IgG Purity/Heterogeneity and SDS-MW Assays with High

IgG Purity/Heterogeneity and SDS-MW Assays with High-Speed Separation Method and High Throughput Tray Setup
Jose-Luis Gallegos-Perez1, Keith Roby2, Ed Horton2
SCIEX Separations, a part of AB SCIEX
ABSTRACT
The PA800 plus Pharmaceutical System provides a
comprehensive, automated, and quantitative solution for
the characterization and analysis of proteins.
The application menu for the PA 800 plus includes, among
others, the IgG Purity/Heterogeneity (IgG PH) assay and
the SDS-gel molecular weight (SDS-MW) analysis.1,2 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. In this poster is
presented the methodology to carry out both assays using a high speed (HS) method that
compares to the conventional high resolution (HR) one giving similar results and saving method
separation time. This poster also shows a high throughput buffer tray (HTT) setup to maximize the
number of samples per run without causing deterioration in the separation.3
INTRODUCTION
Capillary electrophoresis (CE) has become an effective replacement for manual slab gel
electrophoresis processes due to its automation, quantitation, fast speed, and high efficiency.
Biomolecules, such as proteins, carbohydrates, and nucleic acids can be separated by molecular
sieving electrophoresis using gel matrices, a technique referred to as capillary gel
electrophoresis.1
The PA800 plus Pharmaceutical System allows to separate reduced and non-reduced
immunoglobulins by size and subsequently quantify the heterogeneity and impurities that may be
present in IgG preparations. This system can also be used for the separation of protein-SDS
complexes using a replaceable gel matrix. The gel is formulated to provide an effective sieving
range of approximately 10 kDa to 225 kDa and unknown protein molecular weights can be
estimated from a standard calibration curve of known protein sizes.2
Reagents: Reagents contained in the IgG purity (Beckman Coulter, cat. A 10663) and SDS-MW
Analysis (Beckman Coulter, cat. 390953) kits were used as indicated in the corresponding application
guides.1,2 Iodoacetamide (IAA), 2-mercaptoehanol. Bovine Serum Albumin (BSA), and Myoglobin
(Myo) 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.
Capillary: A Bare Fused Silica capillary was used; 50 mm i.d. x 360 mm 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.
Sample Preparation: Samples were prepared as is indicated in correspondent Applications Guides
(see references 1 and 2)
HS and HR method with conventional tray setups: Trays were set up according to the instructions
in the Application Guides (see references 1 and 2)
HTT configuration:
•HR method: Figure 1 shows the position of the buffer vials in each inlet and outlet buffer tray using the
HTT setup. In this configuration is possible to run up to 48 individual samples.
•HS method: If less than 40 individual samples are being analyzed, buffer trays must be set up as shown in
Figure 1 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.
•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 2). All columns in
the sample tray (from A to F) can be loaded with samples.
6
H2O
6
Gel-R
Gel-S
NaOH
HCl
H2O
H2O
Gel-R
Gel-S
NaOH
HCl
H2O
H2O
5
H2O
H2O
Gel-R
Gel-S
NaOH
HCl
H2O
H2O
Gel-R
Gel-S
NaOH
HCl
H2O
H2O
Both separation methods (HR and HS) are used with a tray configuration that allows running up to
24 samples before having to change reagents. However, a high throughput buffer tray (HTT) setup
can be used to maximize the number of samples per run without causing deterioration in the
separation.1,2,3
Gel-S
NaOH
HCl
H2O
Gel-S
Waste Waste Waste
Waste
Gel-S
Waste Waste Waste
Waste
H2O
1
A
Waste
Gel-S
Waste Waste Waste
B
Method
Gel-S
NaOH
HCl
C
D
E
INLET BUFFER TRAY
Tray on
the left
H2O
Waste
F
H2O
A
Gel-S
7
6
6
5
5
4
4
Waste Waste Waste
3
Sample
A
Gel-R
B
NaOH
C
2Gel-S
D
HCl
E
1
F
H2O
Sample
Gel-S
A
Waste Waste Waste
C
D
E
OUTLET BUFFER TRAY
B
F
C
D
6
H2O
Waste
Waste
A
5
H2O
Tray on
the right
E
HGel-S
2O
Waste
Waste
Gel-S
Waste
Waste
Waste
Waste
H2O
Waste
H2O
Waste
F
A
* Use only rows 1-5 (see text)
Figure 1: Scheme of the inlet and outlet buffer vial tray
set-up for the HTT configuration using the HR or the HS
method (HS method FOR LESS than 40 individual
samples, see text). Gel-S = Gel used for separation, GelR = Gel used for rinsing, both are of the same gel type.
