IgG Purity/Heterogeneity and SDS-MW Assays with High

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IgG Purity/Heterogeneity and SDS-MW Assays with HighSpeed Separation Method and High Throughput Tray Setup
High Throughput Methods to Maximize the Use of the PA 800 Plus system
Jose-Luis Gallegos-Perez
SCIEX Separations, Framingham, MA
Introduction
The PA 800 Plus Pharmaceutical System provides a
comprehensive, automated, and quantitative solution for the
characterization and analysis of therapeutic proteins. The
application menu for the PA 800 Plus includes, among others,
the IgG Purity/Heterogeneity assay and the SDS-gel molecular
1,2
weight (SDS-MW) analysis. The first method allows for the
resolution of reduced and non-reduced immunoglobulins by size
and to subsequently quantify the heterogeneity and impurities
that may be present in IgG preparations. The SDS-MW analysis
is designed to provide an estimation of the molecular weight of a
protein in a sample.
There are two types of analysis methods that can be used with
these assays:
1)
The high resolution (HR) method with an inlet to
detection window capillary length of 20.0 cm, using the
capillary cartridge in a left-to-right configuration.
2)
The high-speed (HS) method that uses the capillary
cartridge in the right-to-left configuration with a sample
introduction inlet to detection window capillary length of
10.0 cm.
Both methods are used with a tray configuration that allows
running up to 24 samples before having to change reagents.
However, other tray setups are possible to optimize and
maximize the number of sample runs.
This report illustrates the use of the HS method and proposes a
High Throughput Tray (HTT) setup to maximize the number of
samples per run. We demonstrate that HS and HR analysis
methods can be used with this optimized tray array.
Advantages of the HS Analysis Method with
the High Throughput Tray Setup

The HS method provides faster separation time (15-20
min vs. 30 min of the HR method); it represents 72
samples/day instead of 48/day.
PA 800 Plus system

The use of an HTT setup allows for analysis of up to 48
samples (24 using the conventional tray array) before
changing chemical reagents. It represents savings in
consumables and time.

Using the HS method and high throughput tray setup
saves time and reagents and helps maximize the use of
the PA 800 Plus.
Experimental Procedure
Reagents: Separations using the IgG Purity Heterogeneity
Assay Kit and SDS-MW Kit were performed as described in their
1,2
respective
manuals.
Iodoacetamide
(IAA)
and
2mercaptoethanol were purchased from Sigma-Aldrich and used
without any further purification. Ultrapure water (type 1) was
obtained from a Milli-Q Direct Ultrapure Water System. Bovine
Serum Albumin (BSA) and Myoglobin (Myo) were purchased
from Sigma-Aldrich.
Capillary: A bare fused-silica capillary was used for separation
of monoclonal antibody samples; 50 m i.d. x 360 m o.d. x 30.2
cm total length. Inlet-to-detection window equal to 20 cm for the
HR method and 10 cm for the HS method.
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Sample Preparation:

IgG control standard: A vial containing 95 L of the IgG
control standard was combined with 2 L of the 10 kDa
internal standard and 5 L of 2-mercaptoethanol in a
fume hood. The vial was mixed thoroughly and
centrifuged at 300g for 1 min. The vial was sealed with
Parafilm and heated to 70°C for 10 minutes, after which
the vial was cooled for 3 minutes in a room-temperature
water bath.

Non-reduced IgG sample preparation: A vial containing
95 L of the IgG control was combined with 2 L of the
10 kDa internal standard and 5 L of a 250 mM IAA
solution. The vial was mixed thoroughly and centrifuged
at 300g for 1 minute. The vial was sealed with Parafilm
and heated to 70°C for 10 minutes, after which the vial
was cooled for 3 minutes in a room-temperature water
bath.

SDS-MW Size Standard: 10 L of molecular weight size
standard, 85 L of sample buffer, 2 L of internal
standard, and 5 L of 2-mercaptoethanol were
combined in a vial. The mixture was heated to 100°C
for 3 minutes and placed into a room-temperature water
bath to cool down for five minutes before injection.

