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Contichrom® Discovery
Application note D03. V 1.2 - © ChromaCon 2019 1 /4
Automated three-step purification of Antibodies
Step 1: Protein A Step 2: CIEX Step 3: Desalting (buffer exchange) Utilized resins: Merck Millipore – 1 mL Eshmuno A
Merck Millipore – 5 mL Eshmuno CPX
Merck Millipore – 10 mL Fractogel BioSEC
The Contichrom Discovery is an easy-to-use, multi-
dimensional protein purification system. This application
note describes how to purify a monoclonal antibody using a
predefined method template incorporating capture,
polishing and buffer exchange steps with no intermediate
handling required. The three-step purification protocol is
ideal for the fast isolation of antibodies at very high purity for
downstream applications such as biological assays, stability
studies, and protein crystallography.
Figure 1. Workflow for purification of the target antibody. Step 1: protein is subjected to Protein A affinity chromatography Step 2: product is directly transferred to CIEX column for polishing Step 3: polished antibody is transferred to a desalting column for buffer exchange. The Contichrom Discovery is designed to run all three purification steps in a single integrated process.
Introduction
The Contichrom Discovery is designed for automated three
column antibody purification, combining a Protein A capture,
CIEX polishing and buffer exchange steps into one continuous
process with no intermediate handling. For ease of use, the
Discovery operating software, ChromIQ®, is pre-loaded with
methods optimized for use with high performance resins that
are available with the system.
The three-column Protein A/CIEX/desalting purification has
additional benefits compared to a Protein A purification alone.
The automated CIEX polishing step is excellent for the removal
of dimers and aggregates thereby guaranteeing a high purity.
The automated buffer exchange step using a desalting column
means the product is immediately stored in an optimal buffer
and ready to use without additional sample manipulation. This
1) greatly reduces handling time & sample handling
inconsistency 2) minimizes the potential for sample
degradation from intermediate storage in sub-optimal buffer
conditions 3) improves the reliability of antibody
characterization due to fewer dimers and aggregates 4)
reduces the use of expensive consumables, such as disposable
desalting columns.
Method
The ChromIQ® operating software is pre-loaded with a simple
to use Protein A/CIEX/desalting method for getting started
with purification. This includes buffer recipes which give
excellent results for most antibodies in combination with the
supplied columns.
The duration and flow rates for equilibration, wash, elution
and cleaning phases have been optimized and, in most cases,
require no additional user adaptation (advanced method
creation tools are included if customized methods are
needed).
Method setup and buffer preparation
Automatic direct transfer to a desalting column
Product quality analyzed by SE-HPLC & SDS-PAGE
Purification over Protein A column
Automatic direct transfer to a CIEX column
One process
Main product peak collected as one fraction
Contichrom® Discovery
Application note D03. V 1.2 - © ChromaCon 2019 2 /4
The table below shows specific method information:
A Discovery purification run is initiated as follows (Figure 2):
1. Select “3-step Purification” from the menu
2. Choose “Protein A – CIEX – Desalting” and press start
3. Define sample names and data location
4. Define volume of feed to be processed
- The predicted buffer consumption and run time is
calculated for the user
5. Prepare buffers according predefined recipe
6. Load buffers and samples in the predefined inlet positions
7. Update tank levels to monitor buffer consumption
8. Attach columns (Position 1 - Protein A, Position 2 - CIEX,
Position 3 - desalting)
9. Press start
- A detailed report is automatically generated after each
run
Figure 2. Initiation of the three-step purification on the Contichrom Discovery system.
Results
A three-column Protein A/CIEX/buffer exchange purification
was carried out. The resulting chromatogram is shown in
Figure 3. In step 1 the Protein A affinity capture was carried
out (blue line). Impurities in the feed are seen in the first broad
peak (20-29 min) during the loading step. Elution was initiated
and a specific peak corresponding to the monoclonal antibody
(mAb) is seen (32-33 min). The mAb from step 1 was
automatically transferred to step 2 (red line) for polishing by
CIEX and the product was eluted using a salt gradient (dotted
grey line) starting at 47 min. The mAb from step 2, now
depleted of aggregates, was automatically transferred to the
desalting column and eluted after a total run time of 52 min
(black line). An increase in conductivity after the product peak
corresponds to the CIEX elution buffer which was completely
removed from the product (Figure 3, dotted orange line).
A single fraction was captured and then analyzed for purity by
an analytical SE-HPLC (Figure 4). Compared to a two-step
process (see Application note on “Automated two-step
purification of Antibodies”) an 8-fold reduced aggregate
content could be achieved, reducing it to 0.5%. Analysis by
SDS-PAGE (Figure 5) revealed the removal of slight low
molecular weight impurities around 25 kDa. The aggregates
are not visible in the gel, because these are outside the
analytical range. Taken together, the gel and the
chromatogram evidence a high product purity.
