ÄKTA™ pure
Contents
1. pane presents current data in numerical values while the Chromatogram pane illustrates data as curves during the entire method run The Process Picture pane displays the current flow path during the run and can be used to control the run Fig 10 Color indication incorporated in the process picture shows the current open flow path with flow closed flow path or open flow path without flow Real time data from monitors are also displayed in the process picture pane Prec 0 00 MPa 0 000 mau Alimin Aal r Lum Uutiet Frai E ete Fig 10 The process picture in UNICORN 6 shows currently active and inactive flow paths and provides a fast and easy way to control the system 29 0211 96 AD 7 Column logbook To increase operational safety an optional feature of the software is the Column logbook The practical Column logbook tool keeps track of important run data related to individual columns to provide traceability and operational security Many prepacked columns from GE Healthcare are barcode labeled and individual columns are identified using a 2 D barcode scanner or the information may be entered manually into UNICORN 6 UniTag sticker labels with preprinted 2 D barcodes are available for labeling columns that are not already barcode labeled e g empty columns By tracking individual columns information regarding run data such as total number of runs and maximum pressures is recorded for each run Notification limits c2. Accuracy 2 Precision RSD lt 0 5 conditions 0 25 to 50 mL min lt 3 MPa 0 8 to 3 cP AKTA pure 150 Accuracy 2 Precision RSD lt 0 5 conditions 1 0 to 150 mL min lt 3 MPa 0 8 to 3 cP KTA pure 25 0 to 10 MPa 1450 psi AKTA pure 150 0 to 5 MPa 725 psi 0 7 to 10 cP Chamber with a magnetic stirrer AKTA pure 25 0 6 1 4 mounted on system or 5 mL AKTA pure 150 1 4 mounted on system 5 mL included with system or 15 mL AKTA pure 25 0 1 to 25 mL min AKTA pure 150 0 5 to 150 mL min AKTA pure 25 0 6 conditions 5 to 95 B 0 6 to 25 mL min 0 2 to 2 MPa 0 8 to 2 cP AKTA pure 150 0 8 conditions 5 to 95 B 2 to 150 mL min 0 2 to 2 MPa 0 8 to 2 cP Rotary valves Up to 12 Standard injection valve Options inlet selection mixer by pass loop selection column selection pH outlet versatile 29 0211 96 AD 9 Pressure sensors Placement of sensors Range Accuracy Module options Inlet valves Inlet A Inlet B Sample inlet Additional inlets UV monitors Number of monitors Wavelength range Absorbance range Linearity Operating pressure Flow cells U9 L Flow cells U9 M Standard after system pump Options after sample pump precolumn post column O to 20 MPa 2900 psi 0 02 MPa or 2 whichever is greater 1 2 or 7 inlets 1 2 or 7 inlets O 1 or 7 inlets Up to 16 Up to 2 two U9 L or one U9 L and one U9 M UV monitor U9 L 280
3. Limited Amersham Place Little Chalfont Buckinghamshire HP7 9NA UK GE Healthcare Europe GmbH Munzinger Strasse 5 D 79111 Freiburg Germany 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 29 0211 96 AD 06 2014
4. increased capacity two units of fraction collector F9 R or one F9 R and one F9 C can be connected together Fraction collector F9 R provides a basic option with high capacity A variety of racks is available to allow the use of 3 8 15 and 50 mL tubes To minimize spillage the Drop sync function can be used for flow rates up to 2 mL min Drop sync minimizes spillage by timing fraction changes between drops r Tl kiara roa i e oe A eat FE za i i PE E i T flf ti Fig 7 Fraction collector F9 R allows collection in 3 8 15 or 50 mL tubes Fraction collector F9 C provides flexibility high capacity and security The fraction collector is equipped with a variety of cassettes that can hold tubes 3 8 15 and 50 mL as well as deep well plates 24 48 and 96 well which means that samples can be collected in any format needed Six cassettes can be loaded into the fraction collector in any combination that fits the user s needs Fig 8 As an alternative to using six cassettes loading capacity can be maximized by using a large tube rack for 50 mL tubes or a bottle rack for 250 mL bottles Upon loading the type of cassette is automatically detected by a sensor and the tube bottle configuration is confirmed eliminating mistakes in sample handling Cassettes designed for tubes are equipped with a function that locks tubes into place when discarding liquid waste Later the tubes can be easily unlocked and disc
5. nm UV monitor U9 M 190 to 700 nm in steps of 1 nm up to 3 simultaneous wavelengths 6 to 6 AU Within 2 at 0 to 2 AU O to 2 MPa 290 psi Standard Optical path length 2 mm Cell volume 2 uL Options Optical path length 5 mm Cell volume 6 UL Standard Optical path length 2 mm Cell volume 2 uL Options Optical path length 10 mm Cell volume 8 uL Optical path length 0 5 mm Cell volume 1 uL Conductivity monitor C9n Conductivity reading range Accuracy Operating pressure Flow cell volume Temperature monitor range Temperature monitor accuracy pH monitor V9 pH OH reading range Accuracy Operating pressure Flow cell volume 0 01 mS cm to 999 99 mS cm 0 01 mS cm or 2 whichever is greater within 0 3 to 300 mS cm 0 to 5 MPa 725 psi 22 uL 0 C to 99 C 1 5 C within 4 C to 45 C Oto 14 0 1 pH unit within pH 2 to 12 O to 0 5 MPa 72 psi AKTA pure 25 76 UL AKTA pure 150 129 uL Round fraction collector F9 R Number of F9 R Number of fractions Vessels Fraction volumes Spillage free mode Flammable liquids Dimensions W x H x D Weight Delay volume UV dispenser head 10 29 0211 96 AD Up to 2 two Round fraction collector F9 R or one F9 R and one Flexible fraction collector F9 C Up to 175 per fraction collector 175 3 mL tubes 85 8 or 15 mL tubes 40 50 mL tubes 0 1 to 50 mL Drop sync Yes 320 x 250 x 400 mm 5 kg AKTA pure 25 205 uL 86 u
