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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 : [email protected]
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 : [email protected]
2 /GE /
Introduction of ÄKTA design &
software UNICORN
3 /GE /
AKTA Systems
ÄKTAprime plus
ÄKTApurifier
ÄKTAexplorer ÄKTApilot
ÄKTAprocess
ÄKTAoligo
ÄKTAcrossflow
ÄKTAxpress
4 /GE /
Basic Liquid Chromatography System
HiLoad pump P-50
Pump
COLUMNS
Monitor UVis-920
Conductivity monitor
Frac-920 fraction collector
Peristaltic Pump P-1sample
5 /GE /
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
6 /GE /
ÄKTA Design
7 /GE /
ÄKTA DesignÄKTA
System control
Data/record
Evaluation
Monitor
8 /GE /
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
9 /GE /
ÄKTApurifier UPC
10 /GE /
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
11 /GE /
System SpecificationsInstrument
•Pressure limit :25 MPa, 250 bar, 3625 psi
•Flow rate range :0.001-10 ml/min
•Wavelength :254, 280 nm
12 /GE /
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
13 /GE /
ÄKTAdesign automation kits
14 /GE /
UNICORN
Method editor System control Data evaluation & report
15 /GE /
UNICORN FunctionNew method (Method Editor)
System control
Evaluation
Real-time monitoring System control
Software
16 /GE /
Methods Results
SoftwareUNICORN Manager
17 /GE /
New Method System Control Evaluation
UNICORN Manager (continue)Software
18 /GE /
Optimization
New method
Run method
evaluationUnsatisfied result
Satisfied result
Modify parameters(Scouting)
Data evaluation
19 /GE /
UNICORN
Method editor System control Data evaluation & report
20 /GE /
UNICORN 5.xx
21 /GE /
New Method
Create a new methodUNICORN 5
22 /GE /
WizardUNICORN 5
23 /GE /
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
24 /GE /
Different flow rate required at different step
Flow regulation
ÄKTAexplorer/column valve
Main selectionTechnique, column & optional…
1.
25 /GE /
Chromatography technique 1.
26 /GE /
Column selection 1.
27 /GE /
Flow regulation
Pressure limit
The minimum flow rate you can tolerate
1.
28 /GE /
BufferPrep
1.ÄKTAexplorer2.ÄKTApurifier (old version)3.ÄKTApurifier (new version) and ÄKTApurifier UPC with BufferPrep kit (A1 A2 B1 B2)
1.
29 /GE /
Multi-buffer choose
A2A1 B2B1
Pump BPump A
Buffer Acid/Base
water Salt
Mixer
1.
30 /GE /
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
31 /GE /
Choose buffer inlet
Buffer A inlet
System with BufferPrep
ÄKTAexplorer
3 wavelength at one time(ÄKTAbasic/ÄKTApuriifer/ÄKTAexplorer)
2.
32 /GE /
B buffer conc. at equilibration step
Equilibration buffer volume (sample buffer or binding buffer)
Equilibration stableWatch at equilibration By UV, conductivity, pH
3.
33 /GE /
Injection technique
Sample volume injected into column
Manual : for sample loop & Superloop
4.
34 /GE /
Flowthrough (unbound sample) collection mode
5.
35 /GE /
Wash out unbound sample or not
Fraction volume
5.
36 /GE /
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
37 /GE /
Elution Frac-900
6.
38 /GE /
Collection volume
Fractionation types :Fixed volume and peak fractionation : also collect non-peakFixed volume fractionationPeak fractionation : only collect peak
6.
39 /GE /
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
40 /GE /
Peak fractionation
-UV Level-UV Slope
7.
41 /GE /
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.
42 /GE /
Peak fractionation-UV level
Threshold of UV absorption
Peak collection volume
7.
43 /GE /
Peak fractionation-UV slop
Start to collect peak slop
End to collect peak slop
Peak collection volume
7.
44 /GE /
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.
45 /GE /
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
46 /GE /
Gradient segment number (max. to 9 segments)
Re-equilibration with buffer at equilibration
8.
47 /GE /
Gradient types
Elution buffer conc.
Elution volume
System with BufferPrep kit
ÄKTAexplorer/ A11-A18
8.
48 /GE /
Non-peak fraction volume
Peak fraction volume
Buffer exchange (priming) when using step elution mode
ÄKTAexplorer 100
8.
49 /GE /
Complete method creation
8.
