Getting started

with automated
two-step
protein
purification
Benefits of automated two-step purification

Typically, manual intervention is needed Reduce manual interactions

• Increase throughput
• Reduce labor
• Improve process consistency
Eliminate hold steps
• Ensure product stability
• Eliminate or reduce hold containers

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Apply multistep purification on a wide range of purification
schemes

Polishing
Size exclusion chromatography
Ion exchange chromatography
Hydrophobic interaction chromatography
Purity

Intermediate purification
Ion exchange chromatography
Hydrophobic interaction chromatography

Capture
Affinity chromatography
Ion exchange chromatography
Hydrophobic interaction chromatography

Preparation, extraction, clarification

Step
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Example application: Automated two-step purification using
predefined methods
1. Capture 2. Polishing

Automatic transfer of
target protein (peak) and
start of second method

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Ways of working

1. Select purification protocol

2. Set up ÄKTA™ pure with components to support selected protocol
3. Set up capture method in UNICORN™ Method Editor
4. Set up polishing method in UNICORN Method Editor
5. Set up Method queue
6. Run the methods first with buffer and then with sample
7. Evaluate results

The following slides will demonstrate a workflow for a

two-step purification with intermediate loop collection.

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1. Select purification protocol

Select purification protocol

• Discuss with colleagues
• Web searches
• Reference literature
• Discuss with suppliers
Set up the protocol to combine a capture and a
polishing step in one run.

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2. Set up ÄKTA pure with components to support selected
protocol using intermediate loop collection
The flexible system configuration allows for an
optimized flow path
Components needed Name
Sample inlet valve V9-IS‡
Inlet valve V9-IAB
Inlet valves* V9-IA‡/ V9-IB‡
Injection valve V9-Inj‡
Column valve V9-C‡
UV monitor 1 U9-M‡/U9-L
Conductivity C9n‡
Outlet valve V9-O V9-Os‡
Versatile Valve† V9-V
Fraction collector F9-R‡/F9-C
Sample pump P9S‡
* Enables additional inlet buffers
† Use a versatile valve in front of injection valve if syringe position is needed for manual loop filling
‡ Used in the example described in the following slides

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2. Set up ÄKTA pure with components to support selected
protocol (cont.)
Optimized flow path with intermediate loop
V9-Inj collection used in the application example
V9-C
A sample pump facilitates sample loading in
capture step.
Connect outlet position from Outlet valve to
V9-IA
V9-IB Syringe port of injection valve.
V9-IS
V9-Os When using V9-Os a fraction collector must be used.
Note: As an alternative, the sample can be loaded
using the system pump and a mixer by-pass valve.
Fraction collector

F9-C
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3. Method setup: Automated two-step purification with
intermediate loop collection
Include first and second method in a 1. Set up your methods for the two steps as
Method queue individual methods using the predefined
protocols
1 2 • Capture step
Use peak fractionation (via the outlet valve) to
direct the eluted peak from the capture step to
the loop
• Polishing step
Fractionate normally to Fraction collector
or V9-O
Automatic transfer of
sample and start of 2. Create Method queue and start the run in
second method System Control

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3. Method setup

Method 1: Capture step Method 2: Polishing step

Eluted peak is
Sample (peak)
automatically Method queue
from capture is
directed from function Target
Load sample for stored in loop
outlet valve via automatically molecule is
the capture step between first
syringe port starts second fractionated
and second
to injection method
method
valve loop

1
2

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3. Method setup: Capture
Select adsorption technique
Select predefined method in Method editor: Different phases (steps) will be displayed in
Method outline

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3. Method setup: Capture
Adjust Phase properties
Define parameters for each phase in Settings available in Phase properties
Phase properties tab • Flow rate and pressure limits for selected column
• Column position
• Inlets to be used
• Base unit
• Monitor settings

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3. Method setup: Capture
Sample application and Elution properties

Sample application
Load sample using a
sample pump.

Elution
Use peak fraction to collect
the eluting peak in a loop.

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4. Method setup: Polishing
Select SEC*/Desalting in Method editor
Select predefined method: SEC*/Desalting in Method will be displayed in Method outline
Method editor

*Size exclusion chromatography, also called gel filtration (GF)

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4. Method setup: Polishing
Method settings, Sample application, and Elution properties
Re-inject sample
Select Manual load.
Note that the sample is
already in the loop after
the capture step.

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5. Method setup: Create Method queue
Include the capture and polishing step in Method queue
Method queue is set in UNICORN™ Method editor

Cond.
Capture Polishing UV
step step

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6. Running the methods
Automated two-step purification, example 1
Capture: Affinity chromatography with Polishing: SEC* Superdex™ 75 Increase
HisTrap™ HP 1 mL

Method Peak Retention (mL) Area (mL*mAU) Volume (mL) Purple peaks are similar in
AC Peak A 13.990 313.2 1.549 area indicating minimal
sample loss.
SEC Peak A 12.070 295.4 13.329
Sample: GFP-His (clarified homogenate, 5.3 mg GFP-His/g E. coli cell pellet)
*Size exclusion chromatography, also called gel filtration (GF)
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6. Running the methods
Automated two-step purification, example 2
Capture: Affinity chromatography with Polishing: SEC* HiLoad™ 16/600
HisTrap™ HP 1 mL Superdex™ 75 pg
Peak in non-linear
range (column
over loading)

Method Peak Retention (mL) Area (mL*mAU) Volume (mL)

In the capture step UV is measured
AC Peak A 31.246 1094 2.076 outside of the linear range of the
SEC Peak B 65.158 2496 22.000 monitor, therefore peak areas
cannot be compared.
Sample: GFP-His (clarified homogenate, 5.3 mg GFP-His/g E. coli cell pellet)
*Size exclusion chromatography, also called gel filtration (GF)

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7. Evaluation
Integration results from example 1 and 2
Example 2
Peak area
Sample loading amount up to column capacity (“scaleup”). Difference in area between steps
mL*mAU due to area from eluting peak from Method 1 is in the nonlinear region of the UV monitor.
2500

2000

1500

Example 1
1000 Sample loading amount below column capacity
to show complete transfer between steps and
reproducibility
500

0
Method 1: Method 2: Method 1: Method 2: Method 1: Method 2:
HisTrap™ HP Superdex™ 75 Increase 10/300 HisTrap HP, duplicate Superdex 75 Increase 10/300 GL, HisTrap HP, sample load HiLoad™ 16/600 Superdex 75 pg
GL duplicate

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7. Verification
Reference analysis with electrophoresis
Results form electrophoresis Reference analysis to verify identified peaks

Sample ID:
1. Blank
2. Amersham™ WB molecular weight markers
3. Two-step purification protocol AC-GF HisTrap™ 1 mL +
Superdex™ 75 Increase 10/300 GL
4. Two-step purification protocol AC-GF HisTrap 1 mL +
Superdex 75 Increase 10/300 GL, duplicate
5.–7. Blanks
8. Two-step purification protocol AC-GF HisTrap 1 mL +
HiLoad™ 16/60 Superdex 75 pg, scaleup
9. Start material His-GFP, E. coli
10. Amersham WB molecular weight markers

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Utilize standard hardware and predefined UNICORN methods for
automated two-step purification
ÄKTA™ systems in automated multistep Combine different chromatography steps in
purification automated multistep purification to save time and
add consistency.
Set up automated, two-step purification using
predefined methods and Method queue function
to quickly get started.
Store peak from step one in loop before
automatically continuing to second step.
ÄKTA pure was used in the example application
shown. ÄKTA avant can also be used in the
same way.

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Thank you

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