BSA and Myo were diluted with the SDS-MW sample
buffer to a final concentration of 0.5 mg/mL each. For
non-reduced protein preparation, a vial containing 95
L of sample solution was combined with 2 L of 10
kDa internal standard and 5 L of 250 mM IAA solution.
The vial was mixed thoroughly and centrifuged at 300g
for 1 minute. The vial was sealed with Parafilm and
heated to 70°C for 3 min. For the reduced protein
sample, 95 L of the sample was combined with 2 L of
the 10 kDa internal standard, and 5 L of 2mercaptoethanol. The mixture was heated to 100°C for
3 min.
HS method with conventional tray setups
Because the HS method uses the capillary cartridge in the rightto-left configuration, the buffer tray on the right must be used as
the inlet tray while the buffer tray on the left must be used as the
outlet tray (see Figure 1). The polarity for injection and
separation should be normal. Using the conventional tray
configuration, a maximum of 24 individual samples can be
analyzed without reloading the trays with fresh solutions.
HTT configuration:

HR method: Figure 2 shows the position of the buffer
vials in each inlet and outlet buffer tray using the HTT
setup. In this configuration the buffer vials in each row
are enough for 8 consecutive experiments, therefore it
is possible to run up to 48 individual samples. (8 runs
per row x 6 rows). Method parameters are shown in
Figure 4.
Outlet
Inlet
Figure 1: Image of the inlet and outlet buffer vial tray set-up
for the HS method in the PA800 Plus.
6
H2O
6
Gel-R
Gel-S
NaOH
HCl
H2O
H2O
5
H2O
Gel-R
Gel-S
NaOH
HCl
H2O
H2O
4
H2O
Gel-R
Gel-S
NaOH
HCl
H2O
H2O
Gel-R
Gel-S
NaOH
HCl
H2O
H2O
Gel-R
H2O
Waste Waste
Waste
Gel-S
Waste
Waste Waste
Waste
Gel-S
Waste
Waste Waste
Waste
Gel-S
Waste
Waste Waste
Waste
Gel-S
Waste
Waste Waste
Waste
Gel-S
Waste
Waste Waste
2
Gel-S
NaOH
HCl
H2O
H2O
1
A
Waste
3
2
H2O
Gel-S
4
3
H2O
Waste
5
1
Gel-R
B
Method
Gel-S
NaOH
HCl
C
D
E
INLET BUFFER TRAY
Tray on
the left
H2O
F
H2O
A
B
C
D
E
OUTLET BUFFER TRAY
F
Tray on
the right
HR
HS*
* Use only rows 1-5 (see text)
Figure 2: Scheme of the inlet and outlet buffer vial tray setup for the HTT configuration using the HR or the HS
method. Ensure the position of the buffer trays in the
instrument according to the method being used. (HS
method for no more than 40 individual samples, see text).
Gel-S = Gel used for separation, Gel-R = Gel used for
rinsing, both are of the same gel type.
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

HS method: If less than 40 individual samples are
being analyzed, buffer trays must be set up as shown in
Figure 2 using rows from 1 to 5 only. IMPORTANT: In
this case, the F-column in the SAMPLE TRAY must be
kept empty, with no samples to avoid a tray collision
during analysis. Follow the method parameters shown
in Figure 5.
Table 1 indicates the volume of reagents used in each buffer vial
and their number for all 48 runs using the HTT setup.
HS method and HTT setup to maximize the number
of sample analyses: To use the HTT configuration with
the HS method for analysis of up to 48 samples
requires switching reagents in positions C and D in the
outlet and inlet buffer trays to avoid tray collision when
samples are injected (see Figure 3). All columns in the
sample tray (from A to F) can be loaded with samples.
For this specific case, use the method parameters show
in Figure 6.
8
7
7
6
6
5
5
4
4
3
2
1
3
Sample
A
Gel-R
B
NaOH
C
2Gel-S
D
HCl
E
1
F
Vol (mL)
Number of vials
(48 runs)
Water
1.5
24
Gel
1.5
6
Water
1.5
18
Waste (water)
0.8
24
NaOH
1.5
6
HCl
1.5
6
Reagents