6. of up to 50 mL min Sample pump S9 or up to 150 mL min Sample pump S9H 4 29 0211 96 AD The optional sample inlet valve V9 IS or V9H IS is intended to be used with the sample pump Inlet valve allows fast automatic loading of up to 7 different samples The integrated air sensor enables complete sample application without the need to preprogram the sample volume The valve has seven sample inlet positions plus a dedicated buffer inlet for filling the sample pump with solution before the sample is introduced and for washing out the valve and pump between runs During sample application the air sensor detects when sample has been completely loaded so that the method can continue to the next step without air being introduced into the flow path or column Fig 4 AKTA pure sample pump Buffer selection AKTA pure can be equipped with two different types of inlet valves that allow selection of buffers and wash solutions Valves with multiple inlets enable cleaning reagents to be permanently online which means that columns and system can be cleaned conveniently at regular intervals Inlet selection valve V9 IAB or V9H IAB comprises two A and two B inlet positions in a single valve offering a convenient solution for automation of buffer application and post run cleaning of columns and system when performing basic chromatography Any A inlet can be combined with any B inlet to generate gradients The inlet automation valves A a
7. the need for programming skills as creation of chromatography methods is done by simple drag and drop operations In addition the software is modular allowing the addition of features such as Column Logbook and Design of Experiments DoE functionality for method development Licensing options for remote access to the system and or for creating methods or evaluating results give even greater convenience If preferred the system can be set up so that it enters power save mode after method end which enables reduction of power consumption by around 80 AKTA pure system components and available options are described in the following sections in more detail AKTA pure standard components System pump The two system pumps are based on the modern technology developed for AKTA avant systems The robust construction delivers reproducible flow rates at both low and high back pressures allowing short separation times Each pump consists of one pair of pump heads which deliver low pulsation flow to the mixer The continuous and accurate flow rates generated enable reproducible isocratic or gradient elution For AKTA pure 25 the system pumps provide a flow rate range of up to 25 mL min at maximum operating pressure of 20 MPa For AKTApure 150 the flow rate is up to 150 mL min at maximum operating pressure of 5 MPa For column packing AKTA pure 25 and 150 can be used at flow rates up to 50 mL min and 300 mL min respectively A system pre
8. 1 5 and 5 mL O ring 22 1 x 1 6 mm for Mixer chamber 15 mL Valves valve kit include necessary tubing AKTA pure 25 V9 IS 29 0277 46 V9 IA 29 0122 63 V9 IB 29 0123 70 V9 IAB 29 0113 57 V9 X1 28 9572 27 V9 X2 28 9572 34 V9 M 29 0113 54 V9 L 29 0113 58 V9 Cs 29 0113 55 V9 C 29 0113 67 V9 pH 29 0113 59 V9 V 29 0113 53 V9 O 29 0122 61 V9 Os 29 0113 56 Sample inlet valve kit Inlet valve kit A Inlet valve kit B Inlet valve kit AB Inlet valve X1 Inlet valve X2 Mixer valve kit Loop valve kit Column valve Column selection valve pH valve kit Versatile valve Outlet valve kit 10 outlets Outlet valve kit 1 outlet Code number 29 0182 24 29 0182 26 29 0162 25 29 0162 2 7 29 0162 26 29 0466 65 29 0466 94 29 0466 97 29 2827 26 29 0561 02 29 0670 44 29 0670 45 29 0670 46 29 0670 47 29 0561 00 29 0670 74 28 9561 86 26 9562 25 28 9562 46 28 9803 09 18 1027 11 2o O113 lt 26 28 9535 45 20 9016 57 KTA pure 150 V9H IS 29 0509 43 V9H IA 29 0509 45 V9H IB 29 0509 46 V9H IAB 29 0896 52 V9H X1 28 9793 26 V9H X2 28 9793 28 V9H M 29 0906 92 V9H L 29 0906 89 V9H Cs 29 0906 93 V9H C 29 0509 51 V9H pH 29 0516 84 V9H V 29 0906 91 V9H O 29 0509 49 V9H Os 29 0906 94 The valves for KTA pure 25 and KTA pure 150 are compatible with both systems but for optimal performance the specific valve type should be used UV monitor UV moni
9. A pure with UV monitor U9 M The column used was Superdex 200 10 300 GL The peaks observed on the chromatogram are 1 ferritin M 440 000 2 aldolase M 158 000 3 conalbumin M 75 000 4 ovalbumin M_44 000 5 carbonic anhydrase M 29 000 6 ribonuclease A M13 700 and 7 aprotinin M 6500 Conductivity monitor The conductivity monitor measures conductivity of buffer and samples for online monitoring of the true gradient An integrated temperature sensor corrects for variations in conductivity due to the temperature The conductivity monitor has a broad reading range and is therefore able to monitor conductivity in all different chromatographic techniques KTA pure optional modules for enhanced automation Sample application options The optional sample pump Fig 4 is designed to allow automatic sample application directly to a column or indirectly via a sample loop or Superloop Using the sample pump saves time by eliminating laborious sample application steps and is especially useful when handling large sample volumes The pump consists of two pump heads and is based on the same pump principle as the system pumps Pump purging and air removal can easily be performed automatically The sample pump is equipped with a pressure sensor for control of the sample flow rate to protect the column while preventing pressure stops and minimizing the time for sample loading Using the sample pump samples can be loaded at flow rates
