UNICORN軟體操作暨應用課程 ÄKTA purifier UPC

UNICORN軟體操作暨應用課程ÄKTA purifier UPC

GE Healthcare Life-Sciences Ltd., Taiwan Branch奇異亞洲醫療設備股份有限公司

Protein Separation Christine Hsu 徐瑤蓉

TEL : (886 2)25165280 ext 72124FAX : (886 2)25165260E-mail : christine.hsu@ge.comwww.gelifesciences.com

UNICORN軟體操作暨應用課程

Christine Hsu 徐瑤蓉Sr. Product Sales Leader奇異亞洲醫療設備股份有限公司

TEL : (886 2)25165280 ext 72124FAX : (886 2)25165260E-mail : christine.hsu@ge.comwww.gelifesciences.com

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Introduction of ÄKTA design &

software UNICORN

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AKTA Systems

ÄKTAprime plus

ÄKTApurifier

ÄKTAexplorer ÄKTApilot

ÄKTAprocess

ÄKTAoligo

ÄKTAcrossflow

ÄKTAxpress

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Basic Liquid Chromatography System

HiLoad pump P-50

Pump

COLUMNS

Monitor UVis-920

Conductivity monitor

Frac-920 fraction collector

Peristaltic Pump P-1sample

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pH UVcond

Fraction collection

A

MIXER

W1W2

B

sample

Pump

valve FV-903

different buffers

ColumnsGF, AF, IEX, HIC

MonitorUV, conductivity, pH

A versatile set-up for adsorptive chromatography

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ÄKTA Design

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ÄKTA DesignÄKTA

System control

Data/record

Evaluation

Monitor

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ÄKTA SystemsLab scale

ÄKTAFPLC

ÄKTApurifier UPCÄKTApurifierÄKTAbasic

ÄKTAexplorer

Optimized purification of proteins

High performance purification and characterization of proteins

The industry standard for fast method development and automated multistep protein purification

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ÄKTApurifier UPC

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ÄKTApurifier UPC

A

Systempumps

Mixer

2

4

7

3

16

5

FractioncollectorFrac-950

On-linefilter UV

Injectionvalve

B

Column

Flow restrictor

Instrument

UV detector

Conductivitymonitor

Cond.

Default flow scheme :A (or A1) buffer inlet – mixer – on-line filter – injection valve (Load) – (Column) – UV – Cond. - SV908 (Waste)

Buffer A Buffer B4 buffer inlet w/t BufferPrep kit :

A1, A2, B1, B2Waste

Waste

pH detector (optional)pH

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System SpecificationsInstrument

•Pressure limit :25 MPa, 250 bar, 3625 psi

•Flow rate range :0.001-10 ml/min

•Wavelength :254, 280 nm

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Design your own FPLC purification systemÄKTApurifier 10/100Pump & Base

Fraction collectors

Automation kits

BufferPrep kit

Autosamplers

ÄKTApurifier 10/100Pump & Base

MonitorUV-900

MonitorpH/C-900

MonitorUPC-900

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ÄKTAdesign automation kits

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UNICORN

Method editor System control Data evaluation & report

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UNICORN FunctionNew method (Method Editor)

System control

Evaluation

Real-time monitoring System control

Software

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Methods Results

SoftwareUNICORN Manager

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New Method System Control Evaluation

UNICORN Manager (continue)Software

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Optimization

New method

Run method

evaluationUnsatisfied result

Satisfied result

Modify parameters(Scouting)

Data evaluation

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UNICORN

Method editor System control Data evaluation & report

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UNICORN 5.xx

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New Method

Create a new methodUNICORN 5

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WizardUNICORN 5

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Sampleapplicationand wash

ElutionEquilibration clean

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-- -

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--

- --

- -

-

--

-

--

--

- -

-- --

- -- --

-- -

-- --

-- -

-

---

- -- -

-

-- -

-

+

+

+

+++

+

+

+++ +

+

+

++++ +

+

+

++ + +

+

+

++ +--

- ---

- -

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Purification theory

Conc. B = 0%

Conc. B =100%

A : low-salt buffer

B : high-salt buffer

ConcUV

re-equilibrium

What happens in adsorption chromatography?

mAU

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Different flow rate required at different step

Flow regulation

ÄKTAexplorer/column valve

Main selectionTechnique, column & optional…

1.

