Download Protein A Affinity Column for Monoclonal Antibody (MAb) Titer Analysis

Protein A Affinity Column for Monoclonal
Antibody (MAb) Titer Analysis
Shanhua Lin, Kelly Flook, Yuanxue Hou, Hongmin Zhang, Charanjit Saini, Srinivasa Rao,
Yury Agroskin, and Chris Pohl
Thermo Fisher Scientific, Sunnyvale, CA, USA
Overview
Results
Purpose: Demonstrate the capability of a Thermo
Scientific™ MAbPac™ Protein A column for titer analysis
of monoclonal antibodies (MAb).
Staphylococcal pro
in immunology and
interaction with the
from many mamma
domain exposed on
bacterium, Staphylo
different regions; S
processed during s
domains E, D, A, B
XM. SPA and the s
been used widely fo
Methods: The MAbPac Protein A column has been
analyzed on a Biocompatible HPLC system.
Chromatographic conditions such as flow rate, column
temperature, and sample loadings were tested to
evaluate the column performance.
Results: The MAbPac Protein A column can accurately
determine the MAb titer in the range of 0.025 mg/mL to
5 mg/mL. The total analysis time is less than 2 minutes.
Introduction
Early in the development of recombinant monoclonal
antibodies (MAbs), a large number of harvest cell culture
(HCC) samples must be screened for IgG titer. Affinity
chromatography employing a Protein A ligand is often
used to determine the MAb concentration as well as to
purify it for downstream aggregate and charge variant
analysis. The challenge facing the analytical laboratories
in the pharmaceutical industry is to develop highthroughput and robust titer assay.
Recombinant Prote
as opposed to the n
Staphylococcus au
containing the sam
mass as the native
is used as the affini
column.
FIGURE 1. Schem
fragment crystalli
antigen binding re
Fab
In the current study, we are presenting a Protein A column
for fast MAb titer analysis. The column is developed based
on a novel polymeric resin. The hydrophilic surface is
designed to accommodate protein conjugation. A
recombinant Protein A ligand is covalently attached onto
the hydrophilic resin surface. The functionalized resin with
recombinant Protein A is then packed into a 4 × 35 mm
PEEK™ column body.
The hydrophilic nature of the backbone minimizes
non-specific binding and therefore enables accurate
quantification of the MAb titer. In addition, the non porous
particle produces a highly efficient column at high flow
rates. When injecting 20 µg of rabbit IgG, the IgG peak
width at half height is about 0.01 min. The sharp peak
shape also provides great sensitivity. As little as 0.25 µg
of MAb can be easily detected. The Protein A column has
very low back pressure and this allows high flow rate for
fast analysis. At 2.5 mL/min, the entire analysis, including
equilibration, takes only 1.6 min. Ruggedness testing
shows that this column can go through more than 2,000
cycles with very little loss of performance. The HPLC
compatibility of this column allows automation, provides
accurate and high throughput analysis.
Methods
Sample Preparation
2
Monoclonal antibody harvest cell culture was a gift from
a local biotech company.
The HCC was filtered through a 0.22 µm membrane prior
Protein A Affinity Column for Monoclonal Antibody (MAb) Titer Analysis
to sample injection.
Disulfide
bonds
Dynamic loading c
The dynamic loadin
pooled normal seru
MAbPac Protein A
2 mL/min. Figure 2
load for rabbit IgG w
A column. This corr
column to be used
culture over a wide
FIGURE 2. Area de
Gradient: 0% B for
for 1.2 mins.
60
50
40
Column:
Flow rate:
Gradient:
Detection:
Temperature:
Inj. volume:
Sample:
M
2
0
2
2
2
R
compatibility of this column allows automation, provides
accurate and high throughput analysis.
Methods
FIGURE 2. Area depend
Gradient: 0% B for 0.2 m
for 1.2 mins.
Sample Preparation
60
Monoclonal antibody harvest cell culture was a gift from
a local biotech company.
The HCC was filtered through a 0.22 µm membrane prior
to sample injection.
