chromatographytech note 2026

Evaluation of Different Approaches for the
Chromatographic Purification of Monoclonal Antibodies
Andrew Stapleton, Xiao Y. Zhang*, Howard Petrie*,
Joseph E. Machamer
Summary
Purification of monoclonal antibodies is one of the major
applications of chromatography in the life sciences. Protein A
and Protein G supports provide an efficient means to purify
various classes of immunoglobulins. However, the species orsubclass specificities, high costs, and relatively harsh elution
conditions associated with such supports are often
incompatible with the overall purification strategy. Potential
leakage of Protein A or G from these supports can also be
problematic for antibodies destined for therapeutic applications.

•••

The recently introduced series of analytical and preparative
Bio-Scale chromatography columns provides several options
for the purification of immunoglobulins in a cost effective
manner under gentle elution conditions. The methods detailed
in this study for monoclonal antibody purification include:
Tandem use of DEAE Affi-Gel® blue mixed mode anion
exchange/dye ligand followed by Bio-Scale CHT-I ceramichydroxyapatite chromatography column
Cation exchange chromatography using a Bio-Scale S strong
cation exchange column
Anion exchange chromatography using a Bio-Scale DEAE
weak anion exchange column
All three methods provide cost effective alternatives to affinity
based methods for monoclonal antibody purification. The
results, advantages, and disadvantages of the different
methods are discussed in this study.
Introduction
The Monoclonal Antibody Core Facility at the Memorial Sloan
Kettering Cancer Center produces monoclonal antibodies
used in the center’s research of diseases such as AIDS andcancer. In an effort to develop a single step, non-Protein A
based chromatographic monoclonal antibody (MAb)purification, three different approaches were investigated on
three different MAbs.

Materials and Methods
All samples were filtered through a 0.22 µm filter and thendialyzed against the equilibration buffer for the column used.
The system used was the BioLogic Chromatography
Workstation. All of the columns were from Bio-Rad
Laboratories. The columns used in Approach I: TandemColumns for Human MAb, were a 5 ml Econo-Pac® DEAE bluecartridge (catalog number 732-0031) (mixed mode anion
exchange/dye ligand) and a 2 ml Bio-Scale CHT2-I Column(catalog number 751-0021) packed with 10 µm Macro-Prep®
ceramic hydroxyapatite Type I media. The column used forApproach II: Strong Cation Exchange for Human MAb, was the2 ml Bio-Scale S2 column (catalog number 751-0011). Thiscolumn is packed with a 10 µm Macro-Prep S strong cationexchange support. The column used for Approach III: WeakAnion Exchange for GK 1.5 Anti-mouse CD MAb and PC-61
Murine CD 25 (IL-2 Receptor P 55 alpha chain), was the20 ml Bio-Scale DEAE20 column (catalog number 751-0037),
packed with 10 µm Macro-Prep DEAE (diethylaminesubstituted methacrylate) support.
Approach I: Tandem Columns for Human MAb
The first approach evaluated was the use of tandem
chromatography on an Econo-Pac DEAE blue cartridge and a
Bio-Scale CHT-I column. This approach involves more than
one chromatographic column, but was evaluated as a single
step because the elution buffer from the Econo-Pac DEAE
blue cartridge had the potential to act as a binding buffer forthe Human MAb on the CHT-I column. The BioLogic
Workstation was used to automatically load the peak fromthe Econo-Pac DEAE blue cartridge onto the CHT-I column.
The peaks from the CHT-I column could then be collected via
a fraction collector. The purification thus operated as a single
step because no intervention was required to obtain purified
MAb, after the method had been started from the BioLogic
System controller (Figures 1 and 2).
When the peak from the Econo-Pac DEAE blue cartridge wascollected in the Dynamic Loop, the valves were re-positioned asshown in Figure 2. This allowed the peak from the Econo-Pac
DEAE blue cartridge, which had been collected in the Dynamic
Loop, to be injected onto the CHT-I column.

Valve 2
AV7-3
Shown inLoadposition

Valve 2
AV7-3
Shown in
Injectposition

1

72

Valve 2
AV7-3
shown in
Injectposition

Valve 1
AV7-3
shown inPurge
position

Column 2
61

76Column 1

3454

72

Bio-Rad DynaLoop

35Column 1

Sample Loop

Valve 3
AV9-8863A5UV-Conddetectors
To Model 2128
fraction collector
Waste or Collect0.03

8000.02400

Conductivity (µS)

A280 (AU)Fig. 1. The protein sample is loaded onto column 1 via the loop of valve 1.
The column is then eluted normally. The output of column 1 is routed throughthe detector. Valve 4 then diverts the flow either to the fraction collector or to
the Bio-Rad DynaLoop ready for re-injection onto column 2. This diagramshows the liquid path from column 1 to the dynamic loop.

