5 2 5 Saving the Method plan on page 106 e p 105 5 Method Wizard 5 2 Creating a method plan 5 2 5 Saving the method plan 9 2 9 Saving the method plan Introduction This section describes how to save the method plan Saving the method To save the method plan plan On the Last Page page e click Finish to complete the method plan go to step 2 or e click Next to enter the Advanced Zone for changing parameter values See 5 5 Advanced zone on page 109 Enter a name of the method plan and click OK Result The method plan is saved 2 On the Save As page select destination folder in the Method plan tree e Click Create Folder to create a new folder if preferred In the dialog that appears enter the name Click OK Additional information can be typed in the Notes field Useful for example for identifying old method plans by the Method Notes to reconstruct them after changing the strategy Method plan t CIP Regular 12 aii Purification Protocol E Affinity Step Desalting lon Exchange Gel Filtration Normal Affinity Column Sample Loading Level Running Condition Room Temperature Columns HisTrap_HP_1_ml Global HiPrep_26 10_Desalting Global RESOURCE_ _1_ml Global
187. o receive them at Amersham Biosciences AB SE 751 84 Uppsala Sweden Copyright Amersham Biosciences AB 2004 All rights reserved 1 2 CE certification Class A product Terms and condi tions of sale Trademarks License statement Office addresses Preface 1 Product information This product meets all requirements of applicable CE directives A copy of the corresponding Declaration of Conformity is available on request The CE mark and corresponding declaration of conformity is valid for the instrument when it is e used as a standalone unit or e connected to other CE marked Amersham Biosciences instruments or e connected to other products recommended or described in this manual and e used in the same state as it was delivered from Amersham Biosciences except for alterations described in this manual This is a Class A product In a domestic environment it might cause radio interference in which case the user may be required to take suitable measures Unless otherwise agreed in writing all goods and services are sold subject to the terms and conditions of sale of the company within the Amersham Biosciences group which supplies them A copy of these terms and conditions is available on request Drop Design GSTrap HiLoad HiPrep HisTrap HiTrap Mono Q Mono S MonoBeads RESOURCE PreScission Sephadex Sepharose SOURCE Superdex Superloop AKTA AKTAxpress and UNICORN are trademarks of Amersham B
188. od When a watch condition is met a predefined action is issued for example start collecting a peak Another watch is then activated for detecting the peak maximum and so on The table below describes the basic watch instructions available in the AKTAxpress Method Wizard Other instructions are combinations of these Greater Than The signal exceeds a certain value The signal falls below a certain value Slope_Greater_Than Less Than_Or_Valley Peak Max Stable Baseline Stable Plateau The slope of the the signal curve ex ceeds a specified value The signal falls below a specified value or a valley is detected A valley is de tected only after a Peak_Max has been detected and the valley is defined by a local minimum followed by an in crease to 102 of the local minimum plus the Delta_Peak value The signal falls to a specified fraction of the most recent peak maximum minus the Delta_Peak value The signal is stable within the limits of the Delta_Base value for a specified time interval The signal is stable within the limits of the Delta_Plat value for a specified time interval Note If the flow rate is changed the shape of the peak will change as well Hence the slope values might need to be changed accordingly For more information on watch instructions se UNICORN User Reference Manual for AKTAxpress This example shows how peak collection is performed in AKTAxpress using watch conditions The fo
189. on 321 Fractions How to pool fractions 214 Functional description 37 G GST tag 59 H Handbooks 83 Help online 26 Hints and directions for optimizing a method plan 140 His tags 59 Import files 331 Indicators and controls 43 indicators 44 status Indication 44 Inlet supply 152 Index ep 34 7 Index Installation requirements 17 Installation test 240 Isoelectric point 186 L Labels 46 Leakage 180 Leakage 297 Liquid flow path 34 M Materials 328 Metal ion charging of affinity columns 166 Metal ion stripping of affinity columns after a run 206 creating a method plan 206 Method introducing 85 Method plan introducing 85 create a new 89 main selections 90 using Prepare 92 using Purify 96 using Maintain 102 installation test 103 strip affinity columns 103 CIP Columns Regular 103 CIP Columns Rigorous Customized 104 customized equilibration 104 clean system 105 saving 106 editing 107 deleting 108 types 148 create 148 save 148 18 1178 00 AA ep 348 Starting 186 Method Wizard introducing 85 buttons 87 Methodology 55 Microplate recommended 173 Microplate 173 Molecular weight 186 Monitors description 318 0 Online help 26 Operation work flow 143 Ordering information 336 Outlet tubing 155 P Peak fractionation prepare system 119 Peak handling 116 Pool 213 Pooling fractions 213 Pooling protocol add pools 216 printing
190. on parameters The peak collection options and parameters are described below The parameters define how the peaks are collected in the loops Method Wizard Ea Affinity Peak Collection Advanced Zone Max Volume in Each Loop i 0 5 10 ml Fage Mas Number of Loops 4 i 1 Note Mas two different peaks will be collected 2 3 Allow Collection of Single Peaks in Several Loops 4 Yes No 5 6 Start Collection 7 Watch Level Greater than g 6000 6000 mall B Watch Slope Greater than ae 50000 50000 mAL min g 10 Stop Collection 11 Peak Max Factor Oe 0 1 12 Watch Level Less than p 6000 6000 mal 13 Watch Stable Plateau 14 Stable Time jas 0 9999 min 15 Delta Plateau f 0 6000 m l 16 17 Peak Selection Affinity gt Desalking 18 Select Largest Peak Based on Area Height Max Volume in Each Loop The maximum volume allowed in each loop Using volumes above 7 5 ml might cause sample loss due to band broadening Max Number of Loops The maximum number of loops used to collect peaks in the affinity step Maximum two peaks will be collected in loops but more than two loops might be required to collect large peaks Allow Collection of Single Peaks in Several Loops To collect single peaks with a volume larger than Max Volume in Each Loop in several loops Start Collection The peak collection starts when both the UV level exceeds the Watch Level Greater than value and the UV slope exceeds the Watch Sl
191. on 6 6 6 Starting a run About this section This section describes how to start a run using a method plan and how to runa system manually In this section This section contains the following sub sections Starting a run using a method plan ep 185 6 Operation 6 6 Starting a run 6 6 1 Starting a run using a method plan 6 6 1 Starting a run using a method plan Introduction This section describes how to start a run using a saved method plan Selecting amethod Follow the steps below to select a method plan plan Click the System Control button located on the task bar Result The System Control module appears Click the Instant Run button located on the tool bar Alternatively select File Instant Run Instant Run Result The Method Wizard in System Control appears 3 Select the requested method plan in the Method plan list Click Next Method Wizard x Run Method Plan Method plan ay AC OS IE Clean System H AC DS Ex DS Number of cleaning Solutions 1 Inlets to clear FJ 51 A GF 7 a Maintain cA orctic Al ee svete A Ag Ad AS J Prepare AB Af If selecting a Prepare or a Maintain method plan 4 Select on which System s the method plan shall be used It can be used on any or all selected systems Click Next x Systems Systems Free Text Systems Result Name Iv SYS 1 Summary Iv SYS 2 sys 3 M sys 4 SelectAll Systems C Deselect All Systems 18 1178 00 AA ep 186 O
192. on page 290 Leakage problems Problems related to physical problems with the procedure flow path see 9 4 Leakage problems on page 297 Alarms and connection problems Alarms in UNICORN and error codes on the separation systems see 9 5 Alarms and connection problems on page 298 Make the recommended corrective actions If problem persists after corrective actions contact a local Amersham Biosciences representative 18 1178 00 AA ep 282 General check list Troubleshooting and corrective actions 9 Check the following items before starting troubleshooting System checks Is the correct system selected in UNICORN System Control see 6 7 During a run on page 193 Is the fan blowing at the back of the system Monitor checks Is the UV monitor set to the correct wavelength see 8 6 4 Replacing UV lamp on page 264 Is the UV cell locking nut properly tightened to the stop position For more details see 8 6 4 Replacing UV lamp on page 264 Is the air sensor sensitivity set to Jow in UNICORN System Control to avoid unnecessary stops due to minor air bubbles Flow path checks Is all tubing connected correctly Is there leakage at any of the connections Is the inlet tubing immersed in correct buffer solutions Is the gel filtration column located in the left hand column holder If located in the right hand superloop holder it will hinder the movement of the fraction collector Is any tubing interfering wi
193. on supply 153 creating s method plan 202 Cleavage conditions 64 Cleavage time 64 Column choice AC DS 70 AC GF 71 AC DS IEX 72 AC DS IEX DS 73 AC DS IEX GF 75 DS 76 GF 76 Column heater 64 Column heater 179 Column types 66 Columns alternatives 66 descriptions 66 for affinity chromatography 66 for desalting chromatography 67 for ion exchange chromatograpy 6 7 for gel filtration chromatography 68 choosing 70 connecting 163 unions 164 conditioning 166 blank run 168 equilibrating 169 Index ep 345 Index cleaning in place 202 Component specifications 325 Conductivity curve 292 Conductivity cell calibration 243 Connect a separation system 146 Connection guide 164 Connectors fingertight 249 M6 249 Content description 21 Cutting tool 249 D Description of protocols 55 affinity tags 59 proteases 61 affinity tag removal process 62 columns 66 buffers 78 Description of the process steps 49 Documentation 20 Drip plate emptying 196 emptying 242 E Eject 173 Error codes 300 Evaluation 209 Evaluation process normal 209 optional 209 Evaluation process overview 209 Exporting 18 1178 00 AA ep 346 pooling protocol 216 Extinction coefficient 186 F Fault codes 300 Features 14 Filling Superloop 177 Flask holder set 32 Flow control 184 Flow restrictor checking 223 checking 262 Fraction collector loading a microplate 173 descripti
194. oncentration affects TEV activity negatively The table below provides guidance on appropriate conditions for on column cleavage using AKTAxpress If at then with units of protease per mg of protein using 4 C cleave 8 h 20 PreScission Protease using ACTEV room temperat cleave 8h 200 from Invitrogen ure 1 Unit definitions One unit ACTEV cleaves gt 85 of 3 ug control substrate in 1 h at 30 C One unit PreScission will cleave gt 90 of 100 pg of a test Glutathione S transferase fusion protein in cleavage buffer 50 mM Tris HCl 150 mM NaCl 1 mM EDTA 1 mM DTT pH 7 0 at 25 C at 5 C for 16 h If AKTAxpress is used in a cold room it is possible to raise the temperature in the column by using a column heater during the affinity and cleavage steps The column heater is not controlled from UNICORN but can be placed on top of the system Note Raise the temperature carefully if the protein is temperature sensitive See 4 3 2 Buffer suggestions on page 80 for guidelines on buffers for cleavage See Instructions and Application notes from Amersham Biosciences 4 4 Documents for further information on page 83 and other suppliers Methodology 4 4 2 Column alternatives About this section This section contains general descriptions of the columns currently supported by AKTAxpress guidelines for selection of appropriate column combinations for different applications and schedu
