Automated Glycan sequencing from tandem MS automated Glycan Sequencing From Tandem MS
N-glycan analysis on UPLC and CE EUassets.thermofisher.com/TFS-Assets/BPD/posters/fully...N-glycan...
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Thermo Fisher Scientific • 5791 Van Allen Way • Carlsbad, CA 92008 • www.thermofisher.com Nan Liu, Johnie Young and Peter Bell Pharma Analytics Group, BioProduction Division, Thermo Fisher Scientific, 180 Oyster Point Blvd, South San Francisco, California 94080 Fully automated GlycanAssure AutoXpress N-Glycan sample preparation for N-glycan analysis on UPLC and CE RESULTS Figure 3. How it is Done? Figure 2. Automated Hands-free N-glycan Sample Prep Average relative areas of 6 samples of human IgG glycan Excellent inter-sample repeatability for both automated and manual workflow SDS-PAGE and HPLC analysis demonstrated that RNase B deglycosylation is complete at the PNGase F concentration (1x) on the automated platform. Figure 1. Design of the automated glycan prep workflow Figure 6. Comparable N-glycan Relative Area Percent (UHPLC) Values from the Automated and Manual Workflows Human IgG N-glycan profiles from automated and manual workflows are comparable (Overlay of 3 independent sample preps) NIST mAb glycans prepared on the automated platform showed 24 peaks on UHPLC. Figure 8. NIST mAb UHPLC glycan profile from the automated platform Figure 9. Human IgG glycan profile analyzed on CE 3500xL ABSTRACT We have developed a fully automated platform and workflow for N-glycan sample preparation using Thermo Fisher GlycanAssure reagents. The magnetic beads-based, automated procedure provides streamline workflow for glycoprotein denaturation and deglycosylation, APTS labeling of the released glycans and cleaning of the excess free dyes. The hand-on time for 13 samples is as short as ~5 minutes because the operators only need to add their glycoprotein to micro-tubes before they start the automated run. The setup of the instrument is simple and easy as all of the reagents are prefilled in the prefilled cartridges, which are stable at -20C for at least one year. The total sample preparation time on the instrument is 1 hour and 45 min and the APTS labeled glycans are ready to be analyzed on both both Ultra High Performance Liquid Chromatography (UPLC) and Capillary electrophoresis (CE) analytical platforms. The fully automated platform is demonstrated to result in high quality of glycan profiles analyzed on both UPLC and CE. The glycan profiles of NIST mAb, human serum IgG, RNase B and Fetuin are comparable to the GlycanAssure manual kits, as well as comparable to other glycan sample preparation kits in the market. INTRODUCTION The glycans or polysaccharides attached to proteins after protein post-translation modification play critical roles in eukaryotic cell protein functions, such as protein assembly and folding stability, signal transduction, ligand binding, protein interaction, etc. In the therapeutic immunoglobulin, the N-glycosylation on amide nitrogen of asparagine is a critical quality attribute in the pharmacology, affecting immunogenicity, pharmacokinetics and pharmacodynamics. The great challenges for glycan analysis are the glycan sample preparation and analysis platform that can generate high quality data in a high throughput manner. We have developed a fully automated platform and workflow for N-glycan sample preparation using Thermo Fisher GlycanAssure reagents. The glycan profiles of NIST mAb, human serum IgG, RNase B and Fetuin are comparable to the GlycanAssure manual kits, as well as comparable to other glycan sample preparation kits in the market. MATERIALS AND METHODS Reagents and plastics Glycoprotein GlycanAssure AutoXpress kits (Prefilled Cartridges and magnetic beads) Sample preparation methods 1. Add 10 μl of glycoproteins to a 1.5-ml micro-tube. 2. Place the cartridges, tips, elution tubes, and the tube of magnetic beads on the instrument. 3. Start the run on the instrument. The run takes 1 h and 45 min. 4. Mix 15μl eluted glycans with 45μl Acetonitrile, and analyze on UHPLC (Thermo Fisher Vanquish or Waters Acquity). 