A
H2O
A
B
E
F
H2O
2
5
H2O
D
Waste
Waste
Gel-S
Waste
Waste
H2O
Gel-S
Waste
Waste
H2O
Waste
C
Gel-S
Waste
D
Waste
H2O
Gel-R
Waste
Gel-S
Waste
Waste
E
F
Gel-R
NaOH
Gel-S
HCl
H2O
Gel-R
NaOH
Gel-S
HCl
H2O
Gel-R
NaOH
Gel-S
HCl
H2O
NaOH
Gel-S
HCl
H2O
NaOH
Gel-S
HCl
H2O
4
3
2Waste
B1
D
NaOH
HO
Gel-R Gel-S
NaOH
Gel-S HCl
HCl
C
3
H2O
C
6
Gel-R
Waste
Waste
B
B
4
H2O
HR
HS*
7
1
Waste
B
8
2
1
Gel-R
8
3
2
Gel-R
H2O
Waste Waste Waste
3
2
Two types of analysis methods have been optimized:
Gel-S
4
3
H2O
Sample Tray
Waste
5
4
H2O
•The HR method uses the capillary cartridge in the left to right configuration with a sample
introduction inlet to detection window distance of 20.0 cm and a separation time of about 30 min.
•The HS method provides faster separation with some reduction in resolution but only 15 min for
separation. It method uses the capillary cartridge in the right to left configuration, with an inlet to
detection window distance of 10 cm.
C
Waste
D
E
Gel-S
F
Outlet Buffer Tray
Waste
2
E
1
H2O
A
Waste
F
Gel-R
B
C
D
E
H2O
F
Inlet Buffer Tray
Figure 2. 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.
Results and Discussion
HR and HS methods using the HTT setup
Experimental
Instrument: Separations were performed using a Beckman Coulter PA 800 plus Pharmaceutical
Analysis System sold through SCIEX Separations. PDA detector settings were set according to
the application guides. Instrument was controlled using 32 Karat software v.10. The sample and
capillary storage temperatures were set at 15ºC and 25ºC respectively
Figure 3: Repeatability of HR and
HS Methods using Conventional-tray
and HTT setups. Each graph
contains the stacking of eight
separation electropherograms per
tray setup with the a) HR method or
b) HS method. Each
electropherogram is offset by 0.025
AU for clarity. 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.
To demonstrate assay repeatability using the HTT setup, the SDS-MW Size Standard was used as
a sample. 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. In Figure 3, the stacked
electropherograms show 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 no ghost peaks,
artifacts or any other spurious signal are observed with the HTT configuration.
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, using the HTT setup, were evaluated and
compared. Eight replicates of the IgG control standard under non-reducing and reducing
conditions were injected into the system. In Figures 4 and 5 a qualitative profile comparison of the
HS and HR methods is shown. Under non-reducing conditions (see Figure 4) the observed peaks
in the electropherograms correspond to the 10 kDa internal standard, the intact antibody (IgG) and
the non-glycosylated heavy chain (NG). In addition, impurities such as heavy-heavy (HH) and 2
heavy 1 light chain (2H1L) are resolved from the whole antibody with both methods. Under
reducing conditions, the observed peaks (see Figure 5) correspond to the 10 kDa internal
standard (IS), the light chain (LC), the non-glycosylated heavy chain (NG) and the heavy chain
(HC). As in the previous case, all peaks can be easily identified with either method.
Figure 4: 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 nonglycosylated heavy chain, HH and 2H1L are the heavyheavy (HH) and 2 heavy 1 light chain impurities.
Molecular Weight Estimation of Myoglobin (Myo) and Bovine Serum Albumin (BSA)
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. Figure 6 shows
the stacked electropherograms for the MW-standard and sample mixture runs. In all cases,
analyte peaks were well resolved with either the high speed or the high resolution method.
Molecular weights for Myo and BSA were estimated using the qualitative analysis of the 32 Karat
Control and Analysis Software. Results showed a very 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.
CONCLUSIONS
•The IgG Purity/Heterogeneity and SDS-MW assays can be carried out using a high speed (HS) method using
the capillary cartridge in the right to left configuration. This method compares to the conventional High
Resolution (HR) method giving similar results and saving method separation time.
•The use of a high throughput tray (HTT) setup to maximize the number of samples per run does not cause
deterioration in the separation.
•The combination of the HS method with the HTT setup is an alternative for a more efficient use of the PA800
plus system running continuously on a daily basis.
Figure 5: 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 Nonglycosylated heavy chain and HC the Heavy
Chain.
Figure 6: Determination of Protein Molecular
Weight using HR and HS methods. Peak
identification: 10 kDa is the Internal Standard,
Myo es 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.
REFERENCES
1. PA800 plus Pharmaceutical Analysis System-IgG Purity/Heterogeneity Assay Application Guide. A51967AD, Jan 2014.
2. PA800 plus Pharmaceutical Analysis System-SDS-MW-Analysis Assay Application Guide. A51970AD, Jan 2014
3. Milbrandt C. Increasing Sample Throughput in Process Analytical Support (PAS) on the Beckman Coulter PA800. GDO,
May 2011.
TRADEMARKS/LICENSING
For Research Use Only. Not for use in diagnostic procedures.
© 2014 AB SCIEX. SCIEX is part of AB SCIEX. The trademarks mentioned herein are the property of AB Sciex
Pte. Ltd. or their respective owners. AB SCIEX™ is being used under license.

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