Sample Tray
8
Table 1. Volume in Buffer Vials.
H2O
Instrument: Separations were performed using a PA
800 Plus Pharmaceutical Analysis System from SCIEX.
PDA detector settings were configured according to the
application guides. The instrument was controlled using
32 Karat software v10. The sample and capillary storage
temperatures were set at 15ºC and 25ºC respectively.
CE Instrument setups for the HR and HS methods with
the HTT configuration are shown in Figures 4, 5 and 6.
Sample
A
B
C
D
6
H2O
Waste Waste
A
5
H2O
E
HGel-S
2O
Waste Waste
F
A
Gel-S
Waste Waste
A
H2O
A
F
C
5
H2O
D
E
H2O
F
Gel-R
NaOH
Gel-S
HCl
H2O
Gel-R
NaOH
Gel-S
HCl
H2O
Gel-R
NaOH
Gel-S
HCl
H2O
E H O 1 Gel-R FNaOH
Gel-S
HCl
H2O
Gel-S
HCl
H2O
4
Waste Waste
Gel-S
Waste Waste
H2O
Waste Waste
Gel-S
Waste Waste
H2O
3
2Waste
B1 Waste
Waste
Gel-S
H2O
Waste
C
Gel-S
Waste Waste
H2O
Waste Waste
Gel-S
Waste Waste
B
E
2
3
H2O
D
NaOH
H O Gel-R Gel-S
NaOH Gel-S HCl
HCl
4
H2O
C
6
Gel-R
Waste Waste
B
B
C
D
D
E
F
Outlet Buffer Tray
Waste
Waste
2
2
H2O
A
Gel-R
B
NaOH
C
D
E
H2O
F
Inlet Buffer Tray
Figure 3: Scheme of the inlet and outlet buffer vial tray
setup for the HTT configuration using the HS method to
inject up to 48 individual samples.
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Figure 4: High Resolution (HR) Method Parameters with HTT configuration.
p4
Figure 5: High-Speed (HS) Method with HTT configuration for up to 40 individual samples
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Figure 6: High-Speed Method with HTT configuration for up to 48 individual samples
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Results and Discussion
HR and HS methods using the HTT setup
To demonstrate assay repeatability using the HTT setup, the
SDS-MW Size Standard was used. For the HR and HS methods,
8 sample replicates were injected using the conventional (C)-tray
array and 8 more samples for the HTT configuration. Figure 7
shows profiles obtained in the experiments with a) HR method
and b) HS method. It is evident that both tray setups produce the
same analyte profile and that no ghost peaks, artifacts, or any
other spurious signal is present in the HTT configuration. This
result demonstrates that the HTT setup can be implemented with
high-sensitivity or high-resolution methods, therefore the IgGpurity/heterogeneity or the SDS-MW applications could be run
under these experimental conditions.
Figure 7: Repeatability of HR and HS Methods using
Conventional-tray and HTT setups. Stacked data illustrating 8
separations per tray setup using the a) HR method or b) HS
method. To facilitate easier analysis, electropherograms were
offset by 0.025 AU. Samples were alternately injected using the
conventional (C) or the HTT setup. The order in which the
samples were injected is indicated by the number.
Application of the HR and HS Methods with the IgG/Purity
Heterogeneity Assay
Using the HTT configuration, separation reproducibility of the HR
and HS methods was evaluated. Eight replicates of the IgG
control standard were injected under non-reducing and reducing
conditions. Under non-reducing conditions (Figure 8) the peaks
observed correspond to the 10 kDa internal standard, the intact
antibody (IgG) and the non-glycosylated heavy chain (NG). In
addition, impurities that are known to be present in the IgG
1
control standard , such as heavy-heavy (HH) and 2 heavy 1 light
chain (2H1L) are resolved from the whole antibody using either
method. Under reducing conditions (Figure 9), the peaks
observed correspond to the 10 kDa internal standard (IS), the
IgG light chain (LC), IgG non-glycosylated heavy chain (NG) and
the IgG heavy chain (HC). As in the previous case, all peaks can
be easily identified using either method.
Figure 8: Comparison of HR and HS methods with the HTT
setup for IgG Control Standard under Non-reducing
Conditions. Peak identification: 10 kDa is the Internal Standard,
IgG is the intact antibody, NG the non-glycosylated heavy chain,
HH and 2H1L are the heavy-heavy (HH) and 2 heavy 1 light
chain impurities.
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Table 2. Accuracy and Repeatability of the HS and HR Methods
under Non-Reducing Conditions
Parameter
Migration Time
Method
Injection
HR
Area Percent
HS
HR
HS
NG
IgG
NG
IgG
NG
IgG
NG
IgG
1
26.62
27.10
14.32
14.55
7.91
88.46
7.75
88.27
2
26.66
27.14
14.35
14.58
7.88
88.52
7.71
88.28
3
26.70
27.19
14.38
14.62
7.87
88.51
7.87
88.11
4
26.77
27.25
14.42
14.65
7.89
88.45
7.74
88.25
5
26.83
27.32
14.44
14.68
7.88
88.48
7.73
88.26
6
26.89
27.38
14.48
14.71
7.89
88.53
7.73
88.37
7
26.91
27.40
14.50
14.74
7.79
88.63
7.65
88.35
8
26.60
27.07
14.29
14.53
7.88
88.49
7.77
88.24
Mean
26.75
27.23
14.40
14.29
7.87
88.51
7.74
14.29
SD
0.12
0.13
0.08
0.08
0.04
0.06
0.06
0.08
%CV
0.45
0.46
0.52
0.53
0.46
0.06
0.80
0.55
Table 3. Accuracy and Repeatability of the HS and HR Methods
under Reducing Conditions
Figure 9: Comparison of HR and HS methods with the HTT
setup for IgG Control Standard under Reducing Conditions.
Peak identification: 10 kDa is the Internal Standard, LC is the
Light Chain, NG the Non-glycosylated heavy chain and HC the
Heavy Chain.
Migration times and area percent were determined for the main
identified peaks (NG, intact IgG, LC, and HC) (Tables 2 and 3).
These results indicate that the evaluated parameters show high
reproducibility for 8 separations using either the HR or the HS
method. Reproducibility for migration time was always less 0.6
%CV and lower than 1.7 %CV for the peak area percent. As
expected, resolution was slightly lower in the HS mode due to
the somewhat shorter capillary length; however adjacent peak
pairs with close migration times, such as NG/IgG or NG/HC, can
be resolved and unequivocally identified and quantified. This
demonstrates that the HS method can be used with complete
confidence under standardized and validated conditions.
Molecular Weight Estimation of Myoglobin (Myo) and Bovine
Serum Albumin (BSA)
The SDS-MW analysis kit is designed for the separation and
estimation of molecular weight (MW) of a protein in a range
between 10 and 225 kDa. The use of the HS method, for this
experiment, was compared to the conventional HR method. The
HTT setup was used for all experiments. The molecular weight of
an unknown protein can be estimated by making a comparison
against a standard curve created using proteins of known sizes.
For this reason, a protein sizing standard mixture was injected in
parallel to the Myo and BSA samples. The sizing standard
reagent contained six proteins: 10, 20, 35, 50, 100 and 225 kDa.
Sample mixtures containing the internal standard (IS), Myo and
BSA were treated under reducing or non-reducing conditions and
three replicates of each condition were analyzed using the
PA800 Plus instrument (Figure 10). In all cases, analyte peaks
were well resolved using either the high speed or the high
resolution method. Molecular weights for Myo and BSA were
estimated using the qualitative analysis feature in the 32 Karat
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Table 4. Determination of Molecular Weight for Myoglobin and
Bovine Albumin Serum (in kDa)
Control and Analysis Software. Results listed in Table 4 indicate
high reproducibility (%CV < 0.52, n = 3) for the MW estimation of
Myo or BSA, under non-reducing and reducing conditions.
Similarly, the difference of the MW values determined by either
method (HS or HR) was minimal with an absolute value
difference lower than 0.2 kDa. Therefore, both methods can be
used under optimized experimental conditions producing
comparable results.
Non-reduced
HR method
Reduced
HS method
HR method
HS-Method
Injection
Myo
BSA
Myo
BSA
Myo
BSA
Myo
BSA
1
14.954
64.394
15.014
64.720
14.989
68.715
14.945
68.366
2
14.919
64.394
15.014
64.520
14.989
68.715
14.876
68.829
3
14.919
64.394
14.945
64.299
14.989
68.834
14.808
69.062
Mean
14.931
64.394
14.991
64.513
14.989
68.755
14.876
68.752
SD
0.020
0.000
0.040
0.211
0.000
0.069
0.069
0.354
%CV
0.135
0.000
0.266
0.326
0.000
0.100
0.460
0.515
Conclusions