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Chromatography technique 1.

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Column selection 1.

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Flow regulation

Pressure limit

The minimum flow rate you can tolerate

1.

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BufferPrep

1.ÄKTAexplorer2.ÄKTApurifier (old version)3.ÄKTApurifier (new version) and ÄKTApurifier UPC with BufferPrep kit (A1 A2 B1 B2)

1.

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Multi-buffer choose

A2A1 B2B1

Pump BPump A

Buffer Acid/Base

water Salt

Mixer

1.

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Select a IEX media (continue)

V V V

Cation

Anion

pH0

+

-

V

VVVV

Abs Abs Abs Abs

Surf

ace

net c

harg

e

Abs Abs Abs Abs

Titration curve

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Choose buffer inlet

Buffer A inlet

System with BufferPrep

ÄKTAexplorer

3 wavelength at one time(ÄKTAbasic/ÄKTApuriifer/ÄKTAexplorer)

2.

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B buffer conc. at equilibration step

Equilibration buffer volume (sample buffer or binding buffer)

Equilibration stableWatch at equilibration By UV, conductivity, pH

3.

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Injection technique

Sample volume injected into column

Manual : for sample loop & Superloop

4.

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Flowthrough (unbound sample) collection mode

5.

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Wash out unbound sample or not

Fraction volume

5.

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Outlet valve fractionation

1.Flowthrough :fixed volume fractionation - - - Frac-900large volume fractionation - - - Outlet valve F3

2.Elution :fixed volume fractionation - - - Frac-900peak fractionation - - - Frac-900

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Elution Frac-900

6.

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Collection volume

Fractionation types :Fixed volume and peak fractionation : also collect non-peakFixed volume fractionationPeak fractionation : only collect peak

6.

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Fractionation types

1 2 3 4 5 6 7 8 9 10 11

Fixed volume fractionation

Peak fractionation

1 2 3 4 5 6 7 8 9 10 11

Time/mlTime/ml

mAU mAUFixed volume fractionation

Peak fractionation

Instrument

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Peak fractionation

-UV Level-UV Slope

7.

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Peak identification

UV Level (mAU)UV吸光值達到所設定的值, 即認定是peak的開始及結束

UV Slope (mAU/min)UV斜率值達到所設定的值, 即認定是peak的開始及結束

mAU

min/ml

min/ml

mAU

7.

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Peak fractionation-UV level

Threshold of UV absorption

Peak collection volume

7.

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Peak fractionation-UV slop

Start to collect peak slop

End to collect peak slop

Peak collection volume

7.

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Elution mode

Elution technique- Isocratic (gel filtration)- Isocratic with Delayed Fractionation

(gel filtration reduced collection volume)- Linear Gradient

(adsorption, increase elution buffer gradually)- Segmented Gradient Advanced

(adsorption, different gradients including step elution)- Segmented Gradient Basic

(adsorption, different gradients NOT including step elution)

8.

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Interval collection

Clean column using 100%B

Re-equilibration using buffer at equilibration

Start or stop fractionation by Cond. or Conc.B

8.

Complete method creation

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Gradient segment number (max. to 9 segments)

Re-equilibration with buffer at equilibration

8.

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Gradient types

Elution buffer conc.

Elution volume

System with BufferPrep kit

ÄKTAexplorer/ A11-A18

8.

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Non-peak fraction volume

Peak fraction volume

Buffer exchange (priming) when using step elution mode

ÄKTAexplorer 100

8.

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Complete method creation

8.

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Click here to select page

Run setup window

Show detail parameters

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Save method

NameTechnique

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Save method (continue)

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How to create a new column database

method edit / Edit / column list

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Input column information

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UNICORN

Method editing System control Data evaluation & report

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System Control

SoftwareEnter system control

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Run Data

Real-time Curves

Flow scheme

Logbook

Softwaresystem control window

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SoftwareManual control

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Manual run (system control)Software

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Manual instructionPump

Flowpath

Software

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Manual instructionAlarm&Mon

Frac Other

Software

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SoftwareExecute existing method

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Hold

Continue

End

Hold method time but pump keep running

Pause both program & pump operation

Stop a program or operation

Advance the fraction collector one position

SoftwareFunction buttons

Pause

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Know more about system components and optional functions