Method information
Three-step Protein A/CIEX/desalting
Total Run Time 70 min
Step 1 – Protein A
Buffer A – wash 25 mM phosphate, 150 mM NaCl, pH 7.0
Buffer B – elution 100 mM citrate, pH3.5
Buffer C – cleaning 0.1 M sodium hydroxide
Feed 10 mL CHO supernatant with monoclonal antibody, titer = 1.0 mg/mL
Step 2 – CIEX
Buffer D – wash 25 mM phosphate, pH 6.0
Buffer E – elution 25 mM phosphate + 0.5 M NaCl, pH 6.0
Buffer C – cleaning 0.1 M sodium hydroxide
Step 3 –Buffer exchange
Buffer A – elution/equilibration 25 mM phosphate, 150 mM NaCl, pH 7.0
Contichrom® Discovery
Application note D03. V 1.2 - © ChromaCon 2019 3 /4
Figure 3. Chromatogram of a Protein A/CIEX/desalting purification for isolation of a monoclonal antibody. Blue line – UV1 – Protein A step; red line – UV2 – CIEX step; black line – UV1 – desalting step; dotted grey line – relative conductivity – CIEX step; dotted range line – relative conductivity – desalting step.
Results Product concentration [mg/mL] 0.91 mg/mL
Yield 6.7 mg
% Monomer 99.5%
% Aggregate 0.5%
Figure 4. Analysis of the three-step purified antibody by analytical SEC. A major monomeric peak is seen at 16 min. Dimers are evident at 14 min. Monodispersity = 99.5%.
Figure 5. Evaluation of the antibody product by SDS-PAGE (non-reducing). Lane 1: Protein standard; Lane 2: Feed; Lane 3: Purified target product.
Summary
An automated three-column Protein A/CIEX/desalting
purification was successfully carried out on the Contichrom
Discovery system and the main product peak was captured as
a high purity single fraction ready for downstream
applications. After completion the columns were immediately
ready for the next run due to integrated washing and
equilibration phases. Repeated sample injections can be done
in series without user intervention. With the Contichrom
Discovery HT, which uses additional sample loading valves and
a software extension, up to 18 sample inlets are available. Due
to the greater automation, unattended processing of 18
different samples in a single day becomes possible. ChromIQ,
the Contichrom Discovery operating software, facilitates rapid
method creation. The buffer management system predicts
buffer consumption in advance preventing mistakes in setup.
The Contichrom Discovery automatically scales to your needs
in both sample number and feed volume that can be
processed.
0
200
400
600
800
1000
1200
0
500
1000
1500
2000
2500
15 25 35 45 55
UV
2 A
28
0n
m(m
AU
)UV
1 A
28
0n
m(m
AU
)
Time (min)
Step 3
Load Feed
Wash
Protein A Elution
Transfer to CIEX column
Equilibration
Fin
al p
rod
uct
Des
alti
ng
Transfer to desalting column
Step 1 Step 2
0
10
20
30
40
50
60
0 5 10 15 20 25
A2
80
nm
(mA
U)
Time (min)
0.75
1.25
1.75
0.75
1.25
1.75
3-step protocol = 0.5% dimer2-step protocol without CIEX = 4% dimer
250 -150 -100 -75 -
50 -
37 -
25 -20 -
15 -
10 -
MW
[kD
a]
Purified mAb
1 2 3
Contichrom® Discovery
Application note D03. V 1.2 - © ChromaCon 2019 4 /4
About the Contichrom® Discovery system.
The Contichrom Discovery system is a versatile FLPC system
capable of running automated multi-dimensional purifications
with inline dilution. The system offers fixed recipes and that
can also be customized. If any additional purification is needed
beyond a three-step purification sequence, the ChromIQ batch
wizard can be used to purify the sample even further.
System specifications (see brochure for more details)
Flow rate range 0.1 – 36 mL/min
Pressure rating 50 bar (5 MPa) / 725 psi
Number of buffers inlets Up to 16
Number of sample inlets 1 (HT version: 18)
Fractionation 4 fractions (valve), optional fraction collector
UV Detectors Simultaneously 260 nm and 280 nm, detection behind each column
Conductivity/pH detectors 1 each included
Column type Model
Protein A Eshmuno A – 1 mL Included
CIEX Eshmuno CPX, Fractogel SO3(M) – 5 mL Included
AIEX Eshmuno Q – 5 mL Included
IMAC Fractogel Chelate – 1 mL Included
Desalting Fractogel BioSEC– 10 ml Included
SEC Superdex 75 (10 – 100 kDa) – 23.5 mL Optional
SEC Superdex 200 (> 100 kDa) – 23.5 mL Optional
ChromaCon AG Technoparkstrasse 1 CH-8005 Zurich Switzerland www.chromacon.ch
ChromaCon, Contichrom, ChromaCon monograms, AutomAb, CaptureSMB, ChromIQ are trademarks of ChromaCon AG. Any use of ChromIQ software is subject to ChromaCon Standard Software End-User License Agreement. A copy of this Standard Software End-User License Agreement is available upon request. © 2019 ChromaCon AG. First published September 2017. All goods and services are sold subject to the terms and conditions of sale of the company within ChromaCon AG which supplies them. A copy of these terms and conditions is available on request. Contact your local ChromaCon representative for the most current information.