195. onents Fraction collector s ca 18 1178 00 AA e p 308 10 1 1 Introduction Pump Reference information 10 Pump and related components This section describes the pump and mixer in AKTAxpress Mixer a VEZ Pump heads General The AKTAxpress pump is a high performance laboratory pump for applications where accurately controlled liquid flow is required The pump consists of two parallel pump heads with a cylinder piston Each pump head has two check valves and one purge valve The liquid is drawn into the inlet manifold by the action of the pump Twin reciprocating pump heads work in unison to deliver a low pulsation flow from the pump outlet Pressure sensor Restrictor 0 2 MPa Eccentric eee Timing belt Pump Stepper heads motors ep 309 Each piston is driven by a simple robust cam eccentric These cams are driven by stepper motors via timing belts The motor speed is varied to achieve linear movement This produces the particular motor sound This system guarantees an accurate low pulsation flow over the entire flow rate range independent of the back pressure When an increase in flow rate is programmed the motor speed accelerates gradually giving a soft start and building up speed to the flow rate required When a decrease in flow rate is programmed the motor speed reduces rapidly to the lower flow rate Pump heads The individual heads are identical but are actuated in opposite ph
196. ons the affinity tag has to be removed from the target protein If a protease recognition sequence has been introduced between the affinity tag and the protein then the affinity tag can be removed during the purification process of the protein When the tagged protein is subjected to its specific protease cleavage occurs at the recognition site Cleavage can be performed in batch or on column All AKTAxpress multi step purification protocols can be combined with automatic on column affinity tag cleavage gt Protease Cleavage site Tags supported by AKTAxpress protocols His polyhistidine tag Metal ions with n histidines Example Ni2 Zn2 Cu Co2 GST glutathione S Glutathione 26 kDa transferase depending on the number of histidines and the used linker between the tag and the target protein His tags ep 59 4 Methodology 4 1 Protocols and affinity tag removal 4 1 2 Description of affinity tags Purification prop erties 18 1178 00 AA ep60 His tags are the most widely used affinity tags They are normally composed of a sequence of 6 histidines that can bind with high affinity to metal ions They are small and therefore often considered to have little effect on the structure of the proteins The tag is also useful if purification under denatured conditions is required GST tags GST tags often improve the expression and solubility of the target protein The interaction with glutathione is hig
197. ope Greater than value Note If the flow rate is changed the shape of the peak will change as well Hence the slope values might need to be changed accordingly Stop Collection The Peak_Max condition occurs when the UV signal has fallen to a fraction specified by Peak Max factor of the most recent peak maximum The Peak end is detected when the Peak_ Max condition is met AND e the signal falls below the Watch Level Less than value OR e a Valley is detected OR e a Watch StablePlateau condition is met The condition is met when the signal is stable within the limits of the Delta Plateau value during the Stable Time interval Peak Selection Select Largest Peak Based on either peak height or peak area to be transferred to the next column Area is the default value 18 1178 00 AA ep 122 Tag cleavage parameters Method Wizard 5 For more information on peak handling and watch conditions see 5 5 3 Peak handling and watch conditions on page 116 The tag cleavage parameters are shown only when tag cleavage is selected In addition a few other standard parameters are used differently Flow rate Pressure parameters Method Wizard Equilibration Flow rate when equilibrating the affinity columns Sample Loading Sample loading and protease injection flow rate 2 nd Wash Elution Cleaved Protein Flow rate when washing out the unbound sample from the column s before the protease is injected onto the column s Also the
198. open The valves have UNF 5 16 connectors The valves have no user replaceable parts 2 2 i Default position at startup inject The two positions of the valve gives the following flow paths A1 A2 position B1 B2 position Buffer Buffer Buffer Buffer Al B2 Al B2 Inlet Inlet Inlet Inlet valve valve valve valve Buffer Buffer Buffer Buffer Bl A2 Bl A2 ep 313 10O Reference information 10 1 System description 10 1 2 Valves Inlet valve The inlet valve is a 12 way 13 port valve For each 12 positions an inlet port is connected to the central outlet port The valve has M6 connectors The valve has no user replaceable parts Default position at Startup The valve is used for buffer selection and sample loading For flow path details see 3 2 2 Liquid flow path on page 34 Injection valve The injection valve is a 4 way 8 port valve used for main flow direction in the system The valve is also used for manually injection of samples and filling superloop via a syringe The valve has fingertight connectors The valve has no user replaceable parts Center Default position at startup inject 18 1178 00 AA ep 314 Column valve Reference information 10 The four positions of the valve gives the following flow paths Inject position Reinject position Manual Manual inject Waste inject Waste Loop valve Loop valve upper port Waste upper port Waste Outlet Loop valve Outlet Loop valve valve lower p
199. or UV monitor Conductivity monitor ep 39 3 AKTAxpress overview 3 2 Separation system overview 3 2 4 Functional description 18 1178 00 AA ep 40 UV monitor The UV cell is connected in the flow path directly after the column valve It provides online detection of the absorbance of the eluted protein The optical unit is equipped with a Hg lamp and a filter with a wavelength of 280 nm If required a wavelength of 254 nm can be used by turning the filter wheel and adjusting the housing O L y z EN E ity W Ji a 2 om H in System Control by selecting Monitors UV lamp The warm up time is 60 minutes m Instructions C Alarms UVLamp Parameters Averagingl imeU Mode vTimeU Normal A SetCondScale0 OFF Cond alue 0 00 mS cm Specials SetCondScale1 00 Cond alue 100 00 mS cm UV Lam Monitors AirSensor Mode High_Sensitivity WatchPar UV M Nelta Peak N NNN m l l Set Selected Parameter To Strategy Default Value Curves Cancel Help Conductivity monitor The conductivity cell connected directly after the UV cell is primarily used for verifying gradients during the elution The measurement range is 1 pS cm to 999 9 mS cm Flow restrictor A flow restrictor is connected in the flow path directly after the conductivity cell to prevent air from precipitating or getting
200. ort valve lower port Column Inlet from Column Inlet from valve pump valve pump Load position Waste position Manual Manual inject Waste inject Waste Loop valve Loop valve upper port Waste upper port Waste Outlet Loop valve Outlet Loop valve valve lower port valve lower port Column Inlet from Column Inlet from valve pump valve pump The column valve is a 2x6 way 14 port valve used for directing a flow to any of up to five columns and one bypass route The valve has fingertight connectors The valve has no user replaceable parts C3in C4in C2in Coin Clin UV monitor Bypass Bypass Injection valve Clout C5out C2out CAout Default position C3out at startup ep 315 10O Reference information 10 1 System description The six positions gives the following flow paths 10 1 2 Valves Column 1 position C4in C3in C2in C5in Clin Clout C5out C4out C2out C3out Column 4 position C4in C3in C2in C5in Clin Clout C5out C4out C2out C3out Loop valve Column 2 position C4in C3in C2in C5in Clin Clout C5out C4out C2out C3out Column 5 position C4in C3in C2in C5in Clin Clout C5out C4out C2out C3out Column 3 position C4in C3in C2in C5in Clin Clout C5out C4out C2out C30ut Bypass position default C4in C3in C2in C5in Clin Bypass Bypass Clout C5out C200 ggg O The loop valve is a 2x6 way 14 port valve used for directing a flow to any of up to five sample capillary loops and one bypass route The loop v
201. osen column The first gel filtration page that appears contains options and parameters related to flow rate pressure and volumes Method Wizard Gel Filtration Flow Rate 7 Pressure 7 Yolumes Maaned Aane uer E a Fage 1 Pressure Limit jas 0 3 MPa 2 3 Elution volume before Fractionation 0 3 D 999999 Cv Elution Volume with Fractionation fs 0 999999 CV Flow Rate Flow rate during the gel filtration Pressure Limit Upper pressure limit during the gel filtration Elution Volume before Fractionation Volume used for elution of the gel filtration column before the peak fractionation starts Elution Volume with Fractionation Volume used for elution of the gel filtration column after the peak fractionation has started The peak injection options and parameters for the protocols A and E are described below Method Wizard Fa Gel Filtration Peak Injection 7 Peak Fractionation Advanced one Peak Injection Page Injection Flush Yolurme E 0 100 ml 1 Mas Injection Volume z 0 100 ml 2 3 Peak Fractionation Final 4 Peak Fraction Size 20 0 2 2 mi 5 Peak Fractionation Algorithm Level AND_Slope x E Start Level 20 6000 6000 rd 7 Start Slope 10 0 010 S0000 rmAL min 9 Peak Max Factor U9 0 1 g Minimum Peak Width Jas 0 15 1500 min Stop Level 20 6000 6000 m U Stop Slope 20 0 010 50000 m U min L Cc Injection Flush Volume The extra volume besides the peak
202. ount Which target protein tag Which target protein tag F Ue imua Gatep HisTrap HF GSTrap FF HisTrap HP GSTrap HP 1 ral 1 ml o ml 5 ral Which Why has the target protein HiLoad 16 60 HiLoad 16 60 Superdex FS pg Superdex 200 pg Comments on alternative column choices Comments on alternative column choices Overloading of an affinity column can be performed to minimize non specific binding often results in a purer end product Use in GF step clogging or protein degradation To get sufficient separation it is important to choose a gel filtration column with appropriate selectivity AC If there is a low expressed target protein in a large sample volume consider using a 5 ml HiTrap column to reduce the risk of column ep l 4 Methodology 4 2 Column alternatives 4 2 2 Choosing columns Choosing columns Recommended column combinations when running an AC DS IEX protocol for AC DS IEX His or GS T tagged target protein amount high Which target protein tag Use in AC step HisTrap HP GSTrap FF HisTrap HP GSTrap FF 1 ml 1 ral D mil D mi HiPrep 26 10 Desalting f Which pl has the Which pl has the target protein target protein RESOURCE A 5 mil i RESOURCE 5 RESOURCE 1 ral AIT 6 ml p or any ane of or any one of or any ane of or any one of HiTrap Q HP 1 mi HiTrap SP HP 1 ml 2xHiTrap Q HP 1 mil 2xHiTra
203. overview 3 Tubing and connectors This section describes the tubing and connectors included in the separation system All tubing located before the pump in the flow path i e the sample and buffer inlet tubing has inner and outer diameters i d and o d of 1 6 mm and 1 8 respectively This tubing is connected with M6 fittings not flanged see illustration below except for tubings connected to the pump air sensor and switch valves where 5 16 fittings are used The tubing material is Teflon All tubing located after the pump in the flow path 1 e the system and outlet tubing has i d and o d of 1 0 mm and 1 16 respectively This tubing is connected with narrow headed fingertight connectors The tubing material is Tefzel except the outlet tubing to the fraction collector which is made of PEEK The system also includes stop plugs to be used in non used ports in the valves and the column adapter There are always a specific sample tubing S1 S4 a flowthrough outlet tubing F3 F6 and an outlet tubing for unselected peaks F7 F10 dedicated for each one of the samples These three tubings are marked with a label which has a unique color Sample no Tubing designation S1 F3 F7 Red Samplenos e The tubing for buffers A1 A8 B1 B2 fraction outlet F3 F11 and waste Waste 1 3 are 1 5 m long e The tubing for samples S1 S4 are 0 5 m long Note The tubing should never exceed these lengths e