5. For 3500xL CE analysis, dilute the glycans with HPLC water to 40-fold or 80-fold before loading them onto 96-well plate. CONCLUSIONS • High performance of a fully automated APTS labeling kits for N-glycan analysis are developed and demonstrated. • The glycan profiles of the automated glycan sample prep workflow is consistent with those from the manual workflow. • Automate Express cartridge based hands-free N- glycan sample prep removes analyst error and offers consistent N-glycan results. • The magnetic bead based procedure provides an unbiased streamlined workflow for excess dye removal. • APTS labeled N-glycans from the automated sample perp can be analyzed on both high throughput (3500xL) and UHPLC instruments. • Liquid transfer in cartridges prefilled with the reagents • In-tip mixing and magnetic beads processing Figure 4. Complete and Robust Glycosylation Human IgG N-glycan relative percent areas are comparable among the automated and manual workflows Table 1. Consistent repeatability between the manual and automated workflows Figure 7. Consistency in Results: Between Automation Platforms • High resolution of glycan peaks resolved on CE. • High repeatability among injections. • Relative areas of Glycan species are comparable to those from LC analysis. For Research Use Only, Not for use in diagnostic procedures. © 2017 Thermo Fisher Scientific Inc. All rights reserved. All trademarks are the property of Thermo Fisher Scientific and its subsidiaries unless otherwise specified (27999). No PNGase F PNGase F 1x dilution PNGase F 32x dilution Glyco-Rnase B Deglyco-Rnase B Deglyco-RNase B Glyco-RB Undigested Digested No P PNGaseF dilution fold 1x 2x 4x 8x 16x 32x M12 0.0 20.0 40.0 60.0 80.0 100.0 120.0 1x 2x 4x 8x 16x 32x % Deglycosylation HPLC analysis) Control PNGaseF 4C w10 SDS-PAGE Fully automated denaturation, deglycosylation, dye labeling and cleaning, and glycan elution. Heating (denaturation/deglycosylation and dye labeling) Magnetic beads cleaning APTS labeled glycan elution Minutes 17.00 18.00 19.00 20.00 21.00 22.00 23.00 24.00 25.00 26.00 27.00 28.00 29.00 30.00 31.00 32.00 33.00 34.00 35.00 36.00 37 Minutes 00 18.00 19.00 20.00 21.00 22.00 23.00 24.00 25.00 26.00 27.00 28.00 29.00 30.00 31.00 32.00 33.00 34.00 35.00 36.00 Automated Manual Figure 5. Comparable N-glycan Profiles from Manual and Automated Workflows 0.0 5.0 10.0 15.0 20.0 25.0 G0 G0F G0FB G1 G1F G1F' G1FB G1FB' G2 G2F G2FB G1FS1 A1 A1F A1FB A2F A2FB Relative areas Automated Manual Relative Areas Name Ave CV% G0 0.43 3.41 G0F 17.21 4.32 G0FB 4.01 4.51 G1 1.09 4.32 G1F 19.92 2.45 G1F' 9.67 1.44 G1FB 5.92 0.71 G1FB' 2.09 2.17 G2 0.70 3.73 G2F 16.61 3.26 G2FB 2.01 4.39 G1FS1 1.92 5.17 A1 0.26 5.72 A1F 8.84 6.40 A1FB 2.64 5.78 A2F 1.35 7.79 A2FB 1.44 7.60 Relative Areas Name Average CV% G0 0.5 1.7 G0F 18.9 1.5 G0FB 4.2 1.3 G1 0.6 5.5 G1F 21.3 0.9 G1F' 9.7 1.0 G1FB 5.9 0.6 G1FB' 1.6 2.3 G2 0.3 8.9 G2F 16.4 1.5 G2FB 1.8 1.5 G1FS1 1.9 7.1 A1 0.2 4.0 A1F 8.8 1.5 A1FB 2.0 2.6 A2F 1.2 7.9 A2FB 1.2 8.4 Automated Manual 0.00 5.00 10.00 15.00 20.00 25.00 % Areas Instrument 1 Instrument 2 Human IgG Glycan samples prepared on two instruments demonstrated consistent glycan profiles.. Minutes 19.00 20.00 21.00 22.00 23.00 24.00 25.00 26.00 27.00 28.00 29.00 30.00 31.00 32.00 33.00 34.00 35.00 36.0 Minutes 00 20.00 21.00 22.00 23.00 24.00 25.00 26.00 27.00 28.00 29.00 30.00 31.00 32.00 33.00 34.00 35. Zoom-in view 1 2 3 4 1 2 3 4 5 7 6 8 6 7 9 10 11 10 11 12 13 12 13 15 14 14 15 16 16 17 17 18 18 19 19 20 20 21 22 21 22 23 23 24 24 NIST mAb 0 5 10 15 20 25 A2F A2FB G1FS1 A1 A1F A1FB G0F G1 G0FB G1F G1F' G1FB G2F G2FB % Areas CE LC Human IgG Overlay of 10 injections Consistent % areas between CE and LC Add sample Start run Analyze Glycan 1 2 3 Protein denatura tion Glycan release Glycan labeling Free dye clean up Automate Express Glycan analysis on CE or LC - Add protein samples to a tube - Start the run Automated Glycan Sample Preparation Fully automated and streamline workflow for glycoprotein denaturation and deglycosylation, APTS labeling of the released glycans and cleaning of the excess free dyes.