10. Data file 29 0211 96 AD AKTA pure AKTA pure is a flexible and intuitive chromatography system Fig 1 for fast purification of proteins peptides and nucleic acids from microgram levels to tens of grams of target product AKTA pure is a reliable system where hardware and UNICORN system control software are designed to work together with columns and chromatography media to meet any purification challenge AKTA pure is available in two versions AKTA pure 25 designed for a broad range of research applications and purification tasks in a multiuser environment KTA pure 150 is well suited for optimizing resource utilization and productivity in routine large scale preparative purification The system supports a wide range of chromatography techniques and meets the automation requirements needed to deliver the highest purity The system is configurable and can be upgraded at any time with a wide range of options to further increase its capabilities depending on your purification needs AKTA pure is the product of over fifty years expertise in protein research and three decades of experience in the development of AKTA purification systems AKTA pure offers the following benefits for you e Modular system design with a large range of options to allow flexibility in purification of proteins and peptides e Customizable system that is easy to upgrade as your research needs develop e Reliable system with components and integrated feature
11. L with optional tubing kit i d 0 25 mm KTA pure 150 473 uL 278 uL with optional tubing kit i d 0 5 mm Flexible fraction collector F9 C Number of F9 C Number of fractions Number of cassettes Number of cassette trays Vessel types Fraction volumes Spillage free mode Flammable liquids Dimensions W x H x D Weight Delay volume UV dispenser head Outlet valves Number of outlets Fraction volumes Delay volume UV outlet valve Air sensors Number of sensors Placement of built in sensors Placement of additional sensors Sensing principle O box E9 Number of I O boxes Number of ports per box Analog range 1 if needed add one Round fraction collector F9 R Up to 576 6 1 Tubes per cassette 40 3 mL tubes total per tray 240 24 8 mL tubes total per tray 144 15 15 mL tubes total per tray 90 6 50 mL tubes total per tray 36 Plates per cassette 1 deep well plate 24 48 or 96 wells 6 plates per tray Tubes per cassette tray 55 50 mL tubes Bottles per cassette tray 18 250 mL bottles of squared shape 0 1 to 250 mL Drop sync Accumulator or Automatic No 390 x 320 x 585 mm 21kg KTA pure 25 435 uL 214 uL with optional tubing kit i d 0 25 mm KTA pure 150 876 uL 508 uL with optional tubing kit i d 0 5 mm Valve V9 Os or V9H Os 3 waste fraction collector 1 outlet position Valve V9 O or V9H O 12 waste fraction collector 10 outlet p
12. an be set for instance to define the number of times the column may be run between cleanings and the user is notified when it is time for column maintenance In the Column logbook clicking on the Column History icon for a particular column provides a list of all runs that have been performed with that specific column Design of Experiments DoE functionality DoE functionality can be added as an option DoE is a powerful tool for method optimization DoE provides an efficient and structured approach where selected parameters are varied simultaneously so that a large data set can be obtained from few experiments Fig 11 Since DoE is integrated seamlessly in UNICORN 6 scouting methods are automatically generated from DoE schemes allowing for fast and efficient method optimization 100 DoE structured approach Information O 16 32 64 128 Number of experiments Fig 11 The DoE module which is optional in UNICORN 6 provides an efficient approach to optimization capturing more information in fewer experiments Accessories KTA pure accessories include column holders and clamps for attaching columns flasks and tubing to the system Fig 12 A selection of tubing kits allow a optimization of the flow path for various objectives and the connection of any laboratory scale column from GE Healthcare 8 29 0211 96 AD 1 Flexible column holder for HiScreen columns Column holder rod Bottle and airsensor holder Tubing hol
13. arded 6 29 0211 96 AD The cassettes can also be used for convenient storage of fractions or holders for sample tubes and are easy to handle and clean The fraction collector is covered protecting samples from dust contamination The top of the fraction collector can be used for placement of accessories and equipment i J ll l I llll Fig 8 Fraction collector F9 C holds cassettes for a variety of tubes from 3 to 50 mL as well as 24 48 and 96 deep well plates Fraction collector F9 C has two beneficial features that minimize cross contamination and spillage during fraction collection Drop sync can be used for flow rates up to 2 mL min and minimizes spillage by timing fraction changes to occur between drops At higher flow rates the Accumulator function provides spillage free fractionation without sample loss up to 150 mL min The system can automatically change between the two modes for optimal performance Additional module options KTA pure is a fully modular system that can be further expanded to increase system capability and productivity Due to the accessibility and design of the modules they are easily changed which allows quick and efficient customization Versatile valve V9 V or V9H V is a general four position valve that can be used to tailor the system to specific tasks for example for multistep purification schemes for more information see Cue Card Two step purification with KTA pure usin
14. bration sample application or wash The Method Editor contains predefined methods for different chromatography techniques and maintenance procedures as well as a library of predefined phases for creating or editing your own methods 5 000 mi min 0 0 8 sut r ink a n Manual load A i E l A z5 A Ss E J Wi B1 ft P zm Sa syp F W g Bf 4 lt eee By pass i mmm LIV Method Outline Phase Library Phase Properties pane and Text Instructions pane i Gradient pane gt m a _ m nn a a Fig 9 Method Editor has customizable panes that give a comprehensive overview of the method A method is created or edited simply by dragging and dropping phases from the Phase Library into the Method Outline In the Phase Properties pane specific run parameters are set and these settings are automatically programmed in the Text Instructions pane When selecting a column type parameter settings for that column type e g default flow rates and pressure limits are automatically set The Phase Properties pane allows for easy editing of the method while more advanced users may program the method directly in the Text Instructions pane For quick editing the toolbar contains useful icons such as Undo Redo and Save System Control The System Control module is used to start view and control a method run The module consists of three panes that provide an overview of the status of the run The Run Data