50
MAbPac Protein A column, 12 µm, 4 × 35 mm
Buffers
Detection:
Temperature:
Inj. volume:
Sample:
40
Area, mAu.min
Columns
Column:
Flow rate:
Gradient:
Eluent A: 50mM Sodium Phosphate, 150 mM NaCl, 5%
acetonitrile, pH 7.5
MAbPac P
2 mL/min
0%B for 0
0.60 mins,
280 nm
25°C
20 µL
Rabbit IgG
30
20
10
Eluent B: 50mM Sodium Phosphate, 150 mM NaCl, 5%
acetonitrile, pH 2.5
0
0
20
40
A
Liquid Chromatography
HPLC experiments were carried out using an hybrid
system equipped with:
Influence of flow rate on
and peak area
Thermo Scientific™ Dionex™ ICS-3000 Dual Gradient
Pump System
The MAbPac Protein A co
rate up to 2.5 mL/min. Th
2 mL/min. Figure 4 show
rate on column backpres
increase the flow rate the
of IgG that binds to the co
(Table 1) is due to the us
of the detector.
Thermo Scientific™ Dionex™ TCC-100 Column
Compartment
Thermo Scientific™ WPS-3000 Pull-Loop AutoSampler
Thermo Scientific™ Dionex™ VWD-3400RS UV Detector
equipped with a 2.5 µL Micro Flow Cell
20 µg rabbit IgG was injected every 100 cycles over the
course of 2,000 runs, see Figure 6. Retention time, peak
area, and peak width of IgG from each chromatographic
run are listed on the inserted table.
FIGURE 3. Effect of flow
700
0
TABLE 1. Effect of Flow Rate on IgG peak area.
Flow Rate
(mL/min)
Total Area
(mAu.min)
Unbound
Area
(mAu.min)
Unbound
Relative
Area, %
IgG Area
(mAU.min)
IgG
Relative
Area, %
2.5
7.381
1.196
16.20
6.185
83.80
2.0
8.826
1.419
16.08
7.407
83.92
1.5
12.111
2.068
17.08
10.043
82.92
1.0
17.895
2.937
16.41
14.958
83.59
0.5
34.219
5.582
16.31
28.637
83.69
2.5 mL/min 0%B for 0.2 mins,
100% B for 0.48 mins, 0%B for
700
mAu
0
2.0 mL/min 0%B for 0.2 mins,
700 100% B for 0.60 mins, 0%B for
0
700
0
700
0
1.5 mL/min 0%B for 0.3 mins, 1
1.0 mL/min 0%B for 0.4 mins, 1
0.5 mL/min 0%B for 0.8 mins, 1
0
1
Thermo Scientific Poster Note • PN20806_e 06/13S 3
2
A column has been
PLC system.
ch as flow rate, column
gs were tested to
ce.
A column can accurately
ange of 0.025 mg/mL to
e is less than 2 minutes.
ombinant monoclonal
ber of harvest cell culture
ed for IgG titer. Affinity
otein A ligand is often
centration as well as to
ate and charge variant
he analytical laboratories
s to develop highy.
Staphylococcal protein A (SPA) plays an important role
in immunology and biochemistry owing to its specific
interaction with the Fc part of immunoglobulin G (IgG)
from many mammals. SPA is a cell wall associated protein
domain exposed on the surface of the Gram-positive
bacterium, Staphylococcus aureus. SPA consists of three
different regions; S, being the signal sequence that is
processed during secretion, five homologous IgG binding
domains E, D, A, B, and C, and a cell-wall anchoring region
XM. SPA and the smaller ligands derived from SPA have
been used widely for the affinity purification of antibodies.1
Recombinant Protein A (rSPA), is expressed in E. coli
as opposed to the native protein extracted from
Staphylococcus aureus. rSPA is a 45 kDa protein
containing the same amino acid sequence and molecular
mass as the native Protein A sourced from S. aureus. rSPA
is used as the affinity ligand for the MAbPac Protein A
column.
FIGURE 1. Schematic of an antibody showing the
fragment crystallizable region (Fc) and the fragment
antigen binding region.