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On-line filter

P-901 ( 100 ml/min Pump )High Flow Fibre Filter(ÄKTAFPLC, ÄKTApurifier 100, ÄKTApurifier UPC 100, ÄKTAexplorer 100)

Polymer Support

P-903 ( 10 ml/min Pump )Low Flow HPLC Filter(ÄKTAbasic 10, ÄKTApurifier 10, ÄKTApurifier UPC 10, ÄKTAexplorer 10)

Instrument

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Online filter

Online filter kit (10 ml/min)18-1120-94

Online filter kit (20, 50 & 100 ml/min)18-1027-11

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Injection valve 3 position

7

6

5

2

3

4

PUMPLOADSAMPLE

Waste

COLUMN

Waste

7

6

5

2

3

4

PUMP

Waste

COLUMN

Waste

7

6

5

2

3

4

PUMP

Waste

COLUMN

Waste

LOAD, pos 1 INJECT, pos 2

WASTE, pos 3

Loading sample into sample loop Injecting sample into column

Washing pump, column bypass

Instrument

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Sample application

Sample loop0.1, 0.5, 1.0, 2.0, 5.0 ml

Superloop10, 50, 150 ml

System pumpSample pump> 150 ml

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Sample loop

10 l 100 l 500 l

1 ml 2 ml

10 l 18-1120-39100 l 18-1113-98500 l 18-1113-991 ml 18-1114-012 ml 18-1114-025 ml 18-1140-53

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Superloop

Superloop, 1/16” fittings 10 ml18-1113-81

Superloop,1/16” fittings 50 ml18-1113-82

Superloop, M6 fittings 10 ml19-7585-01

Superloop, M6 fittings 50 ml19-7850-01

Superloop, 150 ml, cpl. M6 fitting18-1023-85

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Superloop

Plunger

Buffer

Sample

EtchedGroove

Pos 2 Pos 6Upside

Downside

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10 ml

150 ml

50 ml

BUFFERFLUSH

SAMPLELOAD

SAMPLEINJECT

SAMPLEFLUSH

WASTEPUMP orSYRINGE

COLUMN COLUMN

Plunger

Buffer

Sample

EtchedGroove

Superloop

4 MPa18-1113-81

4 MPa18-1113-82

2 MPa18-1023-85

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Monitor UPC-900Instrument

UV unitMercury lamp254 & 280 nm filtersLife time 7000 h

Conductivity unitpH unit

1 S/cm-999.9 mS/cmCell volume 24 lpH range 0-14

Cell volume 88 l

(ÄKTAFPLC, ÄKTApurifier UPC)

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Flow restrictorFR-902/ FR-904-after the conductivity or pH monitor

Instrument

-offer a constant pressure to prevent bubble production

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ÄKTAdesign automation kits

Media selection

Sample screen

Buffer selectionBuffer prep and pH scouting

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Multi-buffer choose

A2A1 B2B1

Pump BPump A

Buffer Acid/Base

water Salt

Mixer

Instrument

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BufferPrep

BufferPrep automatically calculates and prepares the composition of the buffer on-line, from 4 stock solutions

pH scouting

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Scouting

pH scouting Media scouting

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AKTAs - Your partner for optimizationUNICORN Controlled Chromatography – Scouting

Media selection (column valve)

Elution scheme Sample loads (superloop)

Sample screen (sample valve)

pH (buffer prep kit)

Flow rate

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System maintenance1. Flush the flow path with ddH20 using System Wash Method,

by the way exchange the buffer in the column to ddH202. Rinse sample loop by injecting ddH2O (5 volume of sample

loop)3. If you are not using the system for a few days (more than 3

days), flush the flow path with ddH20 first, then wash the system with 20% EtOH and store it in 20% EtOH

4. Long-term storage, clean system with 1 M NaOH for 20 minutes (1 ml/min) monthly

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System maintenance5. Long-term storage,

1) clean system with 1 M NaOH for 20 minutes (1 ml/min)monthly. And then flush system with neutral buffer toneutralize pH.

2) Flush the flow path with ddH20 first 20 minutes (1 ml/min), then wash the system with 20% EtOH and

store it in 20% EtOH.