15. der spool Tubing holder comb Column and bottle holder o d 10 50 mm Column clamp o d 10 21 mm Loop holder for five 10 mL loops Multipurpose holder O Rail extension OM FW PO e OO CO Fig 12 AKTA pure accessories include holders and clamps for attaching columns flasks and tubing to the system To use as an air sensor holder the adapter 28 9563 42 is also needed T To be used to attach for example fraction collector cassettes on the side of the system Prepacked columns complete the package GE Healthcare offers an extensive range of prepacked columns for purification from microgram levels to hundreds of milligrams of target protein and for almost every chromatography technique Fig 13 The range includes HiTrap HiPrep HiScreen and HiLoad columns for preparative chromatography Tricorn columns are also available for high resolution semipreparative purifications at microgram scale as well as for protein characterization In addition to prepacked columns empty columns for packing with chromatography media of your choice are available Columns for microgram scale purification Tricorn GL and PE columns are high performance columns prepacked with media for a variety of chromatography techniques Fig 13 The column design allows even distribution of liquid eluent over the entire column cross section which enables high resolution purification at micro and milligram scale Tricorn GL co
16. e applied to the column directly using an optional sample pump or the system pump UV monitoring AKTA pure is equipped with either a fixed wavelength UV monitor or a variable multiwavelength UV and visible spectrum monitor The fixed wavelength 280 nm UV monitor U9 L incorporates LED technology which is durable and reliable and is ready to use at start up Moreover UV monitor U9 L does not heat the sample The monitor is available with a 2 mm flow cell as standard included at delivery and an optional 5 mm flow cell when higher sensitivity measurements are required To determine protein separation at different wavelengths UV monitor U9 M is designed for multiwavelength detection in the UV and visible spectrum from 190 to 700 nm UV monitor U9 M allows monitoring of up to three wavelengths simultaneously Fig 3 and 6 For optimized performance when purifying samples with different protein concentrations there are three flow cell path lengths available 0 5 2 included at delivery and 10 mm The flow cell design together with fiber optic technology provides a high signal to noise ratio without causing any local heating of the UV flow cell The monitor contains a high intensity xenon lamp with a long lifetime that requires minimal start up time Every time the instrument is switched on the monitor is automatically calibrated All U9 M UV cells are calibrated at manufacturing The UV signal is automatically normalized which hel
17. figuration and 53 kg when fully equipped with options The relatively low weight enables easier placement in the lab and the system dimensions allow it to fit conveniently into a standard cold cabinet for work with labile samples A Rails for attachment of column holders and accessories also on the left hand side Verify gradients with Conductivity monitor Round fraction collector F9 R for collection in various tube types B Sample pump S9 and Sample inlet valve V9 IS for automated sample loading of multiple samples Flexible fraction collector F9 C for collection in deep well plates and or tubes Pressure wie Z Le monitor r TRS Bice Column i njection valve Mixer valve pump Column gt Mixer e gt Pressure A wn monitor 2 WLS gt System L pumps 2 sampi gt Pressure valve Sample monitor Column valve V9 Cs with built in bypass and reverse flow functionality UV monitor U9 L for detection at 280 nm Tubing organizer Inlet valve V9 IAB allows selection between two A and two B positions in a single valve High performance system pumps Conductivity UV monitor Column valve monitor Outlet a valve i a 5 GA 1 outlet Waste position Fraction collector Column valve V9 C enables media screening for up to 5 columns and measures the pressure drop over the column UV monitor U9 M for triple wavelength detection pH
18. g loop collection code no 29 0908 05 Up to four versatile valves can be connected to the system Mixer bypass valve V9 M or V9H M is used for bypassing the mixer if samples are loaded through the system pump Loop valve V9 L or V9H L allows the use of up to five loops and can be used for collection of intermediate fractions when performing multistep purification or for automated purification of up to five different samples The loop valve can for example also be used for holding reagents or different samples Up to two extra eight position inlet valves can be deployed to expand on buffer and sample inlet capacity Up to four additional air sensors can be placed in the flow path to enhance security for example before the inlet valves or before the column I O box E9 provides a means of connecting external interfacing equipment such as detectors I O box E9 receives analog or digital signals from or transfers analog or digital signals to external equipment that needs to be incorporated in the system Two I O box E9 units can be connected to KTA pure A list of available additional valves and other options is found in Ordering information UNICORN 6 control software UNICORN control software is used on all AKTA systems and can be used from lab scale up to full scale production UNICORN software contains the tools needed for beginners and experienced users to perform all types of chromatography from setting up and running a
