A Holistic Approach to Understanding cIEF
Ingrid D. Cruzado-Park, Scott Mack and Chitra K. Ratnayake
Discovery Products, Beckman Coulter, Inc., Fullerton, CA, USA
0.10
Hb F
0.08
Hb A
AU
0.07
0.06
0.06
0.05
0.05
0.04
0.04
Hb A1c
0.03
0.02
(b) Chemical Mobilization
0.01
0.00
6
8
10
12
14
23
24
25
26
27
28
29
Minutes
30
31
32
33
34
35
36
37
15.5
16.0
16.5
17.0
17.5
18.0
Minutes
18.5
19.0
19.5
20.0
20.5
21.0
21.5
0.13
HbF
0.13
Hb A
0.12
0.12
Hb A1c
Hb Fac
0.11
0.10
8.4a,b
(b) Low Concentrations
0.08
AU
Because the cIEF gradient is homogeneous at the start of the
separation, the focusing time must be sufficient to allow the
cathodic and anodic peaks for each component to merge. Failure to
achieve complete focusing will result in partial detection and/or
unmerged sample peaks (Fig. 5).
0.09
(a) 800 µm aperture
0.08
HbF
HbF
0.07
HbHb
A
A
0.07
0.06
0.06
0.05
0.05
Hb A1c
HbA1ac
0.04
0.03
0.04
0.03
HbFac
Hb Fac
0.02
0.01
Focusing Time
AU
0.09
0. 9
Cathodic
Peak
0.02
0. 8
0.01
(b) 200 µm aperture
0.00
12 min 21 kV
Focusing
0. 7
-0.01
14.5
15.0
15.5
16.0
16.5
17.0
17.5
18.0
Minutes
18.5
19.0
19.5
20.0
20.5
21.0
21.5
-0.02
22.0
Fig. 2: Separation of hemoglobins A and F by cIEF using chemical mobilization and
different apertures: (a) 800 and (b) 200 µm. Experimental conditions are as described
on Fig. 1.
Peptide pI Markers
The use of synthetic oligopeptides as pI markers increases the
precision and accuracy of the pI determination since they are not
complicated with post-translational modifications as proteins can
be. The peptides used in this work are listed on Table 1. In a
previous work [Shimura et al, Electrophoresis 21, 603 (2000)],
8.40a and 8.40b peptide markers could not be separated. However,
we were able to resolve them by cIEF (Fig. 3).
0. 6
0. 5
AU
-0.02
Merged
Peak
Cathodic
Peak
0.00
-0.01
30
32
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
10 min 21 kV
Focusing
Merged
Peak
10
30 mM
Arg
8 min 21 kV
Focusing
Cathodic
Peak
0. 4
6 min 21 kV
Focusing
0. 3
0. 2
4 min 21 kV
Focusing
Anodic
Peak
Anodic
Peak
0. 1
2 min 21 kV
Focusing
0. 0
1
2
3
4
5
6
7
8
9
10
11
12
Minutes
13
14
15
16
17
18
19
20
21
22
23
4.1
5.5
0.25
0.20
0.20
10
8.4a,b
7.0 6.7
4.1
5.5
0.15
40 mM
Arg
0.10
0.10
9.5
10
0.05
8.4a,b
7.0 6.7 5.5
4.1
0.05
10
0.00
0
2
0.00
4
6
8
10
12
14
16
18
20
Minutes
22
24
26
28
30
32
24
25
Fig. 5: Separations of peptide pI 8.40a marker by cIEF using different focusing times.
Experimental conditions were as described in Fig. 4, except that pI 8.40a was the only
peptide present in the sample and focusing was performed at 21 kV.
0.06
4
2
2
0
0
2
0
4
Minutes
6
8
(e) 35 °C
0
2
4
6
8
10
12
14
16
Minutes
18
20
22
24
26
28
30
32
Fig. 10: Separation of mouse IgG1κ by cIEF at different separation temperatures: (a)
15, (b) 20, (c) 25, (d) 30 and (e) 35 °C. IgG peaks are labeled A - F. Separation
conditions are as described in Fig. 8 with 3 M urea in the cIEF sample. IgG1κ peaks
are labeled A-F.
Results of an Intermediate Precision Study
6
4
(d) 30 °C
0.04
0.00
8
6
(c) 25 °C
0.02
10
8
(b) 20 °C
0.08
(b) With urea
(a) No urea
A
E
F
D
0.10
12
12
An intermediate precision study was carried out on the separation of
mouse IgG1κ by cIEF. Experimental conditions were as described in
Fig. 10 at a separation temperature of 20°C. This study was
performed by 4 operators using 5 PA 800 systems, 4 lots of neutral
capillary and 2 lots of Pharmalyte 5-8 carrier ampholytes over a
period of 8 days. Fig. 11 shows peak integration. The results of the
study are summarized in Table 3.
