Tandem two-step purification using ÄKTA™ pure or ÄKTA avant
Contents
1. 40 0 and target value for pressure control 90 Tandem two step purification using AKTA pure or AKTA avant 29090806 AB 5 Structure and content of a method used for purification step one continued Peak watch flowdirect Elution step 2 Elution step 1 activate watch Delay peak end 4 User defined phase for defining e Watch instructions during the elution used for peak detection e Valve configuration to direct transfer detected peak to the second column the valve configuration is defined and initiated V9 V 1 V9 V 2 f 80 00 Phase Peak watch flowdirect Hution step 2 0 00 Base Volume Any 0 00 Watch UV Greater than 50 0 imAU Peak start be 0 00 Base Volume Any 0 20 System flow 0 000 ml min Off 4 0 20 Versatile valve 1 4 amp 2 3 0 20 Versatile valve 2 14 amp 2 3 0 20 System flow 4 000 mL min Pre column pressure 0 20 Watch UV Less than 50 0 mAU 0 20 End_Bi k 0 00 End Block Delay volume Watch instruction that detects peak from UV end terminates the elution phase monitor to the when peak end is detected second column Note To calculate delay volumes the volume of each component can be found in AKTA pure User Manual and 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 e Elution phase e Previously set2. 29090806 AB 7 Ordering information For ordering information on columns valves and tubing visit www gelifesciences com AKTApure or www gelifesciences com AkTAavant 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 Electric 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 2014 2015 General Electric Company All rights reserved First published Mar 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
3. Japan Corporation Sanken Bldg 3 25 1 Hyakunincho Shinjuku ku Tokyo 169 0073 Japan 29090806 AB 06 2015
4. of the eluted peaks Description The sample is eluted from column two and the column is equilibrated The Elution and Equilibration phases are preceded by user defined phases to set the valve configuration to Wash amp Elution step two position The UV monitor is located after the second column in order to monitor the peak elution 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 A user defined phase can be saved in the Phase Library under Global Phases or Personal Phases for future use For easy identification the modified phases used in this application were renamed starting with a symbol The T symbol is a software generated indication for a text edited phase Note For a comprehensive guide to creating methods that can be run on AKTA pure and AKTA avant systems refer to the UNICORN Method Manual For an explanation of the used methods see next pages The illustration below shows an example of a UNICORN method that can be used for purification step one n tg ag es Peak watch fhowdirect Elution step 2 Versatile valve pos Eguc 1 27 Versitale valve pos Eque JOFEB eR poja Equilibration Column 1 Versatile valve pos Sample Appl iT R Pressure control parameters
5. ET Sample Application Versatile valve pos Column Wash T Column Wash Versatile valve pos Elution iT F Peak watch flowdirect Elution step 2 F Elution step 1 activate watch Duration amp Variables 4 Tandem two step purification using KTA pure or KTA avant 29090806 AB Structure and content of a method used for purification step one Method Settings Versatile valve pos EquC1 T User defined phases to set the valve configuration to the Wash and Elution steps F Equilibration Column 1 V9 V 2 Versatile valve pos Sample Appl i T F Pressure control parameters 7 F Sample Application i 0 00 Versatile valve 14 amp 2 3 E F Versatile valve 2 1 2 amp 34 F Versatile valve pos Column Wash T Optional user defined phase can be used to set new pressure control parameters to facilitate crude sample loading and minimize oscillating behaviour of the flow rate Column Wash Tip Lower the value of and P to get a slower smoother response in flow rate adjustment See an example below a 0 00 Base SameAsMain 0 00 Pressure control parameters 2 0 10 0 75 0 010 ml min 0 00 End Block Versatile valve pos Elution EF In this Pressure control parameters example P 2 0 l 10 0 and target value for pressure control 75 The values for P and I can vary greatly The default values are P 8 0 I
6. Tandem two step purification using AKTA pure or AKTA avant Cue Card Contents Principles Setup for tandem two step purification Valve positions and functionality UNICORN methods How to add user defined phases to a method Structure and content of a method used for purification step one Structure and content of a method used for purification step two NNO fF F amp F WN KN 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 method is suitable for fast buffer exchange for example after eluting the first column with low pH Example methods for two step purifications can be downloaded from www gelifesciences com AKTA 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 two 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 that column log book features can be utilized if pr
7. e or KTA avant 29090806 AB 2 Purification step one loading and wash of column one First column and UV monitor are inline Second column is offline UV monitor measures after the first column Valve positions e V9 C 1 e V9 V 1 1 482 3 e V9 V 2 1 2 amp 3 4 4 Purification step two wash and elution of column two Second column and UV monitor are inline First column is offline UV monitor measures after the second column _ gt V9 V 2 Valve positions e V9 C By pass e V9 V 1 1 2 amp 3 4 e V9 V 2 1 2 amp 3 4 UNICORN methods Method one Objective Perform the first purification step After sample loading and wash the eluted peak of interest is directed onto the second column Description The sample is loaded onto column one and during elution and when the watch condition for peak start is fullfilled valves turn into position Loading column two The UV monitor is located after column one When the peak has passed the UV monitor the flow is directed onto the second column Note The example below allows one detected peak to be loaded onto the second column Note The second column has to be equilibrated and ready for use prior to the method start Method two Objective Perform the second purification step on column two with the protein fraction loaded on column two in the first purification step and collect fractions