19. graphy columns for AKTA systems 28 9317 78 Selection guide Two step purification with AKTA pure using loop valve 29 0908 05 Cue card Tandem two step purification using AKTA pure Cue card 29 0908 06 Connect Alias autosampler to AKTA pure Instructions 29 0404 27 AKTA laboratory scale chromatography systems 29 0108 31 Instrument management handbook Design of experiments DoE in protein production 29 1038 50 and purification Handbook A range of Service agreements and Validation support offerings are available Please contact your GE Healthcare Sales or Service representative for details Purifying proteins Get help from the Purify and AKTA apps www gelifesciences com purify GE and GE monogram are trademarks of General Electric Company AKTA BioProcess Capto HiLoad HiPrep HiScale HiTrap MabSelect RESOURCE SOURCE Superdex Superloop Tricorn and UNICORN are trademarks of General Electric Company or one of its subsidiaries Alias is a trademark of Spark Holland BV All third party trademarks are the property of their respective owners 2012 2014 General Electric Company All rights reserved First published Aug 2012 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 UK
20. ion AC IEX HIC IEX HIC GF Easy to use purifications for a broad with good range of resolution applications IEX HIC RPC AC DS IEX HIC Preparative gel filtration resolution GF IEX HIC Fig 13 Columns for use with KTA pure system for different scales of purification AC affinity chromatography DS desalting GF gel filtration IEX ion exchange chromatography HIC hydrophobic interaction chromatography RPC reversed phase chromatography Pack your own columns for gram scale purification The column packing instruction in KTA pure enables convenient column packing at constant pressure and high flow rates Both A and B pumps are used to generate the flow making it possible to set flow rate up to 50 mL min and 300 mL min for KTA pure 25 and KTA pure 150 respectively Several empty column types are avaliable Which empty column that is best suited will depend upon media type and running conditions i e flow pressure etc HiScale empty columns are developed for standard liquid chromatography optimized for process development and preparative protein purification The columns are designed to withstand high pressures and high flow rates making them compatible with modern BioProcess chromatography media such as MabSelect and Capto media XK columns are user friendly and robust columns for standard protein purification The columns are designed for liquid chromatography at low
21. is calculated by measuring the difference between the two pressure readings and can be used to protect the packed media bed Fig 5 System pump pressure 0 6 MPa Column top hardware pressure 0 5 MPa Column media pressure Ap 0 5 MPa Column bottom pressure 0 2 MPa E Fig 5 For increased operational safety the column selection valve enables continuous measurement of precolumn Pre CP and post column pressure Post CP during runs The pressure difference over the packed media bed Ap is calculated from the two signals The flexibility of the column selection valve for connection of up to five columns was demonstrated in a column scouting study using columns for hydrophobic interaction chromatography HIC Five columns from HiTrap HIC Selection Kit were connected to AKTA pure and used for column scouting for optimization of purification conditions of S aminotransaminase in clarified E coli extract UV monitor U9 M was used for detection of the protein at two wavelengths Chromatograms of the five separate HIC runs are shown in Figure 6 Eluted fractions were analyzed using GF and SDS PAGE data not shown The A signal specifically monitors the target protein The columns giving the sharpest and most symmetrical peaks at A oo as well as the highest possible purity were selected for subsequent optimization and scale up experiments HiTrap Phenyl FF high sub 1 mL and HiTrap Butyl FF 1 mL gave the most pr
22. lumns are manufactured in glass to facilitate visual inspection of the media bed while the tube and filter of PE columns are designed to withstand greater pressure Columns for milligram scale purification HiTrap 1 and 5 mL columns are prepacked with a wide range of media for purification using various chromatography techniques Fig 13 The columns can be connected in series for greater capacity Further scale up can be achieved with HiPrep 20 mL columns RESOURCE columns are designed for high resolution purification of proteins at high flow rates The columns are prepacked with SOURCE media that have high particle Size uniformity and stability to allow high flow rates at low back pressure HiScreen columns are prepacked with a wide range of robust BioProcess media to allow repeated use with highly reproducible results Designed for scalable method optimization HiScreen columns have a 10 cm bed height and can easily be connected in series to achieve a 20 cm bed height HiLoad columns are prepacked glass columns with Superdex prep grade media designed for high resolution GF applications z e gt SN p g _ arzai Gim Sample load capacity aa Semi preparative Preparative lt Characterization RESOURCE HiScreen HiTrap HiPrep High quality Fast and high Optimized Fast and for method easy and process scale up with high development AC DS GF resolut