2
0
4
Minutes
6
8
IgG b
0.07
Fig. 9: Electrical current profiles from cIEF separations (a) with and (b) without
precipitates and aggregates inside the capillary. Separations were carried out as
described in Fig. 8 using chemical mobilization, but with (a) 5 min and (b) 6 min of
focusing.
The urea concentration needs to be optimized for each sample
analyzed by cIEF. For example, the use of urea has been found to
be detrimental to the cIEF analysis of metalloproteins, such as
transferrin and hemoglobin (poster entitled “The Effects of Urea
Concentration on cIEF Analysis of IgG1κ and other Proteins,” Scott
Mack et al., Beckman Coulter, presented at CE Pharm 2007).
Other proteins, such as erythropoietin (EPO), require 6 M urea in
order to minimize their aggregation and precipitation (poster
entitled “Innovating cIEF at the Extreme,” Scott Mack et al.,
Beckman Coulter, presented at CE Pharm 2008).
Wide- vs. Narrow-Range Carrier Ampholytes
50 mM
Arg
34
36
38
40
42
Fig. 7: Separations of peptide markers by cIEF using different Arg concentrations
within the sample. Experimental conditions were as described in Fig. 4, except
chemical mobilization was for 30 min.
0.07
6.38
0.06
0.06
IgG c
0.05
0.05
[Arginine] (mM)
Resolution
Linearity
20
1.02
0.9639
30
1.01
0.9881
40
0.96
0.9893
50
0.73
0.9892
Proteins can aggregate and precipitate as they are focused near
and at their pI values. Protein precipitation and aggregation can be
minimized by adding a protein solubilizer, such as urea, to the cIEF
sample (Fig. 8). Reproducible current profiles can be obtained by
using urea in the cIEF sample and by rinsing with 4.3 M urea
solution between runs (Fig. 9).
IgG
(a) Narrow-Range
7.0
0.07
0.06
5.5
6.7
(b) Wide-Range
6.7
7.0
15
16
17
18
19
20
21
Minutes
pI 5.5
6.47
pI 6.7
0.03
IgG d
0.02
0.02
6.23
0.01
0.01
0.00
0.00
15
16
17
18
19
20
21
22
23
24
25
26
27
28
Minutes
Fig. 11: Peak integration of mouse IgG1κ separated by cIEF.
n = 48
Peaks
IgG a
IgG b
IgG c
IgG d
Average
6.47
6.38
6.31
6.23
Std Dev
0.01
0.01
0.01
0.01
CV
0.13%
0.13%
0.14%
0.17%
22
23
Average
16.29%
31.80%
29.52%
22.39%
Std Dev
0.57%
1.03%
0.82%
1.21%
CV
3.49%
3.25%
2.76%
5.42%
Conclusions
5.5
0.00
14
0.03
Table 3: Results of an intermediate precision study of the separation of
mouse IgG1κ by cIEF.
0.01
13
pI 7.0
Group
IgG a
IgG b
IgG c
IgG d
IgG
0.04
0.02
0.04
IgG a
Isoform Group Percent Composition
0.05
0.03
6.31
0.04
Calculated pI
The choice of carrier ampholytes can affect resolution in cIEF.
Resolution can be increased by the use of narrow-range
ampholytes, after optimization of other cIEF variables. (Fig. 10).
0.08
Protein Solubilizer
Merged
Peak
Cathodic
Peak
7.0 6.7
B
5.5
C
0.35
Table 2: The effect of Arg concentration on cIEF resolution of the pI 8.4a and 8.4b
peptide markers and in the linearity of the pH 4-10 gradient. The optimum
concentration was found to be 40 mM Arg.
Merged
Peak
Cathodic
Peak
5.5
9.5
8.4a,b
0.12
34
0.30
9.5
Fig. 4: Twelve consecutive separations of two peptide markers (pI 7.0 and pI 6.7) by cIEF
at (a) high and (b) low concentrations of anolyte, catholyte and chemical mobilization
solution. Sample: 200 µL of cIEF gel, 4 µL Fluka 3-10 carrier ampholytes with 6 %
TEMED, and 2.0 µL of each peptide pI marker. Focusing was 6 min at 25 kV. Chemical
mobilization was 24 min at 30 kV. Concentrations: (a) High: 200 mM phosphoric acid
(anolyte), 300 mM sodium hydroxide (catholyte) and 350 mM acetic acid (chemical
mobilization solution); and (b) Low: 91 mM phosphoric acid (anolyte), 20 mM sodium
hydroxide (catholyte) and 20 mM acetic acid (chemical mobilization solution).
