Two-step purification with ÄKTA™ pure, or ÄKTA avant, using loop
Contents
1. Block Loop collect flowpath wash Set Base to Volume Any Note If the Base is connected to column volume and used flow rate for the wash is higher than the column s max flow rate a warning will be issued pa 0 00 Base 0 00 System flow 0 000 ml min Off oy 0 00 Inlet A Al inlet A_1 oy 0 00 Inlet B B1 inlet B_1 oy 0 00 Injection valve Sample pump load 3 ve 0 00 Column position By pass Down flow Position on Outlet valve where the tubing from Mixer valve V9 M port Re inj is connected 3 ME ee Er Flow rate used during Loop collect flow path wash j 0 00 Loop valve By pass L 000 System flow 20 000 TAAA Pre column pressure ia 5 00 System flow 0 000 ml min Pre column pressure ay 5 00 Injection valve Manual load oy 5 00 Outlet valve Waste a 5 00 Column position oy 5 00 System flow 4_000 How rate_1 Ta 5 00 End Block Column position variable used as set in Method Settings Down flow Variable for the flow rate used as set in Method Settings Note The use of the same variable allows the changes in Method Settings to be reflected in the Loop collect flow path wash block For a detailed description of variables see UNICORN Method Manual 4 Two step purification with AKTA pure or AKTA avant using loop collection 29090805 AB Edit Sample Application phase in method two Phase Sample Application a Column CIP Method Settings a ae Colum2. for a text edited phase Note For a comprehensive guide to creating methods that can be run on AKTA pure or AKTA avant systems refer to the UNICORN Method Manual Method one Loop valve collect phase e The flow path for peak collection will be washed prior to activating the watch functions for peak collection e During elution the watch functions will be active and if fulfilled will direct the peak to the loop Details are presented in the following pages e After peak collection the flow path used for peak collection will be washed The illustration below shows an example of a UNICORN method that can be used for purification step one Loop valve collect phase column pos Method Settings eN POY Equilibration water Equilibration Loop valve collect SystemP sarnple load v Sample Application send sample to loop v Column Wash Loop valve collect SystemP sample iT load v Elution v Column Wash v Equilibration Predefined Phases Personal Phases Duration amp Variables Two step purification with KTA pure or KTA avant using loop collection 29090805 AB 3 Se 0 00 Block Loop collect flowpath wash Description of the user defined phase in method one User defined phase Loop valve collect Watch instructions Note The phase in this example has been designed for sample loading using the System pump and for collecting one peak Multiple peaks can be coll
3. stop volume count function By inserting the instruction hold counted volume the injection volume of step two can be equal to the collected peak volume The instruction is added at the end of the sample load phase Due to the laminar flow a larger volume than the one collected should be used during sample loading on the second column to empty the loop Ideally a volume that is 1 3 times larger than the collected peak volume should be used Besides the laminar flow compensation a compensation volume equal to the volume of the flow path from loop valve to column valve should also be added These two volumes constitute the chase volume The instruction Empty loop with is used to set preferred chase volume laminar flow compensation volume flow path delay volume Note The desalting and size exclusion columns have limitations in the sample volumes that can be added A chase volume is the volume of buffer that is used to push i e How to add user defined phases to a method Create and edit phases The UNICORN Method Editor software is used when creating and editing phases Follow the steps below to create a user defined phase e Rename a global phase e Add new text instructions e Auser defined phase can be saved in the Phase Library under Global Phases or Personal Phases for future use For easy identification modified phases can be renamed starting with a symbol The T symbol is a software generated indication