Fab
enting a Protein A column
olumn is developed based
hydrophilic surface is
ein conjugation. A
covalently attached onto
e functionalized resin with
acked into a 4 × 35 mm
ckbone minimizes
re enables accurate
n addition, the non porous
nt column at high flow
abbit IgG, the IgG peak
1 min. The sharp peak
tivity. As little as 0.25 µg
The Protein A column has
allows high flow rate for
entire analysis, including
. Ruggedness testing
hrough more than 2,000
ormance. The HPLC
ws automation, provides
nalysis.
ll culture was a gift from
Fab
variable
region
400
350
Column:
Temperature:
300
250
200
150
100
0
0.0
0.5
Influence of tempera
It is recommended to
at temperatures no hi
consistency and to pr
recommended to use
temperature to 25˚C.
binding is minimally a
different binding asso
FIGURE 5. Temperat
800
light chain
700
600
Disulfide
bonds
500
constant
region
Fc
heavy chain
400
300
200
35 ºC
100
30 ºC
0
Dynamic loading capacity
-100
The dynamic loading capacity of rabbit IgG, isolated from
pooled normal serum, is no less than 100 µg on the
MAbPac Protein A column, analyzed at a flow rate of
2 mL/min. Figure 2 shows the linearity of area to sample
load for rabbit IgG when loaded onto the MAbPac Protein
A column. This correlation allows the MAbPac Protein A
column to be used for quantitation of MAb in harvest cell
culture over a wide range of concentrations.
-300
FIGURE 2. Area dependence on rabbit IgG Loading.
Gradient: 0% B for 0.2 mins, 100% B for 0.6 mins, 0% B
for 1.2 mins.
60
50
0.22 µm4 membrane
Protein A Affinityprior
Column for Monoclonal Antibody
40
Column:
Flow rate:
Gradient:
MAbPa
25°C
50
mAu
ability of a Thermo
A column for titer analysis
.
FIGURE 4. Example
backpressure.
Column Pressure (Psi)
Results
MAbPac Protein A, 4 × 35 mm
2 mL/min
0%B for 0.2 mins, 100% B for
0.60 mins, 0%B for 1.20 mins
Detection:
280 nm
(MAb)
Titer Analysis
Temperature: 25°C
Inj. volume:
20 µL
25 ºC
20 ºC
-200
15 ºC
0.00
0.25
0.50
0
Column Ruggednes
The MAbPac Protein
continuously for 2,000
of calibration standard
was analyzed. As sho
peak area, and peak
the upper range, ther
in the lower range sen
FIGURE 6. Chromato
the MAbPac Protein
Column:
Flow rate:
Gradient:
Temperature:
Detection:
Inj. volume:
MAbPac Pro
2 mL/min
0%B for 0.2
25 ºC
280 nm
20 µL
more than 2,000
ce. The HPLC
mation, provides
of calibration standards (from
was analyzed. As shown in F
peak area, and peak width o
the upper range, there is no
in the lower range sensitivity
column to be used for quantitation of MAb in harvest cell
culture over a wide range of concentrations.
FIGURE 2. Area dependence on rabbit IgG Loading.
Gradient: 0% B for 0.2 mins, 100% B for 0.6 mins, 0% B
for 1.2 mins.
60
e was a gift from
Column:
Flow rate:
Gradient:
50
m membrane prior
Detection:
Temperature:
Inj. volume:
Sample:
Area, mAu.min
40
MAbPac Protein A, 4 × 35 mm
2 mL/min
0%B for 0.2 mins, 100% B for
0.60 mins, 0%B for 1.20 mins
280 nm
25°C
20 µL
Rabbit IgG
R² = 0.9998
1,400
30
1,200
1,000
20
10
50 mM NaCl, 5%
0
0
20
40
60
80
Amount rabblit IgG, µg
100
120
ing an hybrid
0 Dual Gradient
The MAbPac Protein A column can be used at a flow
rate up to 2.5 mL/min. The recommended flow rate is
2 mL/min. Figure 4 shows the effect on increasing flow
rate on column backpressure. Figure 3 shows that as you
increase the flow rate there is little effect on the amount
of IgG that binds to the column. The increase in total area
(Table 1) is due to the use of the same data collection rate
of the detector.
00RS UV Detector
0 cycles over the
tention time, peak
chromatographic
FIGURE 3. Effect of flow rate on IgG binding.
700
Column:
Gradient:
2.5 mL/min 0%B for 0.2 mins,
700 100% B for 0.48 mins, 0%B for 0.92 mins Temperature:
Detection:
Inj. Volume:
Sample:
0
2.0 mL/min 0%B for 0.2 mins,
700 100% B for 0.60 mins, 0%B for 1.20 mins
0
peak area.
