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.