204. ow liquid may enter the system causing a short circuit 18 1178 00 AA ep 32 AKTAxpress overview 3 Control panel The separation system is mainly controlled and monitored via UNICORN The separation system is though also equipped with a control panel with controls and indicators on the horizontal front bar The control panel allows for limited control of the system as well as information feedback O RUN ornus tt 8885 SS For more information about the indicators and controls see 3 2 5 Indicators and controls on page 43 ep 33 3 AKTAxpress overview 3 2 Separation system overview 3 2 2 Liquid flow path 3 2 2 Liquid flow path Introduction This section contains an overview of the tubing and components configuration of the liquid flow path in AKTAxpress The liquid flow The illustration below shows the liquid flow path with the valves in the default path positions E Waste Sas Outlet Valve Fraction Collector L1 O O BP v o Bypass 6P Y o L5 F 6 i T Loop Valve Manual LWW LoopV2 inject ColV Column Valve MixInj Inlet Valve Press s3 CA Samples NO EFA AAA AirS en Air sensor Pump 18 1178 00 AA ep 34 3 2 3 Introduction Sample and buffer inlet tubing System and outlet tubing Colored labels on the tubing Length of inlet and outlet tubing AKTAxpress
205. ow describes the Prepare options fill the buffer and System Can also be performed within a Purify sample inlets with buf method plan See 5 2 3 Using the Purify option on fer page 96 equilibrate the columns Columns Can also be performed within a Purify method plan See 5 2 3 Using the Purify option on page 96 metal ion charge of af finity column s perform a blank run to prepare the affinity and or the ion ex change column s 18 1178 00 AA ep 92 Method Wizard 5 oo e o o prepare both system System and Columns and columns The Pr diabe Sys If selecting System follow the instructions below If only selecting the Columns tem d option go to step 1 in 5 2 2 The Prepare Columns dialog on page 94 for further instructions Select the appropriate options on the Prepare System page The table below describes the options Method Wizard Prepare System Main Selections Prepare System Assisted Manual Loading of Superloop Tag Cleavage Prepare Columns Yes C No Last Page Fill Sample Inlets with Butter Yes No Fill Butter Inlets C Yes No Motel After the inlet fill instructions the system will be washed with 41 buffer to remove remaining air bubbles Click Next Selecting Prepare The table below describes the Prepare System options and the related actions during System options ernn manually fill the superloop with pro Yes default under Assisted Manual tease solution thro
206. p 35 3 AKTAxpress overview 3 2 Separation system overview 3 2 3 Tubing and connectors Tubing fitting The illustration below shows the three tubing fitting types used in AKTAxpress types 5 16 fitting M6 fitting Narrow headed fingertight connector ene the e When connecting an M6 or a 5 16 fitting the orientation of the ferrule must ittings be correct see the illustration above e When connecting a fitting the tubing must be inserted fully into the connection port before tightening the fitting 18 1178 00 AA ep 36 3 2 4 Introduction Sample and buffer handling AKTAxpress overview 3 Functional description This section briefly describes the function of the components in the liquid flow path of AKTAxpress The functions of AKTAxpress can be divided into six categories e Sample and buffer handling e Pump and related items e Column handling e UV and conductivity detection e Intermediate fraction collection e Final fraction collection For a more detailed description of the components see 10 1 System description on page 308 The parts for handling samples and buffers are located at the lower part of the separation system They comprise two switch valves for buffer selection and gradient a general inlet valve for samples and buffers an injection valve and an air sensor Injection valve orf Switch valves Inlet valve Air sensor Buffers Buffers Samples A1 A2 A3 A8 S1 to S4 B1
207. p A Superloop can be attached in the right hand column holder Note Do not put gel filtration columns in the right hand side column holder It will block the fraction collector and might get des troyed ep3l 3 AKTAxpress overview 3 2 Separation system overview 3 2 1 Location of the components Location of liquid A tubing holder and tube holder are included in the system at delivery The tubing oe and contain holder is used for organizing the tubing The tube holder can take tubes of two different sizes used for samples S1 S4 The tubes can also be used for flow through fractionation F3 F6 and nonselected peaks F7 F10 if small volumes are expected A flask holder is available as an option and can be mounted on the front of the system The flask holder can be used for samples bottles S1 S4 flow through fractionation bottles F3 F6 and bottles for nonselected peaks F7 to F10 The flask holder can take a maximum weight of 3 5 kg The holders can be used independently of each other 4 o A POTEET s khi ial H Tubing holder Tube holder aA Say Ary Flask holder Buffer and waste containers are usually large and may be stored directly on the floor or on suitable shelves Example of location of different liquid containers Buffers waste Samples fraction outlets WARNING Never place liquid containers on the top of the separation system If they become full and overfl
208. p SP HP 1 ml 2xHiTrap Q HP 1 mi 2xHiTrap SP HP 1 ml Mono O 5 50 GL 1 mill Mono S 5 50 GL 1 ml Mono O 5 50 GL 1 ml Mono 5 5 50 GL 1 ml Which target protein tag Use in OSstep Use in IEX step RESOURCE G T 1 ral i Comments on alternative column choices Comments on alternative column choices Overloading of an affinity column can be performed to minimize non specific binding often results in a purer end product If there is a low expressed target protein in a large sample volume consider using a 5 ml HiTrap column to reduce the risk of column clogging or protein degradation 1 ml HiTrap affinity columns can be combined with 2 x HiTrap desalting columns Band broadening effects in the system combined with a limited sample loading volume on the 2 x HiTrap desalting column might however reduce protein recovery 5 ml HiTrap affinity columns should not be combined with 2 x HiTrap desalting columns since the eluted affinity peak might be larger than the volume possible to load on the 2 x HiTrap desalting column A longer IEX column or a column with smaller bead size can be used to increase resolution 18 1178 00 AA e p72 Methodology 4 Comments on alternative column choices IEX Make sure to use an IEX column with sufficient capacity for the protein amount Choosing columns Recommended column combinations when running an AC DS IEX DS protocol for AC DS IEX
209. peaks are broadened due to e g a more flat gradient Note If the level values are decreased for very large or broad peaks the peak volume collected might be too big for the loop available or exceed the loading volume of the next column tee injection This applies to protocols that include tag cleavage volume The default value for the protease injection is 0 7 CV If using a larger value the target protein might be lost since it will pass through the column before peak collection is activated ep 141 6 Operation 6 Operation About this chapter This chapter describes the normal work flow when operating AKTAxpress from starting the system creating a method plan and preparing the system to cleaning the system and columns after the run In this chapter This chapter contains the following sections Topic Operation overview Starting the system Connecting a system Creating a method plan Preparing the system for a run Starting a run During a run Procedures after a run 18 1178 00 AA ep 142 Operation 6 6 1 Operation overview Introduction This section gives an overview of the operation of AKTAxpress Work flow The typical work flow in AKTAxpress after turning on a system and connecting it can be divided into a number of steps Step 1 5 are described in separate sections in this chapter Step 6 and 7 are described in chapter 7 Evaluation on page 208 1 Creating a method plan 2 Preparing the sys
210. peration 6 Enter any optional text for example running data Click Next The text can be viewed later in the result file as Notes in Evaluation by selecting View Documentation x Free Text System Free Text Result Hame Summary This is written by John Friday Amersham Proceed to 6 6 1 Final setup and starting the run on page 191 If selecting a Purify method plan e Select on which System s the method plan shall be used e Select the Number of Samples for each system s e Click Next Method Wizard ee _ l Systems Samples Systems Samples samplelD System 1 4 Systems Number of S amples Settings SYS 1 Iw SYS Settings SYS 2 Iw SyS 2 Settings SYS 3 Iv sys 3 Settings SYS 4 lf sys 4 Result Mame Summary SelectAll Systems C Deselect All Systems Set same number of samples for all systems ep 187 6 Operation 6 6 Starting a run 6 6 1 Starting a run using a method plan 5 Enter identification name s for the sample s A bar code reader can also be used An import file with sample information can be l nked to this sample ID See 10 3 Import file format on page 331 Click Next xi Sample ID System 1 4 Systems Samples sys 1 SamplelD System 1 4 Sample 1 Sample ID ouster Settings SYS 1 Sample 2 Sample 210 System 1 Settings SYS 2 Sample 3 Sample 31D System 1 Settings SYS 3 Settings S5 4 ST e Result Name Sample 1 Sample TID System 2 Summary Sample 2
211. plan and save it under a new name Creating a new The first page in the Method Wizard is the Create or Change Method Plan page method plan The Method Plan pane contains a list of saved method plans if any and a New option The Note pane displays the contents in the selected method plan x Create or Change Method Plan Method plan Note Use thie Method plan if you want to create a new plan ay GF FL Maintain H 0 Prepare Method plan selected Delete Back Emish Cancel Help Set Defaut e p 89 5 Method Wizard 5 2 Creating a method plan 5 2 1 Creating a new method plan To create a new method plan 1 In the Method plan list select New and click Next Result The Main Selections page appears Main Selections Main Selections Prepare C Purify Maintain o AKTAxpress lt Back Finish Cancel Help Note Always when entering the Main Selections page the parameter values on the subsequent pages are set to de fault Choose the requested type of method plan The Method Wizard provides three main selections The table below describes the method plan types that can be created from each selec tion Main selections Option Use to create a method plan for Prepare Preparation of the system and or the columns before the actual purification run Example of actions Filling the buffer and sample in lets with buffer Equilibrating the columns Instructions for
212. position both are set to the wavelength to be used 280 nm or 254 nm see 8 6 4 Replacing UV lamp on page 264 The theoretical extinction coefficient too low Calculate the theoretical extinction coefficient of the protein If it is zero or very low at 280 nm the protein cannot be detected ep 291 9 Troubleshooting and corrective actions 9 3 Monitoring problems Faulty conductiv Find the possible cause and action for a specific conductivity curve fault in the SO table below If the problem remains contact the local Amersham Biosciences representative Possible cause and action Baseline drift or noisy Leaking tubing connections signal Check for leaking tubing connections and correct see 6 5 9 Checking the tubing on page 180 Air in the conductivity flow cell There may be air in the flow cell Check that the flow restrictor gives a back pressure of 0 2 MPa see 8 2 1 Checking flow restrictor on page 223 Column not equilibrated Equilibrate the column If necessary clean the columns using a method plan for Column cleaning see 6 8 3 Cleaning columns on page 202 Air might be trapped in the pump Purge the pump according to the procedure in 6 5 3 Purging the pump and inlet tubing on page 156 Dirty conductivity cell Clean the conductivity cell see 8 3 3 Cleaning conductivity cell on page 231 Waves on the conductiv Air might be trapped in the pump ity curve Purge the pump according to the p