50 /GE /
Click here to select page
Run setup window
Show detail parameters
51 /GE /
Save method
NameTechnique
52 /GE /
Save method (continue)
53 /GE /
How to create a new column database
method edit / Edit / column list
54 /GE /
Input column information
55 /GE /
UNICORN
Method editing System control Data evaluation & report
56 /GE /
System Control
SoftwareEnter system control
57 /GE /
Run Data
Real-time Curves
Flow scheme
Logbook
Softwaresystem control window
58 /GE /
SoftwareManual control
59 /GE /
Manual run (system control)Software
60 /GE /
Manual instructionPump
Flowpath
Software
61 /GE /
Manual instructionAlarm&Mon
Frac Other
Software
62 /GE /
SoftwareExecute existing method
63 /GE /
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
64 /GE /
Know more about system components and optional functions
65 /GE /
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
66 /GE /
Online filter
Online filter kit (10 ml/min)18-1120-94
Online filter kit (20, 50 & 100 ml/min)18-1027-11
67 /GE /
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
68 /GE /
Sample application
Sample loop0.1, 0.5, 1.0, 2.0, 5.0 ml
Superloop10, 50, 150 ml
System pumpSample pump> 150 ml
69 /GE /
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
70 /GE /
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
71 /GE /
Superloop
Plunger
Buffer
Sample
EtchedGroove
Pos 2 Pos 6Upside
Downside
72 /GE /
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
73 /GE /
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)
74 /GE /
Flow restrictorFR-902/ FR-904-after the conductivity or pH monitor
Instrument
-offer a constant pressure to prevent bubble production
75 /GE /
ÄKTAdesign automation kits
Media selection
Sample screen
Buffer selectionBuffer prep and pH scouting
76 /GE /
Multi-buffer choose
A2A1 B2B1
Pump BPump A
Buffer Acid/Base
water Salt
Mixer
Instrument
77 /GE /
BufferPrep
BufferPrep automatically calculates and prepares the composition of the buffer on-line, from 4 stock solutions
pH scouting
78 /GE /
Scouting
pH scouting Media scouting
79 /GE /
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
80 /GE /
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
81 /GE /
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
82 /GE /
UNICORN
Method editor System control Data evaluation & report
83 /GE /
Result Evaluation& Reporting
84 /GE /
Open a result fileResult evaluation
85 /GE /
Curve properties Result evaluation
Right-click
86 /GE /
Choose curve, curve style & color Result evaluation
87 /GE /
Adjust X- & Y-scale Result evaluation
88 /GE /
Peak integration Result evaluation
89 /GE /
Peak integration (continue)
Peak window
Peak window…
Result evaluation
90 /GE /
Peak integration (continue)
Reject Peaks
Result evaluation
91 /GE /
Peak integration (continue)
Peak table
Save and Edit Peak Table
Result evaluation
92 /GE /
Peak integration (continue)
Peak name
Result evaluation
93 /GE /
Curve differentiate Result evaluation
Differentiate
94 /GE /
Activity histogram Result evaluation
95 /GE /
Compare chromatograms/curve Result evaluation
Open to compare
chromatograms
96 /GE /
Compare chromatograms/curve (continue)Result evaluation
97 /GE /
Compare chromatograms/curve Result evaluation
Open to caompare
curves
Curve options
98 /GE /
Curve options Result evaluation
Overlay MirrorStack
99 /GE /
Export curve Result evaluation
100 /GE /
Export curve (continue)Result evaluation
101 /GE /
Customized report Result evaluation
102 /GE /
Customized report (continue)Result evaluation
103 /GE /
Customized report (continue)Result evaluation
Right-click
104 /GE /
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
105 /GE /
Customized report (continue)Result evaluation
106 /GE /
Next generation of AKTA
107 /GE /
• 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
108 /GE /
ÄKTA Design
ÄKTAavantfast method developmentÄKTApure
high performanceÄKTAprime plus
ÄKTApilotFlexible scale-up ÄKTAprocess
Pilot scale manufacturing
ÄKTApurifierhigh performance
UNICORN controlledÄKTAstart
109 /GE /
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
110 /GE /
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
111 /GE /
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
112 /GE /
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
113 /GE /
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
114 /GE /
Advanced configuration - outlet valves
V9-Os V9-O
115 /GE /
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
116 /GE /
Flow path for high level ÄKTApure system
Integrated air sensorIntegrated pressure sensor
117 /GE /
Drag-and-drop programming
Fast method setupPredefined methods
118 /GE /
Fast method setupPredefined methods
New interactive process pictureSelected run data
Active flow path green
119 /GE /
Pop-up window Detailed valve pictures
Sliders
Drop-down lists Direct selection buttons
Execute buttons
Radio buttons
Numerical input
120 /GE /
Tailor ÄKTA™ pure to fit your needs
121 /GE /
Thank for your attention
122 /GE /
PracticeHow to start a run on AKTA?
123 /GE /
Pure simplicity in action
1.Turn on ÄKTApurifier & computer2.Create a new method3.Equipment setup4.Execute method5.During the run6.Data evaluation7.System maintenance
124 /GE /
START-Turn on system & computer
-Double click UNICORN iconon desktop
-Computer enter windows
125 /GE /
Equipment setup
Purge & pump washA.
B. Connect column onto system
C. Fraction collector setup
D. Rinse sample loop
126 /GE /
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
127 /GE /
Pump wash (system control)Equipment setup
Change the buffer from bottle to injection valve
128 /GE /
Pump wash (continue)Equipment setup
PumpWash (ÄKTAFPLC)
PumpWashPurifier (ÄKTApurifier)
PumpWashBasic (ÄKTApurifier UPC, ÄKTAbasic)
PumpWashExplorer (ÄKTAexplorer)
129 /GE /
Pump wash continueEquipment setup
130 /GE /
B. Connect column onto systemSet flow rate & pressure limit
Equipment setup
Set a low flow rate
131 /GE /
B. Connect column onto system (continue)
Set flow rate & pressure limit
Equipment setup
132 /GE /
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
133 /GE /
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
134 /GE /
Run existing methodExecute method
135 /GE /
Change parameterDuring the run
136 /GE /
During the run
Marker on real-time graph
Marker
137 /GE /
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)
138 /GE /
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
139 /GE /
Reference -columns and connection
140 /GE /
1/16“, male
You need this connector to connect your column to ÄKTAprime
SizeShape
141 /GE /
Pre-packed columns for AKTA system
Tricorn(GF)
RESOURCE(IEX/HIC)
HiLoad
HiPrep
142 /GE /
Empty columns Tricorn
XK
PD-10 for gravity
C
HiScale
143 /GE /
RESOURCE
Tricorn
HiPrep
Column upside
Injection valve
Column downside
System monitor
1/16” female
Union 1/16” male/1/16” male18-1120-93
HiLoad
144 /GE /
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
145 /GE /
Thank for your attention