IgG Area
(mAU.min)
IgG
Relative
Area, %
6.185
83.80
7.407
83.92
10.043
82.92
14.958
83.59
28.637
83.69
mAu
0
700
0
700
0
#2011
#1811
#16
#1411
11
#1211
400
#1011
#811
#611
200
#411
#211
#30
-50
0.00
0.25
0.50
0.75
600
Influence of flow rate on column pressure, cycle time
and peak area
op AutoSampler
MAbPac Protein A, 4 × 3
2 mL/min
0%B for 0.2 mins, 100%
25 ºC
280 nm
20 µL
Rabbit IgG, 1 mg/mL
800
50 mM NaCl, 5%
0 Column
Column:
Flow rate:
Gradient:
Temperature:
Detection:
Inj. volume:
Sample:
mAu
35 mm
FIGURE 6. Chromatogram
the MAbPac Protein A colu
MAbPac Protein A, 4 × 35 mm
Adjusted according to flow rate
25 ºC
280 nm
20 µL
Rabbit IgG, 1 mg/mL
1.00
Conclusions
  The MAbPac Protein
loading capacity of at
of quantifying MAbs i
to 5 mg/mL.
  The MAbPac Protein
time. At 2 mL/min, a
2 min.
  The MAbPac Protein
successfully tested th
loss of binding capac
References
1.  Hober, S., Nord, K., a
Chromatography for A
of Chromatography B
PEEK is a trademark of Victrex Inc. All other trademarks
subsidiaries.
1.5 mL/min 0%B for 0.3 mins, 100% B for 0.90 mins, 0%B for 1.80 mins
This information is not intended to encourage use of the
property rights of others.
PO20806_E 05/13S
1.0 mL/min 0%B for 0.4 mins, 100% B for 1.2 mins, 0%B for 2.40 mins
0.5 mL/min 0%B for 0.8 mins, 100% B for 2.40 mins, 0%B for 4.80 mins
0
1
2
3
4
5
6
7
8
Minutes
Thermo Scientific Poster Note • PN20806_e 06/13S 5
s expressed in E. coli
extracted from
a 45 kDa protein
sequence and molecular
urced from S. aureus. rSPA
he MAbPac Protein A
tibody showing the
(Fc) and the fragment
b
variable
region
400
350
Column Pressure (Psi)
plays an important role
owing to its specific
munoglobulin G (IgG)
ell wall associated protein
of the Gram-positive
us. SPA consists of three
gnal sequence that is
homologous IgG binding
a cell-wall anchoring region
s derived from SPA have
purification of antibodies.1
FIGURE 4. Example of effect of flow rate on column
backpressure.
Column:
Temperature:
MAbPac Protein A, 4 × 35 mm
25°C
300
250
200
150
100
50
0
0.0
0.5
1.0
FIGURE 5. Temperature effect on binding efficiency.
Column:
Flow rate:
Gradient:
MAbPac Protein A, 4 × 35 mm
2 mL/min
0% B for 0.2 mins, 100% B for
0.60 mins, 0%B for 1.20 mins
Detection:
280 nm
Inj. volume: 20 µL
Sample:
Rabbit IgG, 1 mg/mL
700
600
500
constant
region
mAu
400
300
200
35 ºC
100
30 ºC
0
-100
-300
25 ºC
20 ºC
-200
n rabbit IgG Loading.
0% B for 0.6 mins, 0% B
3.0
It is recommended to use the MAbPac Protein A column
at temperatures no higher than 35˚C. To ensure data
consistency and to prolong the column life time, it is
recommended to use a column oven to control the
temperature to 25˚C. Figure 5 shows how rabbit IgG
binding is minimally affected by temperature. Proteins with
different binding association may be affected differently.
800
rabbit IgG, isolated from
than 100 µg on the
yzed at a flow rate of
nearity of area to sample
onto the MAbPac Protein
s the MAbPac Protein A
on of MAb in harvest cell
centrations.