213. precalibrated on delivery but can be measured with a separate calibration procedure see 8 5 2 Calibration of conductivity cell on page 243 WENN LY BZ Liquid flow D A The pressure in the flow path is continuously monitored by a pressure sensor The liquid chamber in the pressure sensor housing is equipped with a titanium membrane A strain gauge is attached to the rear side of the membrane When the liquid pressure increases the membrane bulges which is detected by the strain gauge e p 319 10 Reference information 10 1 System description 10 1 3 Monitors The pressure sensor housing is made of PEEK other wetted parts are made of titanium and FFKM perfluororubber Air sensor The air sensor is a high precision monitor designed for continuous monitoring of air bubbles in the inlet flow path When air is detected the system is either paused or performs an action that is set in the method plan The flow path has i d 1 5 mm The air sensor is made of PEEK 18 1178 00 AA ep 320 Reference information 10 10 1 4 Fraction collector Introduction This section describes the fraction collector in AKTAxpress The fraction collector is used for e Peak fractionation e Elution fractionation Fraction collector The fraction collector is an X Y collector with a free arm The design gives a design compact unit with efficient operation ep 321 10 Reference informat
214. r Internal error Restart system If problem remains contact service System error Internal error Restart system If problem remains contact service ep 301 9 Troubleshooting and corrective actions 9 6 Error code list 416 417 418 No 422 423 427 428 429 430 431 480 481 500 501 The UV lamp is broken The UV lamp is broken Replace the lamp System error Internal error Restart system If problem remains contact service System error Internal error Restart system If problem remains contact service Filter size error The filter size in the UV lamp is out of range Check that the method is made for AKTAxpress System error Internal error Restart system If problem remains contact service Measurement warning The UV lamp has been switched off or the conductivity cell discon nected when measuring the absorbance or the conductivity Try to switch on the UV lamp If it does not help restart the system If the problem still remains contact service Lamp intensity warning The intensity of the UV lamp is too low Replace the lamp Dark current warning The dark current in the UV lamp is higher than the allowed limit Check if the seal is broken UV auto zero warning The UV value was out of the allowed range 0 2 to 2 AU when trying to set the relative absorbance signal to zero No peak found No peak was found by the Peak_Select instruction No peak
215. rective actions 9 7 Checking USB CAN driver In the Device Manager click the plus sign to expand the CAN Hard ware KVASER item Action wiew Jes emela S ESEUPPWHS0D2E6 Eg CAN Hardware KY ASER 3 Amersham UR J45 a KYASER Virtual CON Driver 2 Channels BE Simulated CAN Driver For Kvaser Creator c Computer od Disk drives Eee Display adapters If the Amersham URJ45 icon looks like the image above the install ation was successful If so go to step 10 If the Amersham URJ45 icon has an exclamation or warning sign you must reinstall the driver Continue to step 4 Right click the Amersham URJ45 icon and choose Properties Result The Amersham URJ45 Properties dialog is opened Click the Driver tab Amersham URI45 Properties General Driver BS Amersham UR 45 Driver Provider EVASER AB Driver Date Not available Driver Version 6 6 3990 0 Digital Signer Mot digitally signed To view details about the driver tiles loaded for this device click Driver Details To uninstall the driver files for this device click Uninstall To update the driver files for this device click Update Driver Uninstall Update Driver OF Cancel e Click the Update Driver button Result The Upgrade Device Driver Wizard is opened Click Next 18 1178 00 AA ep 304 Troubleshooting and corrective actions 9 Step Action 6 e Select the option Search for a suitable driver Upgrade Device Driver Wizard
216. required The installation test will check o The liquid delivery by pumping liquid from buffer inlets to waste e The gradient formation by producing a linear gradient and a series of step gradients of acetone The principle flow path of the installation test ne Waste 1 Acetone Deionized water Procedure To run the installation test follow the instructions in AKTAxpress Installation Guide 18 1178 00 AA ep 240 Maintenance 8 8 5 Calibration procedures About this section This section describes some calibration procedures of AKTAxpress In this section This section contains the following sub sections Topic Calibration of zero pressure Calibration of conductivity cell ep 241 8 Maintenance 8 5 Calibration procedures 8 5 1 Calibration of zero pressure 8 5 1 Calibration of zero pressure Introduction Problems in the pressure calibration can cause abnormal pressure readings in chromatograms For specification of the technical data and signal drift of the pressure sensor see 10 Reference information on page 307 The zero pressure reading of the pressure sensor can be calibrated The amplification is factory calibrated and cannot be changed ea inter When required and after replacing a pressure sensor va Calibrate zero To calibrate the zero pressure pressure In the UNICORN System Control module select the system to be calibrated by clicking the system name System Control S
217. rocedure in 6 5 3 Purging the pump and inlet tubing on page 156 Conductivity measure Dirty conductivity cell ment with the same buf fer appears to change over time Clean the conductivity cell see 8 3 3 Cleaning conductivity cell on page 231 The ambient temperature might have decreased or increased The conductivity of the solution changes with temperature Since there is no temperature com pensation the measured conductivity will be dif ferent at different temperatures The buffer might loose its characteristics over time Change buffer 18 1178 00 AA ep 292 Troubleshooting and corrective actions 9 Possible cause and action Ghost peaks in gradient Air bubbles are passing through the flow cell profile Check for leaking tubing connections and correct see 6 5 9 Checking the tubing on page 180 Air might be trapped in the pump Purge the pump according to the procedure in 6 5 3 Purging the pump and inlet tubing on page 156 Non linear gradients Air might be trapped in the pump Purge the pump according to the procedure in 6 5 3 Purging the pump and inlet tubing on page 156 Dirt or residues in the flow path from previous run Clean the system using a method plan for system cleaning see 6 8 2 Cleaning system on page 198 Organic solutions have been used The mixer is designed for non organic solutions only Faulty switch valve Flush through to clear any blockage by running e g System Wash Slow respo
218. rom the pump heads to prevent loss of any internal components Unscrew the two white knurled screws under the pump to release the support bracket locking the inlet manifold into the inlet valve P gt K 4 e p 235 8 Maintenance 8 4 Maintenance when required 8 4 2 Cleaning check valves Gently lower and remove the complete manifold wy Loosen the lower valve from the pump head using the 13 mm wrench CAUTION Handle the check valves with care when they have been re moved from the pump heads to prevent loss of any internal components Gently remove the check valve Immerse the complete valves in methanol and place them in an ultra sonic bath for some minutes Then repeat the ultrasonic bath with deionized water Refit the check valves The inlet check valve with a lip for the manifold and a larger diameter opening is fitted to the side marked IN of the pump head Tighten the valves until fully finger tight and then use the wrench to tighten a further 1 3rd 110 of a turn Note Do not overtighten the valves as damage to the internal components can occur 11 Refit the outlet tubing and the inlet manifold Purge the pump carefully and check that the pumping action has N been corrected see 6 5 3 Purging the pump and inlet tubing on page 156 18 1178 00 AA ep 236 Maintenance 8 8 4 3 Checking pump pressure Introduction Problems in the pump can cause abnormal pre
219. rotein degradation epol 4 Methodology 4 1 Protocols and affinity tag removal 4 1 4 Description of the affinity tag removal process 4 1 4 Description of the affinity tag removal process Introduction This section describes an on column affinity tag removal process The process is performed automatically in AKTAxpress Process overview The table below describes the stages in an affinity tag removal method The software assists the user to manually fill the superloop with the required protease solution at the beginning of the automated affinity tag removal method 3 end pci and orm whl of wes 1 3 6 The system is set to pause in order to allow on column cleavage in cubation The protease cleaves the target protein from the tag 7 The free target protein is washed out with AC binding buffer or Wash 2 buffer from the AC column and is collected in the capillary loop s Tagged protease tags and non cleaved protein stays on the column If non tagged protease has been used the protease will also be re leased with the target protein The target protein can be further purified Automatic affinity tag removal can be combined with any of the AKTAxpress multi step protocols 9 Substances still bound to the column i e the affinity tags tagged protease and non cleaved target protein are eluted with elution buffer and collected in the appropriate outlet tubing sample 1 F7 sample 2 F8 etc 18 1178 00 AA ep 62
220. rs excl flow cell Hg lamp amp housing complete UV flow cell 2 mm Filter 254 nm Filter 280 nm UV test kit 2 mm flow cell 280 nm Conductivity monitor Item 18 1128 20 18 1128 22 Conductivity flow cell complete 18 1111 05 Air sensor Item Air sensor complete 915N 11 0003 08 Flow restrictor The following spare part is available for the flow restrictor Item Flow restrictor FR 902 Cables The following cables are available Item UniNet 0 7 m UniNet 1 5 m UniNet 3 0 m UniNet 15 m 18 1121 35 ep 337 10O Reference information 10 5 Ordering information UniNet Inline coupler 11 0003 79 Mains cable EU 240 V 19 2448 01 Mains cable US 120 V 19 2447 01 Fraction collector The following spare part is available for the fraction collector Capillary loops The following capillary loops are available Capillary loop 11 0003 02 Filters The following filters are available Connectors and The following connectors and unions are available unions 18 1178 00 AA ep 338 Reference information 10 Union M6 male to 1 16 female Union M6 female to 1 16 male Union M6 female to 1 16 female 18 1123 94 PEEK Union 1 16 male to 1 16 male 18 1120 93 Union 1 16 female to 1 16 female 18 3855 01 Union Luer female to 1 16 male 18 1112 51 Tubing The following tubing is available Tefzel tubing i d 1 mm o d 1 16 3 18 1142 38 Teflon tubing i
221. rt a syringe with gt 5ml buffer in the Injection Valve Press CONTINUE A new dialog box appears requesting the buffer to be injected e Inject the buffer and click Continue Do not remove the syringe before clicking Continue System Control SYS_1 Message Inject at least 5 ml buffer Do not remove the syringe Press CONTINUE A new dialog box appears requesting a syringe with protease solution to be fitted e Remove the syringe from the fill port e Fill the syringe with the requested amount of protease solution re fit it in the fill port and click Continue Do not inject the pro tease before clicking Continue System Control SYS_1 Message Insert syringe with protease solution Press CONTINUE Q gt gt N AD 3 vill Y EE II WEF Vy 3 i i _ gt cs Ij lt w g Ea T gt 18 1178 00 AA p 178 Using a column heater Operation 6 6 A new dialog box appears requesting the protease solution to be injected e Inject the protease solution and click Continue to proceed the run Do not remove the syringe The protease will automatically be loaded onto the affinity columns during the purification run System Control SYS_1 Message miei Inject protease solution Do not remove the syringe Press CONTINUE X Continue Help Filling the superloop manually The protease can also be injected directly into the superloop before starting the run by connecting the syringe