23. method to evaluating the data Operation of AKTA pure is controlled by UNICORN 6 system control software which provides built in knowledge for planning controlling and analyzing the system and chromatography results For greater convenience licenses enabling remote control of AKTA pure are also available Key features of UNICORN 6 are e Intuitive software design simple intuitive and flexible method creation using predefined phases steps e Database storage robust data storage allows easy access to data data security and data integrity e Interactive process picture shows the current open flow path and offers an intuitive way to control the run manually e Column logbook optional valuable tool to keep track of individual column and run data for traceability and operational security e DoE optional integrated tool for experimental design provides more precise information in fewer experiments for cost and time efficient method development Method Editor The Method Editor contains all the instructions used for controlling the chromatographic run In UNICORN 6 the Method Editor has a user friendly graphical interface for easy viewing and editing of the method and run properties Figure 9 shows a screenshot of the Method Editor with customizable panes that provide a comprehensive overview of the run In UNICORN 6 methods are built by using phases Each phase reflects a step in the chromatography run such as equili
24. nd B provide up to 2 x 7 inlets Multiple inlets enable automatic screening of buffer and reagent conditions Each of the inlet automation valves is equipped with an integrated air sensor which helps in excluding air from the system If air is detected the system can be paused so that the air can be purged before it enters the flow path Column control A column valve can be connected to the system and used to control the flow to the column AKTA pure can be equipped with one of two different column valves Column control valve V9 Cs or V9H Cs allows connection of one column and has an integrated bypass function which enables washing of the system without the need to remove the column The column control valve also allows reverse flow through the column for fast and effective elution of strongly bound proteins sharper bands and a concentrated target molecule eluent Column selection valve V9 C or V9H C also has the integrated bypass and reverse flow functions Connection of up to five columns for automatic column switching is possible using this valve Connection of multiple columns minimizes manual intervention and reduces further the risk of introducing air into the column The column selection valve has two integrated pressure sensors the first sensor measures pressure before the column enabling protection of the column hardware while the second measures the pressure after the column The pressure drop over the column Ap
25. omising results under the conditions used and HiTrap Phenyl FF high sub 1 mL was selected for further optimization in this case Columns Sample Five columns from HiTrap HIC Selection Kit Supernatant after precipitation with 2 M ammonium sulfate AS at room temperature of extract of E coll expressing S aminotransaminase adjusted to 1 5 M AS Sample volume 2 mL Buffer A 1 5 M ammonium sulfate 50 mM sodium phosphate pH 7 0 Buffer B 50 mM sodium phosphate pH 7 0 Flow rate 1 mL min UV cell 10 mm System AKTA pure 25 equipped with Column selection valve V9 C and Loop valve V9 L HiTrap Butyl S FF 1 mL Conductivity mS cm Buffer B A420 nm MAU WG O Buffer B HiTrap Octyl FF 1 mL Conductivity mS cm A420 nm MAU Buffer B 0 10 20 30 40 50 Ay20 nm MAU WN Buffer B HiTrap Butyl FF 1 mL Conductivity mS cm Buffer B Fig 6 Column scouting for purification of S aminotransaminase expressed in E coli Column selection valve V9 C allowed the connection of five HiTrap HIC columns to AKTA pure for this evaluation UV monitor U9 M was used for multiwavelength detection From this scouting HiTrap Phenyl FF high sub 1 mL was selected for use in further scale up studies pH monitoring An optional pH valve with an integrated pH electrode not included enables in line pH monitoring during the run The pH m
26. onitor is easily calibrated by injection of calibration buffer directly into the valve with the pH electrode mounted A flow restrictor is connected to the pH valve and can be automatically included in the flow path to generate a back pressure that prevents the formation of air bubbles in the UV flow cell The pH valve is used to direct the flow to the pH electrode and flow restrictor or alternatively to bypass one or both Bypassing the pH electrode means that it can be stored and kept in place on the valve at all times 29 0211 96 AD 5 Outlet valves Two different valve options are available to direct the flow to the fraction collector waste or other outlet ports Outlet control valve V9 Os or V9H Os allows connection of one or two fraction collectors If only one is connected the other port can be used for outlet fractionation for example to collect flowthrough Outlet fractionation valve V9 O or V9H O enables connection of up to two fraction collectors and up to 10 available outlets allow collection of large fractions Fraction collection AKTA pure can be equipped with the Round fraction collector F9 R Fig 7 or with the Flexible fraction collector F9 C Fig 8 Both fraction collectors are controlled through UNICORN control software Fraction collection can be based on time volume or automatic peak recognition Automatic peak recognition minimizes cross contamination and unwanted eluent can be diverted to the waste For