6. Keep the fraction arm outside or central the fraction collector to prevent the tube sensor from touching on tube for long term

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UNICORN

Method editor System control Data evaluation & report

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Result Evaluation& Reporting

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Open a result fileResult evaluation

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Curve properties Result evaluation

Right-click

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Choose curve, curve style & color Result evaluation

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Adjust X- & Y-scale Result evaluation

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Peak integration Result evaluation

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Peak integration (continue)

Peak window

Peak window…

Result evaluation

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Peak integration (continue)

Reject Peaks

Result evaluation

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Peak integration (continue)

Peak table

Save and Edit Peak Table

Result evaluation

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Peak integration (continue)

Peak name

Result evaluation

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Curve differentiate Result evaluation

Differentiate

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Activity histogram Result evaluation

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Compare chromatograms/curve Result evaluation

Open to compare

chromatograms

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Compare chromatograms/curve (continue)Result evaluation

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Compare chromatograms/curve Result evaluation

Open to caompare

curves

Curve options

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Curve options Result evaluation

Overlay MirrorStack

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Export curve Result evaluation

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Export curve (continue)Result evaluation

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Customized report Result evaluation

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Customized report (continue)Result evaluation

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Customized report (continue)Result evaluation

Right-click

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Customized report (continue)

Example Result001:1_UV1_215nm Example Result001:1_UV2_280nm Example Result001:1_UV3_0nm Example Result001:1_Cond Example Result001:1_Conc Example Result001:1_Fractions Example Result001:1_Inject Example Result001:1_UV1_215nm@01,BASEM

0

100

200

300

400

mAU

10.0

20.0

30.0

40.0

50.0

60.0

mS/cm

0

100

200

300

400

mAU

0

20

40

60

80

100

%B

0.0 5.0 10.0 15.0 min

A1 A4 A7 A10 B11 B8 B5 B2 C2 C5 C8 C11 D10 Waste

5.83

6.52

7.04

7.74

Result evaluation

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Customized report (continue)Result evaluation

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Next generation of AKTA

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• Incorporates over 50 years of experience in protein research

• Backed by 30 years of experience in developing protein purification systems

• ÄKTA systems are used by 100,000 researchers globally

2012—ÄKTA pure: protein purification your way

Proven technology

1982—FPLC™ system released, predecessor to ÄKTA design

1996—ÄKTAexplorer: single platform for all chromatography techniques

1959—Sephadex™, the world’s first gel filtration medium

2009—ÄKTA avant: a new generation of ÄKTA for process development

2013—ÄKTA start: protein purification your way

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ÄKTA Design

ÄKTAavantfast method developmentÄKTApure

high performanceÄKTAprime plus

ÄKTApilotFlexible scale-up ÄKTAprocess

Pilot scale manufacturing

ÄKTApurifierhigh performance

UNICORN controlledÄKTAstart

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ÄKTA™ pure to fit your needs

Automate system and column cleaning using inlet and column control valves

Verify gradients with conductivity monitor

Detect proteins at 280 nm

Collect fractions using 3 to 50 ml tubes

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Basic configuration - valves

Buffer inlet valve Automatic column and system cleaning

Injection valve No need to re-plumb when changing

sample application technique

Column control valve Reverse flow for

1) CIP

2) a concentrated target molecule eluent

Bypass for system cleaning

Outlet valve Waste/Fraction collector/1 outlet position

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Basic configuration - UV monitor optionsTriple- or single-wavelength UV monitors

190-700 nm

280 nm

Flash lamp and fibre optics - no warm up time- no sample warming

LED technology- no warm up time- no sample warming- long life time

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Advanced configuration – buffer inlet valve

V9-IAB V9-IA / V9-IB

Integrated air sensor

B pump

A pump

B pump

A pump

Buffer selections Automation of column and system cleaning

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Advanced configuration - column valve

V9-Cs V9-C

PreC Pressure

PostC Pressure

Reversed flow

By-passBy-pass

Reversed flow

Increased operation safety Column selection

Integrated pressure sensor

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Advanced configuration - outlet valves

V9-Os V9-O

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Advanced configuration - other monitors