4. Two step purification with AKTA pure or AKTA avant using loop collection Cue Card Contents Principles Setup for multi step purification UNICORN methods How to add user defined phases to a method Description of the user defined phase in method one Edit Sample Application phase in method two Hom FP WW DY PDN Recommendations Introduction This cue card describes how to configure AKTA pure set up methods and perform a fully automated two step purification using two one step UNICORN methods in a method queue AKTA avant can be configured applying the same principle The fully automated two step purification is suitable for most columns and elution combinations Example methods for two step purifications can be downloaded from www gelifesciences com AKITA select AKTA pure or AKTA avant link under the heading Featured Products Then click on Related Documents tab and find the heading Cue Card The purpose of the cue card is to help users get started and to Inspire further multi step method development Principles Two step purification using a method queue with two one step methods By using one method for each purification step column information from UNICORN can easily be used in the method This means for example that pressure and flow rate limits are correct for each column and the column log book features can be utilized Using a method queue allows full automation Method queue outline Method 1 Affinity a
5. ected using the same principle if additional watch commands are added When using the Sample pump with AKTA pure the methods needs to be adjusted accordingly The sample pump is always present in AKTA avant which is reflected in the example method for AKTA avant Volume from UV monitor to Outlet valve In this example 0 20 ml Note The volume of each component is found in AKTA pure User Manual or AKTA avant User Manual The volume of tubing can be calculated using the formula Volume ml Length mm x i d mm x 1 4 Column Wash Position on Outlet valve where the tubing from Mixer valve V9 M port Re Inj is connected H I k Loop collec Note If a Sample pump is used to apply the sample the H 0 0 gt UW Greater than U 4 Peak start instruction where the flow is directed to V9 V Outlet valve i ASE Zi Outlet 3 needs to be followed by an instruction that sets V9 V to i 3 w U 000 unl mins Pre position 1 4 amp 2 3 Injection valve Sample pump load 2 RR pte wane 0 20 nett Position of the loop where the peak will be collected 7 0 20 System flow 4 000 How rate_1 ml min Pre column pressure Pressure control_1 bee 0 20 Start volume count Gill 0 20 Watch UV 1 Less than 400 0 mAU Peak enc 0 00 Base Volume Any Variable for the flow rate used as set in Method Settings oss algi Volume from UV monitor to Loop valve In this example 0 54 ml m 0 00 End Block
6. lectric Company or one of its subsidiaries All other third party trademarks are the propery of their respective owner Any use of UNICORN is subject to GE Healthcare Standard Software End User License Agreement for Life Sciences Software Products A copy of this Standard Software End User License Agreement is available on request Loop selection Use at least 1 3 times the expected peak volume to collect Recommended to use Loop holder 29 0113 50 Column CIP and Equilibration To minimize the time the material in the eluted peak from step one spends in the loop before being re injected during step two you can create dedicated methods for cleaning and equilibration procedures e CIP and re equilibration of column one can be methods executed after the method running step two of the purification e Equilibration of column two can be a method executed prior to starting method one Download Example methods for two step purifications for AKTA pure either equipped with Sample pump or not and AKTA avant can be downloaded from www gelifesciences com AKTA select KTA pure or KTA avant link under the heading Featured Products Then click on Related Documents tab and find the heading Cue Card Ordering information For ordering information on columns valves and tubing visit www gelifesciences com AKTApure or www gelifesciences com AkTAavant 2014 2015 General Electric Company All rights reserved First published Ma
7. n Performance Test Equilibration es Column Preparation kd The Sample Application phase can be edited to utilize the Hold counted volume UNICORN instruction Column Wash Note When performing text editing of the Sample aie Application phase the Phase Properties pane will be ai replaced with a list of phase variables Equilibration Manual Loop Fill Miscellaneous Sample Application System CIP System Preparation User Defined Global Phases Personal Phases Delete Insert Delete Save Phase Duration amp Variables Block Inject f 0 00 Phase Sample Application A 0 00 Base SameAsMain 0 00 Block Prepare sample loading_1 B F 0 00 Base SameAsMain a 0 00 Set mark 0 Sample_ID_1 F 0 00 Loop wash By pass Al Loop wash inlet sample application by pass_1 5 ml E 0 00 End Block 2 0 00 Loop valve 1 Loop position_1 H 0 00 Block Inject_1 Be 0 00 Base Volume ColumnSameAsMain 2 0 00 Inlet A Al inlet A fop 0 00 Inlet B B1 inlet B A 0 00 Gradient 0 0 Percent B Sample Appl _1 4B 0 00 base op 0 00 Injection valve Inject System flow 5_000 How rate ml min Pre column pressurePressure control 1 00 4Empty loop with_1 End_Block 0 00 injection valve Manual load Additional instruction to Sample Application phase if injection volume should equal collected loop volume i et 0 00 Loop valve Bypass 0 00 End Block It is possible to