27. ositions 0 01 to 100 000 mL KTA pure 25 125 uL 66 uL with optional tubing kit i d 0 25 mm KTA pure 150 296 uL 245 uL with optional tubing kit i d 0 5 mm Up to 7 Inlet valve V9 IA Inlet valve V9 IB Sample inlet valve V9 IS After the injection valve sensors Before the system pumps Before the sample pump Ultrasonic 2 2 analog in 2 analog out 4 digital in 4 digital out In 2V Out 1V The delay volume will change if a different tubing length between the system and the fraction collector is used t The fraction collector can hold either up to six cassettes or one cassette tray Ordering information Product KTA pure 25 L KTA pure 25 M KTA pure 25 L1 V9 IAB V9 Os KTA pure 25 M1 V9 IAB V9 Os KTA pure 25 M2 V9 IA V9 IB V9 C V9 O AKTA pure 150 L AKTA pure 150 M KTA pure 150 M3 V9H IA V9H C V9H O AKTA pure User Manual printed copy digital included UNICORN 6 4 workstation License UNICORN 6 4 Remote License without DVD UNICORN 6 4 Dry License without DVD UNICORN 6 4 DoE concurrent license UNICORN 6 4 Column logbook network license UNICORN 6 4 DVD package UNICORN 6 4 Manual package System modules and accessories Mixer Mixer chamber 0 6 mL Mixer chamber 1 4 mL incl with all systems Mixer chamber 5 mL incl with AKTA pure 150 Mixer chamber 15 mL Online filter kit O ring 13 1 x 1 6 mm high resistance O ring 13 1 x 1 6 mm for Mixer chamber 0 6
28. ps when comparing data from different systems Monitoring with multiple wavelengths can be used to detect contaminants specifically labeled proteins or target molecules that do not absorb light at 280 nm To demonstrate this molecular weight standards were monitored at 214 280 and 340 nm wavelengths Detection at 214 nm reveals peptide bonds of all proteins and may be useful if the concentration and extinction coefficient at 280 nm is low for the target protein Ferritin a multimeric iron storage protein showed stronger absorbance at 340 nm than the other proteins due to the high number of ferric ions in the center of the molecule Fig 3 Both UV monitor U9 L and UV monitor U9 M can be combined with a second UV monitor U9 L to give increased application capabilities such as multistep applications or when using small and large flow cells simultaneously to detect both low and high protein concentrations 29 0211 96 AD 3 Column Superdex 200 10 300 GL Sample Molecular weight standards for gel filtration Sample volume 100 uL Eluent PBS 10 mM sodium phosphate 140 mM NaCl 2 7 mM KCI pH 7 4 Flow rate 0 5 mL min System AKTA pure 25 5 5000 1504 Adgonm T A214 nm A340 nm 4000 _ 125 a5 oo 3000 Ee gt 75 1 3 5 2000 lt Ss 50 x 1000 25 0 PIAA NAS 0 O 5 10 15 20 25 Volume mL Fig 3 Gel filtration GF with multiwavelength detection 214 280 and 340 nm of proteins using KT
29. s based on the proven design of AKTA avant e UNICORN 6 software provides simple intuitive and flexible preprogrammed method templates and total system control to simplify your job e Predefined method settings for all GE Healthcare Life Sciences lab scale chromatography columns System overview AKTA pure chromatography system is a highly versatile modular system with a number of design features to facilitate reliable purification gelifesciences com Chromatography systems Fig 1 AKTA pure is a flexible chromatography system for the reliable purification of proteins peptides and nucleic acids at laboratory scale The system consists of the AKTA pure instrument and UNICORN 6 control software The system is modular in design with all valves monitors and columns mounted on the forward facing wet side of the system to allow easy interaction with the instrument modules Fig 2 Additional components such as valves monitors and sensors from the wide range of optional modules can easily be added to the available positions Multiple rails for attachment of column holders and equipment are located at the front and on the side of the instrument A buffer tray on the top of the instrument provides a large storage area for vessels and bottles The instrument control panel shows the system state and allows the possibility to interact with the run pause continue at the touch of a button The system weighs only 48 kg in basic con
30. scanner 2 D with USB Code number 29 0113 60 28 9793 86 28 9793 80 28 9563 78 29 0113 25 18 1128 24 29 0277 45 29 0505 95 28 9542 15 28 9564 97 29 0113 63 18 1120 39 18 1113 98 16 1115 99 18 1114 01 18 1114 02 18 1161 24 19 7505 01 18 1115 82 18 1023 85 29 0113 62 19 8684 03 18 3050 03 18 1124 67 29 0277 43 28 9542 09 28 9564 02 28 9564 04 20 9504 25 28 9564 27 28 9542 12 28 9803 19 20 96816 3 28 9565 02 28 9565 00 28 9563 42 29 0113 61 28 9564 91 28 9564 52 29 0211 96AD 11 Tubing kits AKTA pure 25 Tubing kit i d 0 25 mm 29 0113 28 Tubing kit i d 0 5 mm std KTA pure 25 29 0113 27 Tubing kit i d 0 75 mm std KTA pure 150 29 0113 29 Tubing kit i d 1 0 mm incl AKTA pure 150 29 0324 26 Tubing kit for sample inlet valve 7 inlets 29 0353 31 Sample tubing kit for 7 inlets i d 0 75 mm 28 9572 17 Inlet tubing kit for inlet valve IAB 29 0113 30 Tubing kit for pH valve standard 29 0113 31 Tubing kit for inlet valve A 7 ports 29 0113 32 Tubing kit for inlet valve B 7 ports 29 0113 33 Tubing kit for outlet fractionation 10 outlets 29 0113 34 System and sample pump rinse tubing kit 29 0113 48 Cables Jumper D SUB Jumper 1 IEC 1394 F type External module cable short External module cable long 2 5 m cable for F9 C or S9 UniNet 9 D type Holders Column holder rod Tubing holder spool Tubing holder spool i d 2 9 mm tubing for KTA pure 150 Column and bo
31. ssure monitor is connected to the pumps to continuously measure system pressure and enable the flow rate to be automatically adjusted to avoid reaching any defined pressure limit Mixer The mixer enables homogeneous buffer composition during gradient runs The choice of mixer chamber size depends on the flow rate and buffers used with a larger mixer volume required for higher flow rates or difficult to mix buffers Table 1 shows the mixer chamber sizes available for each instrument Table 1 Mixer chamber sizes available System Mixer chamber sizes AKTA pure 25 AKTA pure 150 Included 1 4 mL options 0 6 and 5 mL Included 1 4 and 5 mL option 15 mL An in line filter is mounted on the mixer The filter is easy to change and the mixer is also easily changed by snapping it in or out of the mixer holder The mixer size used for any given run is always noted in the result file Injection valve The injection valve allows for a variety of sample application techniques using sample loops or Superloop sample injection device The novel valve design eliminates the need for replumbing when changing between various sample application techniques A sample loop with a volume of 500 uL is delivered with the system Sample loops can be filled manually via a syringe or with a sample pump the same sample application options apply to the use of Superloop Sample loops can also be filled using the system pump Moreover sample can also b