7.0 6.7
20 mM
Arg
0.25
0.10
28
4.1
0.35
0.30
0.11
26
0.40
10
(a) High Concentrations
0.15
The detection aperture has an effect on the measurement of peak
width. Peak efficiency and resolution increase with decreasing peak
width (Fig. 2).
24
9.5
0.40
* All trademarks are property of their respective owners.
Aperture in Capillary Cartridge
22
The amount of cathodic stabilizer within the sample needs to be
optimized to ensure the sample is focused prior to reaching the
detection window (Fig. 7). In addition, the amount of cathodic
stabilizer has been found to affect resolution and linearity (Table 2).
22.0
Fig. 1: Separation of hemoglobins A and F by cIEF using (a) pressure and (b)
chemical mobilization. Sample: 200 µL of cIEF gel (Beckman Coulter, P/N 477497),
2.0 µL of Hemoglobin AF (Beckman Coulter, P/N 667630), 6.0 µL of Pharmalyte* 5-8
carrier ampholytes (GE Healthcare, P/N 17-0453-01), 9.0 µL of 0.5 M arginine (Arg)
and 4.0 µL of 0.2 M iminodiacetic acid (IDA). Anolyte: 200 mM phosphoric acid.
Catholyte: 300 mM sodium hydroxide. Focusing: 7.5 min at 25 kV. Pressure
mobilization: 0.7 psi at 30 kV for 20 min. Chemical mobilization: 25 min at 30 kV using
350 mM acetic acid at cathodic side.
20
16
Fig. 8: Separations of mouse IgG1κ and three peptide pI markers (7.0, 6.7 and 5.5) by
cIEF: (a) with and (b) without 3 M urea in the sample. Sample: 200 µL of cIEF gel with
and without 3 M urea, 6.0 µL of Pharmalyte 5-8 carrier ampholytes, 2.0 µL of each pI
marker, 9.0 µL of 0.5 M Arg, 5.0 µL of 0.2 M IDA, and 10.0 µL of desalted IgG.
Focusing was 5 min at 25 kV. Chemical mobilization was 30 min at 30 kV. Anolyte: 200
mM phosphoric acid. Catholyte: 300 mM sodium hydroxide. Chemical mobilization
solution: 350 mM acetic acid. Black arrow indicates the boundary between carrier
ampholytes and anodic stabilizer.
The loss and distortion of the pH gradient due to ITP can be
prevented by using stabilizers, which are chemical compounds with
pI values at the extremes of the pH gradient. These compounds act
as buffering zones protecting the pH gradient from ITP distortions.
Stabilizers permit the use of longer focusing times thus preventing
the loss of resolution. Arg (pI 10.7) has been used successfully as
cathodic stabilizer, and IDA (pI 2.2) used as anodic stabilizer.
AU
15.0
18
Minutes
15
AU
AU
22
0.00
14.5
16
14
µA
21
The anolyte, catholyte and chemical mobilization solutions must be
at a high enough concentration to ensure their conductivity values
are stable in repeated use. High conductivity reduces variations in
detection time (Fig. 4), and can reduce isotachophoresis (ITP)
distortions in the pH gradient.
0.03
Hb Fac
0.02
0.01
20
Concentration of cIEF reagents
0.07
4
13
M inutes
pH Gradient Stabilizers
Fig. 3: Separation of synthetic peptide pI markers by cIEF. Sample: 200 µL of 3 M ureacIEF Gel, 2.0 µL of each peptide (1.25 mM), 12.0 µL of Pharmalyte 3-10 carrier
ampholytes, 20 µL of 0.5 M Arg and 2.0 µL of 0.2 M IDA. Focusing was 15 min at 25
kV. Chemical mobilization was 20 min at 30 kV using 350 mM acetic acid. Anolyte and
catholyte were as described in Fig. 1.
0.10
2
12
(a) 15 °C
0.14
25 kV. Chemical mobilization was at 30 kV for 30 min.