0.00

02040

B

60

Time (minutes)

Column: 5 ml Econo-Pac DEAE blue
Gradient: 20 mM Tris, pH 7.2, 0–0.5 M NaCl
Flow Rate: 1 ml/min

Valve 3AV 9-8

Gradient
Pump34

5

UV-Conddetectors

Each pump inlet
may use a SV5-4
valve for selectionof up to 4 buffers

Valve 4
SV 3-2

Valve 4
SV 3-2

To Model 2128
fraction collector
Waste or CollectFig. 2. Valves are re-positioned as shown. Sample in the dynamic loop is
injected onto column 2. Elution can then proceed, with fractions being
collected into fraction collector.

A280 (AU)4

Each pump inlet
may use a SV5-4
valve for selectionof up to 4 buffers

726

Gradient
Pump

81720Bio-Rad DynaLoop

541B546Sample LoopAColumn 260.08

6000.04300

0Conductivity ( S)

17332120010203040

Time (minutes)

Column: Bio-Scale CHT-I
Gradient: 10 to 140 mM sodium phosphate, pH 6.8
Flow Rate: 1 ml/min

Fig. 3. Separation of human monoclonal antibody using DEAE Affi-Gel blue gel.

Fig. 4. Separation of IgG containing fraction from Figure 3 using ceramichydroxyapatite.

This study was divided into two phases. The first phase
involved the search for buffer conditions which would elutethe human MAb from the Econo-Pac DEAE blue cartridge
while still allowing the antibody to bind to the CHT-I column.
The results shown in Figures 3 and 5 are from this phase ofthe project. The second phase involved the implementationof the automation.

Fractions 16–24 from the Econo-Pac DEAE blue cartridge
were pooled and loaded directly onto the Bio-Scale CHT2-I
ceramic hydroxyapatite column. The pooled fractions boundto the CHT-I column, and were eluted using a 10 to 140 mM
sodium phosphate gradient, pH 6.8 (Figure 4).
Figure 5 is the PAGE gel which was run on fractions 16–21
from the CHT-I column. It shows that the antibody has beensignificantly purified.

The DEAE Affi-Gel blue gel is a bifunctional affinity gel
containing Cibacron blue F3GA dye covalently attached toDEAE Bio-Gel® A agarose gel. The dye binds albumin andthe DEAE group binds the remaining acidic proteins.1 This gel
offers several advantages over Protein A. These include theability to bind all IgG subclasses, mild elution conditions,complete removal of all proteases, and significantly lower costthan Protein A. Under the elution conditions of a gradientfrom 0 to 0.125 M NaCl in 20 mM Tris-HCl, pH 7.2, theantibody eluted but the albumin remained bound to thecolumn (Figure 3).

Approach II: Strong Cation Exchange for Human
Monoclonal Antibody
The second approach developed for the purification of hMAb
used the Bio-Scale S2 strong cation exchange column. This
column is packed with a 10 µm methacrylate based bead.
Since the same bead is available in a 50 µm bulk format,
conditions developed for this separation could easily bescaled up to the 50 µm bead for large scale purifications.

The 10 µm bead was chosen because of its high degree ofresolution, which makes it easier to develop and optimize apurification method. DEAE was chosen over a Q-type strong
anion exchanger for historical reasons, as earlier antibody
purification work has used weak anion exchange. Most
immunoglobulins have pIs in the range of 6–8, hence a pH of7.5 was chosen for the work on the DEAE column. Most
immunoglobulins should bind under these conditions andelute early in the gradient.

Cation exchange was investigated at pH 5.5 in MES. At thispH, albumin (pI 4.5–5.0) is negatively charged and does notbind. The human MAb is positively charged at this pH anddoes bind to the column. Elution is done using a sodium
chloride gradient (Figure 6).
A gel was run on Fractions 8–10. As Figure 7 clearly shows,fraction 8 displays heavy and light bands characteristic ofantibodies analyzed via SDS-PAGE with no visible
contaminating proteins.

0.4

1000

0.2

5000Conductivity (µS)

Fig. 5. SDS-PAGE gel of fraction obtained from Figure 4. Lane labeled with theasterisk is fraction from chromatogram in Figure 4.