8. eferred Using a method queue allows full automation Method queue outline Method 1 Affinity and peak elution to column two Method 2 Desalting or Gel filtration Size exclusion Method one The user defined phase in method one defines all functionality for peak detection and redirection of the eluted peak to column two Method two The redirected sample peak on column two is eluted in a desalting or gel filtration size exclusion method Setup for tandem two step purification dy System configuration Several different AKTA pure and AKTA avant configurations can be used AKTA pure 25 is used in the following example To enable tandem multi step functionality two Versatile valves V9 V and a Column valve V9 C or V9 Cs will be needed See the description below and the illustration for how to connect the modules Flow path connections No From port To port 1 2 7 1 R V9 Cs only applicable for AKTA pure as V9 C is always present in AKTA avant V9 O Note To distinguish between the two versatile valves set the node ID for Versatile valve V9 V 1 to 20 and the node ID for Versatile valve V9 V 2 to 21 See KTA pure User Manual and AKTA avant User Manual for details Important Read AKTA pure Operating Instructions or AKTA avant Operating Instructions before using the instrument The illustration shows the flow path allowing tandem two step purification using the UV monitor U9 L ins
9. erably be a dedicated Manual method in the method queue executed as the last The peak volume should be larger than the delay volume method between the UV monitor and the second column The delay volume is typically 0 1 to 0 3 ml Other options Column selection e For loading larger sample volumes a Sample pump or the System pump in combination with a Mixer valve in a l ake proper care in selecting the column for the second KTA pure can be used step especially with respect to maximum load volume B but also with respect to pressure limits when both e Ifa Sample pump is available the addition of a sample columns are in the flow path inlet makes it possible to load the whole sample using air sensors and also to load multiple samples i r e For full control of protein elution and simplicity Purification stepone Purification step two a second UV monitor can be added allowing for simultaneous monitoring of both columns columns i me Download Example methods for two step purifications for KTA column i d 16 mm pure either equipped with Sample pump or not and KTA avant can be downloaded from 5 ml HiTrap Gel filtration Size exclusion www gelifesciences com AKTA select KTA pure or KTA column i d 26 mm avant link under the heading Featured Products Then click on Related Documents tab and find the heading Cue Card Good combinations are Tandem two step purification using KTA pure or KTA avant
10. talled on KTA pure t Only applicable for KTA pure since U9 M is always present in KTA avant Note Only some optional modules and tubing are included in this picture Note Select tubing id that matches the current tubing kit used Minimize the tubing length for optimal results Note If the UV monitor U9 M is used instead of U9 L position the two versatile valves as close to the UV flow cell as possible to minimize the delay volumes in the instrument 2 Tandem two step purification using KTA pure or KTA avant 29090806 AB Valve positions and functionality The two versatiles valves UV monitor and second column constitute one unit with a flow path configuration that is dependent on the valve positions used in the versatile valves Below are example flow path configuration shown for AKTA pure Four different flow path configurations are used 1 Priming of the system Both columns and UV monitor are offline V9 V 1 V9 V 2 Valve positions e V9 C By pass e V9 V 1 1 3 WISE IZ 1 3 3 Purification step one elution and loading column two Both columns and UV monitor are inline UV monitor measures after the first column V9 V 1 V9 V 2 GO oS A y j j j Op gt YH NG ge Valve positions e V9 C 1 e V9 V 1 1 482 3 e V9 V 2 1 482 3 Tandem two step purification using KTA pur
11. watch commands will be active during the elution e The Watch instruction for peak end triggers the end of the elution phase Renamed Miscellaneous phase introducing a delay The delay is necessary because the last part of the peak has to leave the UV monitor and enter the second column before method one ends Tandem two step purification using AKTA pure or AKTA avant 29090806 AB Structure and content of a method used for purification step two Instructions to set valve configuration to Wash amp Elution position Illustration shows AKTA pure as an example Method Settings gt v9 V 1 V9 V 2 F i p ZZ I N Versatile valve pos Elution 2 T Elution step 2 Se 60 00 Phase Versatile valve pos Hution 2 Ee 0 00 Base SameAcMain 0 00 Versatile valve 1 7 amp 3 4 0 00 Versatile valve 2 1 7 amp 34 Recommendations poe CE ee ess Peak detection Column CIP and Equilibration Set watch limits so that end peak is not triggered by start It is recommended that equilibration of column two peak values For example set start peak to greater than is performed as a first method in the method queue 100 mAU and end peak to less than 100 mAU executed prior to starting method one This ensures Another way is to use the instruction Peak_start_max that step two is ready to start with the elution step before the peak end instruction see UNICORN Method e CIP of column one can pref
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