19
0.09
0.08
3.4
4.1
(b) No urea
-0 .0 0 5
11
x
0
0.00
0.11
(a) Pressure Mobilization
5.5
15 min
0.00
0.01
AU
0.09
10.0 9.5
4.1
5.5
0.02
0.12
Hb A1c
Hb Fac
0.11
0 .0 0 0
µA
Hb F
0.12
0 .0 0 5
10 min
0.02
7.0 6.7
0.16
Fig. 6: Separations of peptide markers by cIEF using different focusing times. Sample:
200 µL of cIEF gel, 12 µL Pharmalyte 3-10 carrier ampholytes, 18 µL of 0.5 M Arg,
4 µL of 0.2 M IDA, and 2 µL of each peptide pI marker at 1.25 mM. Focusing was at
0.13
Hb A
0.18
AU
AU
In cIEF, detection is achieved by mobilizing the pH gradient across
the capillary window using a variety of strategies including chemical
and pressure mobilization. Chemical mobilization offers higher
peak efficiency and resolution than pressure mobilization due to the
absence of laminar flow (Fig. 1).
0.03
(a) 3 M urea
0 .0 1 5
3.4
0.04
0.06
Mobilization Technique
0 .0 3 0
10.0
x
0.08
4.1
x
6.7
7.0
5.5
6.7
0 .0 1 0
0.07
0.04
0.13
9.5
5 min
0.12
5.5
0.06
10.0
Separation temperature can have significant effect on the cIEF
resolution of IgG1κ (Fig. 10). The IgG peaks D, E, and F were not
as well resolved when the temperature was > 25°C. However, IgG
peak A was better defined at temperatures above 15°C. Overall
detection time decreased with increasing temperature due to
compression of the pH gradient resulting in the loss of resolution.
0.14
9.5
8.4b
8.4a
7.0
0 .0 3 5
0 .0 2 0
0.09
0.05
0 .0 4 0
0 .0 2 5
0.10
0.08
Separation Temperature
IgG1κ
0 .0 4 5
0.16
Table 1: Amino acid sequence and pI values of peptides used as pI markers.
All cIEF separations were carried out using ProteomeLab™ PA 800
Protein Characterization Systems (Beckman Coulter, Fullerton, CA)
each equipped with a UV detector and 280 nm filter. Separations
were performed using Neutral Capillaries (Beckman Coulter, P/N
477441, 50 µm i.d., 375 µm o.d., 30.2/20.0 cm long) and carried
out at 20°C, unless otherwise indicated.
0 .0 5 0
AU
Experimental Set-Up
Variables in cIEF
Amino Acid Sequence
H-Trp-Tyr-Lys-Lys-OH
H-Trp-Tyr-Tyr-Lys-Lys-OH
H-Trp-Glu-Tyr-Tyr-Lys-Lys-OH
H-Trp-Tyr-Lys-OH
H-Trp-Glu-His-Arg-OH
H-Trp-Glu-His-His-OH
H-Trp-Glu-His-OH
H-Trp-Asp-Asp-Arg-OH
pI Marker
9.99
9.50
8.40 a
8.40 b
7.00
6.66
5.52
4.05
There are multiple applications for cIEF in the biotechnology
industry. These include as determination of protein charge
heterogeneity, stability of formulations, determination of lot
consistency, and purity assessment. This poster discusses critical
variables to consider for cIEF separation and describes optimization
of these variables for maximum resolution and reproducibility.
Some regions of the pH gradient focus faster than others. When
using Pharmalyte 3-10 carrier ampholytes, the acidic region is
completely focused after 5 minutes, whereas the basic region
requires 10 minutes of focusing. Extension of the focusing time to 15
minutes results in the loss of the pI 3.4 marker to ITP (Fig. 6).
AU
Introduction
24
25
26
27
28
29
Separations of proteins by cIEF can be achieved with high
resolution, reproducibility and robustness by optimizing key
variables. These variables include: focusing time, concentration of
pH gradient stabilizers, amount of protein solubilizer, separation
temperature, and the type of the carrier ampholytes used.
Fig. 10: Separation of mouse IgG1κ and three pI markers (7.0, 6.7 and 5.5) by cIEF
using (a) narrow-range ampholytes, Pharmalyte 5-8 carrier ampholytes; and (b) widerange ampholytes, Pharmalyte 3-10 carrier ampholytes. Experimental conditions are Understanding the effect of each variable on the cIEF separation
(a): as described in Fig. 8 with 3 M urea in the sample and (b): Sample: 200 μL of 3 M
critical when developing and troubleshooting methods.
urea-cIEF gel, 12 μL of Pharmalyte 3-10 carrier ampholytes, 1.0 μL of each pI marker,
7.5 μL of 0.5 M Arg, 2.0 μL of 0.2 M IDA, and 10.0 μL of desalted IgG. Focusing was
Note: For Research Use Only; not for use in diagnostic procedures.
10 min at 25 kV. Chemical mobilization was 25 min at 30 kV.
is