2.5
Influence of temperature on binding efficiency
ght chain
heavy chain
1.5
2.0
Flow Rate (mL/min)
15 ºC
0.00
0.25
0.50
0.75
1.00
1.25
Minutes
1.50
1.75
2.00
2.24
Column Ruggedness
The MAbPac Protein A column has been tested
continuously for 2,000 cycles. Every hundred cycles, a set
of calibration standards (from 0.01 mg/mL to 5 mg/mL)
was analyzed. As shown in Figure 6, the retention time,
peak area, and peak width of IgG remain unchanged. In
the upper range, there is no loss of binding capacity and
in the lower range sensitivity is maintained.
FIGURE 6. Chromatograms of rabbit IgG analyzed on
the MAbPac Protein A column.
35 mm
% B for
0 mins
6 Protein A Affinity Column for Monoclonal
Column:
MAbPac Protein A, 4 × 35 mm
Flow rate:
2 mL/min
Gradient:
0%B for 0.2 mins, 100% B for 0.60 mins, 0%B for 1.20 mins
Temperature: 25 ºC
Antibody
(MAb) Titer
Detection:
280Analysis
nm
Run #
Ret.Time (min) Area (mAu*min)
Inj. volume: 20 µL
30
0.80
7.96
PWHH (min)
0.01
peak area, and peak width of IgG remain unchanged. In
the upper range, there is no loss of binding capacity and
in the lower range sensitivity is maintained.
bit IgG Loading.
or 0.6 mins, 0% B
FIGURE 6. Chromatograms of rabbit IgG analyzed on
the MAbPac Protein A column.
= 0.9998
1,400
Column:
Flow rate:
Gradient:
Temperature:
Detection:
Inj. volume:
Sample:
MAbPac Protein A, 4 × 35 mm
2 mL/min
0%B for 0.2 mins, 100% B for 0.60 mins, 0%B for 1.20 mins
25 ºC
280 nm
Run #
Ret.Time (min) Area (mAu*min)
20 µL
30
0.80
7.96
Rabbit IgG, 1 mg/mL
211
0.78
7.93
1,200
1,000
800
mAu
#2011
#1811
600
#16
#1411
11
#1211
400
#1011
#811
#611
200
#411
#211
#30
-50
0.00
0.25
0.50
0.75
80
100
120
ssure, cycle time
1.00
1.25
PWHH (min)
0.01
0.01
411
0.80
7.88
0.01
611
0.80
7.97
0.01
811
0.80
7.58
0.01
1011
0.80
7.77
0.01
1211
0.80
7.78
0.01
1411
0.79
7.50
0.01
1611
0.81
7.50
0.01
1811
0.79
7.78
0.01
2011
0.79
7.77
0.01
1.50
1.75
2.00
2.25
2.50
Minutes
used at a flow
ed flow rate is
increasing flow
shows that as you
ct on the amount
crease in total area
data collection rate
Conclusions
  The MAbPac Protein A column has a dynamic
loading capacity of at least 100 µg. It is capable
of quantifying MAbs in the range of 0.01 mg/mL
to 5 mg/mL.
  The MAbPac Protein A column has a fast cycle
time. At 2 mL/min, a complete titer analysis takes
2 min.
  The MAbPac Protein A column has been
successfully tested through 2,000 cycles without
loss of binding capacity.
binding.
MAbPac Protein A, 4 × 35 mm
Adjusted according to flow rate
re: 25 ºC
280 nm
e: 20 µL
Rabbit IgG, 1 mg/mL
References
1.  Hober, S., Nord, K., and Linhult, M. Protein A
Chromatography for Antibody Purification. Journal
of Chromatography B, 848 (2007) 40–47.
PEEK is a trademark of Victrex Inc. All other trademarks are the property of Thermo Fisher Scientific and its
subsidiaries.
%B for 1.80 mins
This information is not intended to encourage use of these products in any manner that might infringe the intellectual
property rights of others.
PO20806_E 05/13S
B for 2.40 mins
%B for 4.80 mins
6
7
8
www.thermoscientific.com/dionex
©2013 Thermo Fisher Scientific Inc. All rights reserved. ISO is a trademark of the International Standards Organization.
PEEK is a trademark of Victrex Inc. All other trademarks are the property of Thermo Fisher Scientific Inc. and its subsidiaries.
This information is presented as an example of the capabilities of Thermo Fisher Scientific Inc. products. It is not intended to
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