Transcript of N-glycan analysis on UPLC and CE EUassets.thermofisher.com/TFS-Assets/BPD/posters/fully...N-glycan...
Thermo Fisher Scientific • 5791 Van Allen Way • Carlsbad, CA 92008 • www.thermofisher.com
Nan Liu, Johnie Young and Peter Bell
Pharma Analytics Group, BioProduction Division, Thermo Fisher Scientific, 180 Oyster Point Blvd, South San Francisco, California 94080
Fully automated GlycanAssure AutoXpress N-Glycan sample preparation for
N-glycan analysis on UPLC and CE
RESULTS
Figure 3. How it is Done?
Figure 2. Automated Hands-free N-glycan Sample Prep
Average relative areas of 6 samples of human IgG glycan
Excellent inter-sample repeatability for both automated and manual
workflow
SDS-PAGE and HPLC analysis demonstrated that RNase B
deglycosylation is complete at the PNGase F concentration (1x) on
the automated platform.
Figure 1. Design of the automated glycan prep
workflow
Figure 6. Comparable N-glycan Relative Area Percent
(UHPLC) Values from the Automated and Manual
Workflows
Human IgG N-glycan profiles from automated and manual workflows
are comparable (Overlay of 3 independent sample preps) NIST mAb glycans prepared on the automated platform showed 24
peaks on UHPLC.
Figure 8. NIST mAb UHPLC glycan profile from the
automated platform
Figure 9. Human IgG glycan profile analyzed on CE
3500xL
ABSTRACT
We have developed a fully automated platform and
workflow for N-glycan sample preparation using
Thermo Fisher GlycanAssure reagents. The magnetic
beads-based, automated procedure provides
streamline workflow for glycoprotein denaturation and
deglycosylation, APTS labeling of the released glycans
and cleaning of the excess free dyes. The hand-on
time for 13 samples is as short as ~5 minutes because
the operators only need to add their glycoprotein to
micro-tubes before they start the automated run. The
setup of the instrument is simple and easy as all of the
reagents are prefilled in the prefilled cartridges, which
are stable at -20C for at least one year. The total
sample preparation time on the instrument is 1 hour
and 45 min and the APTS labeled glycans are ready to
be analyzed on both both Ultra High Performance
Liquid Chromatography (UPLC) and Capillary
electrophoresis (CE) analytical platforms. The fully
automated platform is demonstrated to result in high
quality of glycan profiles analyzed on both UPLC and
CE. The glycan profiles of NIST mAb, human serum
IgG, RNase B and Fetuin are comparable to the
GlycanAssure manual kits, as well as comparable to
other glycan sample preparation kits in the market.
INTRODUCTION
The glycans or polysaccharides attached to proteins
after protein post-translation modification play critical
roles in eukaryotic cell protein functions, such as
protein assembly and folding stability, signal
transduction, ligand binding, protein interaction, etc. In
the therapeutic immunoglobulin, the N-glycosylation on
amide nitrogen of asparagine is a critical quality
attribute in the pharmacology, affecting
immunogenicity, pharmacokinetics and
pharmacodynamics. The great challenges for glycan
analysis are the glycan sample preparation and
analysis platform that can generate high quality data in
a high throughput manner. We have developed a fully
automated platform and workflow for N-glycan sample
preparation using Thermo Fisher GlycanAssure
reagents. The glycan profiles of NIST mAb, human
serum IgG, RNase B and Fetuin are comparable to the
GlycanAssure manual kits, as well as comparable to
other glycan sample preparation kits in the market.