- Integrated in pH valve V9-pH- On-line calibration, by-pass and storage of

electrode- Flow restrictor in/off-line

pH monitor

- Up to four external air sensors- For 5/16” or UNF 10-32 connectors

Air sensor

- Connected via I/O-box E9- Up to two external detectors per I/O box- Up to two I/O boxes per ÄKTA™ pure

Other detectors

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Flow path for high level ÄKTApure system

Integrated air sensorIntegrated pressure sensor

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Drag-and-drop programming

Fast method setupPredefined methods

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Fast method setupPredefined methods

New interactive process pictureSelected run data

Active flow path green

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Pop-up window Detailed valve pictures

Sliders

Drop-down lists Direct selection buttons

Execute buttons

Radio buttons

Numerical input

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Tailor ÄKTA™ pure to fit your needs

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Thank for your attention

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PracticeHow to start a run on AKTA?

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Pure simplicity in action

1.Turn on ÄKTApurifier & computer2.Create a new method3.Equipment setup4.Execute method5.During the run6.Data evaluation7.System maintenance

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START-Turn on system & computer

-Double click UNICORN iconon desktop

-Computer enter windows

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Equipment setup

Purge & pump washA.

B. Connect column onto system

C. Fraction collector setup

D. Rinse sample loop

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Purge & pump washA.

drive away bubbles to make buffer flow into buffer inlet tubing

change the buffer in the pumps to desired solutions (from 20% EtOH to ddH2O, from ddH2O to buffer) & drive away bubbles

purge

Pump wash

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Pump wash (system control)Equipment setup

Change the buffer from bottle to injection valve

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Pump wash (continue)Equipment setup

PumpWash (ÄKTAFPLC)

PumpWashPurifier (ÄKTApurifier)

PumpWashBasic (ÄKTApurifier UPC, ÄKTAbasic)

PumpWashExplorer (ÄKTAexplorer)

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Pump wash continueEquipment setup

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B. Connect column onto systemSet flow rate & pressure limit

Equipment setup

Set a low flow rate

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B. Connect column onto system (continue)

Set flow rate & pressure limit

Equipment setup

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1. Adjust the height of the delivery arm2. Rotate the rack by hand until the rear half

of the tube sensor rests against tube 13. Press feed tube4. Fill the fraction collector with enough tubes

C. Fraction collector setupEquipment setup

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Equipment setup

Keep syringe on luer union after injection

Rinse sample loop with 5 loop volumes of ddH2O or binding buffer before injecting sample

D. Rinse sample loop (sample injection)

Fill the syringe with the volume of sample more than the sample loop

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Run existing methodExecute method

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Change parameterDuring the run

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During the run

Marker on real-time graph

Marker

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C column

Column upside

System monitor

Column downside

Injection valve

M6 female/1/16” female18-3859-01 (Titanium)18-1123-94 (PEEK)

Fingertight union 1/16” male/M6 female18-1112-58

Union M6 female/1/16” male18-3858-01

Tubing connector flangeless/M6 male18-1017-98 (2)19-1993-50 (5)

Tubing connector flangeless/M6 male18-1017-98 (2)19-1993-50 (5)

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PEEK tubing

Peek tubing, 2m, 0.25 mm i.d., 1/16” o.d. (blue)18-1120-95

Peek tubing 1/16”, i.d.0.5 mm (orange) 18-1113-68

Peek tubing 1/16”, i.d.0.75 mm (green) 18-1112-53

Tubing (brown) 18-1115-83

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Reference -columns and connection

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1/16“, male

You need this connector to connect your column to ÄKTAprime

SizeShape

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Pre-packed columns for AKTA system

Tricorn(GF)

RESOURCE(IEX/HIC)

HiLoad

HiPrep

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Empty columns Tricorn

XK

PD-10 for gravity

C

HiScale

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RESOURCE

Tricorn

HiPrep

Column upside

Injection valve

Column downside

System monitor

1/16” female

Union 1/16” male/1/16” male18-1120-93

HiLoad

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XK column (M6 fitting)

HR columnM6 male

Column upside

System monitor

Column downside

Injection valve

M6 female/1/16” female18-3859-01 (Titanium)18-1123-94 (PEEK)

Fingertight union 1/16” male/M6 female18-1112-58

Union M6 female/1/16” male18-3858-01

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Thank for your attention