32. to medium pressure System specifications Control system Dimensions W x H x D UNICORN 6 version 6 3 or later 535 x 630 x 470 mm Weight excluding computer Up to 53 kg sample pump fraction collector Power supply Power consumption Enclosure protective class System pump Pump type Flow rate setting Flow rate specifications Pressure range Viscosity range Sample pump Pump type Dimensions W x H x D Weight Flow rate setting Flow rate specifications Pressure range Viscosity range Mixer Mixing principle Mixer volume Gradient flow rate range Gradient composition accuracy Valves Type Number of valves Functions 100 240 V 50 60 Hz 300 VA typical 25 VA power save IP 21 Piston pump metering type AKTA pure 25 0 001 to 25 mL min up to 50 mL min during column packing AKTA pure 150 0 01 to 150 mL min up to 300 mL min during column packing AKTA pure 25 Accuracy 1 2 Precision RSD lt 0 5 conditions 0 25 to 25 mL min lt 3 MPa 0 8 to 2 cP AKTA pure 150 Accuracy 1 5 Precision RSD lt 0 5 conditions 1 0 to 150 mL min lt 3 MPa 0 8 to 2 cP AKTA pure 25 0 to 20 MPa 2900 psi AKTA pure 150 0 to 5 MPa 725 psi KTA pure 25 0 35 to 10 cP 5 cP above 12 5 mL min AKTA pure 150 0 35 to 5 cP Piston pump metering type 215 x 210 x 370 mm 11 kg AKTA pure 25 0 001 to 50 mL min AKTA pure 150 0 01 to 150 mL min AKTA pure 25
33. tor U9 L UV flow cell U9 0 5 0 5 mm for U9 M UV flow cell U9 2 2 mm for U9 M incl in system with U9 M UV flow cell U9 10 10 mm for U9 M UV flow cell 2 mm for U9 L incl with U9 L UV flow cell 5 mm for U9 L Sample pump Sample pump S9 Sample pump S9H pH and conductivity monitors OH electrode O ring 5 3 x 2 4 mm for pH electrode Conductivity monitor C9 Injection valve accessories Sample loop 10 uL Sample loop 100 uL Sample loop 500 uL incl with all systems Sample loop 1 mL Sample loop 2 mL Sample loop 10 mL Superloop 10 mL Superloop 50 mL Superloop 150 mL Fraction collector F9 R Fraction collector F9 R Tube Rack with 175 positions for 12 mm vials bowl tube support holder and guide Tube Rack with 95 positions for 10 18 mm vials Tube Rack with 40 positions for 30 mm vials bowl tube support holder and guide Fraction collector F9 C Fraction collector F9 C Cassette tray holds up to six cassettes Cassette holds six 50 mL tubes 2 pack Cassette holds fifteen 15 mL tubes 2 pack Cassette holds twenty four 8 mL tubes 2 pack Cassette holds forty 3 mL tubes 2 pack Cassette holds one 96 48 or 24 deep well plate 2 pack Rack holds fifty five 50 mL tubes Rack holds eighteen 250 mL bottles Additional air sensors Air sensor L9 1 2 mm Air sensor L9 1 5 mm Adapter for air sensor O box E9 O box E9 Barcode labels and scanner UniTag 1 sheet with 108 labels Barcode
34. ttle holder o d 10 50 mm Tubing holder comb Flexible column holder for HiScreen columns Inlet filter holder Kit AKTA Column clamp o d 10 21 mm Adapter for air sensor Bottle and airsensor holder Tube holder 5 pack Multipurpose holder Rail extension Loop holder with five 10 mL sample loops Screw lid kit AKTA To use as an airsensor holder the adapter 28 9563 42 is also needed For local office contact information visit www gelifesciences com contact www gelifesciences com aktapure GE Healthcare Bio Sciences AB Bj rkgatan 30 751 84 Uppsala Sweden Code number AKTA pure 150 29 0516 69 29 0482 42 29 0324 26 29 0511 66 26 95 2 17 29 1064 97 29 0516 74 29 0511 97 29 0511 89 29 0486 11 29 0113 48 29 0113 65 28 9564 89 29 0124 74 29 0113 66 29 0324 25 28 9562 70 28 9562 74 29 0142 83 20 9562 82 28 9562 86 26 9562 95 11 0004 07 29 9563 19 28 9563 42 2o 9563 2 28 9543 29 29 0113 49 29 0113 52 29 0113 50 11 0004 10 Related literature Code number UNICORN 6 control software Data file 28 9573 46 Validation Support File UNICORN software Data file 28 9626 50 AKTA pure Protein purification your way Brochure 29 0211 97 Purification of a miniature recombinant spidroin protein 29 0211 98 expressed in E coli using AKTA pure system Application note Purification and immobilization of a transaminase for the 29 0211 99 preparation of an enzyme bioreactor Application note Prepacked chromato
35. valve V9 pH enables in line pH monitoring calibration and storage Inlet automation valves V9 IA and V9 IB gives 14 inlet positions and enables condition screening Air sensors are integrated to protect the columns Position for additional modules Conductivity monitor UV monitor Outlet valve LJ ye en JC TR 10 outlet positions waste Flexible fraction collector Fig 2 Two examples of system configurations for AKTA pure 25 showing positions of modules on the front panel and flow paths for each A A basic system configuration for convenient protein purification B System configured for high level of automation 2 29 0211 96 AD Regardless of configuration AKTA pure always comes with two high performance system pumps system pressure monitor for column protection mixer injection valve and UV monitor AKTA pure has a wide range of optional modules to allow a large number of possibilities The system flow path is designed to minimize band broadening effects and all wetted materials used in the flow path are biocompatible and resistant to commonly used solvents The instrument front is designed with empty module positions where optional valves and monitors can be mounted to enable a flexible configuration of the flow path Examples of two AKTA pure system configurations are shown in Figure 2 UNICORN 6 control software allows a fast and easy start to creating methods and starting runs UNICORN 6 eliminates
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