222. s HiLoad 16 60 Superdex 75 prep grade 120 ml HiLoad 16 60 Superdex 200 prep grade 120 ml Characteristics HiLoad column prepacked with Superdex 75 prep grade For high res olution polishing step for proteins of sizes 3 70 kDa and for sample volumes of 5 ml Columns prepacked with Superdex 200 prep grade For high resolution polishing step for proteins of sizes 10 600 kDa and for sample volumes of 5 ml Methodology 4 Coupled columns Some columns can be used in pairs 2 x 1 ml HiTrap SP Q HP 2 x 5 ml HiTrap Desalting Further informa More information about the columns can be found in the Column list e g flow aon rates and pressure limits used Select Edit Column list in the Method Editor Click Advanced Parameters to view the AKTAxpress column parameters Note The Method Wizard does not support all columns in the Column list For further information see the handbooks in 4 4 Documents for further information on page 83 ep 69 4 Methodology 4 2 Column alternatives 4 2 2 Choosing columns 4 2 2 Choosing columns Introduction This section describes how to choose columns Information on what to consider before choosing columns as well as selection guides for the various supported protocols are included Consider when Questions to be considered before choosing columns choosing columns D o l a e How much purified protein is needed after the final purification step e How much sample i
223. s are emptied from the loops to the collection tubes F7 F10 one for each sample The loops are then flushed with buffer A2 to waste The table below describes the gel filtration procedure of the collected peak volumes System Wash is performed to fill the system with buffer A4 2 The largest IEX peak collected in the loops is loaded onto the GF column The maximum volume is 5 ml a The GF column is eluted with buffer A4 The eluted peaks are collected in the fraction collector The excess of the selected peak from the injection onto the GF column is emptied from the loops to the collection tubes F7 F10 one for each sample The loops are then flushed with buffer A2 to waste 6 Elution of the AC column desalting ion exchange and gel filtration is repeated for each sample epdl 3 AKTAxpress overview 3 3 Purification process overview 3 3 2 Description of the process steps Tag cleavage 18 1178 00 AA ep 52 The table below describes the tag cleavage procedure and includes the elution of the AC column Hence when tag cleavage is used this part replaces the Elution of the AC column part described previously in this section A second wash is performed with buffer A1 B1 to wash out unbound sample The column is filled with cleavage buffer A7 optional The protease in the superloop is loaded onto the column s ation the tags are cleaved from the protein of B1 and the peak volume collected in a
224. s on subsequent pages depends on the choices made on the initial page The Method Wizard can be divided in two different parts e The first part where the main selections that define the method plan are made e g type of purfication protocol column types etc All parameters have pre optimized default values e The second part Advanced Zone where a number of parameter values in the method plan can be viewed and changed if preferred Note Avoid changing default parameter values in a method plan unless the result is clearly understood Changing a parameter value might require adjustments of other parameter values as well A Method plan is a set of method instructions created by the Method Wizard which define the frame of a run Additional information is though needed before running the method plan on a specific system See Using a method plan below The Method Wizard can be used for creating three different types of method plans e Preparation of columns and the system e A protein purification run e Maintenance of columns and the system The first step when creating a method plan is to select the type of method plan to be created Different options are then presented depending on the chosen type When all required selections are made the method plan is saved by a user specified name Before using a method plan in a run some additional information must be added e g selecting the separations systems to use number of samples n
225. s to be loaded e What purity and resolution is required Choosing columns Recommended column combinations when running an AC DS protocol for AC DS His or GS T tagged target protein amount Which target protein tag His T rap HP G5 Trap FF F mil F mil HiPrep 26710 Desalting Comments on alternative column choices Comments on alternative column choices Which target protein tag Use in ACstep HisTrap HP GSTrap FF 1 mil 1 ral Use in D step Overloading of an affinity column can be performed to minimize non specific binding often results in a purer end product 1 ml HiTrap affinity columns can be combined with 2 x HiTrap desalting columns Band broadening effects in the system combined with a limited sample loading volume on the 2 x HiTrap desalting column might however reduce protein recovery AC If there is a low expressed target protein in a large sample volume consider using a 5 ml HiTrap column to reduce the risk of column clogging or protein degradation 18 1178 00 AA ep 70 Methodology 4 Comments on alternative column choices DS 5 ml HiTrap affinity column should not be combined with 2 x HiTrap desalting columns since the eluted affinity peak might be larger than the volume possible to load on 2 x HiTrap desalting columns Choosing columns Recommended column combinations when running an AC GF protocol for AC GF His or GS T tagged target protein am
226. selected No peak has been selected Use the Peak_Select instruction to select a peak System error Internal error Restart system If problem remains contact service 18 1178 00 AA ep 302 9 7 Introduction Checking the driver installation Troubleshooting and corrective actions 9 Checking USB CAN driver If there are connection problems between the computer and the separation systems the USB CAN driver installation can be checked To verify that the CAN drivers have been correctly installed 1 Open the System Properties dialog box e Open the Control Panel and choose System or e press down the Windows key and the PauselBreak key simultan eously In the System Properties dialog box click the Hardware tab System Properties General Network Identification Hardware User Profiles Advanced Hardware Wizard The Hardware wizard helps vou install uninstall repair unplug eject and configure pour hardware Device Manager B The Device Manager lists all the hardware devices installed on your computer Use the Device Manager ta change the properties of any device Driver Signing Device Manager Hardware Profiles cos Hardware profiles provide a way for you to set up and store ey different hardware configurations Hardware Profiles OF Cancel Apply e Click the Device Manager button Result The Device Manager is opened ep 303 9 Troubleshooting and cor
227. sione a a aa aAa 47 Fode HPO oo TOW WOK oaoa N atatateaminue mma teeMer 48 3 3 2 Description ofthe process SLEDS cseiicrsicdaniaidascan a a a aa 49 A VIC UIOUOIOGY aiuSis2G ise ahiic tact cceirawnucudeadentataaswaredemaieaneaumandescaueenunaweautweente sun Gusesdsaamsaucaiaueeuanecads 53 4 1 Protocols and affinity tag removal ssuasunnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnn nnn 54 Alel Protocokdescriptons and COICO einn a bhandaueneainsentunanioteninn 55 A dvi Describthonr or ALL IMIEY LAS Seana anda T ae etiam a 59 A loe DESCKID LION O Dl OVCASCS eerren eai 61 4 1 4 Description of the affinity tag removal PrOCESS cccceeeeseeeeeeeeeeteeeeeeeteatenees 62 A Laos Conditions for atfinity tag cleavage rnn n a E N 64 AZ Columa altemnati veS a a a ae aa RES ESE 65 A Zele DESCrIDLIONS OF COLUM Sorire aan r anaes eteiaet 66 A22 CROON COUM S ern aa a a a 70 4A 3 BURNET ANGMAtIVG Ss cswssnsosgotepsdsaweunemaataba Eia 77 A ele DeScripuUon Ol DUNO Sera ar 78 A Oed BUNE SUS SCSTIONS scsnoossanies vonnntdavendete maraiatagueds ETA 80 4 4 Documents for further INfOrMatiOn cccccececeeeeeeeeeeeeeeeeeeteeeeeeeteeeeeeeteaesneeteaeats 83 ep3 Table of Contents 9 Method Wizard aiiin a e E a 84 Sds Method Wizard IMUFOGUC ION ceriose n E A 85 9 2 GRAINS a method DIAN ceinaren rnia A N 88 5 2 1 Creating a new method DIAN a wdacaseahinuiaorninttaxiweanaiadesnniadiwmnieameeniiiaxicumearadaseses 89
228. ssure readings Examples of pump problems e Air trapped in the pump heads e Leaking connections e Leaking pump piston seal e Check valve malfunctioning e Piston damage Checking the To check the pump function pump function Disconnect the Waste tubing from the injection valve port 1 and connect a capillary tubing giving a counter pressure of about 1 MPa at 1 ml min to port 1 Set the injection valve to position Waste Start a flow of 1 ml min or less and observe the pressure curve over several minutes tubing on page 156 Check the tubing connectors for leakage Check the piston seals check valves and pistons Check the number of piston strokes by selecting System Mainten ance in System Control The life time is 4000 x1000 i e 4 000 000 strokes If the pressure pulsation seems abnormal Purge the pump heads see 6 5 3 Purging the pump and inlet e p 237 8 Maintenance 8 4 Maintenance when required 8 4 4 Checking tubing leakage 8 4 4 Checking tubing leakage Introduction Problems with leaking connectors can be found running a tubing leakage test Checking the To check for leakage in the system tubing tubing leakage Check that the inlet tubing to be tested is properly immersed in a flask with deionized water Start the pump e Select System Control Manual Pump e Select a flow rate of for example 20 ml min SYS_1 Pump Instructions x Instructions Panar rs Pump FlowRate
229. st commonly used chemicals in liquid chromatography Note AKTAxpress is intended to be used with water based solutions only Organic solvents are not recommended due to the mixer design en The ratings are based on the following assumptions ma e The synergistic effects of chemical mixtures have not been taken into account Room temperature and limited overpressure is assumed Note Chemical influences are time and pressure dependent Unless otherwise stated all concentrations are 100 List of chemicals List of chemicals and their compatibility to KTAxpress Chemical Exposure lt 1 day Exposure up to 2 Remarks months T T T i OK FFKM PP and PE swell OK K Acetonitrile Acetone 10 PVDF is affected by long term use Ammonia 30 O OK Silicone is af fected by long term use Ammonium O OK chloride Ammonium bicar O bonate Ammonium ni O trate Ammonium O OK sulphate Trimet OR ORY K K K K K 18 1178 00 AA ep 332 Chemical 2 Butanol Citric acid Chloroform Cyclohexane Detergents Dimethyl sulphox ide 1 4 Dioxane Ethanol Ethyl acetate Ethylene glycol Formic acid Glycerol Guanidinium hy drochloride Hexane Hydrochloric acid 0 1 M Reference information 10 Avoid ECTFE CTFE PP and PE are af fected by long term use Exposure lt 1 day Exposure up to 2 K K K K months x w wo w f Avoid Avoid PVDF is
230. stop when the sample supply is empty and air enters the air sensor See also 6 5 10 Using air detection on page 182 18 1178 00 AA ep 136 Miscellaneous op tions Method Wizard 5 Max Sample Volume The maximum allowed sample volume when air sensor controlled sample loading is enabled Pump Air Removal Volume Between Samples The volume used to remove air from the pump between sample loadings Extinction Coefficient corresponds to Choose either mg ml or M e mg ml When starting a run with the wizard in System Control the absorbance of 1 mg ml solution of the purified protein at 280 nm in a 1 cm cuvette must be entered When using Pool Fraction in Evaluation the unit will be given in mg ml and and the amount of protein in mg e M When starting a run with the wizard in System Control the Molar extinction coefficient 1 M cm of the purified protein at 280 nm in a 1 cm cuvette must be entered When using Pool Fraction in Evaluation the unit will be given in M and and the amount of protein in mmoles Note The extinction coefficient is used for calculating the protein amount and concentration when using Pool Fraction in Evaluation Unit Molecular Weight The unit of the protein molecular weight that will be shown e g during the evaluation Preferred Target Concentration The target concentration of the protein When pooling the fractions in Evaluation the software calculates how each pool should be concentrated or diluted in ord