MATERIALS AND METHODS
Reagents and plastics
Glycoprotein
GlycanAssure AutoXpress kits (Prefilled Cartridges
and magnetic beads)
Sample preparation methods
1. Add 10 µl of glycoproteins to a 1.5-ml micro-tube.
2. Place the cartridges, tips, elution tubes, and the
tube of magnetic beads on the instrument.
3. Start the run on the instrument. The run takes 1 h
and 45 min.
4. Mix 15µl eluted glycans with 45µl Acetonitrile, and
analyze on UHPLC (Thermo Fisher Vanquish or
Waters Acquity).
5. For 3500xL CE analysis, dilute the glycans with
HPLC water to 40-fold or 80-fold before loading
them onto 96-well plate.
CONCLUSIONS
• High performance of a fully automated APTS
labeling kits for N-glycan analysis are developed
and demonstrated.
• The glycan profiles of the automated glycan
sample prep workflow is consistent with those
from the manual workflow.
• Automate Express cartridge based hands-free N-
glycan sample prep removes analyst error and
offers consistent N-glycan results.
• The magnetic bead based procedure provides an
unbiased streamlined workflow for excess dye
removal.
• APTS labeled N-glycans from the automated
sample perp can be analyzed on both high
throughput (3500xL) and UHPLC instruments.
• Liquid transfer in cartridges prefilled with the reagents
• In-tip mixing and magnetic beads processing
Figure 4. Complete and Robust Glycosylation
Human IgG N-glycan relative percent areas are comparable
among the automated and manual workflows
Table 1. Consistent repeatability between the manual
and automated workflows
Figure 7. Consistency in Results: Between Automation
Platforms
• High resolution of glycan peaks resolved on CE.
• High repeatability among injections.
• Relative areas of Glycan species are comparable to those from LC
analysis.
For Research Use Only, Not for use in diagnostic procedures.
© 2017 Thermo Fisher Scientific Inc. All rights reserved. All trademarks are the
property of Thermo Fisher Scientific and its subsidiaries unless otherwise
specified (27999).
No PNGase F
PNGase F 1x dilution
PNGase F 32x dilution
Glyco-Rnase B
Deglyco-Rnase B
Deglyco-RNase B
Glyco-RB
Undigested
Digested
No P
PNGaseF dilution fold
1x 2x 4x 8x 16x 32x M12
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% D
eg
lyco
syla
tio
n
HPLC analysis)
Control PNGaseF 4C w10
SDS-PAGE
Fully automated denaturation, deglycosylation, dye labeling and
cleaning, and glycan elution.
Heating
(denaturation/deglycosylation and dye
labeling)
Magnetic beads cleaning
APTS labeled glycan elution
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Figure 5. Comparable N-glycan Profiles from Manual
and Automated Workflows
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Relative Areas
Name Ave CV%
G0 0.43 3.41
G0F 17.21 4.32
G0FB 4.01 4.51
G1 1.09 4.32
G1F 19.92 2.45
G1F' 9.67 1.44
G1FB 5.92 0.71
G1FB' 2.09 2.17
G2 0.70 3.73
G2F 16.61 3.26
G2FB 2.01 4.39
G1FS1 1.92 5.17
A1 0.26 5.72
A1F 8.84 6.40
A1FB 2.64 5.78
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Name Average CV%
G0 0.5 1.7
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G1F' 9.7 1.0
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G2 0.3 8.9
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Human IgG Glycan samples prepared on two instruments
demonstrated consistent glycan profiles..
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Add sample Start run Analyze
Glycan
1 2 3
Protein
denatura
tion
Glycan
release
Glycan
labeling
Free dye
clean up
Automate Express
Glycan analysis on CE or LC
- Add protein
samples to a tube
- Start the run Automated Glycan Sample Preparation
Fully automated and streamline workflow for glycoprotein denaturation
and deglycosylation, APTS labeling of the released glycans and
cleaning of the excess free dyes.