The load for the GK 1.5 anti-mouse CD 4 MAb was 120 mg.
The gradient was from 0 to 160 mM NaCl. As shown inFigure 8, two small peaks, eluted on both sides of the mainpeak, which contains a shoulder. Figure 9 shows theSDS-PAGE analysis of the fraction 11 from the main peak.

A280 (AU)

*0040

80

120160Time (minutes)

Column: Bio-Scale DEAE20
Gradient: 25 mM Tris, pH 7.5 over 300 ml, followed by astep to 1.0 M NaCl, 0–0.4 M NaCl
Flow Rate: 1–4 ml/min
10005000Fig. 7. Separation of GK 1.5 anti-mouse CD4 MAb using Bio-Scale
DEAE column.

0081624321000

400.06

A280 (AU)Time (minutes)

Column: Bio-Scale S2
Gradient: 50 mM MES, pH 5.5, 0–0.5 M NaCl, followed bya step to 1.0 M NaCl
Flow Rate: 1 ml/min

5000.03

000Fig. 6. Strong cation exchange of human MAb.

Approach III: Weak Anion Exchange for GK 1.5
Anti-mouse CD MAb and PC-61 Murine CD 25(IL-2 receptor p 55 alpha chain)4The purification of Mouse GK 1.5 anti-mouse CD MAb3 andPC-61 murine CD 25 (IL-2 receptor p 55 alpha chain)4 wasevaluated on the Bio-Scale DEAE20 weak anion exchange
column. This column contains 20 ml of 10 µm methacrylate
based weak anion exchange support.

Conductivity (µS)

0.2

Conductivity (µS)

A280 (AU)

0.4

4080

120

160200Time (minutes)

Column: Bio-Scale DEAE20
Gradient: 25 mM Tris, pH 7.5 over 480 ml, followed bya step to 1.0 M NaCl, 0–0.4 M NaCl
Flow Rate: 2–4 ml/min

Fig. 8. Weak anion exchange of PC-61 murine CD 25.4

The load for the murine CD 25 (IL-2 receptor p 55 alphachain) was 154 mg. The gradient was from 0 to 160 mM
NaCl. As shown in Figure 10, peaks are eluting throughout
the gradient. Figure 11 shows the purity.

HSFM

289HS
FM10

28910Fig. 9. SDS-PAGE of fractions from Figure 6.

γgMarker

UnpurifiedConclusion
The purpose of this study was to develop single step,
non-Protein A based MAb purification procedures. Threeapproaches were investigated, the tandem use of DEAE Affi-Gel
blue followed by a Bio-Scale CHT-I column, the use of strong
cation exchange on the Bio-Scale S2 column, and the use of
weak anion exchange on the Bio-Scale DEAE20 column.
The tandem use of DEAE Affi-Gel blue followed by Bio-Scale
CHT-I is useful when the pI of the MAb is close enough to thepI of albumin to cause problems with ion exchange. Inaddition, the use of CHT-I could allow different idiotypes of theMAb to be separated. A two step procedure can be easily
automated using the BioLogic system. The strong cationexchange approach yielded the purest antibody, as shown in
the gel in Figure 7. This approach would be less effective incases where the pI of the MAb is close to the pI of albumin.

Purified Medium

Fig. 10. SDS-PAGE of separation in Figure 8. IgG from PC1.53.

The advantage of the weak anion exchange approach is thegreat number of literature references which employ thistechnique. The disadvantage of using any weak ion
exchanger is the requirement for large equilibration volumes
when changing pH, compared to strong ion exchangers
which have no titratable moieties in the pH region whereprotein separations are conducted.
References
Markerγg

Unpurified

Purified

PurifiedMedium1234

Bruck, C., et al., Methods in Enzymol., 121, 587–596 (1986).
Kadoya T., et al., J. Liq. Chromatogr., 9, 3543 (1986).
Dialynas, et al., J. Immunol., 131, 2445, (1983).
Ceredig, et al., Nature, 314, 98–100.

Fig. 11. SDS-PAGE of fractions from Figure 10. IgG1 from PC61.53.
Presented by J. Machamer at PrepTech ‘96, Industrial Separation Science
Conference, February 5–7, 1996, East Rutherford, NJ, USA.
*Ziao Y. Zhang and *Howard Petrie, Monoclonal Antibody Core Facility, Memorial
Sloan Kettering Cancer Center, 1275 York Avenue, New York, NY 10021.
Andrew Stapleton and Joseph E. Machamer, Bio-Rad Laboratories, 2000
Alfred Nobel Drive, Hercules, CA 94547.

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