231. tadeawaaucausanacaudeenasebenaduceaaneamtedainenneniameiamen 142 Gites Operation OVENVIOW cance ed ia name ataie uote vacant tals bare a a T 143 O57 2 SLAMS TMG SyS le Mra aea E E Eei 144 O3 CONNECTING da SVS lE Nison E E A E 146 Ob Greating a method Placer a S aa 148 6 5 Preparing the system for a FUN ccccceseccceeesseeceseeeseeeseeueeeueeeeeseeeeeeaseeseeueeensesoesenaaes 151 65 14 Preparing D ffers and Sol tiONSsasi nesnenin da oaei eda 152 6 5 2 Preparing outlet and waste tUDING ccccceceseee eet eeteteeteeeeteeteaeateateneaneatanes 155 6 5 3 Purging the pump and inlet tUbing sssseesssesssrrrnsrrrrrrrrrrrrrrrrnrrrrrnrrrresrrne 156 6 024 CONMECTING columns and UDINE ersan nn e tenGalendeadanaiadtunte 163 Ovo Oy CONGITIONING COMMAS gancnttateniaraubasntdunabiies tuvenland a T EEE TN 166 Os 90s Fe pal IS SAMDE Sini oe E EE O S 171 6 5 Preparing the fraction collector esseesesseessnrrrssnrrnrrrrrnrrrrrrrrrrrnrrrrrnrrereerrne 173 6 5 8 Preparing the system for automatic affinity tag removal cccccceeeee eee eee eens 175 18 1178 00 AA ep4 Table of Contents 62939 CHECKING ANG TIDINGS stuvcnaee tesca e EA ESEE Salutes 180 Of Oe USIMGS II detec HON carine E NO 182 6 5 11 Using flow control during sample lOACING cccccceeeeeeeeeteeteeeeteateeeeteaeaees 184 6 6 SCAN UNIV cet PUD lt audecss seaiwacvessnesenececsascecuamncsnecereseacsenneaaes 185 6 6 1 Starting a run using AIMCO Dla i
232. tc Make sure that no air enters the tubing If automatic filling is selected in the method plan a dialog will appear requesting the sample inlet tubing to be moved Fill the sample inlet tubing with affinity buffer A See 6 5 3 Purging The sample inlet tubing must be filled with buffer before starting a run to prevent air from entering the flow path To perform it manually see 6 5 3 Purging the pump and inlet tubing on page 156 It is also possible to do it automatically during the system preparation by selecting it in the method plan All purification protocols have air detection enabled by default during the sample application The sample application stops automatically when the sample supply is empty and air detected The sample volumes can also be set manually if preferred The air detection will then be disabled during sample loading See 6 5 10 Using air detection on page 182 e p 171 6 Operation 6 5 Preparing the system for a run 6 5 6 Preparing samples Manual purifica There are two purification protocols in the Method Wizard which require manual tion protocols sample loading e Protocol F Desalting Manual Sample Loading e Protocol G Gel Filtration Manual Sample Loading When running these protocols the sample is injected with a syringe through the fill port MANUAL INJECTION in the injection valve into the loops in the loop valve Up to four loops can be filled Dialog boxes will automatically appear duri
233. tch Level Greater than value AND the UV slope exceeds the Watch Slope Greater than value Note If the flow rate is changed the shape of the peak will change as well Hence the slope values might need to be changed accordingly Stop Collection The Peak_Max condition occurs when the UV signal has fallen to a fraction specified by Peak Max factor of the most recent peak maximum The Peak end is detected when the Peak_ Max condition is met AND e the signal falls below the Watch Level Less than value OR e a Valley is detected OR e a Watch StablePlateau condition is met The condition is met when the signal is stable within the limits of the Delta Plateau value during the Stable Time interval Peak SelectionSelect Largest Peak Based on either peak height or peak area to be transferred to the next column Area is the default value For more information on peak handling and watch conditions see 5 5 3 Peak handling and watch conditions on page 116 ep 131 5 Method Wizard 5 5 Advanced Zone 5 5 7 Advanced Zone for Purify Gel filtration J 0 Introduction Flow rate Pres sure Volumes parameters Peak injection parameters Advanced Zone for Purify Gel filtration This section describes the parameters used in the gel filtration steps for the protocols A E and G Some of the default parameter values depend on the chosen column Note Make sure that the parameter values do not exceed the specification of the ch
234. te 7 Pressure Advanced one Flow Fate Page Equilibration il 0 65 rolrmin Sample Loading fi 0 65 mlmin 2nd wash fi 0 65 ml min Elution fi D 65 ml min Pressure Limit jas 0 3 MPa IY Enable Flow Control During Sample Loading Minimum Allowed Flow Aate During Sample Loading g D 65 ml min wo co M AA FS A y a Equilibration Flow rate when equilibrating the affinity columns Sample Loading Sample loading flow rate 2nd Wash Flow rate used in the second wash Elution Flow rate during the elution of the affinity columns Pressure Limit Upper pressure limit during the affinity step Enable Flow Control During Sample Loading Automatic flow control when loading the sample Prevents the pressure from exceeding the maximum limit by reducing the flow If the flow falls below the Minimum Allowed Flow rate an Alarm is issued and the system enters Pause mode See also 6 5 11 Using flow control during sample loading on page 184 18 1178 00 AA ep 120 Volume paramet ers 2 nd Wash para meters Method Wizard 5 The volume options and parameters are described below Method Wizard Affinity Yolumes Advanced Zone Equilibration Yolure o 0 335599 Cy 7 jal wo fai Enable NaQ0H Wash Between Samples Tat Wash Volume 20 D 999999 Cv I Enable Watch Stable Baseline Stable Time 0 9999 min Delta Base z D 6000 m u Elution Wolume f 0 999999 Cy Equilibration Volume
235. te to set the valves to the correct positions m Instructions Parameters oo Injection alve Position Insert P ColurnPosition Flowpath OutletValve B Delete C Frac C Alarms amp Mon Other J Auto update If this is checked the parameter fields will be updated during method run LoopSelection Repeat step 3 5 to fill the inlet tubing To fill the inlet tubing B1 and B2 1 Check that the inlet tubing B1 and B2 are properly immersed in the correct flasks e p 157 6 Operation 6 5 Preparing the system for a run 6 5 3 Purging the pump and inlet tubing Instructions Parameters Ha Pump FlowRate 0 00 65 00 inset C Flowpath o m ml min Delete C Frac pede C Alarms amp Mon B Buffer C Sample Other MinFlow 0 01 65 00 0 10 m ml min FlowContr B No C Yes 7 Auto update If this is checked the parameter fields will be updated during method run Click Execute to start the flow To fill the B1 inlet tubing the inlet valve first has to be set e Select System Control Manual Pump e Select Gradient and Target 100 B and Mode A1 B1 e Click Execute to set the valve to position B1 Instructions Parameters Opie Target 0 0 100 0 met C Flowpath 100 XB Delete C Frac Lenath 0 00 9999 99 C AlarmskMon 2 00 a min Other ae ode G ave C A2 B2 J Auto update If this is checked the parameter
236. tem and columns Creating a method To create a method plan plan Click the Method Wizard icon in the Method Editor module oe Result The Method Wizard dialog box appears Click Next Result The Main Selections page appears 2 Select New to create a new method plan where all parameters have optimized default values provided by the Method Wizard or Select an existing method plan Some of the parameter values can be changed in Advanced Zone Method Wizard x Create or Change Method Plan Method plan Note Mew Use this Method plan if you want to create a new AC DS IEX GF plan AC GF no airs CIF reg Note For information on the current page click Help 18 1178 00 AA ep 148 Operation 4 Select the main type of method plan to be created and click Next Method Wizard J Main Selections Hain Selections Purify Prepare Prepare System Column Equilibration Last Page Purity I Include System Preparation iM Include Column Equilibration Maintain Note Selecting Include System Preparation and then Fill Sample Inlets with Buffer on the Prepare System page is recom mended On each new page select the appropriate parameter values and click Next to continue On the Last Page e Click Finish to save the selections as a method plan or Result The Save As page appears e Click Next to enter the Advanced zone if any parameter values needs to be changed See 5 5 Advanced zone on page 109 Note Do not change
237. tem for a run 3 Preparing a method plan for a run and starting the run i 4 During a run viewing and changing parameters 5 After a run cleaning the system and columns 6 Evaluate the results 7 Create a report i ep 143 6 Operation 6 2 Starting the system 6 2 Starting the system Introduction This section describes how to turn on AKTAxpress including the computer equipment and how to log on to UNICORN Turning on separ To turn on the separation systems 1 If the system unit number is shown in the display ation systems RUN a The system is set in Idle mode No action is required because the system was turned on when the AC cable was inserted Two segments might flash to indicate no communication with the UNICORN computer If only one segment is on in the display RUN PAUSE The system is set in Standby mode Turn on the system by pressing the On button on the front panel cA Result The display indicates Idle mode and the system identity 1 12 is shown Check that the system identity is correct The separation system should have a unique identity within the interval 1 12 which might have been set from factory If the identity is incorrect it has to be set see the Installation Guide 18 1178 00 AA ep 144 Operation 6 Turn on the com To turn on the UNICORN computer puter Turn on the monitor printer and computer according to the manu facturer
238. th the ejection and loading of the fraction collector Is there a 2 ml deep 96 well microplate in the fraction collector see 6 5 7 Preparing the fraction collector on page 173 Purification checks Does the positioning of the columns correspond to the selections made in the Method Wizard Refer to the Summary page Have all columns been cleaned and prepared according to the column recommendations Have the samples been adjusted to binding buffer conditions Have the samples been clarified by centrifugation and or filtration prior to sample loading Are the correct buffers used for the chosen columns and proteins Are the chosen columns suitable for the chosen target proteins The pH of some buffers changes with the temperature Note The mixer is designed for use with water based solutions only If organic solvents are used incorrect gradients or incomplete mixing may be the result ep 283 9 Troubleshooting and corrective actions 9 2 Problems during a run 9 2 Problems during a run Introduction This section specifies troubleshooting for problems related to sample loading and elution Pressure problems Find the possible cause and action for a specific problem in the table below High back pressure Possible cause and action Make sure that the samples have been centrifuged and or filtered through a 0 45 um filter and that no precipitation has occurred prior to sample loading Make sure that automati
239. the Prepare option are given in 5 2 2 Using the Prepare option on page 92 18 1178 00 AA ep 90 Method Wizard 5 Option Use to create a method plan for Purification operations Example of actions Performing different types of protein purification runs Instructions for the Purify option are given in 5 2 3 Using the Purify option on page 96 Maintain Maintenance of system and columns Example of actions Performs different types of cleaning operation and customized equilibrations Run installation test Instructions for the Maintain option are given in 5 2 4 Using the Maintain option on page 102 For more information on the options available on the current page click Help e p91 5 Method Wizard 5 2 Creating a method plan 5 2 2 Using the Prepare option 9 2 2 Using the Prepare option Introduction This section describes how to create a method plan for preparation of the systems or the columns before the actual purification run Selecting the type To select the type of preparation On the Main Selections page select Prepare of preparation Note Always when entering the Main Selections page the parameters values on the subsequent pages are set to de fault Select System and or Columns The table below describes the options Method Wizard Hain Selections Hain Selections Prepare System Prepare Prepare Columns I System Last Page Purify Maintain Click Next The table bel