The IgG Purity/Heterogeneity and SDS-MW assays can be
performed using a high speed (HS) separation method with
an inlet to detection window of 10.0 cm, using the capillary
cartridge in the right to left configuration. In comparison to
the more highly resolving conventional High Resolution (HR)
method, results are achieved faster saving assay time.

The use of a high throughput tray (HTT) setup in order to
maximize the number of samples per run sequence does
not cause deterioration in the separation.

The combination of the HS method with the HTT setup is an
alternative when in need of higher throughput sample
analysis.
References
Figure 10: Determination of Protein Molecular Weight using
HR and HS methods. Peak identification: 10 kDa is the Internal
Standard, Myo is myoglobin and BSA is Bovine Serum Albumin.
Three injections under reduced and non-reduced conditions
were analyzed. A size standard mixture was run before to inject
the samples with the HS or HR-methods.
1. PA 800 Plus Pharmaceutical Analysis System-IgG
Purity/Heterogeneity Assay Application Guide. SCIEX part
number A51967AD, January 2014.
2. PA 800 Plus Pharmaceutical Analysis System-SDS-MWAnalysis Assay Application Guide. SCIEX part number
A51970AD, January 2014
© 2015 AB SCIEX. For Research Use Only. Not for use in diagnostic procedures. AB SCIEX is doing business as SCIEX.
The trademarks mentioned herein are the property of AB SCIEX Pte. Ltd. or their respective owners. AB SCIEX™ is being used under license.
Document number: RUO-MKT-02-2335-A
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