8. nd Method one peak elution to loop s The user defined phase in method one defines all functionality for peak detection and loop collection of the eluted peak see Description of the user defined phase in method one Up to 5 loops can be connected to the Loop valve V9 L Method two Method 2 Desalting or The collected peak from method one is loaded onto column two Gel filtration Size exclusion see Edit Sample Application phase in method two Setup for multi step purification System configuration Flow path connections when the System pump is used p for sample loading This is only applicable for KTA pure Several different AKTA pure and AKTA avant f as the sample pump is always present in KTA avant configurations can be used AKTA pure 25 is used in the sonal telly yer following example To enable multistep functionality a Loop valve V9 L a Column valve V9 C and a Mixer valve 9 0 or V9 Os V9 M or a Versatile valve V9 V will be needed No From port To port Sample loading method Modules needed System pump V9 L V9 C V9 M Sample pump V9 L V9 V V9 C Sample pump P9 S 1 i Only applicable for AKTA pure as sample pump is always present in AKTA avant Flow path connections when the Sample pump is used From port Le Pump P9 S_ v v 3 V9 0 or V9 Os v9 V V9 Inj F V9 L LoopE on port 2 is not used Note Connect a suitable loop to the Loop valve V9 L see Recommendations sec
9. r 2014 All goods and services are sold subject to the terms and conditions of sale of the company within GE Healthcare which supplies them A copy of these terms and conditions is available on request Contact your local GE Healthcare representative for the most current information GE Healthcare Europe GmbH Munzinger Strasse 5 D 79111 Freiburg Germany GE Healthcare UK Limited Amersham Place Little Chalfont Buckinghamshire HP7 9NA UK GE Healthcare Bio Sciences Corp 800 Centennial Avenue P O Box 1327 Piscataway NJ 08855 1327 USA GE Healthcare Japan Corporation Sanken Bldg 3 25 1 Hyakunincho Shinjuku ku Tokyo 169 0073 Japan 29090805 AB 06 2015
10. set the maximum volume to be used In this example 5 00 ml Chase volume laminar flow compensation volume flow path delay volume In this example 1 00 ml Two step purification with AKTA pure or AKTA avant using loop collection 29090805 AB Recommendations Peak detection Set watch limits so that end peak is not triggered by start peak values For example set start peak to greater than 100 mAU and end peak to less than 100 mAU Another way is to use the instruction Peak_start_max before the peak end instruction see UNICORN Method Manual The peak volume should be larger than the delay volume between the UV monitor and the Outlet valve The delay volume is typically 0 2 to 0 5 ml Column selection Take proper care in selecting the column for the second step especially with respect to maximum load volume Recommended combinations are Purification step one Purification step two 1 ml HiTrap 2 x 5 ml HiTrap Desalting columns in series 1 ml HiTrap Gel filtration Size exclusion column i d 16 mm 5 ml HiTrap HiPrep Desalting 5 ml HiTrap Gel filtration Size exclusion column i d 26 mm For local office contact information visit www gelifesciences com contact GE Healthcare Bio Sciences AB Bj rkgatan 30 SE 751 84 Uppsala Sweden www gelifesciences com AKTA GE and GE monogram are trademarks of General Electric Company AKTA HiPrep HiTrap and UNICORN are trademarks of General E
11. tion Loop selection A 3 Important 9 05 a Read AKTA pure Operating Instructions or The illustration shows the two additional modules blue AKTA avant Operating Instructions before and the needed connections green installed on KTA pure using the instrument allowing two step purification via re injection System pump configuration Standard flow path shown with dash lines 2 Two step purification with AKTA pure or AKTA avant using loop collection 29090805 AB UNICORN methods Method one Objective Execute the first purification step on column one Use peak fractionation for the collection of the eluted peak in a loop Description During elution when the watch condition for peak start is fullfilled and the set delay volume has passed valves turn into position and the content in the peak Is directed to the loop for collection The volume of the peak is counted during the backwards filling of the loop from port F to E on the loop valve Method two Objective Execute purification step two by forward loading port E to F on the loop valve the collected peak content from method one onto a second column wash the column and elute the sample Description Add functionality to the method so that the sample injection uses the correct loop and injects the volume of the peak collected during step one Loop collection and injection The volume of collected peak is counted in step one using the start
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