240. the pump heads see 8 6 6 Replacing pump piston seal on page 272 Tubing connector leaking Unscrew the connector and check if it is worn or incorrectly fitted If required replace the faulty connectors see 8 6 2 Replacing tubing on page 249 Internal fault The item must be replaced contact a local Amersham Biosciences representative ep 297 9 Troubleshooting and corrective actions 9 5 Alarms and connection problems 9 5 Alarms and connection problems Introduction This section specifies troubleshooting for when there are alarms and connection problems Alarms Find the solution for a specific problem in the table below UNICORN error See UNICORN help files and manuals Error code flashing on Check the meaning of the error code in 9 6 Error code the separation system list on page 300 on prob Find the solution for a specific problem below ems If the connection problem cannot be solved by the instructions below check the log file c unicorn bin p4can_drvX 1log where X is the system unit number Contact a local Amersham Biosciences representative and be prepared to send the log file No text on the system s Check that the power cable is connected at the front display back of the separation system Check that the power socket has voltage present One segment on and If the AKTAxpress program update dialog does system unit no or 256 not appear in the UNICORN computer the instru displayed me
241. the purification e Should the protein be in any specific buffer to keep it stable and suitable for the next application e What purity is required e Is it important to have a charge and or size homogenous sample after the purification The more purification steps that are performed the higher the purity of the target protein will be Protein losses increase with each successive purification step AKTAxpress is optimized to perform the supported automated multi step protocols as efficiently as possible It is however up to the user to combine purification techniques columns and buffers to achieve the best purification for each specific target protein The table below gives guidance on some sample characteristics achieved after the last purification step for each protocol Protocol Buffer ex Charge homo Size homogen Effect on pur change geneity j AC DS AC GF AC DS IEX AC DS IEX G F AC DS IEX x DS Run times and maximum number of samples Methodology 4 The run time mainly depends on the number of samples the column properties and the number of chromatography steps in the protocol The run time is also affected by the temperature the sample viscosity and amount sample loading time and the length of the wash steps after loading the sample onto the AC column The run times in the tables below do not include the sample loading time Room temperature Specifications for the seven pro
242. the run has finished the result files can be processed using standard run procedures or own developed run procedures Automatic pooling of fractions will be performed if single chromatograms are opened e p25 3 AKTAxpress overview 3 1 UNICORN overview Help functions The table below describes how to access the on line help utility included in the UNICORN software the general help utility open theHelp menu in any of the software modules context specific help e click the Help button in the dialog box or topics e press the F1 key on the keyboard 18 1178 00 AA ep 26 3 2 AKTAxpress overview 3 Separation system overview About this section This section shows the location of the components in AKTAxpress as well as the In this section controls indicators and rating labels It also describes the liquid flow path with a functional description of the components This section contains the following sub sections Topic Location of the components Liquid flow path Tubing and connectors Functional description Indicators and controls Labels ep2 3 AKTAxpress overview 3 2 Separation system overview 3 2 1 Location of the components 3 2 1 Location of the components Introduction This section contains an overview of the components in the separation system Fluid handling All fluid handling components are located on the front of the separation system components Outlet valve Fraction
243. tinue with the instructions below the method plan After selecting the requested preparation options click Next Result The Last Page appears Proceed to 5 2 5 Saving the method plan on page 106 e p 95 5 Method Wizard 5 2 Creating a method plan 5 2 3 Using the Purify option 5 2 3 Using the Purify option Introduction This section describes how to create a method plan for a purification run Selecting the Puri To select a protocol for purification fy option 1 On the Main Selections page select Purify e Include System Preparation For assisted manual filling of the superloop for tag cleavage to fill the sample inlets before purific ation or to clean the sample inlets after sample loading e Include Column Equilibration To equilibrate the columns before starting a purification run Click Next Method Wizard Main Selections Main Selections Purify Prepare Prepare System Column Equilibration Last Page Purity I Include System Preparation IM Include Column Equilibration Maintain 18 1178 00 AA ep 96 Method Wizard 5 Select Purification Protocol and the appropriate options on the Purify page The table below describes the options Main Selections Purify Last Page o AKTAxpress lt Back Nert gt Fintsh Cancel Help The Purify page options the run To perform tag cleavage not avail able for Protocol F G Select Tag Cleavage Purify Puri
244. tocols when purifying a His tagged protein in room temperature A AC GF B AC DS C AC DS ae Estimated run times for one sample Estimated run times with max number of samples Maximum num ber of samples D AC DS a DS E AC DS IEX Cold room Specifications for the seven protocols when purifying a His tagged protein at cold room temperature Estimated run times for one sample Estimated run times with max number of samples Maximum num ber of samples C AC DS a D AC DS at DS ep5 4 Methodology 4 1 Protocols and affinity tag removal 4 1 1 Protocol descriptions and choice Protocol Maximum num Estimated run Estimated run ber of samples times with max times for one number of sample samples E AC DS IEX 10 1 h 51h GF 18 1178 00 AA ep 58 4 1 2 Introduction About affinity tags Affinity tag types Methodology 4 Description of affinity tags This section gives a general description of affinity tags and their use as well as a more detailed description of the two affinity tags that can be used with AKTAxpress Affinity tags are often used to facilitate the purification of recombinant proteins An affinity tag is included on DNA level for the recombinant protein normally at the N or C terminus The affinity tag can bind to an affinity chromatography medium with a suitable ligand allowing specific purification For several applicati
245. ttings 2 Change the desired values on Page 2 if Maintain was selected and click Next Hi aintain Advanced ane Page Strip Affinity Columns 0 65 ml min 0 999999 CY Last Page Volume Water 10 D 999999 cy R E 0 5 0 3 MPa Flow Aate 1 z Volume Strip Butter Pressure limit Upper pressure limit during the stripping procedure Flow Rate Flow rate during the stripping procedure Volume Strip Buffer Volume of stripping buffer to be used Volume Water Volume of water used for washing after the stripping procedure 18 1178 00 AA ep 138 Method Wizard 5 Clean system To change parameter values in a Clean System method plan 1 Select the required options on Page 1 and click Next Select General Advance Settings to define a main folder for storing the results Method Wizard Advanced one Select type of variables to change Advanced one Page M Maintain 1 4 IY General Advanced Settings Change the desired values on Page 2 if Maintain was selected and click Next Method Wizard Hi aintain Advanced one Inlet Clean Yolurnie 0 999999 ml Page Loop Wash Volume 10 999 ml 1 2 Inlet Clean Volume Volume of cleaning solution to be used for each selected inlet Loop Wash Volume Volume of cleaning solution to be used for washing each selected loop Defining a folder To define a sub folder under the Home folder for storing the results for storing the res u
246. ttons tok J go back to the previous page tiie J finish creating the method plan and save it poner cancel the settings and close the wizard Nothing is saved Ded open help texts for the active page Set Default restore all settings to the default values Enabled on the first page only tee J delete a selected method plan or folder Changing selec The major selections made in the first part of the Method Wizard will be locked nom and changes not allowed when e the Save As page is entered in the first part of the Method Wizard or e the Last Page in Advanced Zone is entered Note Entering the Main Selections page sets the parameters values on the subsequent pages to default It is though always possible to change parameter values in Advanced Zone parameters included in the major selections e p 87 5 Method Wizard 5 2 Creating a method plan 5 2 Creating a method plan About this section This section provides instructions for creating a new method plan using the Method Wizard In this section This section contains the following sub sections Creating a new method plan Using the Prepare option Using the Purify option Using the Maintain option Saving the method plan 18 1178 00 AA ep 88 Method Wizard 5 5 2 1 Creating a new method plan Introduction This section describes how to create a new method plan and the main selections available It is also possible to open and edit an existing method
247. typed on the Settings page in the Method Wizard Method Wizard E l Settings SYS_1 Systems Samples SamplelD System 1 4 Sample 1 Settings ST5S_ 1 Sample Yolurie m 0 9999 ml Result Name pl 0 14 Summary Ext Coeff corr to mgm hii kDa Note Make sure that the sample supply is sufficient for the given sample volume Air might otherwise enter the column ep 183 6 Operation 6 5 Preparing the system for a run 6 5 11 Using flow control during sample loading 6 5 11 Introduction About flow con trol Enabling dis abling flow con trol Using flow control during sample loading This section describes the flow control function in AKTAxpress The flow control is used to avoid exceeding the maximum pressure limit When flow control is used the flow is automatically decreased when the pressure approaches the set maximum limit After a short while when the pressure has decreased the flow slowly increases towards the set flow rate and so on If the flow rate falls below the set minimum limit 0 1 ml min is default the system will issue an Alarm and enter Pause mode The flow control function is activated throughout the run in all purification protocols The flow control can though be disabled during the sample loading in the affinity step To disable flow control in a method plan go to the Affinity Flow rate Pressure page in Advanced Zone in the Method Wizard 18 1178 00 AA ep 184 Operati
248. ugh the fill port in Loading of Superloop Tag Cleavage the injection valve When starting a run help texts appear to guide through the operation fill the sample inlets with buffer Yes under Fill Sample Inlets with Buffer Select which sample inlets to fill fill buffer inlets Yes under Fill Buffer Inlets Select which buffer inlets to fill ep 93 5 Method Wizard 5 2 Creating a method plan 5 2 2 Using the Prepare o ption The Prepare If selecting Columns follow the instructions below If not proceed to 5 2 5 Saving Columns dialog Selecting Prepare Column options 18 1178 00 AA ep 94 the Method plan on page 106 Prepare Columns Mote Max 5 columns allowed Running Condition 0 Room Temperature Cold Room Affinity Desalting Column E quilibration T lon Exchange Gel Filtration Column E quilibration Tip To customize the columna equilibration select Maintain on the first page and then CIP Columns Rigorous Customized Equilibration Select type of column s to prepare When selecting a column type new options will appear related to the column The options are described in the table below Click Next The table below describes the Prepare Column options and the related actions during the run Column Equilibration available for all columns 1 Specify Running Condition on the Prepare Columns page Method Wizard Main Selections Prepare System Prepare Columns
249. um Peak Width is reached even if the signal is fluctuating Peak collection The peak collection options and parameters for the protocols D and E are described below The parameters define how the peaks are collected in the loops Peak Collection Max Volume in Each Loop fa 0 5 10 ral Allow Collection of Single Peaks in Several Loops 2 3 4 Mas Number of Loops z ba 5 E 7 Yes No 18 1178 00 AA ep 130 Method Wizard 5 Max Volume in Each Loop The maximum volume allowed in each loop Using volumes above 7 5 ml might cause sample loss due to band broadening Max Number of Loops The maximum number of loops used to collect peaks in the ion exchange step Allow Collection of Single Peaks in Several Loops Collect single peaks in several loops To be used when the peak volume is larger than the value in Max Volume in Each Loop Method Wizard lon Exchange Intermediate Peak Collection Advanced one Start Collection Page Watch Level Greater than 20 6000 6000 m l 1 Watch Slope Greater than 20 50000 50000 mAlmin 2 3 Stop Collection 4 Peak Max Factor 0 5 0 1 5 Watch Level Less than 30 6000 S000 md B Watch Stable Plateau 7 Stable Time 05 0 9999 min Last Fage Delta Plateau 5 0 6000 m l Peak Selection lon Exchange gt Gel Filtration Select Largest Peak Based on f Area Height Start Collection The peak collection starts when both the UV level exceeds the Wa
250. ut the tag from the protein Fills the affinity columns with cleavage buffer from inlet A7 before adding protease Select High when over loading the affinity column It will raise the Level values in Watch commands used for peak detection in order to collect the best part of the peak See UNICORN AKTAxpress User Refer ence Manual The actual running con dition room temperat ure or cold room for the purification run If selecting Cold Room the flow rates will be decreased to avoid high pressure due to the higher viscosity of samples and buffers Lists showing the avail able columns Method Wizard 5 To perform Select Description related ac tions equilibration of the affin Reequilibrate Affinity Equilibrates the affinity ity column s after the Columns column s after the puri purification run fication run with 5 column volumes CV of affinity buffer A equilibration of the IEX Reequilibrate lon Ex Equilibrates the ion ex column s after the puri change Column change column after the fication run purification run with 5 CV of appropriate IEX buffer A Continuing with The subsequent pages depend on the selections made on the previous pages Follow or finishing the method plan the requested step below If selecting Purify only click Next The Last page appears Proceed to 5 2 5 Saving the method plan on page 106 Prepare System click Next The Prepare System page
251. volume used to flush the loops when loading the sample onto the gel filtration column 18 1178 00 AA ep 132 Peak fractionation Method Wizard 5 Max Injection Volume The maximum volume to be injected onto the gel filtration column The maximum volume is set up to make sure that the peak volume flush volume do not exceed the maximum sample loading volume of the column The peak fractionation options and parameters for the protocols A and E are described below The parameters define the final fractionation how the final peaks are collected in the fraction collector Peak Fraction Size The maximum volume of each peak fraction Peak Fractionation Algorithm Choose to start and stop the peak fractionation based on the signal curve e Level e Slope e Level_AND Slope e Level_OR_ Slope AND means that both conditions have to be met OR means that only one condition has to be met The fractionation start values are set by Start Level and Start Slope and the stop values by Stop Level and Stop Slope Different start and stop values for level and slope can be set The Stop Slope condition cannot be met unless the Peak_Max condition is met Stop Level can be met before Peak_Max The Peak_Max condition occurs when the UV signal has fallen to a fraction specified by Peak Max factor of the most recent peak maximum Note If the flow rate is changed the shape of the peak will change as well Hence the slope values might need to be changed
252. waste flask Carefully immerse the inlet tubing A1 in the flask containing at least 300 ml of deionized water 18 1178 00 AA ep 160 Operation 6 Set the injection valve to position Waste e Start UNICORN and select System Control Manual Flowpath e Select InjectionValve and position Waste Instructions Parameters C Pump Position inser ColumnPosition Flowpath Outlet alve Vaste Dette c Inlet alve Frac LoopSelection Alarms amp Mon Close C Other Help Run the pump at 20 ml min for 2 minutes e Select System Control Manual Pump e Select a flow rate of 20 ml min SYS_1 Pump Instructions x m Instructions Parameters Pump FlowR ate 0 00 65 00 Flowpath 2q E ml min Frac ie C Alarms amp Mon B Buffer C Sample C Other MinFlow 0 01 65 00 0 10 a ml min FlowContr No Yes J Auto update If this is checked the parameter fields will be updated during method run e Click Execute to start the flow Run the pump for 2 minutes e Stop the pump by clicking Pause in System Control Carefully move the inlet tubing A1 to the flask containing about 200 ml of methanol Run the pump at 50 ml min for 2 minutes e Set the flow rate to 50 ml min and click Execute e Click Continue to start the flow Run the pump for 2 minutes e Stop the pump by clicking Pause ep 161 6 Operation 6 5 Preparing the system for a run 6 5 3 Purging the pump and inlet tub
253. wing sub sections Topic Replacing capillary loops Replacing tubing and connectors Replacing flow restrictor Replacing UV lamp Replacing check valves Replacing pump piston seal Replacing a damaged pump piston 18 1178 00 AA ep 246 Maintenance 8 8 6 1 Replacing capillary loops anne inter When required for example when a capillary loop has clogged va Reason for main Clogged tubings may contaminate or cause high back pressure and hence preventing tenance a successful purification run Required material Capillary loop spare part 11 0003 02 CAUTION Only spare parts approved or supplied by Amersham Biosciences may be used for maintaining and servicing the system Replacing capil To replace the capillary loops lary loops Press the Eject button to put the collector plate in load inserted in system position Set the system in Standby mode by pressing the On button This will disconnect the system from UNICORN RUN 3 On the loop valve disconnect the two fingertight connectors of the capillary loop to be replaced ep 247 8 Maintenance 8 6 Replacement procedures 8 6 1 Replacing capillary loops 4 Slide the loop out of the compartment BF Insert the new loop and tighten the two connectors Press the On button to reconnect the system to UNICORN Verify the function by flushing the replaced loop and check the connectors for leakage Set the injection valve to position R
254. xpress About air detec The separation system is equipped with an air sensor which is located between tion the inlet valve and the pump Automatic air detection is used to e prevent air from entering the flow path during the purification and e detect when the sample supply is empty when applying sample Air sensor sensitiv Check that the air sensorsensitivity on each system used is set to Low_Sensitivity uy by selecting System Settings in System Control and then clicking Monitors The system must be in End mode S S_1 Monitors Instructions xi m Instructions AirSensor Parameters Mode AveragingT ime AvTimeUY Normal SetCondScale0 CondValue 0 00 mS cm SetCondScale100 Cond alue 100 00 mS cm UV Lamp Mode ON AirSensor N C Alarms Specials Monitors r i Curves Nelta Peak NANN mal Set Selected Parameter To Strategy Default Value Cancel Help Air detectionat Air detection during buffer application is used to prevent air from reaching the buffer application column By default air detection is always enabled during buffer transport in a method plan made in the Method Wizard When air is detected for example due to empty buffer supply or a loose tubing end the system enters Pause mode After correcting the error for example by running a wash to remove the air the run can proceed by clicking Continue Note If manually running Pump Wash System Wash or Loop
255. y cell 18 1178 00 AA ep 226 Maintenance 8 8 3 1 Checking UV lamp run time Maintenance inter Every 6 months val Reason for main The UV lamp has a life time of typically re Wavelength 254 nm Wavelength 280 nm tenance Checking UV To check the UV lamp run time lamp run time 1 From UNICORN System Control select System Maintenance 2 In the Maintenance manager open AKTAxpress and Specific Result The UV lamp on time is shown in hours together with other life time parameters Information Info 3 System m piston strokes 1000 10 rs Sm AKT Axpress o gt General Be lamp on time 2301 Hrs MOM Specific Ez lamp intensity 12 Inlet valve total turns 13 Ea Read the run time Take suitable action considering the lamp life time e No action e Check that a new UV lamp is available or order replacement lamp e Replace the lamp see 8 6 4 Replacing UV lamp on page 264 ep 227 8 Maintenance 8 3 Six monthly maintenance 8 3 2 Cleaning UV cell 8 3 2 Cleaning UV cell Maintenance inter Every 6 months val Reason for main A clean UV flow cell is essential for correct operation of the UV monitor tenance CAUTION Do not allow solutions containing dissolved salts proteins or other solid solutes to dry out in the UV cell Do not allow particles to enter the cell Damage to the UV cell might occur There are two procedures for cleaning the UV cell e Inplace c
256. y volumes 0 73 ml for the first column only loop valve to column valve plus delay volume for assisted manual loading of superloop 0 27 ml for all columns column valve to column Protease volume The default protease volume in the Method Wizard is 0 7 CV can be changed Volume needed per column 0 7 x 5 ml 3 5 ml Total volume needed 3 5 ml x 4 columns 0 27 ml x 4 columns 0 73 ml 15 8 ml Protease concentration Amount of protease per column 20 mg x 200 units 4000 units Protease concentration 4000 units 3 5 ml 1143 units ml Conclusion For this example fill the superloop with 15 8 ml of 1143 units ml TEV protease Note When injecting the protease solution into the superloop the protease will be diluted with buffer corresponding to the volume in the capillaries between the injection valve and the superloop In this calculation example and in the Protease Calculator sheet this dilution is overlooked When using small protease volumes e g one sample with 1 ml affinity column the dilution might have to be considered when calculating the protease concentration Note Try to keep the glycerol concentration low in the protease solution since high glycerol concentration affects TEV activity negatively Connecting the Follow the instructions below to connect the superloop superloop CAUTION Make sure that no parts e g tubing or columns are positioned in front of the fraction collector hindering the eje
257. ype and size e Adjust the gradient slope A steeper gradient will narrow the peaks Second wash of A second wash of affinity chelating columns can be used to wash out loosely bound oiy chelating proteins The second wash is an additional step before the elution of the target protein For a powerful wash the imizadole concentration B should be set to above 0 B If no imidazole is used B 0 the volume used in this step should be decreased to minimize the run time Note Do not raise the imidazole concentration too much as it might cause the target protein to come off as well Note The second wash is by default not included for GSTrap columns Flow moa and No warning will be issued if the flow rate or pressure limit are set higher than the Poco values recommended for the columns used Note Changing the flow rate will change the slope of an eluting peak 18 1178 00 AA ep 140 Method Wizard 5 Peak detection Peak detection and collection and collection The default values for detecting peak start and peak end are set to match typical peaks eluting from the supported columns Different values are used for normal and high sample loading The slope and levels values though might need to be adjusted if other parameters are changed e The slope of a peak is changed if the flow rate is changed or the gradient slope is changed e The default level values might need adjustment if small peaks are expected or the

ÄKTAxpress

Contents

Download Pdf Manuals

Related Search

Related Contents

&Auml;KTAxpress

Contents

Download Pdf Manuals

Related Search

Related Contents

ÄKTAxpress